U.S. patent application number 10/281644 was filed with the patent office on 2003-06-26 for compositions and methods for the diagnosis, prevention and treatment of tumor progression.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc., a Delaware corporation. Invention is credited to Shyjan, Andrew W..
Application Number | 20030119740 10/281644 |
Document ID | / |
Family ID | 27021775 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119740 |
Kind Code |
A1 |
Shyjan, Andrew W. |
June 26, 2003 |
Compositions and methods for the diagnosis, prevention and
treatment of tumor progression
Abstract
The present invention relates to methods and compositions for
the diagnosis, prevention, and treatment of tumor progression in
cells involved in human tumors such as melanomas, breast,
gastrointestinal, lung, and bone tumors, various types of skin
cancers, and other neoplastic conditions such as leukemias and
lymphomas. Genes are identified that are differentially expressed
in benign (e.g., non-malignant) tumor cells relative to malignant
tumor cells exhibiting a high metastatic potential. Genes are also
identified via the ability of their gene products to interact with
gene products involved in the progression to, and/or aggressiveness
of, neoplastic tumor disease states. The genes and gene products
identified can be used diagnostically or for therapeutic
intervention.
Inventors: |
Shyjan, Andrew W.; (Nahant,
MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Millennium Pharmaceuticals, Inc., a
Delaware corporation
|
Family ID: |
27021775 |
Appl. No.: |
10/281644 |
Filed: |
October 28, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10281644 |
Oct 28, 2002 |
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08862442 |
May 23, 1997 |
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08862442 |
May 23, 1997 |
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08623679 |
Mar 29, 1996 |
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5674739 |
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08623679 |
Mar 29, 1996 |
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08412431 |
Mar 29, 1995 |
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5633161 |
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Current U.S.
Class: |
506/17 ;
435/320.1; 435/325; 435/6.14; 435/7.23; 514/19.3; 514/19.4;
514/19.6; 530/350; 536/23.5 |
Current CPC
Class: |
C12N 15/1034 20130101;
C07K 14/82 20130101; A61K 38/00 20130101; A01K 2217/075 20130101;
A01K 2217/05 20130101 |
Class at
Publication: |
514/12 ; 435/6;
435/7.23; 435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
A61K 038/17; C12Q
001/68; G01N 033/574; C07H 021/04; C12P 021/02; C12N 005/06; C07K
014/435 |
Claims
What is claimed is:
1. An isolated nucleic acid comprising the nucleotide sequence SEQ
ID NO:2 as depicted in FIG. 3, or the nucleotide sequence of a gene
or gene fragment contained in the following clone as deposited with
the NRRL: pFOMY030 (NRRL accession No. B-21416).
2. An isolated nucleic acid which encodes the amino acid sequence
depicted in FIG. 3 from amino acid residue number 1 to 542, or its
complement.
3. An isolated nucleic acid which hybridizes to an isolated nucleic
acid which encodes the amino acid sequence depicted in FIG. 3 from
amino acid residue number 1 to 542, or its complement.
4. A genetically engineered host cell comprising the nucleotide
sequence of claim 2.
5. An expression vector comprising the nucleotide sequence of claim
2 in operative association with a nucleotide sequence regulatory
element that controls expression of the nucleotide sequence in a
host cell.
6. A substantially pure gene product encoded by the nucleic acid of
claim 3.
7. An isolated nucleic acid comprising the nucleotide sequence SEQ
ID NO:6 as depicted in FIG. 5, or SEQ ID NO:8 as depicted in FIG.
6.
8. An isolated nucleic acid which encodes the amino acid sequence
depicted in FIG. 5 from amino acid residue number 1 to 1497, or its
complement.
9. An isolated nucleic acid which encodes the amino acid sequence
depicted in FIG. 6 from amino acid residue number 1 to 1533, or its
complement.
10. An isolated nucleic acid which hybridizes to a nucleic acid
that encodes the amino acid sequence depicted in FIG. 5 from amino
acid residue number 1 to 1497 (SEQ ID NO:7), or its complement, or
the amino acid sequence depicted in FIG. 6 from amino acid residue
number 1 to 1533 (SEQ ID NO:9), or its complement.
11. A nucleotide vector containing the nucleotide sequence of claim
10.
12. A genetically engineered host cell containing the nucleotide
sequence of claim 10.
13. An expression vector containing the nucleotide sequence of
claim 10 in operative association with a nucleotide sequence
regulatory element that controls expression of the nucleotide
sequence in a host cell.
14. A substantially pure gene product encoded by the nucleic acid
of claim 10.
15. An antibody that immunospecifically binds the gene product of
claim 14.
16. A method of diagnosing tumor progression in a mammal, said
method comprising: obtaining a test sample of tissue cells from the
mammal; obtaining a control sample of known normal cells from the
same type of tissue; and detecting in both the test sample and the
control sample the level of expression of a gene transcript or gene
product of gene 030, wherein a level of expression lower in the
test sample than in the control sample indicates a tumor
progression state in the test sample.
17. A method for treating tumor progression in a mammal, said
method comprising administering to the mammal a compound in an
amount effective to increase the level of expression or activity of
a gene transcript or gene product of gene 030 in cells exhibiting a
tumor progression state, to a level effective to ameliorate
symptoms of the tumor progression state.
18. A method of claim 17, wherein the compound comprises a nucleic
acid whose administration results in an increase in the level of
the differentially expressed gene transcript and gene product in
the cells in the tumor progression state, thereby ameliorating
symptoms of the tumor progression state.
19. A method of claim 18, wherein the nucleic acid comprises a
nucleic acid of gene 030.
20. A method of claim 17, wherein the compound is a 030 gene
product.
21. A method of claim 17, wherein the tumor progression state is
neoplasia.
22. A method of claim 17, wherein the tumor progression state is
metastasis.
23. A method for inhibiting tumor progression in a mammal, said
method comprising administering to the mammal a normal allele of a
030 gene so that the normal gene product is expressed, thereby
inhibiting tumor progression.
24. A method of claim 23, wherein the tumor progression is
metastasis.
25. A method for treating tumor progression in a mammal, said
method comprising administering to the mammal an effective amount
of a 030 gene product.
26. A method of claim 25, wherein the tumor progression is
metastasis.
27. A method of monitoring the efficacy of a compound in clinical
trials for inhibition of tumor progression in a patient, said
method comprising obtaining a first sample of tumor tissue cells
from the patient; administering the compound to the patient; after
a time sufficient for the compound to inhibit tumor progression,
obtaining a second sample of tumor tissue cells from the patient;
and detecting in the first and second samples the level of
expression of a 030 gene transcript or product, wherein a level of
expression higher in the second sample than in the first sample
indicates that the compound is effective to inhibit tumor
progression in the patient.
28. A method of claim 27, wherein the 030 gene transcript or gene
product is differentially expressed in individuals predisposed to a
metastatic neoplastic disease.
Description
[0001] This is a continuation-in-part of U.S. Ser. No.
08/412,431.
1. INTRODUCTION
[0002] The present invention relates to methods and compositions
for the diagnosis, prevention and treatment of tumor progression in
mammals, for example, humans. The different types of tumors may
include, but are not limited to, human melanomas, breast,
gastrointestinal tumors such as esophageal, stomach, duodenal,
colon, colorectal and rectal cancers, prostate, bladder,
testicular, ovarian, uterine, cervical, brain, lung, bronchial,
larynx, pharynx, liver, pancreatic, thyroid, bone, various types of
skin cancers and neoplastic conditions such as leukemias and
lymphomas. Specifically, genes which are differentially expressed
in tumor cells relative to normal cells and/or relative to tumor
cells at a different stage of tumor progression are identified. For
example, genes are identified which are differentially expressed in
benign (e.g., non-malignant) tumor cells relative to malignant
tumor cells exhibiting a high metastatic potential. Genes are also
identified via the ability of their gene products to interact with
gene products involved in the progression to and/or aggressiveness
of neoplastic tumor disease states. The genes identified can be
used diagnostically or as targets for therapeutic intervention. In
this regard, the present invention provides methods for the
identification of compounds useful in the diagnosis, prevention and
therapeutic treatment of tumor progression, including, for example,
metastatic neoplastic disorders. The present invention also
provides methods for the identification of compounds useful in the
diagnosis, prevention and therapeutic treatment of tumor
progression, including, for example, pre-neoplastic and/or benign
states. Additionally, methods are provided for the diagnostic
evaluation and prognosis of conditions involving tumor progression,
for the identification of subjects exhibiting a predisposition to
such conditions, for monitoring patients undergoing clinical
evaluation for the prevention and treatment of tumor progression
disorders, and for monitoring the efficacy of compounds used in
clinical trials.
2. BACKGROUND OF THE INVENTION
[0003] Cancer is the second leading cause of death in the United
States, after heart disease (Boring, C. C. et al., 1993, CA Cancer
J. Clin. 43:7), and develops in one in three Americans, and one of
every four Americans dies of cancer. Cancer is characterized
primarily by an increase in the number of abnormal, or neoplastic,
cells derived from a given normal tissue which proliferate to form
a tumor mass, the invasion of adjacent tissues by these neoplastic
tumor cells, and the generation of malignant cells which spread via
the blood or lymphatic system to regional lymph nodes and to
distant sites. The latter progression to malignancy is referred to
as metastasis.
[0004] Cancer can be viewed as a breakdown in the communication
between tumor cells and their environment, including their normal
neighboring cells. Signals, both growth-stimulatory and
growth-inhibitory, are routinely exchanged between cells within a
tissue. Normally, cells do not divide in the absence of stimulatory
signals, and, likewise, will cease dividing in the presence of
inhibitory signals. In a cancerous, or neoplastic, state, a cell
acquires the ability to "override" these signals and to proliferate
under conditions in which normal cells would not grow.
[0005] Tumor cells must acquire a number of distinct aberrant
traits to proliferate. Reflecting this requirement is the fact that
the genomes of certain well-studied tumors carry several different
independently altered genes, including activated oncogenes and
inactivated tumor suppressor genes. Each of these genetic changes
appears to be responsible for imparting some of the traits that, in
aggregate, represent the full neoplastic phenotype (Land, H. et
al., 1983, Science 222:771; Ruley, H. E., 1983, Nature 304:602;
Hunter, T., 1991, Cell 64:249).
[0006] In addition to unhindered cell proliferation, cells must
acquire several traits for tumor progression to occur. For example,
early on in tumor progression, cells must evade the host immune
system. Further, as tumor mass increases, the tumor must acquire
vasculature to supply nourishment and remove metabolic waste.
Additionally, cells must acquire an ability to invade adjacent
tissue, and, ultimately, cells often acquire the capacity to
metastasize to distant sites.
[0007] The biochemical basis for immune recognition of tumor cells
is unclear. It is possible that the tumorigenicity of cells can
increase when the cells' display of Class I histocompatability
antigens is reduced (Schrier, P. I. et al., 1983, Nature 305:771),
in that these antigens, in conjunction with tumor-specific antigens
are required for the tumor cells to be recognized by cytotoxic T
lymphocytes (CTLs). Tumor cells which have lost one or more genes
encoding tumor-specific antigens seem to escape recognition by the
corresponding reactive CTLs (Van der Bruggen, P. et al., 1991,
Science 25:1643).
[0008] Once a tumor reaches more than about 1 mm in diameter, it
can no longer rely on passive diffusion for nutrition and removal
of metabolic waste. At this point, the tumor mass must make
intimate contact with the circulatory system. Thus, cells within
more advanced tumors secrete angiogenic factors which promote
neovascularization, i.e., the growth of blood vessels from
surrounding tissue into the tumor mass (Folkman, J. and Klagsburn,
M., 1987, Science 235:442; Liotta, L. A. et al., 1991, Cell
64:327). Among these angiogenic factors are the fibroblast growth
factor (FGF) and endothelial cell growth factor (ECGF).
Neovascularization can, in fact, be an essential precursor to
metastasis. First, the process is required for a large increase in
tumor cell number, which in turn, allows the appearance of rare
metastatic variants. Further, neovascularization provides a direct
portal entry into the circulatory system which can be used by
metastasizing cells.
[0009] A variety of biochemical factors have been associated with
different phases of metastases. Cell surface receptors for
collagen, glycoproteins such as laminin, or proteoglycans,
facilitate tumor cell attachment, an important step in invasion and
metastases. Attachment then triggers the release of degradative
enzymes which facilitate the penetration of tumor cells through
tissue barriers. Once the tumor cell has entered the target tissue,
specific growth factors are required for further proliferation.
[0010] It is apparent that the complex process of tumor progression
must involve multiple gene products. It is therefore important to
define the role of specific genes involved in tumor progression, to
identify those gene products involved in the tumor progression
process and to further identify those gene products which can serve
as therapeutic targets for the diagnosis, prevention and treatment
of metastases of various forms of cancers.
[0011] Some attempts have been made to study genes which are
thought to elicit or augment tumor progression phenotypes.
Mutations may drive a wave of cellular multiplication associated
with gradual increases in tumor size, disorganization and
malignancy. For example, a mutation in the tumor suppressor gene
which is a negative regulator of cellular proliferation, results in
a loss of crucial control over tumor growth and progression.
Differential expression of the following suppressor genes has been
demonstrated in human cancers: the retinoblastoma gene, RB; the
Wilms' tumor gene, WT1 (11p); the gene deleted in colon carcinoma,
DCC (18q); the neurofibromatosis type 1 gene, NF1 (17q); and the
gene involved in familial adenomatous polyposis coli, APC (5q)
(Vogelstein, B. and Kinzler, K. W., 1993, Trends Genet.
9:138-141).
[0012] Insight into the complex events that lead from normal
cellular growth to neoplasia, invasion and metastasis is crucial
for the development of effective diagnostic and therapeutic
strategies. The foregoing studies are aimed at defining the role of
particular gene products presumed to be involved in tumor
progression. However, such approaches cannot identify the full
panoply of gene products that are involved in the cascade of steps
in tumor progression. A great need, therefore, exists for the
successful identification of those genes which are differentially
expressed in cells involved in or predisposed to a tumor
progression phenotype. Such differentially expressed gene and/or
gene products can represent useful diagnostic markers and/or
therapeutic targets for tumor progression disorders. With respect
to diagnostic techniques, such genes and/or gene products could
represent useful markers for the diagnosis, especially early
diagnosis, given the correlation between early diagnosis and
successful cancer treatment. With respect to therapeutic
treatments, such differentially expressed genes and/or gene
products could represent useful targets for therapeutic treatment
of various forms of tumor progression disorders, including
metastatic and non-metastatic neoplastic disorders, and for
inhibiting the progression of pre-neoplastic lesions (e.g.,
hyperplastic lesions or other benign tumors) to malignant
tumors.
[0013] Differentially expressed genes involved in tumor metastasis
have been identified using murine melanoma cell lines of varying
metastatic potentials, N-nitroso-methylurea-induced rat mammary
carcinomas, mammary carcinoma cell lines, human breast tumors and
spontaneous colonic and intestinal tumors in mice (Steeg, P. S., et
al., 1988, J. Natl. Cancer Inst. 80:200-204; Qian, F., et al.,
1994, Cell 77:335-347; Leone, A., et al., 1991, 65:25-35; Zou, Z.,
et al., 1994, Science 263:526-529; and Fodde, R., et al., 1994,
Proc. Natl. Acad. Sci. USA 91:8969-8973).
3. SUMMARY OF THE INVENTION
[0014] The present invention relates to methods and compositions
for diagnosis, prevention, and treatment of tumor progression.
Specifically, murine and human genes are identified and described
which are differentially expressed in tumor cells relative to
normal cells and/or to tumor cells at a different stage of tumor
progression. For example, genes are identified which are
differentially expressed in benign (e.g., non-malignant) tumor
cells relative to malignant, metastatic tumor cells. The modulation
of the expression of the identified genes and/or the activity of
the identified gene products can be utilized therapeutically to
treat disorders involving tumor progression, including, for
example, metastatic disorders. As such, methods and compositions
are described for the identification of novel therapeutic compounds
for the inhibition of tumor progression and the treatment of tumor
progression disorders, including metastatic diseases.
[0015] Further, the identified genes and/or gene products can be
used to identify cells exhibiting or predisposed to a disorder
involving a tumor progression phenotype, thereby diagnosing
individuals having, or at high risk for developing, such disorders.
Additionally, the identified genes and/or gene products can be used
to grade or stage identified tumor cells. Still further, the
detection of the differential expression of identified genes can be
used to devise treatments (for example, chemoprevention) before the
benign cells attain a malignant state. Still further, the detection
of differential expression of identified genes can be used to
design a preventive intervention in pre-neoplastic cells in
individuals at high risk.
[0016] "Tumor progression," as used herein, refers to any event
which, first, promotes the transition of a normal, non-neoplastic
cell to a cancerous, neoplastic one. Such events include ones which
occur prior to the onset of neoplasia, and which predispose, or act
as a step toward, the cell becoming neoplastic. These events can,
for example, include ones which cause a normal cell to exhibit a
pre-neoplastic phenotype. Second, such events also include ones
which bring about the transition from a pre-neoplastic state to a
neoplastic one. Such events can, for example, include ones which
promote two hallmarks of the neoplastic state, namely unhindered
cell proliferation and/or tumor cell invasion of adjacent tissue.
Third, tumor progression can include events which promote the
transition of a tumor cell to a metastatic state. Within each
state, (e.g., pre-neoplastic, neoplastic and metastatic) the term
"tumor progression" as used herein can also refer to the disorder
severity or aggressiveness a cell exhibits relative to other cells
within the same state.
[0017] Because multiple tumor progression events occur as a cell
progresses from normal to neoplastic and metastatic states, certain
cells will have undergone a different set of such tumor progression
events. As such, such cells are referred to herein as belonging to
different "tumor progression stages."
[0018] A "disorder involving tumor progression" or a "tumor
progression disorder," as used herein, refers to the state of a
cell or cells which have undergone or are in the process of
undergoing a tumor progression event, as defined above.
[0019] "Differential expression," as used herein, refers to both
quantitative, as well as qualitative, differences in the genes'
temporal and/or cellular expression patterns among, for example,
normal and neoplastic tumor cells, and/or among tumor cells which
have undergone different tumor progression events. Differentially
expressed genes can represent "fingerprint genes," and/or "target
genes."
[0020] "Fingerprint gene," as used herein, refers to a
differentially expressed gene whose expression pattern can be
utilized as part of a prognostic or diagnostic marker for the
evaluation of a disorder involving tumor progression, or which,
alternatively, can be used in methods for identifying compounds
useful for the treatment of such disorders. For example, the effect
of the compound on the fingerprint gene expression normally
displayed in connection with disorders involving tumor progression
can be used to evaluate the efficacy of the compound as a treatment
for such a disorder, or can, additionally, be used to monitor
patients undergoing clinical evaluation for the treatment of the
disorder.
[0021] "Fingerprint pattern," as used herein, refers to the pattern
generated when the expression pattern of a series (which can range
from two up to all the fingerprint genes which exist for a given
state) of fingerprint genes is determined. A fingerprint pattern
can be used in the same diagnostic, prognostic and compound
identification methods as the expression of a single fingerprint
gene.
[0022] "Target gene," as used herein, refers to a differentially
expressed gene involved in tumor progression such that modulation
of the level of target gene expression or of target gene product
activity can act to prevent and/or ameliorate symptoms of the tumor
progression. Compounds that modulate the expression of the target
gene or the activity of the target gene product can be used in the
treatment of neoplastic diseases, including, for example, disorders
involving the progression to a metastatic state. Still further,
compounds that modulate the expression of the target gene or
activity of the target gene product can be used in treatments to
prevent benign cells from attaining a malignant state. Still
further, compounds that modulate the expression of the target gene
or activity of the target gene product can be used to design a
preventive intervention in pre-neoplastic cells in individuals at
high risk.
[0023] Further, "pathway genes" are defined via the ability of
their products to interact with other gene products involved in
tumor progression disorders. Pathway genes can also exhibit target
gene and/or fingerprint gene characteristics.
[0024] The present invention includes the products of such
fingerprint, target, and pathway genes, as well as antibodies to
such gene products. Furthermore, the engineering and use of
cell-based and/or animal-based models of tumor progression
disorders, including disorders involving metastasis, to which such
gene products can contribute, are described.
[0025] The present invention also relates to methods for prognostic
and diagnostic evaluation of tumor progression conditions, and for
the identification of subjects containing cells predisposed to such
conditions. Furthermore, the invention provides methods for
evaluating the efficacy of therapies for disorders involving tumor
progression, and for monitoring the progress of patients
participating in clinical trials for the treatment of such
diseases.
[0026] The tumor progression disorders described herein can include
disorders involved in the progression of such human cancers as, for
example, human melanomas, breast, gastrointestinal, such as
esophageal, stomach, colon, bowel, colorectal and rectal cancers,
prostate, bladder, testicular, ovarian, uterine, cervical, brain,
lung, bronchial, larynx, pharynx, liver, pancreatic, thyroid, bone,
leukemias, lymphomas, and various types of skin cancers.
[0027] The invention also provides methods for the identification
of compounds that modulate the expression of genes or the activity
of gene products involved in tumor progression, including the
progression of metastatic neoplastic diseases, as well as methods
for the treatment of such diseases. Such methods can, for example,
involve the administration of such compounds to individuals
exhibiting symptoms or markers of tumor progression, such as
markers for metastatic neoplastic diseases.
[0028] This invention is based, in part on systematic search
strategies involving in vivo and in vitro paradigms of tumor
progression, including the progression to metastatic disease,
coupled with sensitive and high throughput gene expression assays,
to identify genes differentially expressed in tumor cells relative
to normal cells and/or relative to tumor cells at a different tumor
progression stage. In contrast to approaches that merely evaluate
the expression of a given gene product presumed to play a role in
one or another of the various stages of tumor progression, such as,
for example the progression to a metastatic disease process, the
search strategies and assays used herein permit the identification
of all genes, whether known or novel, which are differentially
expressed in tumor cells relative to normal cells or relative to
tumor cells at a different stage of tumor progression.
[0029] This comprehensive approach and evaluation permits the
discovery of novel genes and gene products, as well as the
identification of an array of genes and gene products (whether
novel or known) involved in novel pathways that play a major role
in the disease pathology. Thus, the present invention makes
possible the identification and characterization of targets useful
for prognosis, diagnosis, monitoring, rational drug design, and/or
other therapeutic intervention of tumor progression disorders,
including disorders involving metastasis.
[0030] The Example presented in Section 6, below, demonstrates the
successful use of tumor progression search strategies of the
invention to identify genes which are differentially expressed
within tumor cells relative to tumor cells at a different stage of
tumor progression. Specifically, the Example identifies a gene
which is differentially expressed in metastatic cell populations
relative to benign, non-malignant tumor cells.
[0031] This gene, referred to herein as the 030 gene (fomyo30 in
the mouse and fohy030 in humans), is a novel gene which is
expressed at a many-fold higher level in non-metastatic tumor cells
relative to its expression in metastatic tumor cells. The gene
appears in mice and has the cDNA sequence shown in FIGS. 3A and 3B
(SEQ ID NO:2). A homologous gene, referred to herein as the fohy030
gene, appears in humans and has the cDNA sequence shown in FIG. 5
(SEQ ID NO:6). An alternative splice form of the human cDNA has the
sequence shown in FIG. 6 (SEQ ID NO:8). Unless stated expressly
otherwise, any general reference to the 030 gene hereinafter refers
to both the murine (fomy030) and human (fohy030) homologs of this
gene.
[0032] The identification of the 030 gene and the characterization
of its expression in particular stages of metastatic spread
provides, therefore, newly identified targets for the diagnosis,
prevention, and treatment of tumor progression disorders, including
metastatic neoplastic diseases.
[0033] Its expression pattern indicates that the 030 gene product
acts to inhibit tumor progression. For example, a reduction in the
level of 030 gene expression correlates with an increase in a
cell's metastatic potential ie., a reduction of 030 gene product in
tumor cells can induce or predispose a cell to progress to a
metastatic state.
[0034] Hence, any method which can bring about an increase in the
amount of 030 gene product can inhibit or slow the progression to
metastasis. In fact, it is possible that the 030 gene product
exhibits general tumor inhibition properties.
[0035] A cDNA clone of the murine homolog, designated fomy030, is
described herein in FIGS. 3A and 3B (SEQ ID NO:2) (nucleotide
sequence and amino acid sequence), and was derived from fomy030
mRNA. However, as used herein, fomy030cDNA refers to any DNA
sequence that encodes the amino acid sequence depicted in FIGS. 3A
and 3B (SEQ ID NO:3).
[0036] A cDNA clone of the human homolog, designated fohy030, is
shown in FIG. 5 (SEQ ID NO:6) (nucleotide sequence and amino acid
sequence). An alternative splice form of fohy030 is shown in FIG. 6
(SEQ ID NO:8). Both were obtained using the entire mouse fomy030
cDNA as a probe. However, as used herein, fohy030 cDNA refers to
any DNA sequence that encodes the amino acid sequences depicted in
FIG. 5 (SEQ ID NO:7) and FIG. 6 (SEQ ID NO:9).
3.1. Definitions
[0037] "Tumor progression," as used herein, refers to any event
which, first promotes the transition of a normal, non-neoplastic
cell to a cancerous, neoplastic one. Such events include ones which
occur prior to the onset of neoplasia, and which predispose, or act
as a step toward, the cell becoming neoplastic. These events can,
for example, include ones which cause a normal cell to exhibit a
pre-neoplastic phenotype. Second, such events also include ones
which bring about the transition from a pre-neoplastic state to a
neoplastic one. Such events can, for example, include ones which
promote unhindered cell proliferation and/or tumor cell invasion of
adjacent tissue, which are viewed as hallmarks of the neoplastic
state. Third, tumor progression can include events which promote
the transition of a tumor cell to a metastatic state. Within each
state, (e.g., pre-neoplastic, neoplastic and metastatic) the term
"tumor progression" as used herein can also refer to the disorder
severity or aggressiveness a cell exhibits.
[0038] Because multiple tumor progression events occur as a cell
progresses from a normal to neoplastic and metastatic states,
certain cells will have undergone a different set of such tumor
progression events. As such, such cells are referred to herein as
belonging to different "tumor progression stages."
[0039] A "disorder involving tumor progression" or a "tumor
progression disorder," as used herein, refers to the state of a
cell or cells which have undergone or are in the process of
undergoing a tumor progression event, as defined above.
[0040] "Differential expression," as used herein, refers to both
quantitative, as well as qualitative differences in the genes'
temporal and/or cellular expression patterns among, for example,
normal and neoplastic tumor cells, and/or among tumor cells which
have undergone different tumor progression events. Differentially
expressed genes can represent "fingerprint genes," and/or "target
genes."
[0041] "Fingerprint gene," as used herein, refers to a
differentially expressed gene whose expression pattern can be
utilized as part of a prognostic or diagnostic marker for the
evaluation of tumor progression, or which, alternatively, can be
used in methods for identifying compounds useful for the treatment
of tumor progression. For example, the effect of the compound on
the fingerprint gene expression normally displayed in connection
with tumor progression can be used to evaluate the efficacy of the
compound as a treatment for tumor progression, or can,
additionally, be used to monitor patients undergoing clinical
evaluation for the treatment of tumor progression.
[0042] "Fingerprint pattern," as used herein, refers to the pattern
generated when the expression pattern of a series (which can range
from two up to all the fingerprint genes which exist for a given
state) of fingerprint genes is determined. A fingerprint pattern
can be used in the same diagnostic, prognostic and compound
identification methods as the expression of a single fingerprint
gene.
[0043] "Target gene," as used herein, refers to a differentially
expressed gene involved in tumor progression such that modulation
of the level of target gene expression or of target gene product
activity can act to prevent and/or ameliorate symptoms of the tumor
progression. Compounds that modulate target gene expression or
activity of the target gene product can be used in the treatment of
tumor progression and tumor progression disorders, including, for
example, disorders involving the progression to a metastatic
state.
[0044] Further, "pathway genes" are defined via the ability of
their products to interact with other gene products involved in
tumor progression. Pathway genes can also exhibit target gene
and/or fingerprint gene characteristics.
4. DESCRIPTION OF THE FIGURES
[0045] FIG. 1 is a Northern blot confirming differential regulation
of the 030 gene. Total RNA (12 .mu.g/lane) obtained from F1 (lanes
1 and 3) and F10 (lanes 2 and 4) melanoma cell cultures was
hybridized with a cDNA probe prepared by random priming of
reamplified romy030 band. (See materials and methods below in
Section 6.1.). The romy030 probe identifies an RNA band of
approximately 3 kb, corresponding to a fomy030 mRNA.
[0046] FIG. 2 is a nucleotide sequence of romy030 band (SEQ ID
NO:1).
[0047] FIGS. 3A and 3B are representations of the nucleotide and
derived amino acid sequences of cDNA clone fomy030 (SEQ ID NOs:2
[nucleotide sequence] and 3 [amino acid sequence]) derived from
fomy030 mRNA.
[0048] FIG. 4 is a Northern blot analysis confirming differential
regulation of the fomy030 gene. Lane 1 is B16 F1, lane 2 is B16
F10, and lanes 3-6 are B16 H5, B16 H6, B16 H7 and B16 H8.
[0049] FIG. 5 is a representation of the nucletide and deduced
amino acid sequences of cDNA clone of fohy030 (SEQ ID NOs:6
(nucleotide sequence) and 7 [amino acid sequence]).
[0050] FIG. 6 is a comparison of the nucletide and deduced amino
acid sequences of another cDNA clone of fohy030 (SEQ ID NOs:8
[nucleotide sequence] and 9 [amino acid sequence]).
[0051] In FIGS. 3A and 3B, the nucleotide sequence is numbered
starting at the first nucleotide, whereas in FIGS. 5 and 6, the
nucleotide sequence is numbered starting at the ATG start
codon.
5. DETAILED DESCRIPTION OF THE INVENTION
[0052] Methods and compositions for the prevention, treatment and
diagnosis of tumor progression, including tumor progression
involving metastatic disorders, in cells involved in human tumors.
Such human tumors may include, for example, human melanomas,
breast, gastrointestinal tumors such as esophageal, stomach,
duodenal, colon, colorectal and rectal cancers, prostate, bladder,
testicular, ovarian, uterine, cervical, brain, lung, bronchial,
larynx, pharynx, liver, pancreatic, thyroid, bone, various types of
skin cancers and other neoplastic conditions such as leukemias,
lymphomas. The invention is based, in part, on the evaluation and
expression and role of all genes that are differentially expressed
in tumor cells relative to normal cells and/or relative to tumor
cells at a different stage of tumor progression. This permits the
definition of disease pathways and identification of targets in
such pathways that are useful for diagnosis, prevention and
treatment of tumor progression, including the tumor progression
disorders involving metastatic neoplastic diseases.
[0053] Genes, termed "target genes" and/or "fingerprint genes" are
described which are differentially expressed in tumor cells
relative to their expression in normal cells or relative to their
expression in tumor cells which are at a different stage of tumor
progression. Additionally, genes, termed "pathway genes" are
described whose gene products exhibit an ability to interact with
gene products involved tumor progression, including tumor
progression disorders involving metastatic neoplastic disorders.
Pathway genes can additionally have fingerprint and/or target gene
characteristics. Methods for the identification of such
fingerprint, target, and pathway genes are also described.
[0054] Further, the gene products of such fingerprint, target, and
pathway genes are described in Section 5.2.2, antibodies to such
gene products are described in Section 5.2.3, as are cell-and
animal-based models of tumor progression disorders to which such
gene products can contribute, in Section 5.2.4.
[0055] Methods for the identification of compounds which modulate
the expression of genes or the activity of gene products involved
in tumor progression are described in Section 5.3. Methods for
monitoring the efficacy of compounds during clinical trials are
described in Section 5.3.5. Additionally described, below, are
methods for treatment of tumor progression disorders, including
metastatic diseases.
[0056] Also discussed, below, are methods for prognostic and
diagnostic evaluation of tumor progression and disorders involving
tumor progression, including metastatic disorders, and, further,
for the identification of subjects exhibiting a predisposition to
such disorders.
5.1 Identification of Differentially Expressed Genes
[0057] Described herein are methods for the identification of
differentially expressed genes which are involved in tumor
progression. There exist a number of levels or stages at which the
differential expression of such genes can be exhibited. For
example, differential expression can occur in tumor cells relative
to normal cells, or in tumor cells within different stages of tumor
progression. For example, genes can be identified which are
differentially expressed in pre-neoplastic versus neoplastic cells.
Such genes can include, for example, ones which promote unhindered
cell proliferation or tumor cell invasion of adjacent tissue, both
of which are viewed as hallmarks of the neoplastic state. Further,
differential expression can occur in benign (e.g., non-malignant)
tumor cells versus metastatic, malignant tumor cells. Still
further, differential expression can occur among cells within any
one of these states (e.g., pre-neoplastic, neoplastic and
metastatic), and can indicate, for example, a difference in tumor
progression severity or aggressiveness of one cell relative to that
of another cell within the same state.
[0058] Methods for the identification of such differentially
expressed genes are described, below, in Section 5.1.1. Methods for
the further characterization of such differentially expressed
genes, and for their categorization as target and/or fingerprint
genes, are presented, below, in Section 5.3.
[0059] "Differential expression" as used herein refers to both
quantitative, as well as qualitative differences in the genes'
temporal and/or tissue expression patterns. Thus, a differentially
expressed gene can qualitatively have its expression activated or
completely inactivated in, for example, normal versus tumor
progression states, in cells within different tumor progression
states or among cells within a single given tumor progression
state. Such a qualitatively regulated gene will exhibit an
expression pattern within a given state which is detectable by
standard techniques in one such state, but is not detectable in
both states being compared. "Detectable," as used herein, refers to
an RNA expression level which is detectable via the standard
techniques of differential display, RT (reverse
transcriptase)-coupled PCR, Northern and/or RNase protection
analyses.
[0060] Alternatively, a differentially expressed gene can exhibit
an expression level which differs, i.e., is quantitatively
increased or decreased in normal versus tumor progression states,
in cells within different tumor progression states or among cells
within a single given tumor progression state.
[0061] The degree to which expression differs need only be large
enough to be visualized via standard characterization techniques,
such as, for example, the differential display technique described
below. Other standard, well-known characterization techniques by
which expression differences can be visualized include, but are not
limited to, quantitative RT (reverse transcriptase)-coupled PCR and
Northern analyses and RNase protection techniques.
[0062] Differentially expressed genes can be further described as
target genes and/or fingerprint genes. "Fingerprint gene," as used
herein, refers to a differentially expressed gene whose expression
pattern can be utilized as part of a prognostic or diagnostic
marker in tumor progression evaluation, or which, alternatively,
may be used in methods for identifying compounds useful for the
prevention or treatment of tumor progression and tumor progression
disorders, including metastatic disorders. A fingerprint gene can
also have the characteristics of a target gene or a pathway gene
(see below, in Section 5.2).
[0063] "Fingerprint pattern," as used herein, refers to the pattern
generated when the expression pattern of a series (which can range
from two up to all the fingerprint genes which exist for a given
state) of fingerprint genes is determined. A fingerprint pattern
can be used in the same diagnostic, prognostic and compound
identification methods as the expression of a single fingerprint
gene.
[0064] "Target gene," as used herein, refers to a differentially
expressed gene involved in tumor progression in a manner by which
modulation of the level of target gene expression or of target gene
product activity can act to prevent and/or ameliorate symptoms of
disorders involving tumor progression. Tumor progression disorders
include, for example, disorders involved in human tumors,
including, but not limited to human melanomas, breast,
gastrointestinal, such as esophageal, stomach, colon, bowel,
colorectal and rectal cancers, prostate, bladder, testicular,
ovarian, uterine, cervical, brain, lung, bronchial, larynx,
pharynx, liver, pancreatic, thyroid, bone, leukemias, lymphomas and
various types of skin cancers. A target gene can also have the
characteristics of a fingerprint gene and/or a pathway gene (as
described, below, in Section 5.2).
5.1.1 Methods for the Identification of Differentially Expressed
Genes
[0065] A variety of methods can be utilized for the identification
of genes which are involved in tumor progression. Described in
Section 5.1.1.1 are experimental paradigms which can be utilized
for the generation of samples which can be used for the
identification of such genes. Material generated in paradigm
categories can be characterized for the presence of differentially
expressed gene sequences as discussed, below, in Section
5.1.1.2.
5.1.1.1. Paradigms for the Identification of Differentially
Expressed Genes
[0066] Paradigms which represent models of tumor progression states
are described herein. These paradigms can be utilized for the
identification of genes which are differentially expressed in
normal cells versus cells in tumor progression states, in cells
within different tumor progression states or among cells within a
single given tumor progression state.
[0067] The paradigms described herein include at least two groups
of cells of a given cell type, preferably genetically matched cells
(e.g., cells derived from variants of the same cell line, or cells
derived from a single individual or biological sample), whose
expression patterns are compared and analyzed for differential
expression. Methods for the analysis of paradigm material are
described, below, in Section 5.1.1.2.
[0068] Once a particular gene has been identified through the use
of one paradigm, its expression pattern can be further
characterized, for example, by studying its expression in a
different paradigm. A gene can, for example, be regulated one way,
i.e., can exhibit one differential gene expression pattern, in a
given paradigm, but can be regulated differently in another
paradigm. The use, therefore, of multiple paradigms can be helpful
in distinguishing the roles and relative importance of particular
genes in tumor progression.
[0069] In one embodiment of such a paradigm, referred to herein as
the "in vitro" paradigm, cell lines can be used to identify genes
which are differentially expressed in tumor progression states.
Differentially expressed genes are detected, as described herein,
by comparing the pattern of gene expression between the
experimental and control conditions. In such a paradigm,
genetically matched tumor cell lines (e.g., variants of the same
cell line) are generally utilized. For example, the gene expression
pattern of two variant cell lines can compared, wherein one variant
exhibits characteristics of one tumor progression state while the
other variant exhibits characteristics of another tumor progression
state. Alternatively, two variant cell lines, both of which exhibit
characteristics of the same tumor progression state, but which
exhibit differing degrees of tumor progression disorder severity or
aggressiveness. Further, genetically matched cell lines can be
utilized, one of which exhibits characteristics of a tumor
progression state, while the other exhibits a normal cellular
phenotype.
[0070] The variant cell lines utilized herein can exhibit such
tumor progression characteristics as, for example, a high or low
metastatic potential, which refers to the likelihood that a cell
will give rise to a distant site tumor mass. Alternatively, one or
more such variant cell lines can exhibit pre-neoplastic
characteristics or can exhibit characteristics generally associated
with one or more neoplastic cell phenotypes, such as, for example,
cell proliferation or invasion phenotypes.
[0071] In accordance with this aspect of the invention, the cell
line variants are cultured under appropriate conditions, the cells
are harvested, and RNA is isolated and analyzed for differentially
expressed genes, as described in detail in Section 5.1.1.2,
below.
[0072] Examples of cell lines that can be used as part of such in
vitro paradigms include but are not limited to variants of melanoma
cell lines, such as, for example, the murine melanoma B16 F1 cell
line which exhibits a low metastatic potential and the melanoma B16
F10 cell line which exhibits a high metastatic potential (Fidler,
I. J., 1973, Nature New Biol 242:148-149); human colon cell lines,
such as, for example KM12c (tumor cell line with low metastatic
potential) and the KM20L4 (tumor cell line with high metastatic
potential; Morikawa K., et al., 1988, Cancer Research
48:1943-1948); prostatic tumor cell lines, such as, for example, DU
145 (non metastatic tumor cell line) and PC-3-M (high metastatic
potential tumor cell line; Karmali, R. A. et al., 1987, Anticancer
Res. 7:1173-1180, and Koziowski, J. M. et al., 1984, Cancer
Research 44:3522-3529); and breast carcinoma tumor cell lines, such
as, for example, MCF-7 (non metastatic tumor cell line) and
MDA-MB-435 (high metastatic potential tumor cell line; Watts C. K.
et al., 1994, Breast Cancer Res. Treat. 31:95-105 and Rose, D. P.
et al., 1993, J. Natl. Cancer Inst. 85:1743-1747).
[0073] As presented in the Example presented in Section 6, below,
this paradigm has been successfully utilized to identify a gene,
referred to herein as the 030 gene, which is differentially
expressed in cells exhibiting a high metastatic potential relative
to cells exhibiting a low metastatic potential. Specifically, the
030 gene is expressed at a many-fold higher level in low metastatic
potential cells relative to cells exhibiting a high metastatic
potential.
[0074] In a second paradigm, referred to herein as the in vivo
paradigm, animal models of tumor progression disorders can be
utilized to discover differentially expressed gene sequences. The
in vivo nature of such tumor progression models can prove to be
especially predictive of the analogous responses in living
patients.
[0075] A variety of tumor progression animal models can be used for
as part of the in vivo paradigms. For example, animal models of
tumor progression may be generated by passaging tumor cells in
animals (e.g., mice), leading to the appearance of tumors within
these animals.
[0076] Additional animal models, some of which may exhibit
differing tumor progression characteristics, may be generated from
the original animal models described above. For example, the tumors
which result in the original animals can be removed and grown in
vitro. Cells from these in vitro cultures can then be passaged in
animals and tumors resulting from this passage can then be
isolated. RNA from pre-passage cells, and cells isolated after one
or more rounds of passage can then be isolated and analyzed for
differential expression. The differential expression can be
compared to the metastatic potential expression of such cells.
These cells can now represent cells from different tumor
progression states, or cells within a given tumor progression state
exhibiting differing degrees of severity or aggressiveness. Such
passaging techniques can utilizing any of the variant cell lines
described, above, for the in vitro paradigms.
[0077] Additionally, animal models for tumor progression which can
be utilized for such an in vivo paradigm include any of the animal
models described, below, in Section 5.7.1. Other models include
transgenic mouse model for melanoma (Mintz, B. and Silvers, W. K.,
1993, Proc. Natl. Acad. Sci. USA 90:8817-8812), transgenic mice
which carry specific adenomatous polyposis coli (APC) gene
mutations (Fodde, R., et al., 1994, Proc. Natl. Acad. Sci. USA
91:8969-8973) and the transgenic mouse in which the mammary tumor
virus LTR/c-myc gene is anomalously expressed (Leder, A., et al.,
1986, Cells 45:485-495).
[0078] A third paradigm, referred to herein as the "specimen
paradigm," utilizes samples from surgical and biopsy specimens.
Such specimens can represent normal tissue, primary, secondary or
metastasized tumors obtained from patients having undergone
surgical treatment for disorders involving tumor progression such
as, for example, melanomas, colon carcinomas, lung carcinomas,
prostatic cancers and breast cancers.
[0079] Surgical specimens can be procured under standard conditions
involving freezing and storing in liquid nitrogen (see, for
example, Karmali, R. A., et al., 1983, Br. J. cancer 48:689-696.)
RNA from specimen cells is isolated by, for example, differential
centrifugation of homogenized tissue, and analyzed for differential
expression relative to other specimen cells, preferably cells
obtained from the same patient.
[0080] In paradigms designed to identify genes which are involved
in tumor progression, compounds known to have an ameliorative
effect on the tumor progression symptoms can also be used in
paradigms to detect differentially expressed genes. Such compounds
can include known therapeutics, as well as compounds that are not
useful as therapeutics due to their harmful side effects. For
example, tumor cells that are cultured as explained in this
Section, above, can be exposed to one of these compounds and
analyzed for differential gene expression with respect to untreated
tumor cells, according to the methods described below in Section
5.1.1.2. In principle, however, according to the paradigm, any cell
type involved in tumor progression and disorders thereof can be
treated by these compounds at any stage of the tumor progression
process.
[0081] Cells involved in tumor progression can also be compared to
unrelated cells (e.g., fibroblasts) which have been treated with
the compound, such that any generic effects on gene expression that
might not be related to the disease or its treatment may be
identified. Such generic effects might be manifest, for example, by
changes in gene expression that are common to the test cells and
the unrelated cells upon treatment with the compound.
[0082] By these methods, the genes and gene products upon which
these compounds act can be identified and used in the assays
described below to identify novel therapeutic compounds for
inhibition of tumor progression and the treatment of tumor
progression disorders, including metastatic diseases.
5.1.1.2. Analysis of Paradigm Material
[0083] In order to identify differentially expressed genes, RNA,
either total or mRNA, can be isolated from cells utilized in
paradigms such as those described earlier in Section 5.1.1.1. Any
RNA isolation technique which does not select against the isolation
of mRNA can be utilized for the purification of such RNA samples.
See, for example, Ausubel, F. M. et al., eds., 1987-1993, Current
Protocols in Molecular Biology, John Wiley & Sons, Inc. New
York, which is incorporated herein by reference in its entirety.
Additionally, large numbers of tissue samples can readily be
processed using techniques well known to those of skill in the art,
such as, for example, the single-step RNA isolation process of
Chomczynski, P. (1989, U.S. Pat. No. 4,843,155), which is
incorporated herein by reference in its entirety.
[0084] Transcripts within the collected RNA samples which represent
RNA produced by differentially expressed genes can be identified by
utilizing a variety of methods which are well known to those of
skill in the art. For example, differential screening (Tedder, T.
F. et al., 1988, Proc. Natl. Acad. Sci. USA 85:208-212),
subtractive hybridization (Hedrick, S. M. et al., 1984, Nature
308:149-153; Lee, S. W. et al., 1984, Proc. Natl. Acad. Sci. USA
88:2825), and, preferably, differential display (Liang, P. and
Pardee, A. B., 1993, U.S. Pat. No. 5,262,311, which is incorporated
herein by reference in its entirety), can be utilized to identify
nucleic acid sequences derived from genes that are differentially
expressed.
[0085] Differential screening involves the duplicate screening of a
cDNA library in which one copy of the library is screened with a
total cell cDNA probe corresponding to the mRNA population of one
cell type while a duplicate copy of the cDNA library is screened
with a total cDNA probe corresponding to the mRNA population of a
second cell type. For example, one cDNA probe can correspond to a
total cell cDNA probe of a cell type or tissue derived from a
control subject, while the second cDNA probe can correspond to a
total cell cDNA probe of the same cell type derived from an
experimental subject. Those clones which hybridize to one probe but
not to the other potentially represent clones derived from genes
differentially expressed in the cell type of interest in control
versus experimental subjects.
[0086] Subtractive hybridization techniques generally involve the
isolation of mRNA taken from two different sources, e.g., control
and experimental tissue, the hybridization of the mRNA or
single-stranded cDNA reverse-transcribed from the isolated mRNA,
and the removal of all hybridized, and therefore double-stranded,
sequences. The remaining non-hybridized, single-stranded cDNAs,
potentially represent clones derived from genes that are
differentially expressed in the two mRNA sources. Such
single-stranded cDNAs are then used as the starting material for
the construction of a library comprising clones derived from
differentially expressed genes.
[0087] The differential display technique describes a procedure,
utilizing the well-known polymerase chain reaction (PCR; the
experimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat.
No. 4,683,202) which allows for the identification of sequences
derived from genes which are differentially expressed. First,
isolated RNA is reverse-transcribed into single-stranded cDNA,
utilizing standard techniques which are well known to those of
skill in the art. Primers for the reverse transcriptase reaction
can include, but are not limited to, oligo dT-containing primers,
preferably of the 3' primer type of oligonucleotide described
below. Next, this technique uses pairs of PCR primers, as described
below, which allow for the amplification of clones representing a
random subset of the RNA transcripts present within any given cell.
Utilizing different pairs of primers allows each of the mRNA
transcripts present in a cell to be amplified. Among such amplified
transcripts can be identified those which have been produced from
differentially expressed genes.
[0088] The 3' oligonucleotide primer of the primer pairs can
contain an oligo dT stretch of 10-13 dT nucleotides at its 5' end,
preferably 11, which hybridizes to the poly(A) tail of mRNA or to
the complement of a cDNA reverse transcribed from an mRNA poly(A)
tail. Second, in order to increase the specificity of the 3'
primer, the primer can contain one or more, preferably two,
additional nucleotides at its 3' end. Because, statistically, only
a subset of the mRNA derived sequences present in the sample of
interest will hybridize to such primers, the additional nucleotides
allow the primers to amplify only a subset of the mRNA derived
sequences present in the sample of interest. This is preferred in
that it allows more accurate and complete visualization and
characterization of each of the bands representing amplified
sequences.
[0089] The 5' primer can contain a nucleotide sequence expected,
statistically, to have the ability to hybridize to cDNA sequences
derived from the tissues of interest. The nucleotide sequence can
be an arbitrary one, and the length of the 5' oligonucleotide
primer can range from about 9 to about 15 nucleotides, with about
13 nucleotides being preferred.
[0090] Additionally, arbitrary primer sequences cause the lengths
of the amplified partial cDNAs produced to be variable, thus
allowing different clones to be separated by using standard
denaturing sequencing gel electrophoresis.
[0091] PCR reaction conditions should be chosen which optimize
amplified product yield and specificity, and, additionally, produce
amplified products of lengths which can be resolved utilizing
standard gel electrophoresis techniques. Such reaction conditions
are well known to those of skill in the art, and important reaction
parameters include, for example, length and nucleotide sequence of
oligonucleotide primers as discussed above, and annealing and
elongation step temperatures and reaction times.
[0092] The pattern of clones resulting from the reverse
transcription and amplification of the mRNA of two different cell
types is displayed via sequencing gel electrophoresis and compared.
Differences in the two banding patterns indicate potentially
differentially expressed genes.
[0093] Once potentially differentially expressed gene sequences
have been identified via bulk techniques such as, for example,
those described above, the differential expression of such
putatively differentially expressed genes should be corroborated.
Corroboration can be accomplished via, for example, such well-known
techniques as Northern analysis, quantitative RT-coupled PCR or
RNase protection.
[0094] Upon corroboration, the differentially expressed genes can
be further characterized, and can be identified as target and/or
fingerprint genes, as discussed, below, in Section 5.1.4.
[0095] Also, amplified sequences of differentially expressed genes
obtained through differential display can be used to isolate the
full length clones of the corresponding gene. The full-length
coding portion of the gene can readily be isolated, without undue
experimentation, by molecular biological techniques well known in
the art. For example, the isolated differentially expressed
amplified fragment can be labeled and used to screen a cDNA
library. Alternatively, the labeled fragment can be used to screen
a genomic library.
[0096] PCR technology can also be utilized to isolate full-length
cDNA sequences. As described in this section above, the isolated
amplified gene fragments (of about at least 10 nucleotides,
preferrably longer, of about 15 nucleotides) obtained through
differential display have their 5' terminal end at some random
point within the gene and have 3' terminal ends at a position
corresponding to the 3' end of the transcribed portion of the gene.
Once nucleotide sequence information from an amplified fragment is
obtained, the,remainder of the gene (i.e., the 5' end of the gene,
when-utilizing differential display) can be obtained using, for
example, RT PCR.
[0097] In one embodiment of such a procedure for the identification
and cloning of full length gene sequences, RNA can be isolated,
following standard procedures, from an appropriate tissue or
cellular source.
[0098] A reverse transcription reaction can then be performed on
the RNA using an oligonucleotide primer complementary to the mRNA
that corresponds to the amplified cloned fragment, for the priming
of first strand synthesis. Because the primer is anti-parallel to
the mRNA, extension will proceed toward the 5' end of the mRNA. The
resulting RNA/DNA hybrid can then be "tailed" with guanines using a
standard terminal transferase reaction, the hybrid can be digested
with RNAase H, and second strand synthesis can then be primed with
a poly-C primer. Using the two primers, the 5' portion of the gene
is then amplified using PCR. Sequences obtained can then be
isolated and recombined with previously isolated sequences to
generate a full-length cDNA of the differentially expressed genes
of the invention. For a review of cloning strategies and
recombinant DNA techniques which can be used, see, e.g., Sambrook
et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs
Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in
Molecular Biology, (Green Publishing Associates and Wiley
Interscience, N.Y.).
5.2. Method for the Identification of Pathway Genes
[0099] Methods are described herein for the identification of
pathway genes. "Pathway gene," as used herein, refers to a gene
whose gene product exhibits the ability to interact with gene
products involved in tumor progression. A pathway gene can be
differentially expressed and, therefore, can have the
characteristics of a target and/or fingerprint gene.
[0100] Any method suitable for detecting protein-protein
interactions can be employed for identifying pathway gene products
by identifying interactions between gene products and gene products
known to be involved in tumor progression and tumor progression
disorders, including metastatic disorders. Such known gene products
can be cellular or extracellular proteins. Those gene products
which interact with such known gene products represent pathway gene
products and the genes which encode them represent pathway
genes.
[0101] Among the traditional methods which can be employed are
co-immunoprecipitation, cross-linking and co-purification through
gradients or chromatographic columns. Utilizing procedures such as
these allows for the identification of pathway gene products. Once
identified, a pathway gene product can be used, in conjunction with
standard techniques, to identify its corresponding pathway gene.
For example, at least a portion of the amino acid sequence of the
pathway gene product can be ascertained using techniques well known
to those of skill in the art, such as via the Edman degradation
technique (see, e.g., Creighton, 1983, Proteins: Structures and
Molecular Principles, W. H. Freeman & Co., N.Y., pp.34-49). The
amino acid sequence obtained can be used as a guide for the
generation of oligonucleotide mixtures that can be used to screen
for pathway gene sequences. Screening can be accomplished, for
example by standard hybridization or PCR techniques. Techniques for
the generation of oligonucleotide mixtures and the screening are
well known. (See, e.g., Ausubel, supra., and PCR Protocols: A Guide
to Methods and Applications, 1990, Innis, M. et al., eds. Academic
Press, Inc., New York).
[0102] Additionally, methods can be employed which result in the
simultaneous identification of pathway genes which encode the
protein interacting with a protein involved in tumor progression
and tumor progression disorders, including metastatic diseases.
These methods include, for example, probing expression libraries
with labeled protein known or suggested to be involved in
metastatic diseases using this protein in a manner similar to the
well known technique of antibody probing of .lambda.gt11
libraries.
[0103] One method which detects protein interactions in vivo, the
yeast two-hybrid system, is described in detail for illustration
only and not by way of limitation. One version of this system has
been described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA,
88:9578-9582) and is commercially available from Clontech (Palo
Alto, Calif.).
[0104] Briefly, utilizing such a system, plasmids are constructed
that encode two hybrid proteins: the first hybrid protein consists
of the DNA-binding domain of a transcription factor (e.g.,
activation protein) fused to a known protein, in this case, a
protein known to be involved in tumor progression, and the second
hybrid protein consists of the transcription factor's activation
domain fused to an unknown protein that is encoded by a cDNA which
has been recombined into this plasmid as part of a cDNA library.
The plasmids. are transformed into a strain of the yeast
Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ)
whose expression is regulated by the transcription factor's binding
site. Either hybrid protein alone cannot activate transcription of
the reporter gene. The DNA binding hybrid protein cannot activate
transcription because it does not provide the activation domain
function and the activation domain hybrid protein cannot activate
transcription because it lacks the domain required for binding to
its target site (e.g., it cannot localize to the transcription
activator protein's binding site). Interaction between the DNA
binding hybrid protein and the library encoded protein
reconstitutes the functional transcription factor and results in
expression of the reporter gene, which is detected by an assay for
the reporter gene product.
[0105] The two-hybrid system or related methodology can be used to
screen activation domain libraries for proteins that interact with
a known "bait" gene product. By way of example, and not by way of
limitation, gene products (e.g., 030 gene products) known to be
involved in tumor progression and tumor progression disorders, such
as metastatic diseases, can be used as the bait gene products.
Total genomic or cDNA sequences are fused to the DNA encoding an
activation domain. This library and a plasmid encoding a hybrid of
the bait gene product fused to the DNA-binding domain are
cotransformed into a yeast reporter strain, and the resulting
transformants are screened for those that express the reporter
gene. For example, and not by way of limitation, the bait gene can
be cloned into a vector such that it is translationally fused to
the DNA encoding the DNA-binding domain of the GAL4 protein. The
colonies are purified and the (library) plasmids responsible for
reporter gene expression are isolated. The inserts in the plasmids
are sequenced to identify the proteins encoded by the cDNA or
genomic DNA.
[0106] A cDNA library of a cell or tissue source which expresses
proteins predicted to interact with the bait gene product can be
made using methods routinely practiced in the art. According to the
particular system described herein, the library is generated by
inserting the cDNA fragments into a vector such that they are
translationally fused to the activation domain of GAL4. This
library can be co-transformed along with the bait gene-GAL4 fusion
plasmid into a yeast strain which contains a lacZ gene whose
expression is controlled by a promoter which contains a GAL4
activation sequence. A cDNA encoded protein, fused to GAL4
activation domain, that interacts with the bait gene product will
reconstitute an active GAL4 transcription factor and thereby drive
expression of the lacZ gene. Colonies which express lacZ can be
detected by their blue color in the presence of X-gal. cDNA
containing plasmids from such a blue colony can then be purified
and used to produce and isolate the bait gene product interacting
protein using techniques routinely practiced in the art.
[0107] Once a pathway gene has been identified and isolated, it can
be further characterized as, for example, discussed below, in
Section 5.3.
5.3. Characterization of Differentially Expressed and Pathway
Genes
[0108] Differentially expressed genes, such as those identified via
the methods discussed, above, in Section 5.1, and pathway genes,
such as those identified via the methods discussed, above, in
Section 5.2, above, as well as genes identified by alternative
means, can be further characterized by utilizing, for example,
methods such as those discussed herein. Such genes will be referred
to herein as "identified genes."
[0109] Analyses such as those described herein, yield information
regarding the biological function of the identified genes. An
assessment of the biological function of the differentially
expressed genes, in addition, will allow for their designation as
target and/or fingerprint genes.
[0110] Specifically, any of the differentially expressed genes
whose further characterization indicates that a modulation of the
gene's expression or a modulation of the gene product's activity
can inhibit tumor progression will be designated "target genes," as
defined, above, in Section 5.1. Such target genes and target gene
products, along with those discussed below, will constitute the
focus of the compound discovery strategies discussed, below, in
Section 5.8. Further, such target genes, target gene products
and/or modulating compounds can be used as part of the tumor
progression disorder treatment methods described, below, in Section
5.9.
[0111] Any of the differentially expressed genes whose further
characterization indicates that such modulations does not
positively affect tumor progression, but whose expression pattern
contributes to a gene expression "fingerprint" pattern correlative
of, for example, tumor progression will be designated a
"fingerprint gene." "Fingerprint patterns" will be more fully
discussed, below, in Section 5.11.1. It should be noted that each
of the target genes can also function as fingerprint genes, as can
all or a portion of the pathway genes.
[0112] It should further be noted that the pathway genes can also
be characterized according to techniques such as those described
herein. Those pathway genes which yield information indicating that
they are differentially expressed and that modulation of the gene's
expression or a modulation of the gene product's activity can
inhibit tumor progression or ameliorate tumor
progression-associated symptoms will also be designated "target
genes." Such target genes and target gene products, along with
those discussed above, will constitute the focus of the compound
discovery strategies discussed, below, in Section 5.8 and can be
used as part of the treatment methods described in Section 5.9,
below.
[0113] It should be additionally noted that the characterization of
one or more of the pathway genes can reveal a lack of differential
expression, but evidence that modulation of the gene's activity or
expression can, nonetheless, ameliorate symptoms of tumor
progression. In such cases, these genes and gene products would
also be considered a focus of the compound discovery strategies of
Section 5.8, below and can be used as part of the treatment methods
described in Section 5.9, below.
[0114] In instances wherein a pathway gene's characterization
indicates that modulation of gene expression or gene product
activity cannot retard the tumor progression diseases of interest,
but is differentially expressed and contributes to a gene
expression fingerprint pattern correlative of, tumor progression
states or disorders, such as metastatic diseases, such pathway
genes can additionally be designated as fingerprint genes.
[0115] A variety of techniques can be utilized to further
characterize the identified genes. First, the nucleotide sequence
of the identified genes, which can be obtained by utilizing
standard techniques well known to those of skill in the art, can be
used to further characterize such genes. For example, the sequence
of the identified genes can reveal homologies to one or more known
sequence motifs which can yield information regarding the
biological function of the identified gene product.
[0116] Second, an analysis of the tissue and/or cell type
distribution of the mRNA produced by the identified genes can be
conducted, utilizing standard techniques well known to those of
skill in the art. Such techniques can include, for example,
Northern analyses, RT-coupled PCR and RNase protection techniques.
Such analyses provide information as to whether the identified
genes are expressed in tissues expected to contribute to tumor
progression. Such analyses can also provide quantitative
information regarding steady state mRNA regulation, yielding data
concerning which of the identified genes exhibits a high level of
regulation in, preferably, tissues which can be expected to
contribute to tumor progression. Additionally, standard in situ
hybridization techniques can be utilized to provide information
regarding which cells within a given tissue express the identified
gene. Such an analysis can provide information regarding the
biological function of an identified gene relative to given tumor
progression in instances wherein only a subset of the cells within
the tissue is thought to be relevant to the disorder.
[0117] Third, the sequences of the identified genes can be used,
utilizing standard techniques, to place the genes onto genetic
maps, e.g., mouse (Copeland, N. G. and Jenkins, N. A., 1991, Trends
in Genetics 7:113-118) and human genetic maps (Cohen, D., et al.,
1993, Nature 366:698-701). Such mapping information can yield
information regarding the genes' importance to human disease by,
for example, identifying genes which map within genetic regions to
which known genetic tumor progression disorders map.
[0118] Fourth, the biological function of the identified genes can
be more directly assessed by utilizing relevant in vivo and in
vitro systems. In vivo systems can include, but are not limited to,
animal systems which naturally exhibit symptoms of tumor
progression, such as metastatic disease, or ones which have been
engineered to exhibit such symptoms. For example, tumor progression
animal models may be generated by injecting animals, such as mice,
with tumor cells, some of which will give rise to tumors within the
injected animals. Among the cells which may be utilized for such a
purpose are cells listed, above, in Section 5.1.1.1, such as the
B16 cell variants.
[0119] The role of identified gene products (e.g., 030 gene
products) can be determined by transfecting cDNAs encoding these
gene products into appropriate cell lines, such as, for example, a
B16 cell line variant, and analyzing the effect on tumor
progression characteristics. For example, the role/function of
genes important in the progression of human colorectal cancers are
assessed using the KM12c (low metastatic potential) and KM12L4
(highly metastatic) cells implanted into nude mice spleens and the
number of hepatic tumors that develop are determined. The function
of genes isolated using human colorectal tumors and their hepatic
metastases are assessed by expressing the gene in the appropriate
KM12 variant. Additionally, the role/function of genes important in
the progression of prostatic and breast cancers are assessed using
appropriate cell lines described above in Section 5.1.1.1.
Importantly, the role/function of genes important in the
progression of melanoma, colon, prostate and breast cancers in
humans are assessed using biopsy specimens from patients having
undergone surgical treatment, as described in Section 5.1.1.1.
above.
[0120] Further, such systems can include, but are not limited to
transgenic animal systems such as those described, above, in
Section 5.7.1 below. in vitro systems can include, but are not
limited to, cell-based systems comprising cell types known or
suspected of contributing to tumor progression. Such cells can be
wild type cells, or can be non-wild type cells containing
modifications known to or suspected of, contributing to tumor
progression. Such systems are discussed in detail, below, in
Section 5.7.2. The procedure to identify and isolate the human
homologue of the fomy030 gene is described, below, in Section
5.7.3.
[0121] In further characterizing the biological function of the
identified genes, the expression of these genes can be modulated
within the in vivo and/or in vitro systems, i.e., either over- or
under-expressed, and the subsequent effect on the system then
assayed. Alternatively, the activity of the product of the
identified gene can be modulated by either increasing or decreasing
the level of activity in the in vivo and/or in vitro system of
interest, and its subsequent effect then assayed.
[0122] The information obtained through such characterizations can
suggest relevant methods for the treatment of tumor progression and
tumor progression disorders involving the gene of interest.
Further, relevant methods for controlling the spread of tumor cells
involving the gene of interest can be suggested by information
obtained from such characterization. For example, treatment can
include a modulation of gene expression and/or gene product
activity. Characterization procedures such as those described
herein can indicate where such modulation should involve an
increase or a decrease in the expression or activity of the gene or
gene product of interest. Such methods of treatment are discussed,
below, in Section 5.9.
5.4. Differentially Expressed and Pathway Genes
[0123] Differentially expressed genes, such as those identified in
Section 5.1.1, above, and pathway genes, such as those identified
in Section 5.2, above, are described herein.
[0124] The differentially expressed and pathway genes of the
invention are listed below, in Table 1. The nucleotide sequence for
the differentially expressed fomy030 gene is shown in FIGS. 2 and
3A and 3B. Specifically, FIG. 2 depicts the nucleotide sequence
(SEQ ID NO:1) of the amplified cDNA band initially identified via
differential display analysis, which is referred to herein as
romy030. FIGS. 3A AND 3B depict the nucleotide sequence (SEQ ID
NO:2) of a fomy030 cDNA clone which was isolated using a romy030
probe. The deduced amino acid sequence also is shown in FIGS. 3A
and 3B (SEQ ID NO:3). FIG. 5 shows the nucleotide (SEQ ID NO:6) and
deduced amino acid sequences (SEQ ID NO:7) of a fohy030 cDNA clone
which was isolated using the entire mouse fomy030 cDNA as a probe.
FIG. 6 shows an alternative splice form of fohy030 (SEQ ID NOs:8
and 9).
[0125] Table 1 summarizes information regarding the further
characterization of the differentially expressed fomy030 gene of
the invention. Table 2 lists E. coli clones, deposited with the
Agricultural Research Service Culture Collection (NRRL), which
contain sequences found within the genes of Table 1.
[0126] In Table 1, the paradigm used initially to detect the
differentially expressed gene is described under the column headed
"Paradigm of Original Detection." In this column, ".Arrow-up bold."
indicates that gene expression is higher (i.e., there is a greater
steady state amount of detectable mRNA produced by a given gene) in
the indicated cell type relative to the other cell type, while
".dwnarw." indicates that gene expression is lower (i.e., there is
a lower steady state amount of detectable mRNA, produced by a given
gene) in the indicated cell type relative to the other cell type.
As indicated under this column, the 030 gene was initially
identified via a differential screen between B16 F1 (low metastatic
potential cells) and B16 F10 (high metastatic potential cells) in
which 030 gene expression is lower in the high metastatic potential
B16 F10 cell line than in the low metastatic potential B16 F1 cell
line.
[0127] The Table 1 column headed "Paradigm Expression Pattern"
lists the cell type in which gene expression was initially
detected. In the case of the 030 gene, gene expression was first
detected in melanoma (ie., B16) cells. "Detectable" as used herein,
refers to levels of mRNA which are detectable, via standard
differential display, Northern, RT-coupled PCR and/or RNase
protection techniques which are well known to those of skill in the
art.
[0128] Cell types in which differential expression was detected are
summarized in Table 1 under the column headed "Cell Type Detected
in." In the case of the 030 gene, expression has additionally been
detected within melanocyte cells.
[0129] Additionally, in instances wherein the genes contain
nucleotide sequences similar or homologous to sequences found in
nucleic acid databases, references to such similarities are listed.
Because the 030 gene is a novel gene, i.e., no homologous gene
sequences are present in the published databases, no such reference
is listed.
[0130] Finally, nucleotide sequences contained within the
differentially expressed genes are listed in the Figures indicated
under the heading "Seq." In the case of the fomy030 gene, such
sequences are listed in FIGS. 2 and 3A and 3B, and for fohy030, in
FIGS. 5 and 6.
[0131] The genes listed in Table 1 can be obtained using cloning
methods well known to those skilled in the art, including, but not
limited to, the use of appropriate probes to detect the genes
within an appropriate cDNA or gDNA (genomic DNA) library. (See, for
example, Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, which is incorporated by
reference herein in its entirety). Probes for the novel sequences
reported herein can be obtained directly from the isolated clones
deposited with the NRRL, as indicated in Table 2, below.
Alternatively, oligonucleotide probes for the novel genes can be
synthesized, using techniques well known to those of skill in the
art, based on the DNA sequences disclosed herein in FIGS. 2, 3A,
3B, 5, and 6.
[0132] The probes can be used to screen cDNA libraries prepared
from an appropriate cell or cell line in which the gene is
transcribed. For example, the genes described herein that were
detected in melanocyte cells can be cloned from a cDNA library
prepared from melanocyte cells such as, for example, melan-c
(Hodgkinson, C. A., et al., 1993, Cell 74:395-404), the cDNA
libraries developed from the human melanoma cell line A2058
(Clontech, Palo Alto, Calif.) and cDNA libraries developed from the
murine melanoma cell line K1735 (Stratagene, La Jolla, Calif.).
Genomic DNA libraries can be prepared from any source.
1TABLE 1 Differentially Expressed and Pathway Genes Paradigm of
Original Paradigm Cell Type Sequence Detection Expression Detected
GENE ID (.Arrow-up bold./.dwnarw.) Pattern in Ref. Seq. fomy030 2
B16 .Arrow-up bold. F1 melanoma melanocyte B16 .dwnarw. F10 cells
3A & 3B fohy030 6 & 8 benign biopsy melanocyte FIG. nevi
.Arrow-up bold. samples 5 & 6 malignant melanoma .dwnarw.
[0133] Table 2, below, lists an E. coli strain as deposited with
the NRRL, which contains an isolated plasmid fomy030 clone. The
clone contains a fomy030 cDNA in a pBlueScript SK- (Stratagene, La
Jolla, Calif.) vector which was isolated from a mouse melanocyte
cDNA library screened with a romy030probe, as described in Section
6.2, below.
2 TABLE 2 STRAIN DEPOSITED PLASMID CLONE CONTAINED GENE WITH NRRL
WITHIN DEPOSITED STRAIN fomy030 FOMY030 pFOMY030 fohy030
[0134] As used herein, "differentially expressed gene" (i.e.,
target and fingerprint genes) or "pathway gene" refers to (a) a
gene containing: at least one of the DNA sequences disclosed herein
(as shown in FIGS. 2, 3A, 3B, 5, and 6) or contained in the clones
listed in Table 2, as deposited with the NRRL; (b) any DNA sequence
that encodes the amino acid sequence encoded by: the DNA sequences
disclosed herein (as shown in FIGS. 2, 3A, 3B, 5, and 6), contained
in the clones, listed in Table 2, as deposited with the NRRL or
contained within the coding region of the gene to which the DNA
sequences disclosed herein (as shown in FIGS. 2, 3A, 3B, 5, and 6)
or contained in the clones listed in Table 2, as deposited with the
NRRL, belong; (c) any DNA sequence that hybridizes to the
complement of: the coding sequences disclosed herein (as shown in
FIGS. 2, 3A, 3B, 5, and 6), contained in clones listed in Table 2,
as deposited with the NRRL, or contained within the coding region
of the gene to which the DNA sequences disclosed herein (as shown
in FIGS. 2, 3A, 3B, 5, and 6) or contained in the clones listed in
Table 2, as deposited with the NRRL, belong under highly stringent
conditions, e.g., hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. (Ausubel F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc., and
John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a
gene product functionally equivalent to a gene product encoded by a
gene of (a), above and/or (d) any DNA sequence that hybridizes to
the complement of: the coding sequences disclosed herein, (as shown
in FIGS. 2, 3A, 3B, 5, and 6) contained in the clones listed in
Table 2, as deposited with the NRRL or contained within the coding
region of the gene to which DNA sequences disclosed herein (as
shown in FIGS. 2, 3A, 3B, 5, and 6) or contained in the clones,
listed in Table 2, as deposited with the NRRL, belong under less
stringent conditions, such as moderately stringent conditions,
e.g., washing in 0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel
et al., 1989, supra), yet which still encodes a gene product
functionally equivalent to a gene product encoded by a gene of (a),
above.
[0135] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the DNA sequences (a) through (d), in the preceding
paragraph. Such hybridization conditions can be highly stringent or
less highly stringent, as described above. In instances wherein the
nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly
stringent conditions can refer, e.g., to washing in
6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C. (for
14-base oligos), 48.degree. C. (for 17-base oligos), 55.degree. C.
(for 20-base oligos), and 60.degree. C. (for 23-base oligos). These
nucleic acid molecules can act as target gene antisense molecules,
useful, for example, in target gene regulation and/or as antisense
primers in amplification reactions of target, fingerprint, and/or
pathway gene nucleic acid sequences. Further, such sequences can be
used as part of ribozyme and/or triple helix sequences, also useful
for target gene regulation. Still further, such molecules can be
used as components of diagnostic methods whereby tumor progression
disorders can be detected.
[0136] The invention also encompasses (a) DNA vectors that contain
any of the foregoing coding sequences and/or their complements
(i.e., antisense); (b) DNA expression vectors that contain any of
the foregoing coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences; and (c) genetically engineered host cells that contain
any of the foregoing coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences in the host cell. As used herein, regulatory elements
include but are not limited to inducible and non-inducible
promoters, enhancers, operators and other elements known to those
skilled in the art that drive and regulate expression. The
invention includes fragments of any of the DNA sequences disclosed
herein.
[0137] In addition to the gene sequences described above,
homologues of these gene sequences as can, for example be present
in other species, preferably human in instances wherein the above
described gene sequences are not human gene sequences, can be
identified and can readily be isolated, without undue
experimentation, by molecular biological techniques well known in
the art. Further, there can exist genes at other genetic loci
within the genome that encode proteins which have extensive
homology to one or more domains of such gene products. These genes
can also be identified via similar techniques.
[0138] For example, the isolated differentially expressed gene
sequence can be labeled and used to screen a cDNA library
constructed from mRNA obtained from the organism of interest.
Hybridization conditions will be of a lower stringency when the
cDNA library was derived from an organism different from the type
of organism from which the labeled sequence was derived.
Alternatively, the labeled fragment can be used to screen a genomic
library derived from the organism of interest, again, using
appropriately stringent conditions. Such low stringency conditions
will be well known to those of skill in the art, and will vary
predictably depending on the specific organisms from which the
library and the labeled sequences are derived. For guidance
regarding such conditions see, for example, Sambrook et al., 1989,
Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press,
N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular
Biology, (Green Publishing Associates and Wiley Interscience,
N.Y.).
[0139] Further, a previously unknown differentially expressed or
pathway gene-type sequence can be isolated by performing PCR using
two degenerate oligonucleotide primer pools designed on the basis
of amino acid sequences within the gene of interest. The template
for the reaction can be cDNA obtained by reverse transcription of
mRNA prepared from human or non-human cell lines or tissue known or
suspected to express a differentially expressed or pathway gene
allele. The PCR product can be subcloned and sequenced to insure
that the amplified sequences represent the sequences of a
differentially expressed or pathway gene-like nucleic acid
sequence.
[0140] The PCR fragment can then be used to isolate a full length
cDNA clone by a variety of methods. For example, the amplified
fragment can be labeled and used to screen a bacteriophage cDNA
library. Alternatively, the labeled fragment can be used to screen
a genomic library.
[0141] PCR technology can also be utilized to isolate full length
cDNA sequences. For example, RNA can be isolated, following
standard procedures, from an appropriate cellular or tissue source.
A reverse transcription reaction can be performed on the RNA using
an oligonucleotide primer specific for the most 5' end of the
amplified fragment for the priming of first strand synthesis. The
resulting RNA/DNA hybrid can then be "tailed" with guanines using a
standard terminal transferase reaction, the hybrid can be digested
with RNAase H, and second strand synthesis can then be primed with
a poly-C primer. Thus, cDNA sequences upstream of the amplified
fragment can easily be isolated. For a review of cloning strategies
which can be used, see e.g., Sambrook et al., 1989, Molecular
Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and
Ausubel et al., 1989, Current Protocols in Molecular Biology,
(Green Publishing Associates and Wiley Interscience, N.Y.).
[0142] In cases where the differentially expressed or pathway gene
identified is the normal, or wild type, gene, this gene can be used
to isolate mutant alleles of the gene. Such an isolation is
preferable in processes and disorders which are known or suspected
to have a genetic basis. Mutant alleles can be isolated from
individuals either known or suspected to have a genotype which
contributes to tumor progression symptoms. Mutant alleles and
mutant allele products can then be utilized in the therapeutic and
diagnostic assay systems described below.
[0143] A cDNA of a mutant gene can be isolated, for example, by
using PCR, a technique which is well-known to one skilled in the
art. In this case, the first cDNA strand can be synthesized by
hybridizing a oligo-dT oligonucleotide to mRNA isolated from tissue
known or suspected of being expressed in an individual putatively
carrying the mutant allele, and by extending the new strand with
reverse transcriptase. The second strand of the cDNA can then be
synthesized using an oligonucleotide that hybridizes specifically
to the 5'-end of the normal gene. Using these two primers, the
product is then amplified via PCR, cloned into a suitable vector,
and subjected to DNA sequence analysis through methods well-known
to one skilled in the art. By comparing the DNA sequence of the
mutant gene to that of the normal gene, the mutation(s) responsible
for the loss or alteration of function of the mutant gene product
can be ascertained.
[0144] Alternatively, a genomic or cDNA library can be constructed
and screened using DNA or RNA, respectively, from a tissue known to
or suspected of expressing the gene of interest in an individual
suspected of or known to carry the mutant allele. The normal gene
or any suitable fragment thereof can then be labeled and used as a
probe to identify the corresponding mutant allele in the library.
The clone containing this gene can then be purified through methods
routinely practiced in the art, and subjected to sequence analysis
as described, above, in this Section.
[0145] Additionally, an expression library can be constructed
utilizing DNA isolated from or cDNA synthesized from a tissue known
to or suspected of expressing the gene of interest in an individual
suspected of or known to carry the mutant allele. In this manner,
gene products made by the putatively mutant tissue can be expressed
and screened using standard antibody screening techniques in
conjunction with antibodies raised against the normal gene product,
as described, below, in Section 5.2.3. (For screening techniques,
see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A
Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor.)
In cases where the mutation results in an expressed gene product
with altered function (e.g., as a result of a missense mutation), a
polyclonal set of antibodies are likely to cross-react with the
mutant gene product. Library clones detected via their reaction
with such labeled antibodies can be purified and subjected to
sequence analysis as described in this Section, above.
[0146] Taking the fomy030 gene as an example, the fomy030 human
homolog can be isolated by a variety of methods. First, sequences
found in a murine fomy030 cDNA can be utilized as hybridization
probes to detect human fohy030sequences. This can be accomplished,
for example, by probing Southern blots containing total human
genomic DNA with a labelled fomy030 probe. Once it is verified that
the probe being utilized detects the human 030 gene, one of skill
in the art can employ any of several routine approaches to isolate
the human gene without undue experimentation.
[0147] In one approach, appropriate human cDNA libraries can be
screened. Such cDNA libraries can, for example, include human
melanocyte, human retina and fetal human brain cDNA libraries. For
example, panels of human melanoma cells (such as, for example,
SK-MEL-2, ATCC 68-HTB; SK-MEL-5, ATCC 70-HTB; SK-MEL-28, ATCC
72-HTB; G-361, ATCC 1424-CRL; and/or HT-144 [63-HTB] cells) can be
screened for 030 expression by, for example, Northern blot
analysis. Upon detection of 030 transcript, cDNA libraries can be
constructed from RNA isolated from the appropriate cell line,
utilizing standard techniques well known to those of skill in the
art. The human cDNA library can then be screened with a 030 probe
in order to isolate a human romyo30 cDNA. As described below, this
method was used to determine the human fohy030 cDNAs in FIGS. 5 and
6.
[0148] Alternatively, a human total genomic DNA library can be
screened using 030 probes. 030-positive clones can then be
sequenced and, further, the intron/exon structure of the human 030
gene may be elucidated. Once genomic sequence is obtained,
oligonucleotide primers can be designed based on the sequence for
use in the isolation, via, for example RT-coupled PCR, of human 030
cDNA.
[0149] The procedures described in these approaches are routine and
have been described in detail in Sections 5.1.1.2, 5.3 and
5.7.2.
5.5. Differentially Expressed and Pathway Gene Products
[0150] Differentially expressed and pathway gene products include
those proteins encoded by the differentially expressed and pathway
gene sequences described in Section 5.2.1, above, as for example,
the peptide listed in FIG. 3. Specifically, differentially
expressed and pathway gene products can include differentially
expressed and pathway gene polypeptides encoded by the
differentially expressed and pathway gene sequences contained in
the clones listed in Table 2, above, as deposited with the NRRL, or
contained in the coding regions of the genes to which DNA sequences
disclosed herein (in FIGS. 3A, 3B, 5, and 6) or contained in the
clones, listed in Table 2, as deposited with the NRRL, belong, for
example.
[0151] In addition, differentially expressed and pathway gene
products can include proteins that represent functionally
equivalent gene products. Such an equivalent differentially
expressed or pathway gene product can contain deletions, additions
or substitutions of amino acid residues within the amino acid
sequence encoded by the differentially expressed or pathway gene
sequences described, above, in Section 5.2.1, but which result in a
silent change thus producing a functionally equivalent
differentially expressed on pathway gene product. Amino acid
substitutions can be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipatic nature of the residues involved. For example, nonpolar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan, and methionine; polar
neutral amino acids include glycine, serine, threonine, cysteine,
tyrosine, asparagine, and glutamine; positively charged (basic)
amino acids include arginine, lysine, and histidine; and negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid. "Functionally equivalent," as utilized herein, refers to
either a protein capable exhibiting a substantially similar in vivo
activity as the endogenous differentially expressed or pathway gene
products encoded by the differentially expressed or pathway gene
sequences described in Section 5.2.1, above. Alternatively, when
utilized as part of assays such as those described, below, in
Section 5.3, "functionally equivalent" can refer to peptides
capable of interacting with other cellular or extracellular
molecules in a manner substantially similar to the way in which the
corresponding portion of the endogenous differentially expressed or
pathway gene product would.
[0152] The differentially expressed or pathway gene products can be
produced by synthetic techniques or via recombinant DNA technology
using techniques well known in the art. Methods for preparing the
differentially expressed or pathway gene polypeptides and peptides
of the invention by expressing nucleic acid encoding differentially
expressed or pathway gene sequences are described herein. Methods
which are well known to those skilled in the art can be used to
construct expression vectors containing differentially expressed or
pathway gene protein coding sequences and appropriate
transcriptional/translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y. which is incorporated by reference
herein in their entirety, and Ausubel, 1989, supra. Alternatively,
RNA capable of encoding differentially expressed or pathway gene
protein sequences can be chemically synthesized using, for example,
synthesizers. See, for example, the techniques described in
"Oligonucleotide Synthesis," 1984, Gait, M. J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0153] A variety of host-expression vector systems can be utilized
to express the differentially expressed or pathway gene coding
sequences of the invention. Such host-expression systems represent
vehicles by which the coding sequences of interest can be produced
and subsequently purified, but also represent cells which can, when
transformed or transfected with the appropriate nucleotide coding
sequences, exhibit the differentially expressed or pathway gene
protein of the invention in situ. These include but are not limited
to microorganisms such as bacteria (e.g., E. coli, B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing differentially expressed
or pathway gene protein coding sequences; yeast (e.g.,
Saccharomyces, Pichia) transformed with recombinant yeast
expression vectors containing the differentially expressed or
pathway gene protein coding sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing the differentially expressed or pathway gene protein
coding sequences; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing differentially
expressed or pathway gene protein coding sequences; or mammalian
cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia
virus 7.5K promoter).
[0154] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
differentially expressed or pathway gene protein being expressed.
For example, when a large quantity of such a protein is to be
produced, for the generation of antibodies or to screen peptide
libraries, for example, vectors which direct the expression of high
levels of fusion protein products that are readily purified can be
desirable. Such vectors include, but are not limited, to the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which the differentially expressed or pathway gene
protein coding sequence can be ligated individually into the vector
in frame with the lacZ coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors can also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
protein can be released from the GST moiety.
[0155] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The
differentially expressed or pathway gene coding sequence can be
cloned individually into non-essential regions (for example the
polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example, the polyhedrin promoter). Successful
insertion of differentially expressed or pathway gene coding
sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking
the proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed (e.g., see Smith et
al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051).
[0156] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, the differentially expressed or pathway gene
coding sequence of interest can be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene can then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing differentially expressed
or pathway gene protein in infected hosts (e.g., See Logan &
Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific
initiation signals can also be required for efficient translation
of inserted differentially expressed or pathway gene coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. In cases where an entire identified gene,
including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals can be needed. However, in cases
where only a portion of the identified coding sequence is inserted,
exogenous translational control signals, including, perhaps, the
ATG initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression can be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc., (see Bittner et al., 1987, Methods in Enzymol.
153:516-544).
[0157] In addition, a host cell strain can be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products can be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cell lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells which possess
the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
can be used. Such mammalian host cells include but are not limited
to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.
[0158] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the differentially expressed or pathway gene
protein can be engineered. Rather than using expression vectors
which contain viral origins of replication, host cells can be
transformed with DNA controlled by appropriate expression control
elements (e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation sites, etc.), and a selectable marker.
Following the introduction of the foreign DNA, engineered cells can
be allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method can advantageously be used to engineer cell
lines which express the identified gene protein. Such engineered
cell lines can be particularly useful in screening and evaluation
of compounds that affect the endogenous activity of the
differentially expressed or pathway gene protein.
[0159] A number of selection systems can be used, including, but
not limited to, the herpes simplex virus thymidine kinase (Wigler,
et al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
in tk.sup.-, hgprt.sup.- or aprt.sup.- cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
dhfr, which confers resistance to methotrexate (Wigler, et al.,
1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol.
150:1); and hygro, which confers resistance to hygromycin
(Santerre, et al., 1984, Gene 30:147) genes.
[0160] An alternative fusion protein system allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA
88:8972-8976). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the gene's open
reading frame is translationally fused to an amino-terminal tag
consisting of six histidine residues. Extracts from cells infected
with recombinant vaccinia virus are loaded onto ni2+ nitriloacetic
acid-agarose columns and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers.
[0161] When used as a component in assay systems such as that
described herein, the differentially expressed or pathway gene
protein can be labeled, either directly or indirectly, to
facilitate detection of a complex formed between the differentially
expressed or pathway gene protein and a test substance. Any of a
variety of suitable labeling systems can be used including but not
limited to radioisotopes such as .sup.125I; enzyme labelling
systems that generate a detectable calorimetric signal or light
when exposed to substrate; and fluorescent labels.
[0162] Where recombinant DNA technology is used to produce the
differentially expressed or pathway gene protein for such assay
systems, it can be advantageous to engineer fusion proteins that
can facilitate labeling, solubility, immobilization and/or
detection.
[0163] Indirect labeling involves the use of a third protein, such
as a labeled antibody, which specifically binds to either a
differentially expressed or pathway gene product. Such antibodies
include but are not limited to polyclonal, monoclonal, chimeric,
single chain, Fab fragments and fragments produced by a Fab
expression library.
5.6. Antibodies Specific for Differentially Expressed or Pathway
Gene Products
[0164] Described herein are methods for the production of
antibodies capable of specifically recognizing one or more
differentially expressed or pathway gene epitopes. Such antibodies
can include, but are not limited to polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies,
single chain antibodies, Fab fragments, F(ab').sub.2 fragments,
fragments produced by a FAb expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the
above. Such antibodies can be used, for example, in the detection
of a fingerprint, target, or pathway gene in a biological sample,
or, alternatively, as a method for the inhibition of abnormal
target gene activity. Thus, such antibodies can be utilized as
tumor progression treatment methods, and/or can be used as part of
diagnostic techniques whereby patients can be tested for abnormal
levels of fingerprint, target, or pathway gene proteins, or for the
presence of abnormal forms of the such proteins.
[0165] For the production of antibodies to a differentially
expressed or pathway gene, various host animals can be immunized by
injection with a differentially expressed or pathway gene protein,
or a portion thereof. Such host animals can include but are not
limited to rabbits, mice, and rats, to name but a few. Various
adjuvants can be used to increase the immunological response,
depending on the host species, including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and Corynebacterium
parvum.
[0166] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as target gene product (e.g., protein encoded by
030), or an antigenic functional derivative thereof. For the
production of polyclonal antibodies, host animals such as those
described above, can be immunized by injection with differentially
expressed or pathway gene product (e.g., 030) supplemented with
adjuvants as also described above.
[0167] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be obtained by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today A:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the BV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies can be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention can be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the presently
preferred method of production.
[0168] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452-454; U.S. Pat. No. 4,816,567)
by splicing the genes from a mouse antibody molecule of appropriate
antigen specificity together with genes from a human antibody
molecule of appropriate biological activity can be used. A chimeric
antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region
derived from a murine mAb and a human immunoglobulin constant
region.
[0169] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) and
for making humanized monoclonal antibodies (U.S. Pat. No.
5,225,539, which is incorporated herein by reference in its
entirety) can be utilized to produce anti-differentially expressed
or anti-pathway gene product antibodies.
[0170] Antibody fragments which recognize specific epitopes can be
generated by known techniques. For example, such fragments include
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries can be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
5.7. Cell- and Animal-Based Model Systems
[0171] Described herein are cell- and animal-based systems which
represent reliable models for tumor progression disorders. These
systems can be used in a variety of applications. For example, the
cell- and animal-based model systems can be used to identify
differentially expressed genes via the paradigms described, above,
in Section 5.1.1.1. Such systems can also be used to further
characterize differentially expressed and pathway genes, as
described, above, in Section 5.3. Such further characterization
can, for example, indicate that a differentially expressed gene is
a target gene, for example. Additionally, such assays can be
utilized as part of screening strategies designed to identify
compounds which are capable of preventing and/or ameliorating
symptoms of tumor progression disorders, including those associated
with metastatic diseases, as described, below. Thus, the animal-
and cell-based models can be used to identify drugs,
pharmaceuticals, therapies and interventions which can be effective
in treating tumor progression disorders, such as, for example,
metastatic diseases. In addition, as described in detail, below, in
Section 5.10.1, such animal models can be used to determine the
LD.sub.50 and the ED.sub.50 in animal subjects, and such data can
be used to determine the in vivo efficacy of potential anti-tumor
progression disorder treatments.
5.7.1. Animal-Based Systems
[0172] Animal-based model systems of tumor progression disorders
can be both non-recombinant animals as well as recombinantly
engineered transgenic animals.
[0173] Non-recombinant animal models for tumor progression can
include, for example, murine models of melanoma, prostate cancer
and colon cancer. Such models may be generated, for example, by
introducing tumor cells into syngeneic mice using techniques such
as subcutaneous injection, tail vein injection, spleen
implantation, intraperitoneal implantation, implantation under the
renal capsule or orthotopic implantation (eq., colon cancer cells
implanted in colonic tissue or prostatic cancer cells implanted in
prostate gland). After an appropriate period of time, the tumors
which result from these injections can be counted and analyzed.
[0174] Among the cells which may be used for the production of such
animal models of tumor progression are cells derived from the cell
lines listed, above, in Section 5.1.1.1. For example, B16 melanoma
cells (Fidler, I. J., 1973, Nature New Biol. 242:148-149),
including cell variants exhibiting high (e.g., B16 F10 cells) and
low (e.g., B16 F1 cells) metastatic potential may be utilized.
Post-injection, pulmonary tumors generally develop in the mouse
models. Thus, these animal serve as models of not only melanoma
tumor progression but also as models of pulmonary metastases.
[0175] For the generation of animal models of colorectal cancers,
colon cancer cells such as, for example, KM12c (low metastatic
potential) and KM12L4 (highly metastatic) cells (Morikawa, K. et
al., 1988, Cancer Research 48:1943-1948) can be implanted into nude
mice spleens. In these cases, the animals generally develop hepatic
tumors. Thus, such animals serve as models of not only colorectal
tumor progression but also as models of hepatic metastases.
[0176] For the generation of animal models of prostate cancer tumor
progression, cells derived from, for example, the high metastatic
potential prostatic cell line PC-3-M or the non-metastatic cell
line DU 145 (Karmali, R. A. et al., 1987, Anticancer Res.
7:1173-1180; Koziowski, J. M. et al., 1984, Cancer Research
44:3522-3529) may be implanted into the prostates of animals and
the resulting tumors may be analyzed and compared to, for example,
normal tissue. In such a manner, genes which are differentially
expressed in neoplastic versus normal cells as well as versus
metastatic cells may be identified.
[0177] The role of identified gene products (e.g., 030 gene
products) can be determined by transfecting cDNAs encoding such
gene products into the appropriate cell line and analyzing its
effect on the cells' ability to induce tumor progression in animal
models such as these. The role of the identified gene products may
be further analyzed by, for example, culturing cells derived from
the tumors which develop in the animal models, introducing these
cultured cells into animals, and subsequently measuring the level
of identified gene product present in the resulting tumor cells. In
this manner, cell line variants are developed which can be useful
in analyzing the role of quantitative and/or qualitative
differences in the expression of the identified genes on the cells'
ability to induce tumor progression. For example, as demonstrated,
below, in the Example presented in Section 6, 030 gene expression
is inversely related to the metastatic potential of the tumor cell
line used to generate such a tumor progression animal model.
[0178] Additionally, recombinant animal models exhibiting tumor
progression characteristics and/or symptoms of tumor progression
disorders, including metastatic diseases, can be utilized, for
example, such well-known animal models as the transgenic mouse
model for human melanoma and transgenic mice which carry specific
mutations which result in multiple intestinal tumors (Mintz, M. and
Silvers W. K., 1993, Proc. Natl. Acad. Sci. USA 90:8817-8821; and
Fodde, R., et al., 1994, Proc. Natl. Acad. Sci. USA 91:8969-8973).
Further, recombinant animal models for tumor progression can be
engineered by utilizing, for example, target gene sequences such as
those described, above, in Section 5.4, in conjunction with
techniques for producing transgenic animals that are well known to
those of skill in the art. For example, target gene sequences can
be introduced into, and overexpressed in, the genome of the animal
of interest, or, if endogenous target gene sequences are present,
they can either be overexpressed or, alternatively, can be
disrupted in order to underexpress or inactivate target gene
expression.
[0179] In order to overexpress a target gene sequence, the coding
portion of the target gene sequence can be ligated to a regulatory
sequence which is capable of driving gene expression in the animal
and cell type of interest. Such regulatory regions will be well
known to those of skill in the art, and can be utilized in the
absence of undue experimentation.
[0180] In order to obtain underexpression of an endogenous target
gene sequence, such a sequence can be introduced into the genome of
the animal of interest such that the endogenous target gene alleles
will be inactivated. Preferably, an engineered sequence comprising
at least part of the target gene sequence is utilized and is
introduced, via gene targeting, such that the endogenous target
sequence is disrupted upon integration of the engineered target
gene sequence into the animal's genome. Gene targeting is
discussed, below, in this Section.
[0181] Animals of any species, including, but not limited to, mice,
rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human
primates, e.g., baboons, monkeys, and chimpanzees can be used to
generate animal models of tumor progression and tumor progression
disorders, such as, for example, metastatic diseases.
[0182] Any technique known in the art can be used to introduce a
target gene transgene into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited to
pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989,
U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci.,
USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson
et al., 1989, Cell 56:313-321); electroporation of embryos (Lo,
1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene
transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a
review of such techniques, see Gordon, 1989, Transgenic Animals,
Intl. Rev. Cytol. 115:171-229, which is incorporated by reference
herein in its entirety.
[0183] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e, mosaic
animals. The transgene can be integrated, either as a single
transgene or in concatamers, e.g., head-to-head tandems or
head-to-tail tandems. The transgene can also be selectively
introduced into and activated in a particular cell type by
following, for example, the teaching of Lasko et al. (Lasko, M., et
al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236). The regulatory
sequences required for such a cell-type specific activation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[0184] When it is desired that the target gene transgene be
integrated into the chromosomal site of the endogenous target gene,
gene targeting is preferred. Briefly, when such a technique is to
be utilized, vectors containing some nucleotide sequences
homologous to the endogenous target gene of interest are designed
for the purpose of integrating, via homologous recombination with
chromosomal sequences, into and disrupting the function of, the
nucleotide sequence of the endogenous target gene. The transgene
can also be selectively introduced into a particular cell type,
thus inactivating the endogenous gene of interest in only that cell
type, by following, for example, the teaching of Gu et al. (Gu, H.,
et al., 1994, Science 265:103-106). The regulatory sequences
required for such a cell-type specific inactivation will depend
upon the particular cell type of interest, and will be apparent to
those of skill in the art.
[0185] Once transgenic animals have been generated, the expression
of the recombinant target gene and protein can be assayed utilizing
standard techniques. Initial screening can be accomplished by
Southern blot analysis or PCR techniques to analyze animal tissues
to assay whether integration of the transgene has taken place. The
level of mRNA expression of the transgene in the tissues of the
transgenic animals can also be assessed using techniques which
include, but are not limited to, Northern blot analysis of tissue
samples obtained from the animal, in situ hybridization analysis,
and RT-coupled PCR. Samples of target gene-expressing tissue, can
also be evaluated immunocytochemically using antibodies specific
for the transgenic product of interest.
[0186] The target gene transgenic animals that express target gene
mRNA or target gene transgene peptide (detected
immunocytochemically, using antibodies directed against target gene
product epitopes) at easily detectable levels should then be
further evaluated to identify those animals which display tumor
progression state characteristics, including tumor progression
disorder symptoms. Such tumor progression disorder characteristics
and/or symptoms can include, for example, those associated with
such tumor cells as found in human melanoma, breast,
gastrointestinal, such as esophageal, stomach, colon, bowel,
colorectal and rectal cancers, prostate, bladder, testicular,
ovarian, uterine, cervical, brain, lung, bronchial, larynx,
pharynx, liver, pancreatic, thyroid, bone, leukemias, lymphomas and
various types of skin cancers.
[0187] Additionally, specific cell types within the transgenic
animals can be analyzed for cellular phenotypes characteristic of
tumor progression. Such cellular phenotypes can include, for
example, differential gene expression characteristic of cells
within a given tumor progression state of interest. Further, such
cellular phenotypes can include as assessment of a particular cell
type fingerprint pattern of expression and its comparison to known
fingerprint expression profiles of the particular cell type in
animals exhibiting tumor progression. Such transgenic animals serve
as suitable model systems for tumor progression disorders.
[0188] Once target gene transgenic founder animals are produced
(i.e., those animals which express target gene proteins in cells or
tissues of interest, and which, preferably, exhibit tumor
progression characteristics), they can be bred, inbred, outbred, or
crossbred to produce colonies of the particular animal. Examples of
such breeding strategies include but are not limited to:
outbreeding of founder animals with more than one integration site
in order to establish separate lines; inbreeding of separate lines
in order to produce compound target gene transgenics that express
the target gene transgene of interest at higher levels because of
the effects of additive expression of each target gene transgene;
crossing of heterozygous transgenic animals to produce animals
homozygous for a given integration site in order to both augment
expression and eliminate the possible need for screening of animals
by DNA analysis; crossing of separate homozygous lines to produce
compound heterozygous or homozygous lines; breeding animals to
different inbred genetic backgrounds so as to examine effects of
modifying alleles on expression of the target gene transgene and
the development of symptoms for tumor progression disorders. One
such approach is to cross the target gene transgenic founder
animals with a wild type strain to produce an Fl generation that
exhibits symptoms for tumor progression disorders. The F1
generation can then be inbred in order to develop a homozygous
line, if it is found that homozygous target gene transgenic animals
are viable.
5.7.2. Cell-Based Assays
[0189] Cells that contain and express target gene sequences which
encode target gene protein, and, further, exhibit cellular
phenotypes associated with tumor progression disorders, can be
utilized to identify compounds that exhibit an ability to prevent
and/or ameliorate tumor progression. Cellular phenotypes which can
indicate an ability to ameliorate symptoms of tumor progression
disorders can include, for example, tumor cells with low or high
metastatic potential.
[0190] Further, the fingerprint pattern of gene expression of cells
of interest can be analyzed and compared to the normal fingerprint
pattern. Those compounds which cause cells exhibiting cellular
phenotypes of tumor progression disorders, including metastatic
diseases, to produce a fingerprint pattern more closely resembling
a normal fingerprint pattern for the cell of interest can be
considered candidates for further testing regarding an ability to
ameliorate the symptoms of such diseases.
[0191] Cells which will be utilized for such assays can, for
example, include non-recombinant cell lines, such as, but not
limited to, melanoma (e.g., B16 F1 and B16 F10 cell lines), human
colon (e.g., KM12c and KM20L4 cell lines), prostate (e.g., DU 145
and PC-3-M cell lines) and breast cancer cell lines (e.g., MCF-7
and MDA-MB-435 cell lines). In addition, purified primary or
secondary tumor cells derived from either transgenic or
non-transgenic tumor cells can be used.
[0192] Further, cells which can be used for such assays can also
include recombinant, transgenic cell lines. For example, the
metastatic disease animal models of the invention, discussed,
above, in Section 5.2.4.1, can be used to generate cell lines,
containing one or more cell types involved in metastatic diseases,
that can be used as cell culture models for these disorders. While
primary cultures derived from the metastasis in transgenic animals
of the invention can be utilized, the generation of continuous cell
lines is preferred. For examples of techniques which can be used to
derive a continuous cell line from the transgenic animals, see
Small et al., 1985, Mol. Cell Biol. 5:642-648.
[0193] Alternatively, cells of a cell type known to be involved in
metastatic diseases can be transfected with sequences capable of
increasing or decreasing the amount of target gene expression
within the cell. For example, target gene sequences can be
introduced into, and over expressed in, the genome of the cell of
interest, or, if endogenous target gene sequences are present, they
can either be overexpressed or, alternatively, be disrupted in
order to underexpress or inactivate target gene expression.
[0194] In order to overexpress a target gene sequence, the coding
portion of the target gene sequence can be ligated to a regulatory
sequence which is capable of driving gene expression in the cell
type of interest. Such regulatory regions will be well known to
those of skill in the art, and can be utilized in the absence of
undue experimentation.
[0195] For under expression of an endogenous target gene sequence,
such a sequence can be isolated and engineered such that when
reintroduced into the genome of the cell type of interest, the
endogenous target gene alleles will be inactivated. Preferably, the
engineered target gene sequence is introduced via gene targeting
such that the endogenous target sequence is disrupted upon
integration of the engineered target gene sequence into the cell's
genome. Gene targeting is discussed, above, in Section 5.7.1.
[0196] Transfection of target gene sequence nucleic acid can be
accomplished by utilizing standard techniques. See, for example,
Ausubel, 1989, supra. Transfected cells should be evaluated for the
presence of the recombinant target gene sequences, for expression
and accumulation of target gene mRNA, and for the presence of
recombinant target gene protein production. In instances wherein a
decrease in target gene expression is desired, standard techniques
can be used to demonstrate whether a decrease in endogenous target
gene expression and/or in target gene product production is
achieved.
5.8. Screening Assays for Compounds that Interact with the Target
Gene Product
[0197] The following assays are designed to identify compounds that
bind to target gene products, bind to other cellular proteins that
interact with a target gene product, and to compounds that
interfere with the interaction of the target gene product with
other cellular proteins.
[0198] Such compounds can include, but are not limited to, other
cellular proteins. Specifically, such compounds can include, but
are not limited to, peptides, such as, for example, soluble
peptides, including, but not limited to Ig-tailed fusion peptides,
comprising extracellular portions of target gene product
transmembrane receptors, and members of random peptide libraries
(see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghton, R.
et al., 1991, Nature 354:84-86), made of D-and/or L-configuration
amino acids, phosphopeptides (including, but not limited to,
members of random or partially degenerate phosphopeptide libraries;
see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778), antibodies
(including, but not limited to, polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and FAb,
F(ab').sub.2 and FAb expression libary fragments, and
epitope-binding fragments thereof), and small organic or inorganic
molecules.
[0199] Compounds identified via assays such as those described
herein can be useful, for example, in elaborating the biological
function of the target gene product, and for ameliorating symptoms
of tumor progression. In instances, for example, whereby a tumor
progression state or disorder results from a lower overall level of
target gene expression, target gene product, and/or target gene
product activity in a cell involved in the tumor progression state
or disorder, compounds that interact with the target gene product
can include ones which accentuate or amplify the activity of the
bound target gene protein. Such compounds would bring about an
effective increase in the level of target gene activity, thus
ameliorating symptoms of the tumor progression disorder or state.
In instances whereby mutations within the target gene cause
aberrant target gene proteins to be made which have a deleterious
effect that leads to tumor progression, compounds that bind target
gene protein can be identified that inhibit the activity of the
bound target gene protein. Assays for testing the effectiveness of
compounds, identified by, for example, techniques such as those
described in Section 5.8.1-5.8.3, are discussed, below, in Section
5.8.4.
5.8.1. In Vitro Screening Assays for Compounds that Bind to a
Target Gene Product
[0200] In vitro systems can be designed to identify compounds
capable of binding the target gene products of the invention.
Compounds identified can be useful, for example, in modulating the
activity of wild type and/or mutant target gene products,
preferably mutant target gene proteins, can be useful in
elaborating the biological function of the target gene product, can
be utilized in screens for identifying compounds that disrupt
normal target gene interactions, or can in themselves disrupt such
interactions.
[0201] The principle of the assays used to identify compounds that
bind to the target gene product involves preparing a reaction
mixture of the target gene protein and the test compound under
conditions and for a time sufficient to allow the two components to
interact and bind, thus forming a complex which can be removed
and/or detected in the reaction mixture. These assays can be
conducted in a variety of ways. For example, one method to conduct
such an assay would involve anchoring target gene product or the
test substance onto a solid phase and detecting target gene
product/test compound complexes anchored on the solid phase at the
end of the reaction. In one embodiment of such a method, the target
gene product can be anchored onto a solid surface, and the test
compound, which is not anchored, can be labeled, either directly or
indirectly.
[0202] In practice, microtitre plates can conveniently be utilized
as the solid phase. The anchored component can be immobilized by
non-covalent or covalent attachments. Non-covalent attachment can
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized can be used to anchor the protein to the solid surface.
The surfaces can be prepared in advance and stored.
[0203] In order to conduct the assay, the nonimmobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
nonimmobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with a labeled anti-Ig antibody).
[0204] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g., using an immobilized antibody
specific for target gene or the test compound to anchor any
complexes formed in solution, and a labeled antibody specific for
the other component of the possible complex to detect anchored
complexes.
5.8.2. Assays for Cellular Protiens that Interact with the Target
Gene Product
[0205] Any method suitable for detecting protein-protein
interactions can be employed for identifying novel target
product-cellular or extracellular protein interactions. These
methods are outlined in Section 5.1.3., supra, for the
identification of pathway genes, and can be utilized herein with
respect to the identification of proteins which interact with
identified target proteins. In such a case, the target gene serves
as the known "bait" gene.
5.8.3. Assays for Compounds that Interfere with Target
Gene/Cellular Product Interaction
[0206] The target gene products of the invention can, in vivo,
interact with one or more cellular or extracellular macromolecules,
such as proteins. Such macromolecules include, but are not limited
to, nucleic acid molecules and those products identified via
methods such as those described, above, in Section 5.8.2. For the
purposes of this discussion, such cellular and extracellular
macromolecules are referred to herein as "binding partners."
Compounds that disrupt such interactions can be useful in
regulating the activity of the target gene product, especially
mutant target gene products. Such compounds can include, but are
not limited to molecules such as antibodies, peptides, and the like
described in Section 5.3.1. above.
[0207] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between the target
gene product and its cellular or extracellular binding partner or
partners involves preparing a reaction mixture containing the
target gene product, and the binding partner under conditions and
for a time sufficient to allow the two products to interact and
bind, thus forming a complex. In order to test a compound for
inhibitory activity, the reaction mixture is prepared in the
presence and absence of the test compound. The test compound can be
initially included in the reaction mixture, or can be added at a
time subsequent to the addition of target gene and its cellular or
extracellular binding partner. Control reaction mixtures are
incubated without the test compound or with a placebo. The
formation of any complexes between the target gene product and the
cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target gene product
and the interactive binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal target gene product can also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene product. This comparison can be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
products.
[0208] The assay for compounds that interfere with the interaction
of the target gene products and binding partners can be conducted
in a heterogeneous or homogeneous format. Heterogeneous assays
involve anchoring either the target gene product or the binding
partner onto a solid phase and detecting complexes anchored on the
solid phase at the end of the reaction. In homogeneous assays, the
entire reaction is carried out in a liquid phase. In either
approach, the order of addition of reactants can be varied to
obtain different information about the compounds being tested. For
example, test compounds that interfere with the interaction between
the target gene products and the binding partners, e.g., by
competition, can be identified by conducting the reaction in the
presence of the test substance; i.e., by adding the test substance
to the reaction mixture prior to or simultaneously with the target
gene product and interactive cellular or extracellular binding
partner. Alternatively, test compounds that disrupt preformed
complexes, e.g. compounds with higher binding constants that
displace one of the components from the complex, can be tested by
adding the test compound to the reaction mixture after complexes
have been formed. The various formats are described briefly
below.
[0209] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface, while the non-anchored
species is labeled, either directly or indirectly. In practice,
microtitre plates are conveniently utilized. The anchored species
can be immobilized by non-covalent or covalent attachments.
Non-covalent attachment can be accomplished simply by coating the
solid surface with a solution of the target gene product or binding
partner and drying. Alternatively, an immobilized antibody specific
for the species to be anchored can be used to anchor the species to
the solid surface. The surfaces can be prepared in advance and
stored.
[0210] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. Where the non-immobilized species
is pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the non-immobilized
species is not pre-labeled, an indirect label can be used to detect
complexes anchored on the surface; e.g., using a labeled antibody
specific for the initially non-immobilized species (the antibody,
in turn, can be directly labeled or indirectly labeled with a
labeled anti-Ig antibody). Depending upon the order of addition of
reaction components, test compounds which inhibit complex formation
or which disrupt preformed complexes can be detected.
[0211] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds which inhibit complex
or which disrupt preformed complexes can be identified.
[0212] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this approach, a preformed complex of the
target gene product and the interactive cellular or extracellular
binding partner product is prepared in which either the target gene
products or their binding partners are labeled, but the signal
generated by the label is quenched due to complex formation (see,
e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this
approach for immunoassays). The addition of a test substance-that
competes with and displaces one of the species from the preformed
complex will result in the generation of a signal above background.
In this way, test substances which disrupt target gene
product-cellular or extracellular binding partner interaction can
be identified.
[0213] In a particular embodiment, the target gene product can be
prepared for immobilization using recombinant DNA techniques
described in Section 5.1.2, supra. For example, the target gene
coding region can be fused to a glutathione-S-transferase (GST)
gene using a fusion vector such as pGEX-5X-1, in such a manner that
its binding activity is maintained in the resulting fusion product.
The interactive cellular or extracellular product can be purified
and used to raise a monoclonal antibody, using methods routinely
practiced in the art and described above, in Section 5.2.4. This
antibody can be labeled with the radioactive isotope .sup.125I, for
example, by methods routinely practiced in the art. In a
heterogeneous assay, e.g., the GST-Target gene fusion product can
be anchored to glutathione-agarose beads. The interactive cellular
or extracellular binding partner product can then be added in the
presence or absence of the test compound in a manner that allows
interaction and binding to occur. At the end of the reaction
period, unbound material can be washed away, and the labeled
monoclonal antibody can be added to the system and allowed to bind
to the complexed components. The interaction between the target
gene product and the interactive cellular or extracellular binding
partner can be detected by measuring the amount of radioactivity
that remains associated with the glutathione-agarose beads. A
successful inhibition of the interaction by the test compound will
result in a decrease in measured radioactivity.
[0214] Alternatively, the GST-target gene fusion product and the
interactive cellular or extracellular binding partner product can
be mixed together in liquid in the absence of the solid
glutathione-agarose beads. The test compound can be added either
during or after the binding partners are allowed to interact. This
mixture can then be added to the glutathione-agarose beads and
unbound material is washed away. Again the extent of inhibition of
the binding partner interaction can be detected by adding the
labeled antibody and measuring the radioactivity associated with
the beads.
[0215] In another embodiment of the invention, these same
techniques can be employed using peptide fragments that correspond
to the binding domains of the target gene product and the
interactive cellular or extracellular binding partner (in case
where the binding partner is a product), in place of one or both of
the full length products. Any number of methods routinely practiced
in the art can be used to identify and isolate the protein's
binding site. These methods include, but are not limited to,
mutagenesis of one of the genes encoding one of the products and
screening for disruption of binding in a co-immunoprecipitation
assay. Compensating mutations in the gene encoding the second
species in the complex can be selected. Sequence analysis of the
genes encoding the respective products will reveal the mutations
that correspond to the region of the product involved in
interactive binding. Alternatively, one product can be anchored to
a solid surface using methods described in this Section above, and
allowed to interact with and bind to its labeled binding partner,
which has been treated with a proteolytic enzyme, such as trypsin.
After washing, a short, labeled peptide comprising the binding
domain can remain associated with the solid material, which can be
isolated and identified by amino acid sequencing. Also, once the
gene coding for the cellular or extracellular binding partner
product is obtained, short gene segments can be engineered to
express peptide fragments of the product, which can then be tested
for binding activity and purified or synthesized.
5.8.4. Asays for Amelioration of Tumor Progression Systems
[0216] Any of the binding compounds, including but not limited to,
compounds such as those identified in the foregoing assay systems,
can be tested for the ability to prevent and/or ameliorate symptoms
of tumor progression and tumor progression disorders, including
metastatic disease. Cell-based and animal model-based assays for
the identification of compounds exhibiting an ability to prevent
and/or ameliorate tumor progression symptoms are described
below.
[0217] First, cell-based systems such as those described, above, in
Section 5.7.2, can be used to identify compounds which can act to
ameliorate symptoms of tumor progression For example, such cell
systems can be exposed to a compound, suspected to exhibiting an
ability to ameliorate tumor progression symptoms, at a sufficient
concentration and for a time sufficient to elicit such an
amelioration in the exposed cells. After exposure, the cells are
examined to determine whether one or more tumor progression state
or tumor progression disorder phenotypes has been altered to
resemble a more normal or more wild-type, non-neoplastic disease
phenotype.
[0218] Taking, as an example, tumor progression involving
metastasis, cell-based systems such as the highly metastatic B16
F10 melanoma cell line can be utilized. Upon exposure to such cell
systems, compounds can be assayed for their ability to reduce the
metastatic potential of such cells. Further, the level of 030 gene
expression within these cells may be assayed. Presumably, an
increase in the observed level of 030 gene expression would
indicate an amelioration of the metastatic tumor progression
state.
[0219] In addition, animal-based systems, such as those described,
above, in Section 5.7.1, can be used to identify compounds capable
of ameliorating symptoms of tumor progression. Such animal models
can be used as test substrates for the identification of drugs,
pharmaceuticals, therapies, and interventions which can be
effective in treating tumor progression disorders. For example,
animal models can be exposed to a compound suspected to exhibit an
ability to ameliorate tumor progression symptoms, at a sufficient
concentration and for a time sufficient to elicit such an
amelioration in the exposed animals. The response of the animals to
the exposure can be monitored by assessing the reversal of
disorders associated with tumor progression. With regard to
intervention, any treatments which reverse any aspect of symptoms
of tumor progression, such as, for example, those associated with
metastatic disease, should be considered as candidates for human
therapeutic intervention in the treatment of tumor progression.
Dosages of test agents can be determined by deriving dose-response
curves, as discussed in Section 5.10, below.
[0220] Further, gene expression patterns can be utilized to assess
the ability of a compound to ameliorate symptoms of tumor
progression and tumor progression disorders. For example,
fingerprint gene expression or a fingerprint pattern can then be
used in such an assessment. Fingerprint gene expression and
fingerprint patterns are described, below, in Section 5.11.
[0221] Fingerprint patterns can be characterized for known states
(e.g., normal or known pre-neoplastic, neoplastic or metastatic
states) within the cell- and/or animal-based model systems.
Subsequently, these known fingerprint patterns can be compared to
ascertain the effect a test compound has to modify such fingerprint
patterns, and to cause the pattern to more closely resemble that of
a more desirable fingerprint pattern.
[0222] For example, administration of a compound can cause the
fingerprint pattern of a metastatic disease model system to more
closely resemble a control, normal system. Administration of a
compound can, alternatively, cause the fingerprint pattern of a
control system to begin to mimic tumor progression states, such as
metastatic disease states.
5.8.5. Monotoring of Effects During Clinical Trials
[0223] Monitoring the influence of compounds on tumor progression
can be applied not only in basic drug screening, but also in
clinical trials. In such clinical trials, the expression of a panel
of genes that have been discovered in any one of the paradigms
discovered in Section 5.1.1.1 can be used as a "read out" of the
tumor progression state of a particular cell.
[0224] For example, and not by way of limitation, the paradigm
describing the B16 melanoma cells provides for the identification
of fingerprint genes (e.g., 030) that are down-regulated in
metastatic tumor cells. For example, in a clinical trial, tumor
cells can be isolated from the primary tumors removed by surgery,
and RNA prepared and analyzed by differential display as described
in Section 6.1. The levels of expression of the fingerprint genes
can be quantified by Northern blot analysis or RT-PCR, as described
in Section 6.1, or alternatively by measuring the amount of protein
produced, by one of the methods described in Section 5.7.2. In this
way, the fingerprint profiles can serve as putative biomarkers
indicative of the metastatic potential of the tumor cell. Thus, by
monitoring the level of expression of romy030, a protocol for
suitable chemotherapeutic anticancer drugs can be developed based
on the metastatic potential of tumor cells in the primary. In cases
of inoperable metastatic disease, patients can have biopsies
removed for measurement of romy030 expression so that the drug's
efficacy can be measured by monitoring the degree of restored
expression of romy030.
5.9. Compounds and Methods for Treatment of Tumor Progression
[0225] Described herein are methods and compositions which can be
used ameliorate symptoms of tumor progression and disorders
involving tumor progression via, first, target gene modulation,
and/or second, via a depletion of the cells involved in tumor
progression. Target gene modulation can be of a positive or
negative nature, depending on the specific situation involved, but
each modulatory event yields a net result in which tumor
progression symptoms are ameliorated.
[0226] "Negative modulation," as used herein, refers to a reduction
in the level and/or activity of target gene product relative to the
level and/or activity of the target gene product in the absence of
the modulatory treatment.
[0227] "Positive modulation," as used herein, refers to an increase
in the level and/or activity of target gene product relative to the
level and/or activity of target gene product in the absence of
modulatory treatment.
[0228] It is possible that tumor progression can be brought about,
at least in part, by an abnormal level of gene product, or by the
presence of a gene product exhibiting abnormal activity. As such,
the reduction in the level and/or activity of such gene products
would bring about the amelioration of tumor progression symptoms.
Negative modulatory techniques for the reduction of target gene
expression levels or target gene product activity levels are
discussed in Section 5.9.1, below.
[0229] Alternatively, it is possible that tumor progression can be
brought about, at least in part, by the absence or reduction of the
level of gene expression, or a reduction in the level of a gene
product's activity. As such, an increase in the level of gene
expression and/or the activity of such gene products would bring
about the amelioration of tumor progression symptoms.
[0230] For example, as demonstrated in the Example presented in
Section 6, below, a reduction in the level of 030 gene expression
correlates with a highly metastatic tumor progression state. A 030
positive modulatory technique which increased 030 gene expression
in cells within a highly metastatic tumor progression state should,
therefore, act to ameliorate the symptoms of such a state. Further,
because the 030 gene product may exhibit general tumor suppressor
features, it is possible that a 030 positive modulatory technique
could ameliorate symptoms of many tumor progression events.
[0231] Positive modulatory techniques for increasing the target
gene expression levels or target gene product activity levels are
discussed in Section 5.9.2, below.
[0232] Additionally, tumor progression treatment techniques whereby
the concentration of cells involved in tumor progression are
depleted are described, below, in Section 5.9.3.
[0233] Among the tumor progression events which may be treated are
those associated with human tumors. Such human tumors may include,
for example, human melanomas, breast, gastrointestinal, such as
esophageal, stomach, colon, bowel, colorectal and rectal cancers,
prostate, bladder, testicular, ovarian, uterine, cervical, brain,
lung, bronchial, larynx, pharynx, liver, pancreatic, thyroid, bone,
leukemias, lymphomas and various types of skin cancers.
5.9.1. Negative Modulatory Technique
[0234] As discussed, above, successful treatment of tumor
progression symptoms and of disorders involving tumor progression
can be brought about by techniques which serve to inhibit the
expression or activity of target gene products.
[0235] For example, compounds such as those identified through
assays described, above, in Section 5.8, which exhibit negative
modulatory activity, can be used in accordance with the invention
to prevent and/or ameliorate symptoms of tumor progression,
including tumor progression involving metastatic disorders. As
discussed in Section 5.8., above, such molecules can include, but
are not limited to peptides, phosphopeptides, small organic or
inorganic molecules, or antibodies (including, for example,
polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or
single chain antibodies, and FAb, F(ab').sub.2 and FAb expression
library fragments, and epitope-binding fragments thereof). Negative
modulatory techniques involving antibody administration are
described, below, in Section 5.9.1.2. Techniques for the
determination and administration of such compounds are described,
below, in Section 5.10.
[0236] Further, antisense and ribozyme molecules which inhibit
expression of the target gene can also be used in accordance with
the invention to reduce the level of target gene expression, thus
effectively reducing the level of target gene activity. Still
further, triple helix molecules can be utilized in reducing the
level of target gene activity. Such techniques are described,
below, in Section 5.9.1.1.
5.9.1.1. Negative Modulatory Antisense, Ribozyme and Triple Helix
Approaches
[0237] Among the compounds which can exhibit the ability to prevent
and/or ameliorate symptoms of tumor progression are antisense,
ribozyme, and triple helix molecules. Such molecules can be
designed to reduce or inhibit either wild type, or if appropriate,
mutant target gene activity. Techniques for the production and use
of such molecules are well known to those of skill in the art.
[0238] Anti-sense RNA and DNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. With respect to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g., between the -10 and +10 regions of the Target gene
nucleotide sequence of interest, are preferred.
[0239] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. (For a review, see, for example,
Rossi, J., 1994, Current Biology A:469-471). The mechanism of
ribozyme action involves sequence specific hybridization of the
ribozyme molecule to complementary target RNA, followed by a
endonucleolytic cleavage. The composition of ribozyme molecules
must include one or more sequences complementary to the target gene
mRNA and must include the well-known catalytic sequence responsible
for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246,
which is incorporated by reference herein in its entirety. As such
within the scope of the invention are engineered hammerhead motif
ribozyme molecules that specifically and efficiently catalyze
endonucleolytic cleavage of RNA sequences encoding target gene
proteins.
[0240] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the molecule of
interest for ribozyme cleavage sites which include the following
sequences, GUA, GUU, and GUC. Once identified, short RNA sequences
of between 15 and 20 ribonucleotides corresponding to the region of
the target gene containing the cleavage site can be evaluated for
predicted structural features, such as secondary structure, that
can render the oligonucleotide sequence unsuitable. The suitability
of candidate sequences can also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using ribonuclease protection assays.
[0241] Nucleic acid molecules to be used in triplex helix formation
for the inhibition of transcription should be single stranded and
composed of deoxynucleotides. The base composition of these
oligonucleotides must be designed to promote triple helix formation
via Hoogsteen base pairing rules, which generally require sizeable
stretches of either purines or pyrimidines to be present on one
strand of a duplex. Nucleotide sequences can be pyrimidine-based,
which will result in TAT and CGC.sup.+ triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarily to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules can
be chosen that are purine-rich, for example, contain a stretch of G
residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC paris, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in GGC triplets across the three strands in the
triplex.
[0242] Alternatively, the potential sequences that can be targeted
for triple helix formation can be increased by creating a so called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizeable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0243] In instances wherein the antisense, ribozyme, and/or triple
helix molecules described herein are utilized to reduce or inhibit
mutant gene expression, it is possible that the technique utilized
can also efficiently reduce or inhibit the transcription (triple
helix) and/or translation (antisense, ribozyme) of mRNA produced by
normal target gene alleles such that the possibility can arise
wherein the concentration of normal target gene product present can
be lower than is necessary for a normal phenotype. In such cases,
to ensure that substantially normal levels of target gene activity
are maintained, nucleic acid molecules that encode and express
target gene polypeptides exhibiting normal target gene activity can
be introduced into cells via gene therapy methods such as those
described, below, in Section 5.9.2 that do not contain sequences
susceptible to whatever antisense, ribozyme, or triple helix
treatments are being utilized. Alternatively, in instances whereby
the target gene encodes an extracellular protein, it can be
preferable to coadminister normal target gene protein into the cell
or tissue in order to maintain the requisite level of cellular or
tissue target gene activity.
[0244] Anti-sense RNA and DNA, ribozyme and triple helix molecules
of the invention can be prepared by any method known in the art for
the synthesis of DNA and RNA molecules. These include techniques
for chemically synthesizing oligodeoxyribonucleotides and
oligoribonucleotides well known in the art such as, for example,
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules can be generated by in vitro and in vivo transcription of
DNA sequences encoding the antisense RNA molecule. Such DNA
sequences can be incorporated into a wide variety of vectors which
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
[0245] Various well-known modifications to the DNA molecules can be
introduced as a means of increasing intracellular stability and
half-life. Possible modifications include but are not limited to
the addition of flanking sequences of ribo- or deoxy-nucleotides to
the 5' and/or 3' ends of the molecule or the use of
phosphorothioate or 2' O-methyl rather than phospho-diesterase
linkages within the oligodeoxyribonucleotide backbone.
5.9.1.2. Negative Modulatory Antibody Techniques
[0246] Antibodies can be generated which are both specific for
target gene product and which reduce target gene product activity.
Such antibodies may, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of tumor progression. Antibodies can be generated using
standard techniques described in Section 5.6, above, against the
proteins themselves or against peptides corresponding to portions
of the proteins. The antibodies include but are not limited to
polyclonal, monoclonal, Fab fragments, single chain antibodies,
chimeric antibodies, and the like.
[0247] In instances where the target gene protein to which the
antibody is directed is intracellular and whole antibodies are
used, internalizing antibodies can be preferred. However,
lipofectin or liposomes can be used to deliver the antibody or a
fragment of the Fab region which binds to the target gene epitope
into cells. Where fragments of the antibody are used, the smallest
inhibitory fragment which binds to the target protein's binding
domain is preferred. For example, peptides having an amino acid
sequence corresponding to the domain of the variable region of the
antibody that binds to the target gene protein can be used. Such
peptides can be synthesized chemically or produced via recombinant
DNA technology using methods well known in the art (e.g., see
Creighton, 1983, supra; and Sambrook et al., 1989, supra).
Alternatively, single chain neutralizing antibodies which bind to
intracellular target gene product epitopes can also be
administered. Such single chain antibodies can be administered, for
example, by expressing nucleotide sequences encoding single-chain
antibodies within the target cell population by utilizing, for
example, techniques such as those described in Marasco et al.
(Marasco, W. et al., 1993, Proc. Natl. Acad. Sci. USA
90:7889-7893).
[0248] In instances where the target gene protein is extracellular,
or is a transmembrane protein, any of the administration techniques
described, below in Section 5.10 which are appropriate for peptide
administration can be utilized to effectively administer inhibitory
target gene antibodies to their site of action.
5.9.2. Positive Modulatory Techniques
[0249] As discussed above, successful treatment of tumor
progression symptoms and of disorders involving tumor progression
can be brought about by techniques which serve to increase the
level of target gene expression or to increase the activity of a
target gene product.
[0250] For example, compounds such as those identified through
assays described, above, in Section 5.8, which exhibit positive
modulatory activity can be used in accordance with the invention to
ameliorate tumor progression symptoms. As discussed in Section 5.8,
above, such molecules can include, but are not limited to,
peptides, phosphopeptides, small organic or inorganic molecules, or
antibodies (including, for example, polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric or single chain antibodies, and
FAb, F(ab').sub.2 and FAb expression library fragments, and
epitope-binding fragments thereof). Positive modulatory techniques
involving antibody administration are described, below, in Section
5.9.2.1.
[0251] For example, a target gene protein, at a level sufficient to
ameliorate tumor progression symptoms can be administered to a
patient exhibiting such symptoms. Any of the techniques discussed,
below, in Section 5.10, can be utilized for such administration.
One of skill in the art will readily know how to determine the
concentration of effective, non-toxic doses of the normal target
gene protein, utilizing techniques such as those described, below,
in Section 5.10.1.
[0252] In instances wherein the compound to be administered is a
peptide compound, DNA sequences encoding the peptide compound can,
alternatively, be directly administered to a patient exhibiting
tumor progression symptoms, at a concentration sufficient to
generate the production of an amount of target gene product
adequate to ameliorate tumor progression symptoms. Any of the
techniques described, below, in Section 5.10, which achieve
intracellular administration, can be utilized for the
administration of such DNA molecules. The DNA molecules can be
produced, for example, by well-known recombinant techniques.
[0253] In the case of peptide compounds which act extracellularly,
the DNA molecules encoding such peptides can be taken up and
expressed by any cell type, so long as a sufficient circulating
concentration of peptide results for the elicitation of a reduction
in tumor progression symptoms.
[0254] In the case of compounds which act intracellularly, the DNA
molecules encoding such peptides must be taken up and expressed by
cells involved in the tumor progression at a sufficient level to
bring about the reduction of tumor progression symptoms.
[0255] Any technique which serves to selectively administer DNA
molecules to a cell involved in tumor progression is, therefore,
preferred for the DNA molecules encoding intracellularly acting
peptides.
[0256] Further, patients can be treated for symptoms of tumor
progression by gene replacement therapy. One or more copies of a
normal target gene or a portion of the gene that directs the
production of a normal target gene protein with target gene
function can be inserted into cells, using vectors which include,
but are not limited to adenovirus, adeno-associated virus, and
retrovirus vectors, in addition to other particles that introduce
DNA into cells, such as liposomes. Techniques such as those
described above can be utilized for the introduction of normal
target gene sequences into human cells.
[0257] In instances wherein the target gene encodes an
extracellular, secreted gene product, such gene replacement
techniques may be accomplished either in vivo or in vitro. For such
cases, the cell types expressing the target gene is less important
than achieving a sufficient circulating concentration of the
extracellular molecules for the amelioration of tumor progression
symptoms to occur. In vitro, target gene sequences can be
introduced into autologous cells. Those cells expressing the target
gene sequence of interest can then be reintroduced, preferably by
intravenous administration, into the patient such that there
results an amelioration of tumor progression symptoms.
[0258] In instances wherein the gene replacement involves a gene
which encodes a product which acts intracellularly, it is preferred
that gene replacement be accomplished in vivo. Further, because the
cell type in which the gene replacement must occur is the cell type
involved in tumor progression, such techniques must successfully
target such tumor progression cells.
[0259] Taking the 030 gene as an example, an increase in 030
expression can serve to ameliorate tumor progression symptoms, such
as, for example, tumor progression symptoms involving metastatic
processes. Therefore, any positive modulatory described herein
which increases the 030 gene product or gene product activity to a
level which is sufficient to ameliorate tumor progression symptoms
represents a successful tumor progression therapeutic
treatment.
5.9.3. Methods for Depleting Cells Involved in Tumor
Progression
[0260] Techniques described herein can be utilized to deplete the
total number of cells involved in tumor progression, thus
effectively decreasing the ratio of the tumor cells to
non-cancerous cells. Specifically, separation techniques are
described which can be used to deplete the total number of tumor
cells present within a cell population, and, further, targeting
techniques are described which can be utilized to deplete specific
tumor cell subpopulations.
[0261] Depending on the particular application, changing the number
of cells belonging to tumor cell population can yield inhibitory
responses leading to the amelioration of cancerous disorders.
[0262] The separation techniques described herein are based on the
presence or absence of specific cell surface, preferably
transmembrane, markers. By way of example, and not by way of
limitation, the techniques described herein utilize tumor specific
cell surface markers or antigens and will describe procedures
whereby tumor cells can be separated from other cells, thus
allowing for selective depletion of tumor cells.
[0263] Separation techniques can be utilized which separate and
purify cells, tumor cells, for example, in vitro from a population
of cells, such as hematopoietic cells autologous to the patient
being treated. For example, an initial tumor cell
subpopulation-containing population of cells, such as hematopoietic
cells, can be obtained from a leukemia patient using standard
procedures well known to those of skill in the art. Peripheral
blood can be utilized as one potential starting source for such
techniques, and can, for example, be obtained via venipuncture and
collection into heparinized tubes.
[0264] Once the starting source of autologous cells is obtained,
tumor cells can be removed, and thus selectively separated and
purified, by various methods which utilize antibodies which bind
specific markers present on tumor cells while absent on other cells
within the starting source. These techniques can include, for
example, flow cytometry using a fluorescence activated cell sorter
(FACS) and specific fluorochromes, biotin-avidin or
biotin-streptavidin separations using biotin conjugated to cell
surface marker-specific antibodies and avidin or streptavidin bound
to a solid support such as affinity column matrix or plastic
surfaces or magnetic separations using antibody-coated magnetic
beads.
[0265] Separation via antibodies for specific markers can be by
negative or positive selection procedures. In negative separation,
antibodies are used which are specific for markers present on
undesired cells, in this case tumor cells, which exhibit, for
example, the tumor specific cell surface marker. Cells bound by an
antibody to such a cell surface marker can be removed or lysed and
the remaining desired mixture retained. In positive separation,
antibodies specific for markers present on the desired cells of
interest, in this case tumor-like cells, are used. Cells bound by
the antibody are separated and retained. It will be understood that
positive and negative separations can be used substantially
simultaneously or in a sequential manner.
[0266] A common technique for antibody based separation is the use
of flow cytometry such as by a florescence activated cell sorter
(FACS). Typically, separation by flow cytometry is performed as
follows. The suspended mixture of cells are centrifuged and
resuspended in media. Antibodies which are conjugated to
fluorochrome are added to allow the binding of the antibodies to
specific cell surface markers. The cell mixture is then washed by
one or more centrifugation and resuspension steps. The mixture is
run through a FACS which separates the cells based on different
fluorescence characteristics. FACS systems are available in varying
levels of performance and ability, including multi-color analysis.
The facilitating cell can be identified by a characteristic profile
of forward and side scatter which is influenced by size and
granularity, as well as by positive and/or negative expression of
certain cell surface markers.
[0267] Other separation techniques besides flow cytometry can also
provide fast separations. One such method is biotin-avidin based
separation by affinity chromatography. Typically, such a technique
is performed by incubating cells with biotin-coupled antibodies to
specific markers, such as, for example, the transmembrane protein
encoded by the tumor-specific marker, followed by passage through
an avidin column. Biotin-antibody-cell complexes bind to the column
via the biotin-avidin interaction, while other cells pass through
the column. The specificity of the biotin-avidin system is well
suited for rapid positive separation. Multiple passages can ensure
separation of a sufficient level of the tumor cell subpopulation of
interest.
[0268] In instances whereby the goal of the separation technique is
to deplete the overall number of cells belonging to the tumor cell
subpopulation, the cells derived from the starting source of cells
which has now been effectively depleted of tumor cells can be
reintroduced into the patient. Such a depletion of the tumor cell
subpopulation results in the amelioration of cancerous disorders
associated with tumor progression.
[0269] In instances whereby the goal of the separation technique is
to augment or increase the overall number of cells belonging to a
non-cancerous cell subpopulation, cells derived from the purified
normal cell subpopulation can be reintroduced into the patient,
thus resulting in the amelioration of cancerous disorders
associated with an under activity of the normal cell
subpopulation.
[0270] The cells to be reintroduced will be cultured and expanded
ex vivo prior to reintroduction. Purified normal cell subpopulation
cells can be washed, suspended in, for example, buffered saline,
and reintroduced into the patient via intravenous
administration.
[0271] Cells to be expanded can be cultured, using standard
procedures, in the presence of an appropriate expansion agent which
induces proliferation of the purified normal cell subpopulation.
Such an expansion agent can, for example, be any appropriate
cytokine, antigen, or antibody.
[0272] Prior to being reintroduced into a patient, the purified
normal cells can be modified by, for example, transformation with
gene sequences encoding gene products of interest. Such gene
products should represent products which enhance the activity of
the purified normal cell subpopulation or, alternatively, represent
products which repress the activity of one or more of the other
normal cell subpopulations. Cell transformation and gene expression
procedures are well known to those of skill in the art, and can be
as those described, above, in Section 5.2.
[0273] Well-known targeting methods can, additionally, be utilized
in instances wherein the goal is to deplete the number of cells
belonging to a specific tumor cell subpopulation. Such targeting
methods can be in vivo or in vitro, and can involve the
introduction of targeting agents into a population of cells such
that the targeting agents selectively destroy a specific subset of
the cells within the population. In vivo administration techniques
which can be followed for such targeting agents are described,
below, in Section 5.10.
[0274] Targeting agents generally comprise, first, a targeting
moiety which, in the current instance, causes the targeting agent
to selectively associate with a specific tumor cell subpopulation.
The targeting agents generally comprise, second, a moiety capable
of destroying a cell with which the targeting agent has become
associated.
[0275] Targeting moieties can include, but are not limited to,
antibodies directed to cell surface markers found specifically on
the tumor cell subpopulation being targeted, or, alternatively, to
ligands, such as growth factors, which bind receptor-type molecules
found exclusively on the targeted tumor cell subpopulation.
[0276] Destructive moieties include any moiety capable of
inactivating or destroying a cell to which the targeting agent has
become bound. For example, a destructive moiety can include, but it
is not limited to cytotoxins or radioactive agents. Cytotoxins
include, for example, plant-, fungus-, or bacteria-derived toxins,
with deglycosylated Ricin A chain toxins being generally preferred
due to their potency and lengthy half-lives.
5.10. Pharmaceutical Preparation and Methods of Administration
[0277] The identified compounds that inhibit target gene
expression, synthesis and/or activity can be administered to a
patient at therapeutically effective doses to prevent, treat or
ameliorate tumor progression. A therapeutically effective dose
refers to that amount of the compound sufficient to result in
amelioration of symptoms of tumor progression.
5.10.1. Effective Dose
[0278] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0279] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (ie., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
5.10.2. Formulations and Use
[0280] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
[0281] Thus, the compounds and their physiologically acceptable
salts and solvates can be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral or rectal administration.
[0282] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration can take the form of, for example, solutions,
syrups or suspensions, or they can be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0283] Preparations for oral administration can be suitably
formulated to give controlled release of the active compound.
[0284] For buccal administration the compositions can take the form
of tablets or lozenges formulated in conventional manner.
[0285] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator
can be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0286] The compounds can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0287] The compounds can also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0288] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation (for
example, subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0289] The compositions can, if desired, be presented in a pack or
dispenser device which can contain one or more unit dosage forms
containing the active ingredient. The pack can for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device can be accompanied by instructions for
administration.
5.11. Diagnosis of Tumor Progression
[0290] A variety of methods can be employed for the diagnosis of
tumor progression and of disorders involving tumor progression,
including metastatic diseases. Such methods can, for example,
utilize reagents such as fingerprint gene nucleotide sequences
described in Sections 5.2.1, and antibodies directed against
differentially expressed and pathway gene peptides, as described,
above, in Section 5.2.1 (peptides) and 5.2.3 (antibodies).
Specifically, such reagents can be used, for example, for the
detection of the presence of target gene mutations, or the
detection of either over or under expression of target gene in
RNA.
[0291] The methods described herein can be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
specific fingerprint gene nucleic acid or anti-fingerprint gene
antibody reagent described herein, which can be conveniently used,
e.g., in clinical settings, to diagnose patients exhibiting
symptoms of metastatic diseases.
[0292] Any cell type or tissue, preferably T-cells, in which the
fingerprint gene is expressed can be utilized in the diagnostics
described below.
5.11.1. Detection of Fingerprint Gene Neucleic Acids
[0293] DNA or RNA from the cell type or tissue to be analyzed can
easily be isolated using procedures which are well known to those
in the art. Diagnostic procedures can also be performed "in situ"
directly upon tissue sections (fixed and/or frozen) of patient
tissue obtained from biopsies or resections, such that no nucleic
acid purification is necessary. Nucleic acid reagents such as those
described in Section 5.1 can be used as probes and/or primers for
such in situ procedures (see, for example, Nuovo, G. J., 1992, PCR
in situ hybridization: Protocols and Applications, Raven Press,
NY).
[0294] Fingerprint gene nucleotide sequences, either RNA or DNA,
can, for example, be used in hybridization or amplification assays
of biological samples to detect gene structures and expression
associated with metastasis. Such assays can include, but are not
limited to, Southern or Northern analyses, single stranded
conformational polymorphism analyses, in situ hybridization assays,
and, polymerase chain reaction analyses. Such analyses can reveal
both quantitative aspects of the expression pattern of the
fingerprint gene, and qualitative aspects of the fingerprint gene
expression and/or gene composition. That is, such techniques can
include, for example, point mutations, insertions, deletions,
chromosomal rearrangements, and/or activation or inactivation of
gene expression.
[0295] Preferred diagnostic methods for the detection of
fingerprint gene-specific nucleic acid molecules can involve for
example, contacting and incubating nucleic acids, derived from the
cell type or tissue being analyzed, with one or more labeled
nucleic acid reagents as are described in Section 5.1, under
conditions favorable for the specific annealing of these reagents
to their complementary sequences within the nucleic acid molecule
or interest. Preferably, the lengths of these nucleic acid reagents
are at least 15 to 30 nucleotides. After incubation, all
non-annealed nucleic acids are removed from the nucleic
acid:fingerprint RNA molecule hybrid. The presence of nucleic acids
from the target tissue which have hybridized, if any such molecules
exist, is then detected. Using such a detection scheme, the nucleic
acid from the tissue or cell type of interest can be immobilized,
for example, to a solid support such as a membrane, or a plastic
surface such as that on a microtitre plate or polystyrene beads. In
this case, after incubation, non-annealed, labeled fingerprint
nucleic acid reagents of the type described in Section 5.1 are
easily removed. Detection of the remaining, annealed, labeled
nucleic acid reagents is accomplished using standard techniques
well-known to those in the art.
[0296] Alternative diagnostic methods for the detection of
fingerprint gene specific nucleic acid molecules can involve their
amplification, e.g., by PCR (the experimental embodiment set forth
in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany, F., 1991, Proc. Natl. Acad. Sci. USA 88:189-193),
self sustained sequence replication (Guatelli, J. C. et al., 1990,
Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional
amplification system (Kwoh, D. Y et al., 1989, Proc. Natl. Acad.
Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al.,
1988, Bio/Technology 6:1197), or any other nucleic acid
amplification method, followed by the detection of the amplified
molecules using techniques well known to those of skill in the art.
These detection schemes are especially useful for the detection of
nucleic acid molecules if such molecules are present in very low
numbers.
[0297] In one embodiment of such a detection scheme, a cDNA
molecule is obtained from an RNA molecule of interest (e.g., by
reverse transcription of the RNA molecule into cDNA). Cell types or
tissues from which such RNA can be isolated include any tissue in
which wild type fingerprint gene is known to be expressed. A
sequence within the cDNA is then used as the template for a nucleic
acid amplification reaction, such as a PCR amplification reaction,
or the like. The nucleic acid reagents used as synthesis initiation
reagents (e.g., primers) in the reverse transcription and nucleic
acid amplification steps of this method are chosen from among the
fingerprint gene nucleic acid reagents described in Section 5.1.
The preferred lengths of such nucleic acid reagents are at least
19-30 nucleotides. For detection of the amplified product, the
nucleic acid amplification can be performed using radioactively or
non-radioactively labeled nucleotides. Alternatively, enough
amplified product can be made such that the product can be
visualized by standard ethidium bromide staining or by utilizing
any other suitable nucleic acid staining method.
[0298] In addition to methods which focus primarily on the
detection of one nucleic acid sequence, fingerprint profiles, as
discussed in Section 5.3.4., can also be assessed in such detection
schemes. Fingerprint profiles can be generated, for example, by
utilizing a differential display procedure, as discussed above in
5.1.1.2, Northern analysis and/or RT-PCR. Any of the gene sequences
described, above, in Section 5.2.1 can be used as probes and/or PCR
primers for the generation and corroboration of such fingerprint
profiles.
5.11.2. Detection of Target Gene Peptide
[0299] Antibodies directed against wild type or mutant fingerprint
gene peptides, which are discussed, above, in Section 5.2.3, can
also be used in tumor progression diagnostics and prognostics, as
described, for example, herein. Such diagnostic methods, can be
used to detect abnormalities in the level of fingerprint gene
protein expression, or abnormalities in the structure and/or
tissue, cellular, or subcellular location of fingerprinting gene
protein. Structural differences can include, for example,
differences in the size, electronegativity, or antigenicity of the
mutant fingerprint gene protein relative to the normal fingerprint
gene protein.
[0300] Protein from the tissue or cell type to be analyzed can
easily be isolated using techniques which are well known to those
of skill in the art. The protein isolation methods employed herein
can, for example, be such as those described in Harlow and Lane
(Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual,"
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),
which is incorporated herein by reference in its entirety.
[0301] Preferred diagnostic methods for the detection of wild type
or mutant fingerprint gene peptide molecules can involve, for
example, immunoassays wherein fingerprint gene peptides are
detected by their interaction with an anti-fingerprint gene
specific peptide antibody.
[0302] For example, antibodies, or fragments of antibodies, such as
those described, above, in Section 5.2.3, useful in the present
invention can be used to quantitatively or qualitatively detect the
presence of wild type or mutant fingerprint gene peptides. This can
be accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody (see below) coupled with
light microscopic, flow cytometric, or fluorimetric detection. Such
techniques are especially preferred if the fingerprint gene
peptides are expressed on the cell surface.
[0303] The antibodies (or fragments thereof) useful in the present
invention can, additionally, be employed histologically, as in
immunofluorescence or immunoelectron microscopy, for in situ
detection of target gene peptides. in situ detection can be
accomplished by removing a histological specimen from a patient,
and applying thereto a labeled antibody of the present invention.
The antibody (or fragment) is preferably applied by overlaying the
labeled antibody (or fragment) onto a biological sample. Through
the use of such a procedure, it is possible to determine not only
the presence of the fingerprint gene peptides, but also their
distribution in the examined tissue. Using the present invention,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0304] Immunoassays for wild type or mutant fingerprint gene
peptides typically comprise incubating a biological sample, such as
a biological fluid, a tissue extract, freshly harvested cells, or
cells which have been incubated in tissue culture, in the presence
of a detectably labeled antibody capable of identifying fingerprint
gene peptides, and detecting the bound antibody by any of a number
of techniques well-known in the art.
[0305] The biological sample can be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
can then be washed with suitable buffers followed by treatment with
the detectably labeled fingerprint gene specific antibody. The
solid phase support can then be washed with the buffer a second
time to remove unbound antibody. The amount of bound label on solid
support can then be detected by conventional means.
[0306] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material can
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration can be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface can be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0307] The binding activity of a given lot of anti-wild type or
mutant fingerprint gene peptide antibody can be determined
according to well-known methods. Those skilled in the art will be
able to determine operative and optimal assay conditions for each
determination by employing routine experimentation.
[0308] One of the ways in which the fingerprint gene
peptide-specific antibody can be detectably labeled is by linking
the same to an enzyme and use in an enzyme immunoassay (EIA)
(Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA),"
Diagnostic Horizons 2:1-7, 1978) (Microbiological Associates
Quarterly Publication, Walkersville, Md.); Voller, A. et al., J.
Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol.
73:482-523 (1981); Maggio, E. (ed.), ENZYME IMMUNOASSAY, CRC Press,
Boca Raton, Fla., 1980; Ishikawa, E. et al., (eds.) ENZYME
IMMUNOASSAY, Kgaku Shoin, Tokyo, 1981). The enzyme which is bound
to the antibody will react with an appropriate substrate,
preferably a chromogenic substrate, in such a manner as to produce
a chemical moiety which can be detected, for example, by
spectrophotometric, fluorimetric or by visual means. Enzymes which
can be used to detectably label the antibody include, but are not
limited to, malate dehydrogenase, staphylococcal nuclease,
delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
calorimetric methods which employ a chromogenic substrate for the
enzyme. Detection can also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0309] Detection can also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect
fingerprint gene wild type or mutant peptides through the use of a
radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles
of Radioimmunoassays, Seventh Training Course on Radioligand Assay
Techniques, The Endocrine Society, March, 1986, which is
incorporated by reference herein). The radioactive isotope can be
detected by such means as the use of a gamma counter or a
scintillation counter or by autoradiography.
[0310] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0311] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0312] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0313] Likewise, a bioluminescent compound can be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
6. EXAMPLE
[0314] Identificatin and Characterization of a Novel Gene that
Inhibits Tumor Progression
[0315] In the Example presented in this Section, the in vitro
paradigm, described, above, in Section 5.1.1.1, was utilized to
identify a gene, designated herein as the 030 gene, which is
differentially expressed in cells with a high metastatic potential
relative to cells having a low metastatic potential. Specifically,
the 030 gene is expressed in high metastatic potential cells at a
rate which is many-fold lower than it is expressed in
non-metastatic cells. Thus, as discussed below, the 030 gene can
encode a product important to a number of neoplastic processes,
including, for example, the progression of a cell to a metastatic
state, the aggressiveness of a cell's metastatic state, and the
ability of a primary tumor cell to invade surrounding tissue. Given
the differential 030 gene expression pattern revealed in this
Section, the 030 gene product can represent a protein having tumor
suppressor or inhibitor function.
6.1. Materials and Methods
6.1.1. Cell Culture
[0316] B16 F1 and B16 F10 melanoma cell lines were maintained in
culture in Eagle's minimal essential medium (MEM) supplemented with
10% fetal calf serum. Cells were harvested from nonconfluent
monolayers by a two minute treatment with 0.25% trypsin and 2 mM
EDTA.
[0317] For further characterization of in vivo activity, each cell
line was injected into mice. Cells were washed two times in MEM,
and the final cell suspension adjusted to 5.times.10.sup.5 cells
per ml in MEM. Two hundred microliters of this cell suspension
(1.times.10.sup.5 cells) was injected i.v. into the lateral tail
vein of C57BL/6J mice. After three weeks, the mice were sacrificed
and their lungs autopsied. The number of pulmonary tumors was
determined by counting surface nodules using a dissecting
microscope.
[0318] The differential expression of the 030 gene in B16 F1
relative to B16 F10 cell lines was compared with the extent of
pulmonary metastases which developed in B16 F1-injected mice
relative to B16 F10-injected mice.
6.1.2. Differential Display
[0319] Differential mRNA display was carried out as described,
above, in Section 5.1.1.2. Details of the differential display are
given, below.
[0320] RNA Isolation
[0321] RNA was isolated, using RNAzol, from nonconfluent monolayers
of B16 F1 and B16 F10 cell lines.
[0322] Isolated RNA was resuspended in DEPC H.sub.2O and
quantitated by spectrophotometry at OD.sub.260. Approximately half
of the RNA samples were then treated with DNAse I to remove
contaminating chromosomal DNA. Each 50 .mu.l RNA sample (50 .mu.g),
5.7 .mu.l 10.times.PCR buffer (Perkin-Elmer/Cetus) and 1 .mu.l
RNAse inhibitor (40 units/.mu.l; Boehringer Mannheim, Germany) were
mixed together. Two microliters of DNAse I (10 units/.mu.l;
Boehringer Mannheim) was added to the reaction which was incubated
for 30 min. at 37.degree. C. The total volume was brought to 200
.mu.l with DEPC H.sub.2O, extracted once with phenol/chloroform and
precipitated by adding 20 .mu.l 3M NaOAc, pH 4.8, (DEPC-treated),
500 .mu.l absolute ETOH and incubated for 1 hour on dry ice. The
precipitated sample was centrifuged for 15 min., and the pellet was
washed with 70% ETOH. The sample was re-centrifuged, the remaining
liquid was aspirated, and the pellet was resuspended in 50 .mu.l
H.sub.2O. The concentration of RNA was measured by reading the
OD.sub.260.
[0323] First Strand cDNA Synthesis
[0324] For each RNA sample, duplicate reverse transcription
reactions were carried out in parallel. Four hundred ng RNA plus
DEPC H.sub.2O in a total volume of 10 .mu.l were added to 4 .mu.l
T.sub.11CC 3' primer (10 .mu.M; Operon). The mixture was incubated
at 70.degree. C. for 5 min. to denature the RNA and then placed at
room temperature. Twenty-six .mu.l of reaction mix containing the
following components was added to each denatured RNA/primer sample:
8 .mu.l 5.times.First Strand Buffer (Gibco/BRL, Gaithersburg, Md.),
4 .mu.l 0.1M DTT (Gibcol/BRL), 2 .mu.l RNAse inhibitor (40
units/.mu.l) (Boehringer Mannheim), 4 .mu.l 200 .mu.M DNTP mix, 6
.mu.l H.sub.2O, 2 .mu.l Superscript reverse transcriptase (200
units/.mu.l; Gibcol/BRL). The reactions were mixed gently and
incubated for 30 min. at 42.degree. C. Sixty .mu.l of H.sub.2O, for
a final volume of 100 .mu.l, was then added and the samples were
denatured for 5 min. at 85.degree. C. and stored at -20.degree.
C.
[0325] PCR Reactions
[0326] The resulting single stranded cDNA molecules were then
amplified by PCR. Specifically, 13 .mu.l of reaction mix was added
to each tube of a 96 well plate on ice. The reaction mix contained
6.4 .mu.l H.sub.2O, 2 .mu.l 10.times.PCR Buffer (Perkin-Elmer), 2
.mu.l 20 .mu.M dNTPs, 0.4 .mu.l .sup.35S DATP (12.5 .mu.Ci/.mu.l;
50 .mu.Ci total; Dupont/NEN), 2 .mu.l 5' primer OPE4
(5'GTGACATGCC-3'; 10 .mu.M; Operon), and 0.2 .mu.l AmpliTaq.TM.
Polymerase (5 units/.mu.l; Perkin-Elmer). Next, 2 .mu.l of 3'
primer (T.sub.11CC, 10 .mu.M) were added to the side of each tube,
followed by 5 .mu.l of cDNA, also to the sides of the tubes, which
were still on ice. Tubes were capped and mixed, and brought up to
1000 rpm in a centrifuge, then immediately returned to ice. A
Perkin-Elmer 9600 thermal cycler was used, and programmed as
follows:
3 94.degree. C. 2 min. *94.degree. C. 15 sec. *40.degree. C. 2 min.
*ramp 72.degree. C. 1 min. *72.degree. C. 30 sec. 72.degree. C. 5
min. 4.degree. C. hold *x 40
[0327] When the thermal cycler initially reached 94.degree. C., the
96 well plate was removed from ice and placed directly into the
cycler. Following the amplification reaction, 15 .mu.l of loading
dye, containing 80% formamide, 10 mM EDTA, 1 mg/ml xylene cyanole,
1 mg/ml bromphenol blue were added. The loading dye and reaction
were mixed, incubated at 85.degree. C. for 5 min., cooled on ice,
centrifuged, and placed on ice. Approximately 4 .mu.l from each
tube was loaded onto a pre-run (60V) 6% denaturing acrylamide gel.
The gel was run at approximately 80V until top dye front was about
1 inch from bottom. The gel was transferred to 3MM paper (Whatman
Paper, England) and dried under vacuum. Bands were visualized by
autoradiography.
6.1.3. Other Technique
[0328] Amplified cDNA Band Isolation and Amplification
[0329] PCR bands determined to be of interest in the differential
display analysis were recovered from the gel and reamplified.
[0330] Briefly, differentially expressed bands were excised from
the dried gel with a razor blade and placed into a microfuge tube
with 100 .mu.l H.sub.2O and heated at 100.degree.0 C. for 5 min.,
vortexed, heated again to 100.degree. C. for 5 min., and vortexed
again. After cooling, 100 .mu.l H.sub.2O, 20 .mu.l 3M NaOAc, 1
.mu.l glycogen (20 mg/ml), and 500 .mu.l ethanol were added and the
sample was precipitated on dry ice. After centrifugation, the
pellet was washed and resuspended in 10 .mu.l H.sub.2O.
[0331] DNA isolated from the excised differentially expressed bands
were then reamplified by PCR using the following reaction
conditions:
4 58 .mu.l H.sub.2O 10 .mu.l 10x PCR Buffer (see above) 10 .mu.l
200 .mu.M dNTPs 10 .mu.l 10 .mu.M 3' primer (see above) 10 .mu.l 10
.mu.M 5' primer (see above) 1.5 .mu.l amplified band 0.5 .mu.l
AMPLITAQ .RTM. polymerase (5 units/.mu.l; (Perkin Elmer)
[0332] PCR conditions were the same as the initial conditions used
to generate the original amplified band, as described, above. After
reamplification, glycerol loading dyes were added and samples were
loaded onto a 2% preparative TAE/Biogel (Bio101, La Jolla, Calif.)
agarose gel and eluted. Bands were then excised from the gel with a
razor blade and vortexed for 15 min. at r.t., and purified using
the MERMAID.TM. kit from Bio101 by adding 3 volumes of MERMAID.TM.
high salt binding solution and 8 .mu.l of resuspended glassfog in a
microfuge tube. Glassfog was then pelleted, washed 3 times with
ethanol wash solution, and then DNA was eluted twice in 10 .mu.l at
50.degree. C.
[0333] Subcloning and Sequencing
[0334] The TA cloning kit (Invitrogen, San Diego, Calif.) was used
to subclone the amplified bands. The ligation reaction typically
consisted of 4 .mu.l sterile H.sub.2O, 1 .mu.l ligation buffer, 2
.mu.l TA cloning vector, 2 .mu.l PCR product, and 1 .mu.l T4 DNA
ligase. The volume of PCR product can vary, but the total volume of
PCR product plus H.sub.2O was always 6 .mu.l. Ligations (including
vector alone) were incubated overnight at 12.degree. C. before
bacterial transformation. TA cloning kit competent bacteria
(INV.alpha.F': enda1, recA1, hsdRl7(r-k, m+k), supE44, .lambda.-,
thi-1, gyrA, relA1, .phi.80lacZ.alpha..DELTA.M15.DELTA- .
(lacZYA-arqF), deoR+, F') were thawed on ice and 2 .mu.l of 0.5 M
.beta.-mercaptoethanol were added to each tube. Two .mu.l from each
ligation were added to each tube of competent cells (50 .mu.l),
mixed without vortexing, and incubated on ice for 30 min. Tubes
were then placed in 42.degree. C. bath for exactly 30 sec., before
being returned to ice for 2 min. Four hundred-fifty .mu.l of SOC
media (Sambrook et al., 1989, supra) were then added to each tube
which were then shaken at 37.degree. C. for 1 hr. Bacteria were
then pelleted, resuspended in approximately 200 .mu.l SOC and
plated on Luria broth agar plates containing X-gal and 60 .mu.g/ml
ampicillin and incubated overnight at 37.degree. C. White colonies
were then picked and screened for inserts using PCR.
[0335] A master mix containing 2 .mu.l 10.times.PCR buffer, 1.6
.mu.l 2.5 mM dNTP's, 0.1 .mu.l 25 mM MgCl.sub.2, 0.2 .mu.l M13
reverse primer (100 ng/.mu.l), 0.2 .mu.l M13 forward primer (100
ng/.mu.l), 0.1 .mu.l AmpliTaq.RTM. (Perkin-Elmer), and 15.8 .mu.l
H.sub.2O was made. Forty .mu.l of the master mix were aliquoted
into tubes of a 96 well plate, and whole bacteria were added with a
pipette tip prior to PCR. The thermal cycler was programmed for
insert screening as follows:
5 94.degree. C. 2 min. *94.degree. C. 15 sec. *47.degree. C. 2 min.
*ramp 72.degree. C. 30 sec. *72.degree. C. 30 sec. 72.degree. C. 10
min. 4.degree. C. hold *x 35
[0336] Reaction products were eluted on a 2% agarose gel and
compared to vector control. Colonies with vectors containing
inserts were purified by streaking onto LB/Amp plates. Vectors were
isolated from such strains and subjected to sequence analysis,
using an Applied Biosystems Automated Sequencer (Applied
Biosystems, Inc. Seattle, Wash.).
[0337] Cloning of Human Gene
[0338] A human retina cDNA library obtained from Clontech was
screened using the entire mouse fomy030 cDNA (FIGS. 3A and 3B) as a
probe. During this screen, one million library phage were screened,
53 of which were found to hybridize with the mouse fomy030 probe.
The cDNA inserts for eight of these positives were isolated,
subcloned, and sequenced.
[0339] Comparison of the murine fomy030 and human fohy030sequences
demonstrated a high degree of sequence similarity (86% identical at
the nucleotide level and 94.4% identical at the amino acid level)
within the 5', 1813 base pairs of their cDNAs. However, beyond this
point the sequences diverge and share no significant similarity.
The sequence of fomy030 at the point of divergence is GTAG, which
corresponds to a consensus splice donor site.
[0340] Three independent library isolated cDNAs, as well as a cDNA
isolated as a 3' RACE product were found to contain the fomy030
sequence. Thus, the most probable explanation for the divergence of
the human and murine sequences is the existence of alternate splice
forms of the fomy030 and fohy030 transcripts. The fomy030 splice
version results in a protein product of 542 amino acids in length,
while the fohy030 splice variant is predicted to encode a protein
of 1497 amino acids in length (FIG. 5).
[0341] Another splice variant is shown in FIG. 6 (SEQ ID NO:8), and
encodes a protein of 1533 amino acids in length (SEQ ID NO:9). The
cDNA of FIG. 5 (SEQ ID NO:6) is missing 34 nucleotides beginning
after 2879 in SEQ ID NO:8, and is missing 74 nucleotides beginning
after 2926 in SEQ ID NO:8. Thus, nucleotides 2880-2892 in SEQ ID
NO:6 are identical to nucleotides 2914-2926 in SEQ ID NO:8, and the
sequences are essentially identical starting at 2893 in SEQ ID NO:6
and 3001 in SEQ ID NO:8. The difference in the respective amino
acid sequences is that the amino acids are identical from 1 to 844,
and then again from 850 to 1497 in SEQ ID NO:7 and from 886 to 1533
in SEQ ID NO:9.
[0342] Within their common 5' sequences, fohy030 was also found to
have an additional three base pairs (GGA) inserted after position
1394 in the mouse cDNA (at positions 1066-1068 in FIGS. 5 and 6).
These additional three base pairs fall within the open reading
frames of both fohy030 and fomy030, and result in an additional
Glycine residue at position 356 within the open reading frame of
fohy030relative to fomy030.
[0343] Northern Analysis
[0344] Northern analysis was performed to confirm the differential
expression of the genes corresponding to the amplified bands, as
described below.
[0345] Twelve micrograms of total RNA sample, 1.5 .times.RNA
loading dyes (60% formamide, 9% formaldehyde, 1.5.times.MOPS,
0.075%.times.C/BPB dyes) at a final concentration of 133 and
H.sub.2O to a final volume of 40 .mu.l were mixed. The tubes were
heated at 65.degree. C. for 5 min. and then cooled on ice. The RNA
samples analyzed were loaded onto a denaturing 1% agarose gel. The
gel was run overnight at 32V in 1.times.MOPS buffer.
[0346] A 300 ml denaturing 1% agarose gel was made as follows.
Three grams of agarose (SeaKem.TM. LE, FMC BioProducts, Rockland,
Me.) and 60 ml of 5.times.MOPS buffer (0.1M MOPS [pH 7.0], 40 mM
NaOAc, 5 mM EDTA [pH 8.0]) were added to 210 ml sterile H.sub.2O.
The mixture was heated until melted, then cooled to 50.degree. C.,
at which time 5 .mu.l ethidium bromide (5 mg/ml) and 30 ml of 37%
formaldehyde were added to the melted gel mixture. The gel was
swirled quickly to mix, and then poured immediately.
[0347] After electrophoresis, the gel was photographed with a
fluorescent ruler, then was washed three times in DEPC H.sub.2O,
for 20 minutes per wash, at room temperature, with shaking. The RNA
was then transferred from the gel to Hybond-N.RTM. membrane
(Amersham), according to the methods of Sambrook et al., 1989,
supra, in 20.times.SSC overnight.
[0348] The probes used to detect mRNA were typically synthesized as
follows: 2 .mu.l amplified cDNA band (.about.30 ng), 7 .mu.l
H.sub.2O, and 2 .mu.l 10 .times. Hexanucleotide mix
(Boehringer-Mannheim) were mixed and heated to 95.degree. C. for 5
min., and then allowed to cool on ice. The volume of the amplified
band can vary, but the total volume of the band plus H.sub.2O was
always 9 .mu.l. 3 .mu.l dATP/dGTP/dTTP mix (1:1:1 of 0.5 mM each),
5 .mu.l .alpha..sup.32P dCTP 3000 Ci/mM (50 .mu.Ci total; Amersham,
Arlington Heights, Ill.), and 1 .mu.l Klenow (2 units;
Boehringer-Mannheim) were mixed and incubated at 37.degree. C.
After 1 hr., 30 .mu.l TE were added and the reaction was loaded
onto a Biospin-6.TM. column (Biorad, Hercules, Calif.), and
centrifuged. A 1 .mu.l aliquot of eluate was used to measure
incorporation in a scintillation counter with scintillant to ensure
that 10.sup.6 cpm/.mu.l of incorporation was achieved.
[0349] For pre-hybridization, the blot was placed into a roller
bottle containing 10 ml of rapid-hyb solution (Amersham), and
placed into 65.degree. C. incubator for at least 1 hr. For
hybridization, 1.times.10.sup.7 cpm of the probe was then heated to
95.degree. C., chilled on ice, and added to 10 ml of rapid-hyb
solution. The prehybridization solution was then replaced with
probe solution and incubated for 16 hours at 65.degree. C. The
following day, the blot was washed once for 20 min. at room
temperature in 2.times.SSC/0.1% SDS and twice for 15 min. at
65.degree. C. in 0.1.times.SSC/0.1% SDS before being covered in
plastic wrap and put down for exposure.
[0350] In Situ Hybridization
[0351] 10 .mu.m sections of formalin fixed/paraffin embedded benign
nevi (non-metastic growths of melanocytes) and malignant melanoma
were post-fixed with 4% PFA/PBS for 15 minutes. After washing with
PBS, sections were digested with 21 .mu.g/ml proteinase K at
37.degree. C. for 15 minutes, and again incubated with 4% PFA/PBS
for 10 minutes. Sections were then washed with PBS, incubated with
0.2 N HCl for 10 minutes, washed with PBS, incubated with 0.25%
acetic anhydride/1 M triethanolamine for 10 minutes, washed with
PBS, and dehydrated with 70% ethanol and 100% ethanol.
[0352] Hybridizations were performed with .sup.35S-radiolabeled
(5.times.10.sup.7 cpm/ml) CRNA probes encoding a 1.1 kB segment of
the coding region of the human cDNA (clone fohy030), and a 1 kB
segment of the coding region of the human H4 histone gene in the
presence of 50% formamide, 10% dextran sulfate, 1.times.Denhardt's
solution, 600 mM NaCl, 10 mM DTT, 0.25% SDS, and 100 .mu.g/ml tRNA
for 18 hours at 55.degree. C. The H4 histone gene was used as a
control to show proper transcription of RNA.
[0353] After hybridization, slides were washed with 5.times.SSC at
55.degree. C., 50% formamide/2.times.SSC at 55.degree. C. for 30
minutes, 10 mM Tris-HCl(pH 7.6)/500 mM NaCl/1 mM EDTA (TNE) at
37.degree. C. for 10 minutes, washed in TNE at 37.degree. C. for 10
minutes, incubated once in 2.times.SSC at 50.degree. C. for 30
minutes, twice in 0.2.times.SSC at 50.degree. C. for 30 minutes,
and dehydrated with 70% ethanol and 100% ethanol. Localization of
mRNA transcripts was detected by dipping slides in Kodak NBT-2
photo-emulsion and exposing for 4 days at 4.degree. C. Controls for
the in situ hybridization experiments included the use of a sense
probe which showed no signal above backgrounds levels.
6.2. Results
[0354] An in vitro paradigm, as described, above, in Section
5.1.1.1, was carried out using the melanoma cell lines, B16 F1 and
B16 F10. The B16 F1 cell line exhibits a low metastatic potential,
while the B16 F10 cell line exhibits a high metastatic potential.
Thus, the two cell lines were grown in vitro as described in
Section 6.1.1, RNA was isolated from these cells and differential
display carried out as described in Section 6.1.
[0355] The differential display analysis identified a band,
designated romy030, which represents a cDNA derived from RNA
produced by a gene which was expressed at a much higher level in
the B16 F1 cells, i.e., the low metastatic potential cells,
relative to the gene's expression in B16 F10 cells, ie., high
metastatic potential cells. The gene corresponding to the romy030
band is referred to herein as the fomy030 or 030 gene.
[0356] The amplified romy030 band was isolated, reamplified,
subcloned, and sequenced, as described, above, in Section 6.1.3.
The romy030 nucleotide sequence (SEQ ID NO:1) is shown in FIG.
2.
[0357] A BLAST (Altschul, S. F. et al., 1990, J. Mol. Biol.
215:403-410) database search with the romy030 nucleotide sequence
revealed no sequences within the database which are similar to that
of romy030. Thus, 030, the gene corresponding to romy030, appears
to represent a novel, previously unknown gene which is
differentially expressed in cells exhibiting a low metastatic
potential relative to those cells exhibiting a high metastatic
potential.
[0358] To confirm this putative differential regulation, amplified
romy030 cDNA was used to probe Northern RNA blots containing RNA
from B16 F1 and B16 F10 cells. FIG. 1 shows the results of one such
Northern blot analysis, in which it is demonstrated that the steady
state levels of fomy030 mRNA are significantly higher in the low
metastatic potential cells (i.e., the B16 F1 cells) relative to the
high metastatic potential cells (i.e., B16 F10 cells). Lanes 1 and
3 represent F1 cells and Lanes 2 and 4 represent F10 cells
respectively. Thus, this Northern analysis confirmed the putative
differential fomy030 regulation which had been suggested by the
differential display results.
[0359] Two specific oligonucleotides were generated based on the
sequence of romy030, romy030 U 5'-GGGGAAGCACATCAAGGAAC-3' (SEQ ID
NO:4) and romy030 L 5'-GCAACTACACTCGGAAAAGC-3' (SEQ ID NO:5), for
use in PCR reactions. cDNA libraries prepared from mRNA isolated
from normal melanocytes and a mouse melanoma cell line were
analyzed for the presence of fomy030 by PCR, utilizing the above
romy030 probes. Fomy030 was detected in the melanocyte library but
not in the melanoma library. The melanoma library was generated
from a highly metastatic mouse melanoma K-1735 m2. This result is
consistent the observation that fomy030 is present at reduced
levels in the metastatic B16 F10 melanoma cell line. A radioactive
DNA probe was generated from the subcloned romy030 DNA. This probe
was used to screen the normal mouse melanocyte cDNA library. Three
independent positive clones were identified and isolated during
this screening. These clones were designated fomyo3oa, fomy030b,
and fomy030c. These cDNAs were sequenced and the overlapping
portions were found to be identical. The nucleotide sequence of all
three fomy030cDNAs, designated as the fomy030 sequence (SEQ ID
NO:2) is depicted in FIGS. 3A and 3B, and contains the sequence of
romy030. The findings described herein suggest a novel role for
fomy030 in tumor progression. A down-regulation of 030 can be used
as a diagnostic marker for tumor progression, especially for the
progression to metastasis. Further, 030 gene products can be used
in the prevention and treatment of tumor progression disorders.
[0360] Fohy030 Expression in Human Tissue Samples
[0361] To determine whether the fohy030 gene product is
differentially expressed in clinically relevant human disease,
fohy030 gene expression was analyzed in biopsy sections of human
benign nevi (non-metastic growths of melanocytes) and malignant
melanoma using in situ hybridization. Fohy030 expression was
detected in small intermittent cells in the basal layer of the
epidermis (likely, melanocytes) and in the majority of nevus cells
in patients diagnosed with benign nevi. No expression of fohy030
was detected in the majority of melanoma cells in patients
diagnosed with metastatic melanoma, though expression was detected
in normal melanocytic cells in the same tissue section. These
results show that the fohy030gene product is associated with
metastasis suppression.
6.3. 030 Gene Expression is Inversely Correlated with Metastatic
Potential
6.3.1. Experimental Protocols and Results
[0362] The relationship between 030 gene expression and tumor
progression was confirmed as described herein. Specifically, the
metastatic potentials of six variants of the B16 cell line were
tested in animals and the metastatic potential was compared to the
level of 030 gene expression observed within the cell variants.
[0363] A single cell suspension of 816 F1 cells (low metastatic
potential) was injected intravenously into syngeneic C57BL/6 mice.
After three weeks, lung tumors were excised and seeded into tissue
culture. The following six cell lines were grown in culture: B16
G1, B16 G2, B16 G3, B16 G4, B16 G9 and B16 G12.
[0364] To test the metastatic ability of the above listed six tumor
cell lines, the same number of cells for each of the six cell lines
intravenously into different groups of syngeneic C57BL/6 mice.
Three weeks later, the mice were killed and the lungs were removed
aseptically. Significantly more number of tumors were observed in
mice injected with the following three cell lines: B16 G4, B16 G9
and B16 G12. These results demonstrate that the B16 G4, B16 G9 and
B16 G12 cell lines have high metastatic potential and the B16 G1,
B16 G2 and B16 G3 cell lines have low metastatic potential.
[0365] The lung tumors produced from these three highly metastatic
cell lines (B16 G4, B16 G9 and B16 G12) were then excised and
seeded into tissue culture to produce the following four cell
lines: B16 H5, B16,H6, B16 H7 and B16 H8.
[0366] Northern analysis was performed to determine the expression
of 030 gene in the above listed cell lines (i.e., B16 H5, B16,H6,
B16 H7 and B16 H8) using procedures described above in Section
6.1.3. FIG. 4 shows the results of one such Northern blot analysis,
in which it is demonstrated that the steady state levels of 030
mRNA are significantly lower in the highly metastatic cells (ie.,
B16 H5, B16,H6, B6 H7 and B16 H8) relative to the B16 F1 low
metastatic potential cells. Lane 1 represents the B16 F1 cells,
lane 2 is B16 F10 metastatic cells and lanes 3-6 represent B16 H5,
B16,H6, B16 H7 and B16 H8.
[0367] Thus, this Northern analysis confirmed the initial finding
in this invention that 030 expression is inversely related to the
metastatic potential of tumor cells and supports the theory that
the 030 gene product plays a role in inhibiting tumor progression,
including the progression to a high metastatic potential state. In
this regard, it is important to note that the tumor cell number and
homogeneity, and the syngeneic recipient did not change from one
cell line to another in the above protocols. Therefore, the
differences in metastatic incidence can only be attributed to
properties intrinsic to the various cell lines used. The clonal
selection of tumors from successive metastases results in cells
better capable of survival, formation and progression of tumor foci
in the lung. This indicates that the decrease in expression of 030
observed in the highly metastatic four cell lines (ie., B16 H5,
B16,H6, B16 H7 and B16 H8) is an intrinsic property of these cell
lines and is related to the development, progression and metastatic
potential of the tumor cells.
7. Example
[0368] Use of Fingerprint Genes as Surrogate Markers in Clinical
Trials
[0369] The expression pattern of the fingerprint genes of the
invention may be utilized as surrogate markers to monitor clinical
human trials of drugs being tested for their efficacy as tumor
progression treatments, or may, additionally, be used to monitor
patients undergoing clinical evaluation for the treatment of tumor
progression. "Fingerprint gene," as used herein is defined as in
Section 3, above. Individual fingerprint gene expression patterns
may be analyzed or, alternatively, fingerprint patterns may be
analyzed. "Fingerprint pattern," as used herein is defined as in
Section 3, above.
[0370] The effect of the compound on the fingerprint gene
expression normally displayed in connection with a disorder
involving tumor progression can be used to evaluate the efficacy of
the compound as a treatment for such a disorder. Additionally,
fingerprint gene expression can be used to monitor patients
undergoing clinical evaluation for the treatment of the
disorder.
[0371] According to the invention, the fingerprint gene expression
and fingerprint pattern derived from any of the paradigms described
in Section 5.1.1.1 can be used to monitor clinical trials of drugs
in human patients. The paradigms described in Section 5.1.1.1, and
illustrated in the Example presented in Section 6, above, for
example, provide the fingerprint pattern of B16 melanoma cells.
This profile gives an indicative reading, therefor, of the
metastatic and non-metastatic states of melanoma cells.
Accordingly, the influence of anticancer chemotherapeutic agents on
the melanoma cells can be measured by performing differential
display on melanoma cells of patients undergoing clinical
tests.
7.1. Treatment of Patients and Procurement of Tumor Cells or
Biopsies
[0372] Test patients can be administered compounds suspected of
antimetastatic activity. Control patients can be given a
placebo.
[0373] Tumor cells or biopsies can be drawn from each patient after
a determined period of treatment and RNA can be isolated as
described in Section 6.6.1, above.
7.2. Analysis of Samples
[0374] RNA can be subjected to differential display analysis as
described in Section 6.6.1, above. A decrease in the metastatic
potential of tumor cells is indicated by an increase in the
intensity of the romy030 band, as described in Section 6.2,
above.
8. Deposit of Microorganisms
[0375] The following microorganism was deposited with the
Agricultural Research Service Culture Collection (NRRL), Peoria,
Ill., on Mar. 3, 1995 and assigned the indicated accession
number:
6 Microorganism NRRL Accession No. E. coli B-21416
Other Embodiments
[0376] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention and
functionally equivalent methods and components are within the scope
of the invention, in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
Sequence CWU 1
1
9 1 186 DNA Mus musculus 1 ggtgctggag tacctcatgg gcggtgccta
ccgctgcaac tacactcgga aaagcttccg 60 gactctctac aacaacttgt
ttggccctaa gagggtagag ctcagcagac acacagtgtc 120 ctgtgcctcc
cagagtaaca tgtggttcct tgatgtgctt ccccaaaagc ccacctgtgc 180 agaatg
186 2 2729 DNA Mus musculus CDS (321)...(1946) 2 aaggaggcta
ggctgcaccc ttcccgcttg ctcagcagct gaggcagggt cagaaagcat 60
ggatagagaa gacattttgc aaaagggaat gcatctttgt aattcccagt acaaaagacc
120 ctaacagatg ttgctgtggt cagctcacta accagcacat cccccctttg
ccgagtgggg 180 ctcccagcac aacaggagag gacaccaagc aggcagacac
gcagtccggg aaatggtctg 240 tcagcaaaca cacccagagc tacccaacag
actcctatgg gattcttgaa ttccagggtg 300 ggggttactc caataaagcc atg tac
atc cga gtc tcc tac gac acc aag cca 353 Met Tyr Ile Arg Val Ser Tyr
Asp Thr Lys Pro 1 5 10 gat tcc ctg ctc cac ctc atg gtg aag gac tgg
cag ctg gag ctc ccg 401 Asp Ser Leu Leu His Leu Met Val Lys Asp Trp
Gln Leu Glu Leu Pro 15 20 25 aag ctc ttg ata tct gtg cac gga ggc
ctc caa agc ttc gag atg cag 449 Lys Leu Leu Ile Ser Val His Gly Gly
Leu Gln Ser Phe Glu Met Gln 30 35 40 tcc aaa ctg aag cag gtg ttt
ggg aaa ggt ctg atc aag gct gcc atg 497 Ser Lys Leu Lys Gln Val Phe
Gly Lys Gly Leu Ile Lys Ala Ala Met 45 50 55 acc acg ggg gcg tgg
atc ttc acc ggg ggt gtg agc act ggt gtc gtc 545 Thr Thr Gly Ala Trp
Ile Phe Thr Gly Gly Val Ser Thr Gly Val Val 60 65 70 75 agc cat gtg
ggg gat gcc ttg aaa gac cac tcc tcc aag tcc aga ggc 593 Ser His Val
Gly Asp Ala Leu Lys Asp His Ser Ser Lys Ser Arg Gly 80 85 90 cgg
ctc tgt gct ata gga att gct ccc tgg ggc atg gtg gag aac aag 641 Arg
Leu Cys Ala Ile Gly Ile Ala Pro Trp Gly Met Val Glu Asn Lys 95 100
105 gaa gac ctg att gga aaa gat gta aca aga gtc tat cag acc atg tcc
689 Glu Asp Leu Ile Gly Lys Asp Val Thr Arg Val Tyr Gln Thr Met Ser
110 115 120 aac cct ctg agc aag ctc tct gtg ctc aac aat tcc cac act
cac ttc 737 Asn Pro Leu Ser Lys Leu Ser Val Leu Asn Asn Ser His Thr
His Phe 125 130 135 atc ttg gct gac aac ggc acc ctg ggc aag tat ggt
gct gag gtg aag 785 Ile Leu Ala Asp Asn Gly Thr Leu Gly Lys Tyr Gly
Ala Glu Val Lys 140 145 150 155 ctt cga aga cag ctg gaa aaa cac atc
tcc ctg cag aag atc aac aca 833 Leu Arg Arg Gln Leu Glu Lys His Ile
Ser Leu Gln Lys Ile Asn Thr 160 165 170 agg ctg ggc cag ggt gta cct
gtc gtg ggc cta gtg gta gaa ggt ggt 881 Arg Leu Gly Gln Gly Val Pro
Val Val Gly Leu Val Val Glu Gly Gly 175 180 185 cct aac gtg gtt tct
atc gtc ctg gag tat ctc aaa gaa gac cct cct 929 Pro Asn Val Val Ser
Ile Val Leu Glu Tyr Leu Lys Glu Asp Pro Pro 190 195 200 gtc cct gtg
gtg gtt tgc gat ggc agt gga cgt gcc tct gac att ttg 977 Val Pro Val
Val Val Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu 205 210 215 tcc
ttc gca cac aaa tac tgc gac gaa gga gga gtc ata aac gag tcc 1025
Ser Phe Ala His Lys Tyr Cys Asp Glu Gly Gly Val Ile Asn Glu Ser 220
225 230 235 ctg cgg gac cag ctt cta gtt acc att cag aaa aca ttt aat
tac agc 1073 Leu Arg Asp Gln Leu Leu Val Thr Ile Gln Lys Thr Phe
Asn Tyr Ser 240 245 250 aag tcc cag tcg tat cag ctg ttt gca att atc
atg gag tgc atg aag 1121 Lys Ser Gln Ser Tyr Gln Leu Phe Ala Ile
Ile Met Glu Cys Met Lys 255 260 265 aag aaa gaa ctc gtc act gtg ttt
cgg atg ggt tcc gag ggt cag caa 1169 Lys Lys Glu Leu Val Thr Val
Phe Arg Met Gly Ser Glu Gly Gln Gln 270 275 280 gat gtc gag atg gca
att tta act gcc ttg ctc aaa gga acc aac gca 1217 Asp Val Glu Met
Ala Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn Ala 285 290 295 tca gct
cca gat cag ctg agc ttg gcc ctg gct tgg aac cgg gtc gac 1265 Ser
Ala Pro Asp Gln Leu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp 300 305
310 315 ata gcg cga agc cag atc ttc gtc ttt ggc cca cac tgg ccg cca
ctg 1313 Ile Ala Arg Ser Gln Ile Phe Val Phe Gly Pro His Trp Pro
Pro Leu 320 325 330 gga agc ctg gcc cct cct gtg gac acc aaa gcc gca
gag aag gaa aag 1361 Gly Ser Leu Ala Pro Pro Val Asp Thr Lys Ala
Ala Glu Lys Glu Lys 335 340 345 aag cca ccc aca gcc acc acc aag ggg
aga gga aaa gga aaa ggc aag 1409 Lys Pro Pro Thr Ala Thr Thr Lys
Gly Arg Gly Lys Gly Lys Gly Lys 350 355 360 aag aaa ggc aaa gtg aaa
gag gaa gtg gag gaa gag acg gac ccc cgg 1457 Lys Lys Gly Lys Val
Lys Glu Glu Val Glu Glu Glu Thr Asp Pro Arg 365 370 375 aag ctt gag
ctg ctc aac tgg gtg aat gcc ctg gag caa gcc atg ctg 1505 Lys Leu
Glu Leu Leu Asn Trp Val Asn Ala Leu Glu Gln Ala Met Leu 380 385 390
395 gat gct ctt gtc cta gat cgg gtg gac ttt gta aag ctc ctg att gaa
1553 Asp Ala Leu Val Leu Asp Arg Val Asp Phe Val Lys Leu Leu Ile
Glu 400 405 410 aac gga gtg aac atg cag cat ttc ctc acc atc ccg agg
ctg gag gag 1601 Asn Gly Val Asn Met Gln His Phe Leu Thr Ile Pro
Arg Leu Glu Glu 415 420 425 cta tac aac acc aga ctg ggc cca cca aac
acc ctt cat ctg ctg gtg 1649 Leu Tyr Asn Thr Arg Leu Gly Pro Pro
Asn Thr Leu His Leu Leu Val 430 435 440 cgg gat gta aag aag agc aac
ctt cca cct gat tac cac atc agc ctc 1697 Arg Asp Val Lys Lys Ser
Asn Leu Pro Pro Asp Tyr His Ile Ser Leu 445 450 455 att gat ata gga
ctg gtg ctg gag tac ctc atg ggc ggt gcc tac cgc 1745 Ile Asp Ile
Gly Leu Val Leu Glu Tyr Leu Met Gly Gly Ala Tyr Arg 460 465 470 475
tgc aac tac act cgg aaa agc ttc cgg act ctc tac aac aac ttg ttt
1793 Cys Asn Tyr Thr Arg Lys Ser Phe Arg Thr Leu Tyr Asn Asn Leu
Phe 480 485 490 ggc cct aag agg gta gag ctc agc aga cac aca gtg tcc
tgt gcc tcc 1841 Gly Pro Lys Arg Val Glu Leu Ser Arg His Thr Val
Ser Cys Ala Ser 495 500 505 cag agt aac atg tgg ttc ctt gat gtg ctt
ccc caa aag ccc acc tgt 1889 Gln Ser Asn Met Trp Phe Leu Asp Val
Leu Pro Gln Lys Pro Thr Cys 510 515 520 gca gaa tgc aac tct tca cct
cac ctg tcc caa act gac atc acc cca 1937 Ala Glu Cys Asn Ser Ser
Pro His Leu Ser Gln Thr Asp Ile Thr Pro 525 530 535 cct ctg ccc
tgacacccag tgcagggcct cctagctttc acatgcagcc 1986 Pro Leu Pro 540
attcacatcg cctctcaaga ctgggccagg cagtgcaacc tgtcaagcat gtctgtcctc
2046 ccctccttcc tacaatagcc ccccctctgg gccccatgcc tctgctctct
cagcccgttc 2106 tcctccccac tgatcactgg cgctcctgtt gtcttccaag
gcaaggaaca aggaaaagca 2166 tctttttgcc cacaaaagtt tagggctccc
cgctgttcaa ccatagccaa cctcactgta 2226 catcggagtc atccaggcca
gctgccacac acaagccttc cccaccctat cccaatagac 2286 cctattcctc
catcaaaatc aaagctaact cctggcctgc cacattgctt cttcttgctc 2346
cagcctgtta aacctccaat aaatgtcaga tctgtgggaa gccttcctca ctctcactcc
2406 acagtttgta cagagagcga gagcctcgtt tggttctact tacaaggaag
gctttgtgtc 2466 tgtctgtcct tcccaactga cttctgttga cagaagcagt
ttccacatga aagcgttgac 2526 tcacctggat gttgtcatta attaatagtg
atacaaaata ttgacacttc ttttcctgct 2586 tctttgttat gcagccgaaa
gcacttaagc ttctgggaat ggaagtaagt aggacatgtt 2646 tgtggcagtt
tatttactat atataccttt gtcattctgt ggaagcaaaa attgcaatgt 2706
tttccatgaa taaagctcgt gcc 2729 3 542 PRT Mus musculus 3 Met Tyr Ile
Arg Val Ser Tyr Asp Thr Lys Pro Asp Ser Leu Leu His 1 5 10 15 Leu
Met Val Lys Asp Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25
30 Val His Gly Gly Leu Gln Ser Phe Glu Met Gln Ser Lys Leu Lys Gln
35 40 45 Val Phe Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly
Ala Trp 50 55 60 Ile Phe Thr Gly Gly Val Ser Thr Gly Val Val Ser
His Val Gly Asp 65 70 75 80 Ala Leu Lys Asp His Ser Ser Lys Ser Arg
Gly Arg Leu Cys Ala Ile 85 90 95 Gly Ile Ala Pro Trp Gly Met Val
Glu Asn Lys Glu Asp Leu Ile Gly 100 105 110 Lys Asp Val Thr Arg Val
Tyr Gln Thr Met Ser Asn Pro Leu Ser Lys 115 120 125 Leu Ser Val Leu
Asn Asn Ser His Thr His Phe Ile Leu Ala Asp Asn 130 135 140 Gly Thr
Leu Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu 145 150 155
160 Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly
165 170 175 Val Pro Val Val Gly Leu Val Val Glu Gly Gly Pro Asn Val
Val Ser 180 185 190 Ile Val Leu Glu Tyr Leu Lys Glu Asp Pro Pro Val
Pro Val Val Val 195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile
Leu Ser Phe Ala His Lys 210 215 220 Tyr Cys Asp Glu Gly Gly Val Ile
Asn Glu Ser Leu Arg Asp Gln Leu 225 230 235 240 Leu Val Thr Ile Gln
Lys Thr Phe Asn Tyr Ser Lys Ser Gln Ser Tyr 245 250 255 Gln Leu Phe
Ala Ile Ile Met Glu Cys Met Lys Lys Lys Glu Leu Val 260 265 270 Thr
Val Phe Arg Met Gly Ser Glu Gly Gln Gln Asp Val Glu Met Ala 275 280
285 Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn Ala Ser Ala Pro Asp Gln
290 295 300 Leu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp Ile Ala Arg
Ser Gln 305 310 315 320 Ile Phe Val Phe Gly Pro His Trp Pro Pro Leu
Gly Ser Leu Ala Pro 325 330 335 Pro Val Asp Thr Lys Ala Ala Glu Lys
Glu Lys Lys Pro Pro Thr Ala 340 345 350 Thr Thr Lys Gly Arg Gly Lys
Gly Lys Gly Lys Lys Lys Gly Lys Val 355 360 365 Lys Glu Glu Val Glu
Glu Glu Thr Asp Pro Arg Lys Leu Glu Leu Leu 370 375 380 Asn Trp Val
Asn Ala Leu Glu Gln Ala Met Leu Asp Ala Leu Val Leu 385 390 395 400
Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val Asn Met 405
410 415 Gln His Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr
Arg 420 425 430 Leu Gly Pro Pro Asn Thr Leu His Leu Leu Val Arg Asp
Val Lys Lys 435 440 445 Ser Asn Leu Pro Pro Asp Tyr His Ile Ser Leu
Ile Asp Ile Gly Leu 450 455 460 Val Leu Glu Tyr Leu Met Gly Gly Ala
Tyr Arg Cys Asn Tyr Thr Arg 465 470 475 480 Lys Ser Phe Arg Thr Leu
Tyr Asn Asn Leu Phe Gly Pro Lys Arg Val 485 490 495 Glu Leu Ser Arg
His Thr Val Ser Cys Ala Ser Gln Ser Asn Met Trp 500 505 510 Phe Leu
Asp Val Leu Pro Gln Lys Pro Thr Cys Ala Glu Cys Asn Ser 515 520 525
Ser Pro His Leu Ser Gln Thr Asp Ile Thr Pro Pro Leu Pro 530 535 540
4 20 DNA Artificial Sequence primer 4 ggggaagcac atcaaggaac 20 5 23
DNA Artificial Sequence primer 5 gcaactacta cactcggaaa agc 23 6
4944 DNA Homo sapiens CDS (346)...(4836) 6 actcattata gggntcgagc
ggccgcccgg gcaggtttga gctgtgccct ctccattcca 60 ctgctgtggc
agggtcagaa atcttggata gagaaaacct tttgcaaacg ggaatgtatc 120
tttgtaattc ctagcacgaa agactctaac aggtgttgct gtggccagtt caccaaccag
180 catatccccc ctctgccaag tgcaacaccc agcaaaaatg aagaggaaag
caaacaggtg 240 gagactcagc ctgagaaatg gtctgttgcc aagcacaccc
agagctaccc aacagattcc 300 tatggagttc ttgaattcca gggtggcgga
tattccaata aagcc atg tat atc cgt 357 Met Tyr Ile Arg 1 gta tcc tat
gac acc aag cca gac tca ctg ctc cat ctc atg gtg aaa 405 Val Ser Tyr
Asp Thr Lys Pro Asp Ser Leu Leu His Leu Met Val Lys 5 10 15 20 gat
tgg cag ctg gaa ctc ccc aag ctc tta ata tct gtg cat gga ggc 453 Asp
Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser Val His Gly Gly 25 30
35 ctc cag aac ttt gag atg cag ccc aag ctg aaa caa gtc ttt ggg aaa
501 Leu Gln Asn Phe Glu Met Gln Pro Lys Leu Lys Gln Val Phe Gly Lys
40 45 50 ggc ctg atc aag gct gct atg acc acc ggg gcc tgg atc ttc
acc ggg 549 Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp Ile Phe
Thr Gly 55 60 65 ggt gtc agc aca ggt gtt atc agc cac gta ggg gat
gcc ttg aaa gac 597 Gly Val Ser Thr Gly Val Ile Ser His Val Gly Asp
Ala Leu Lys Asp 70 75 80 cac tcc tcc aag tcc aga ggc cgg gtt tgt
gct ata gga att gct cca 645 His Ser Ser Lys Ser Arg Gly Arg Val Cys
Ala Ile Gly Ile Ala Pro 85 90 95 100 tgg ggc atc gtg gag aat aag
gaa gac ctg gtt gga aag gat gta aca 693 Trp Gly Ile Val Glu Asn Lys
Glu Asp Leu Val Gly Lys Asp Val Thr 105 110 115 aga gtg tac cag acc
atg tcc aac cct cta agt aag ctc tct gtg ctc 741 Arg Val Tyr Gln Thr
Met Ser Asn Pro Leu Ser Lys Leu Ser Val Leu 120 125 130 aac aac tcc
cac acc cac ttc atc ctg gct gac aat ggc acc ctg ggc 789 Asn Asn Ser
His Thr His Phe Ile Leu Ala Asp Asn Gly Thr Leu Gly 135 140 145 aag
tat ggc gcc gag gtg aag ctg cga agg ctg ctg gaa aag cac atc 837 Lys
Tyr Gly Ala Glu Val Lys Leu Arg Arg Leu Leu Glu Lys His Ile 150 155
160 tcc ctc cag aag atc aac aca aga ctg ggg cag ggc gtg ccc ctc gtg
885 Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly Val Pro Leu Val
165 170 175 180 ggt ctc gtg gtg gag ggg ggc cct aac gtg gtg tcc atc
gtc ttg gaa 933 Gly Leu Val Val Glu Gly Gly Pro Asn Val Val Ser Ile
Val Leu Glu 185 190 195 tac ctg caa gaa gag cct ccc atc cct gtg gtg
att tgt gat ggc agc 981 Tyr Leu Gln Glu Glu Pro Pro Ile Pro Val Val
Ile Cys Asp Gly Ser 200 205 210 gga cgt gcc tcg gac atc ctg tcc ttt
gcg cac aag tac tgt gaa gaa 1029 Gly Arg Ala Ser Asp Ile Leu Ser
Phe Ala His Lys Tyr Cys Glu Glu 215 220 225 ggc gga ata ata aat gag
tcc ctc agg gag cag ctt cta gtt acc att 1077 Gly Gly Ile Ile Asn
Glu Ser Leu Arg Glu Gln Leu Leu Val Thr Ile 230 235 240 cag aaa aca
ttt aat tat aat aag gca caa tca cat cag ctg ttt gca 1125 Gln Lys
Thr Phe Asn Tyr Asn Lys Ala Gln Ser His Gln Leu Phe Ala 245 250 255
260 att ata atg gag tgc atg aag aag aaa gaa ctc gtc act gtg ttc aga
1173 Ile Ile Met Glu Cys Met Lys Lys Lys Glu Leu Val Thr Val Phe
Arg 265 270 275 atg ggt tct gag ggc cag cag gac atc gag atg gca att
tta act gcc 1221 Met Gly Ser Glu Gly Gln Gln Asp Ile Glu Met Ala
Ile Leu Thr Ala 280 285 290 ctg ctg aaa gga aca aac gta tct gct cca
gat cag ctg agc ttg gca 1269 Leu Leu Lys Gly Thr Asn Val Ser Ala
Pro Asp Gln Leu Ser Leu Ala 295 300 305 ctg gct tgg aac cgc gtg gac
ata gca cga agc cag atc ttt gtc ttt 1317 Leu Ala Trp Asn Arg Val
Asp Ile Ala Arg Ser Gln Ile Phe Val Phe 310 315 320 ggg ccc cac tgg
acg ccc ctg gga agc ctg gca ccc ccg acg gac agc 1365 Gly Pro His
Trp Thr Pro Leu Gly Ser Leu Ala Pro Pro Thr Asp Ser 325 330 335 340
aaa gcc acg gag aag gag aag aag cca ccc atg gcc acc acc aag gga
1413 Lys Ala Thr Glu Lys Glu Lys Lys Pro Pro Met Ala Thr Thr Lys
Gly 345 350 355 gga aga gga aaa ggg aaa ggc aag aag aaa ggg aaa gtg
aaa gag gaa 1461 Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Gly Lys
Val Lys Glu Glu 360 365 370 gtg gag gaa gaa act gac ccc cgg aag ata
gag ctg ctg aac tgg gtg 1509 Val Glu Glu Glu Thr Asp Pro Arg Lys
Ile Glu Leu Leu Asn Trp Val 375 380 385 aat gct ttg gag caa gcg atg
cta gat gct tta gtc tta gat cgt gtc 1557 Asn Ala Leu Glu Gln Ala
Met Leu Asp Ala Leu Val Leu Asp Arg Val 390 395 400 gac ttt gtg aag
ctc ctg att gaa aac gga gtg aac atg caa cac ttt 1605 Asp Phe Val
Lys Leu Leu Ile Glu Asn Gly Val Asn Met Gln His Phe 405 410 415 420
ctg acc att ccg agg ctg gag gag ctt tat aac aca aga ctg ggt cca
1653 Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr Arg Leu Gly
Pro 425 430 435 cca aac aca ctt cat ctg ctg gtg agg gat gtg aaa aag
agc aac ctt 1701 Pro Asn Thr Leu His Leu Leu Val Arg Asp Val Lys
Lys Ser Asn Leu 440 445 450 ccg cct gat tac cac atc agc ctc ata gac
atc ggg ctc gtg ctg gag 1749 Pro Pro Asp Tyr His Ile Ser Leu Ile
Asp Ile Gly Leu Val Leu Glu 455 460 465 tac ctc atg gga gga gcc tac
cgc tgc aac tac act cgg aaa aac ttt 1797 Tyr Leu Met Gly Gly Ala
Tyr Arg Cys Asn Tyr Thr Arg Lys Asn Phe 470 475 480 cgg acc ctt tac
aac aac ttg ttt gga cca aag agg cct aaa gct ctt 1845 Arg Thr Leu
Tyr Asn Asn Leu Phe Gly Pro Lys Arg Pro Lys Ala Leu 485 490 495 500
aaa ctt ctg gga atg gaa gat gat gag cct cca gct aaa ggg aag aaa
1893 Lys Leu Leu Gly Met Glu Asp Asp Glu Pro Pro Ala Lys Gly Lys
Lys 505 510 515 aaa aaa aaa aag aaa aag gag gaa gag atc gac att gat
gtg gac gac 1941 Lys Lys Lys Lys Lys Lys Glu Glu Glu Ile Asp Ile
Asp Val Asp Asp 520 525 530 cct gcc gtg agt cgg ttc cag tat ccc ttc
cac gag ctg atg gtg tgg 1989 Pro Ala Val Ser Arg Phe Gln Tyr Pro
Phe His Glu Leu Met Val Trp 535 540 545 gca gtg ctg atg aaa cgc cag
aaa atg gca gtg ttc ctc tgg cag cga 2037 Ala Val Leu Met Lys Arg
Gln Lys Met Ala Val Phe Leu Trp Gln Arg 550 555 560 ggg gaa gag agc
atg gcc aag gcc ctg gtg gcc tgc aag ctc tac aag 2085 Gly Glu Glu
Ser Met Ala Lys Ala Leu Val Ala Cys Lys Leu Tyr Lys 565 570 575 580
gcc atg gcc cac gag tcc tcc gag agt gat ctg gtg gat gac atc tcc
2133 Ala Met Ala His Glu Ser Ser Glu Ser Asp Leu Val Asp Asp Ile
Ser 585 590 595 cag gac ttg gat aac aat tcc aaa gac ttc ggc cag ctt
gct ttg gag 2181 Gln Asp Leu Asp Asn Asn Ser Lys Asp Phe Gly Gln
Leu Ala Leu Glu 600 605 610 tta tta gac cag tcc tat aag cat gac gag
cag atc gct atg aaa ctc 2229 Leu Leu Asp Gln Ser Tyr Lys His Asp
Glu Gln Ile Ala Met Lys Leu 615 620 625 ctg acc tac gag ctg aaa aac
tgg agc aac tcg acc tgc ctc aaa ctg 2277 Leu Thr Tyr Glu Leu Lys
Asn Trp Ser Asn Ser Thr Cys Leu Lys Leu 630 635 640 gcc gtg gca gcc
aaa cac cgg gac ttc att gct cac acc tgc agc cag 2325 Ala Val Ala
Ala Lys His Arg Asp Phe Ile Ala His Thr Cys Ser Gln 645 650 655 660
atg ctg ctg acc gat atg tgg atg gga aga ctg cgg atg cgg aag aac
2373 Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg Met Arg Lys
Asn 665 670 675 ccc ggc ctg aag gtt atc atg ggg att ctt cta ccc ccc
acc atc ttg 2421 Pro Gly Leu Lys Val Ile Met Gly Ile Leu Leu Pro
Pro Thr Ile Leu 680 685 690 ttt ttg gaa ttt cgc aca tat gat gat ttc
tcg tat caa aca tcc aag 2469 Phe Leu Glu Phe Arg Thr Tyr Asp Asp
Phe Ser Tyr Gln Thr Ser Lys 695 700 705 gaa aac gag gat ggc aaa gaa
aaa gaa gag gaa aat acg gat gca aat 2517 Glu Asn Glu Asp Gly Lys
Glu Lys Glu Glu Glu Asn Thr Asp Ala Asn 710 715 720 gca gat gct ggc
tca aga aag ggg gat gag gag aac gag cat aaa aaa 2565 Ala Asp Ala
Gly Ser Arg Lys Gly Asp Glu Glu Asn Glu His Lys Lys 725 730 735 740
cag aga att atc ccc atc gga aca aaa atc tgt aaa ttc tat aac gcg
2613 Gln Arg Ile Ile Pro Ile Gly Thr Lys Ile Cys Lys Phe Tyr Asn
Ala 745 750 755 ccc att gtc aag ttc tgg ttt tac aca ata tca tac ttg
ggc tac ctg 2661 Pro Ile Val Lys Phe Trp Phe Tyr Thr Ile Ser Tyr
Leu Gly Tyr Leu 760 765 770 ctg ctg ttt aac tac gtc atc ctg gtg cgg
atg gat ggc tgg ccg tcc 2709 Leu Leu Phe Asn Tyr Val Ile Leu Val
Arg Met Asp Gly Trp Pro Ser 775 780 785 ctc cag gag tgg atc gtc atc
tcc tac atc gtg agc ctg gcg tta gag 2757 Leu Gln Glu Trp Ile Val
Ile Ser Tyr Ile Val Ser Leu Ala Leu Glu 790 795 800 aag ata cga gag
atc ctc atg tca gaa cca ggc aaa ctc agc cag aaa 2805 Lys Ile Arg
Glu Ile Leu Met Ser Glu Pro Gly Lys Leu Ser Gln Lys 805 810 815 820
atc aaa gtt tgg ctt cag gag tac tgg aac atc aca gat ctc gtg gcc
2853 Ile Lys Val Trp Leu Gln Glu Tyr Trp Asn Ile Thr Asp Leu Val
Ala 825 830 835 att tcc aca ttc atg att gga gca atg gcc acg aga tct
gtg atg atg 2901 Ile Ser Thr Phe Met Ile Gly Ala Met Ala Thr Arg
Ser Val Met Met 840 845 850 att gga aag atg atg atc gac atg ctg tac
ttt gtg gtc atc atg ctg 2949 Ile Gly Lys Met Met Ile Asp Met Leu
Tyr Phe Val Val Ile Met Leu 855 860 865 gtc gtg ctc atg agt ttc gga
gta gcc cgt caa gcc att ctg cat cca 2997 Val Val Leu Met Ser Phe
Gly Val Ala Arg Gln Ala Ile Leu His Pro 870 875 880 gag gag aag ccc
tct tgg aaa ctg gcc cga aac atc ttc tac atg ccc 3045 Glu Glu Lys
Pro Ser Trp Lys Leu Ala Arg Asn Ile Phe Tyr Met Pro 885 890 895 900
tac tgg atg atc tat gga gag gtg ttt gca gac cag ata gac ctc tac
3093 Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp Leu
Tyr 905 910 915 gcc atg gaa att aat cct cct tgt ggt gag aac cta tat
gat gag gag 3141 Ala Met Glu Ile Asn Pro Pro Cys Gly Glu Asn Leu
Tyr Asp Glu Glu 920 925 930 ggc aag cgg ctt cct ccc tgt atc ccc ggc
gcc tgg ctc act cca gca 3189 Gly Lys Arg Leu Pro Pro Cys Ile Pro
Gly Ala Trp Leu Thr Pro Ala 935 940 945 ctc atg gcg tgc tat cta ctg
gtc gcc aac atc ctg ctg gtg aac ctg 3237 Leu Met Ala Cys Tyr Leu
Leu Val Ala Asn Ile Leu Leu Val Asn Leu 950 955 960 ctg att gct gtg
ttc aac aat acc ttc ttt gaa gta aaa tca ata tcc 3285 Leu Ile Ala
Val Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile Ser 965 970 975 980
aac cag gtg tgg aag ttc cag cga tat cag ctg att atg aca ttt cat
3333 Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe
His 985 990 995 gac agg cca gtc ctg ccc cca ccg atg atc att tta agc
cac atc tac 3381 Asp Arg Pro Val Leu Pro Pro Pro Met Ile Ile Leu
Ser His Ile Tyr 1000 1005 1010 atc atc att atg cgt ctc agc ggc cgc
tgc agg aaa aag aga gaa ggg 3429 Ile Ile Ile Met Arg Leu Ser Gly
Arg Cys Arg Lys Lys Arg Glu Gly 1015 1020 1025 gac caa gag gaa cgg
gat cgt gga ttg aag ctc ttc ctt agc gac gag 3477 Asp Gln Glu Glu
Arg Asp Arg Gly Leu Lys Leu Phe Leu Ser Asp Glu 1030 1035 1040 gag
cta aag agg ctg cat gag ttc gag gag cag tgc gtg cag gag cac 3525
Glu Leu Lys Arg Leu His Glu Phe Glu Glu Gln Cys Val Gln Glu His
1045 1050 1055 1060 ttc cgg gag aag gag gat gag cag cag tcg tcc agc
gac gag cgc atc 3573 Phe Arg Glu Lys Glu Asp Glu Gln Gln Ser Ser
Ser Asp Glu Arg Ile 1065 1070 1075 cgg gtc act tct gaa aga gtt gaa
aat atg tca atg agg ttg gaa gaa 3621 Arg Val Thr Ser Glu Arg Val
Glu Asn Met Ser Met Arg Leu Glu Glu 1080 1085 1090 atc aat gaa aga
gaa act ttt atg aaa act tcc ctg cag act gtt gac 3669 Ile Asn Glu
Arg Glu Thr Phe Met Lys Thr Ser Leu Gln Thr Val Asp 1095 1100 1105
ctt cga ctt gct cag cta gaa gaa tta tct aac aga atg gtg aat gct
3717 Leu Arg Leu Ala Gln Leu Glu Glu Leu Ser Asn Arg Met Val Asn
Ala 1110 1115 1120 ctt gaa aat ctt gcg gga atc gac agg tct gac ctg
atc cag gca cgg 3765 Leu Glu Asn Leu Ala Gly Ile Asp Arg Ser Asp
Leu Ile Gln Ala Arg 1125 1130 1135 1140 tcc cgg gct tct tct gaa tgt
gag gca acg tat ctt ctc cgg caa agc 3813 Ser Arg Ala Ser Ser Glu
Cys Glu Ala Thr Tyr Leu Leu Arg Gln Ser 1145 1150 1155 agc atc aat
agc gct gat ggc tac agc ttg tat cga tat cat ttt aac 3861 Ser Ile
Asn Ser Ala Asp Gly Tyr Ser Leu Tyr Arg Tyr His Phe Asn 1160 1165
1170 gga gaa gag tta tta ttt gag gat aca tct ctc tcc acg tca cca
ggg 3909 Gly Glu Glu Leu Leu Phe Glu Asp Thr Ser Leu Ser Thr Ser
Pro Gly 1175 1180 1185 aca gga gtc agg aaa aaa acc tgt tcc ttc cgt
ata aag gaa gag aag 3957 Thr Gly Val Arg Lys Lys Thr Cys Ser Phe
Arg Ile Lys Glu Glu Lys 1190 1195 1200 gac gtg aaa acg cac cta gtc
cca gaa tgt cag aac agt ctt cac ctt 4005 Asp Val Lys Thr His Leu
Val Pro Glu Cys Gln Asn Ser Leu His Leu 1205 1210 1215 1220 tca ctg
ggc aca agc aca tca gca acc cca gat ggc agt cac ctt gca 4053 Ser
Leu Gly Thr Ser Thr Ser Ala Thr Pro Asp Gly Ser His Leu Ala 1225
1230 1235 gta gat gac tta aag aac gct gaa gag tca aaa tta ggt cca
gat att 4101 Val Asp Asp Leu Lys Asn Ala Glu Glu Ser Lys Leu Gly
Pro Asp Ile 1240 1245 1250 ggg att tca aag gaa gat gat gaa aga cag
aca gac tct aaa aaa gaa 4149 Gly Ile Ser Lys Glu Asp Asp Glu Arg
Gln Thr Asp Ser Lys Lys Glu 1255 1260 1265 gaa act att tcc cca agt
tta aat aaa aca gat gtg ata cat gga cag 4197 Glu Thr Ile Ser Pro
Ser Leu Asn Lys Thr Asp Val Ile His Gly Gln 1270 1275 1280 gac aaa
tca gat gtt caa aac act cag cta aca gtg gaa acg aca aat 4245 Asp
Lys Ser Asp Val Gln Asn Thr Gln Leu Thr Val Glu Thr Thr Asn 1285
1290 1295 1300 ata gaa ggc act att tcc tat ccc ctg gaa gaa acc aaa
att aca cgc 4293 Ile Glu Gly Thr Ile Ser Tyr Pro Leu Glu Glu Thr
Lys Ile Thr Arg 1305 1310 1315 tat ttc ccc gat gaa acg atc aat gct
tgt aaa aca atg aag tcc aga 4341 Tyr Phe Pro Asp Glu Thr Ile Asn
Ala Cys Lys Thr Met Lys Ser Arg 1320 1325 1330 agc ttc gtc tat tcc
cgg gga aga aag ctg gtc ggt ggg gtt aac cag 4389 Ser Phe Val Tyr
Ser Arg Gly Arg Lys Leu Val Gly Gly Val Asn Gln 1335 1340 1345 gat
gta gag tac agt tca atc acg gac cag caa ttg acg acg gaa tgg 4437
Asp Val Glu Tyr Ser Ser Ile Thr Asp Gln Gln Leu Thr Thr Glu Trp
1350 1355 1360 caa tgc caa gtt caa aag atc acg cgc tct cat agc aca
gat att cct 4485 Gln Cys Gln Val Gln Lys Ile Thr Arg Ser His Ser
Thr Asp Ile Pro 1365 1370 1375 1380 tac att gtg tcg gaa gct gca gtg
caa gct gag caa aaa gag cag ttt 4533 Tyr Ile Val Ser Glu Ala Ala
Val Gln Ala Glu Gln Lys Glu Gln Phe 1385 1390 1395 gca gat atg caa
gat gaa cac cat gtc gct gaa gca att cct cga atc 4581 Ala Asp Met
Gln Asp Glu His His Val Ala Glu Ala Ile Pro Arg Ile 1400 1405 1410
cct cgc ttg tcc cta acc att act gac aga aat ggg atg gaa aac tta
4629 Pro Arg Leu Ser Leu Thr Ile Thr Asp Arg Asn Gly Met Glu Asn
Leu 1415 1420 1425 ctg tct gtg aag cca gat caa act ttg gga ttc cca
tct ctc agg tca 4677 Leu Ser Val Lys Pro Asp Gln Thr Leu Gly Phe
Pro Ser Leu Arg Ser 1430 1435 1440 aaa agt tta cat gga cat cct agg
aat gtg aaa tcc att cag gga aag 4725 Lys Ser Leu His Gly His Pro
Arg Asn Val Lys Ser Ile Gln Gly Lys 1445 1450 1455 1460 tta gac aga
tct gga cat gcc agt agt gta agc agc tta gta att gtg 4773 Leu Asp
Arg Ser Gly His Ala Ser Ser Val Ser Ser Leu Val Ile Val 1465 1470
1475 tct gga atg aca gca gaa gaa aaa aag gtt aag aaa gag aaa gct
tcc 4821 Ser Gly Met Thr Ala Glu Glu Lys Lys Val Lys Lys Glu Lys
Ala Ser 1480 1485 1490 aca gaa act gaa tgc tagtctgttt tgtttcttta
attttttttt ttaacagtca 4876 Thr Glu Thr Glu Cys 1495 gaaccactaa
tgggtgtcat cttggccatc ctaaacatcc atccaatttc ctaaaaacat 4936
tttccctt 4944 7 1497 PRT Homo sapiens 7 Met Tyr Ile Arg Val Ser Tyr
Asp Thr Lys Pro Asp Ser Leu Leu His 1 5 10 15 Leu Met Val Lys Asp
Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val His Gly
Gly Leu Gln Asn Phe Glu Met Gln Pro Lys Leu Lys Gln 35 40 45 Val
Phe Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55
60 Ile Phe Thr Gly Gly Val Ser Thr Gly Val Ile Ser His Val Gly Asp
65 70 75 80 Ala Leu Lys Asp His Ser Ser Lys Ser Arg Gly Arg Val Cys
Ala Ile 85 90 95 Gly Ile Ala Pro Trp Gly Ile Val Glu Asn Lys Glu
Asp Leu Val Gly 100 105 110 Lys Asp Val Thr Arg Val Tyr Gln Thr Met
Ser Asn Pro Leu Ser Lys 115 120 125 Leu Ser Val Leu Asn Asn Ser His
Thr His Phe Ile Leu Ala Asp Asn 130 135 140 Gly Thr Leu Gly Lys Tyr
Gly Ala Glu Val Lys Leu Arg Arg Leu Leu 145 150 155 160 Glu Lys His
Ile Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly 165 170 175 Val
Pro Leu Val Gly Leu Val Val Glu Gly Gly Pro Asn Val Val Ser 180 185
190 Ile Val Leu Glu Tyr Leu Gln Glu Glu Pro Pro Ile Pro Val Val Ile
195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu Ser Phe Ala
His Lys 210 215 220 Tyr Cys Glu Glu Gly Gly Ile Ile Asn Glu Ser Leu
Arg Glu Gln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys Thr Phe Asn
Tyr Asn Lys Ala Gln Ser His 245 250 255 Gln Leu Phe Ala Ile Ile Met
Glu Cys Met Lys Lys Lys Glu Leu Val 260 265 270 Thr Val Phe Arg Met
Gly Ser Glu Gly Gln Gln Asp Ile Glu Met Ala 275 280 285 Ile Leu Thr
Ala Leu Leu Lys Gly Thr Asn Val Ser Ala Pro Asp Gln 290 295 300 Leu
Ser Leu Ala Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln 305 310
315 320 Ile Phe Val Phe Gly Pro His Trp Thr Pro Leu Gly Ser Leu Ala
Pro 325 330 335 Pro Thr Asp Ser Lys Ala Thr Glu Lys Glu Lys Lys Pro
Pro Met Ala 340 345 350 Thr Thr Lys Gly Gly Arg Gly Lys Gly Lys Gly
Lys Lys Lys Gly Lys 355 360 365 Val Lys Glu Glu Val Glu Glu Glu Thr
Asp Pro Arg Lys Ile Glu Leu 370 375 380 Leu Asn Trp Val Asn Ala Leu
Glu Gln Ala Met Leu Asp Ala Leu Val 385 390 395 400 Leu Asp Arg Val
Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val Asn 405 410 415 Met Gln
His Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr 420 425 430
Arg Leu Gly Pro Pro Asn Thr Leu His Leu Leu Val Arg Asp Val Lys 435
440 445 Lys Ser Asn Leu Pro Pro Asp Tyr His Ile Ser Leu Ile Asp Ile
Gly 450 455 460 Leu Val Leu Glu Tyr Leu Met Gly Gly Ala Tyr Arg Cys
Asn Tyr Thr 465 470 475 480 Arg Lys Asn Phe Arg Thr Leu Tyr Asn Asn
Leu Phe Gly Pro Lys Arg 485 490 495 Pro Lys Ala Leu Lys Leu Leu Gly
Met Glu Asp Asp Glu Pro Pro Ala 500 505 510 Lys Gly Lys Lys Lys Lys
Lys Lys Lys Lys Glu Glu Glu Ile Asp Ile 515 520 525 Asp Val Asp Asp
Pro Ala Val Ser Arg Phe Gln Tyr Pro Phe His Glu 530 535 540 Leu Met
Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Val Phe 545 550 555
560 Leu Trp Gln Arg Gly Glu Glu Ser Met Ala Lys Ala Leu Val Ala Cys
565 570 575 Lys Leu Tyr Lys Ala Met Ala His Glu Ser Ser Glu Ser Asp
Leu Val 580 585 590 Asp Asp Ile Ser Gln Asp Leu Asp Asn Asn Ser Lys
Asp Phe Gly Gln 595 600 605 Leu Ala Leu Glu Leu Leu Asp Gln Ser Tyr
Lys His Asp Glu Gln Ile 610 615 620 Ala Met Lys Leu Leu Thr Tyr Glu
Leu Lys Asn Trp Ser Asn Ser Thr 625 630 635 640 Cys Leu Lys Leu Ala
Val Ala Ala Lys His Arg Asp Phe Ile Ala His 645 650 655 Thr Cys Ser
Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg 660 665 670 Met
Arg Lys Asn Pro Gly Leu Lys Val Ile Met Gly Ile Leu Leu Pro 675 680
685 Pro Thr Ile Leu Phe Leu Glu Phe Arg Thr Tyr Asp
Asp Phe Ser Tyr 690 695 700 Gln Thr Ser Lys Glu Asn Glu Asp Gly Lys
Glu Lys Glu Glu Glu Asn 705 710 715 720 Thr Asp Ala Asn Ala Asp Ala
Gly Ser Arg Lys Gly Asp Glu Glu Asn 725 730 735 Glu His Lys Lys Gln
Arg Ile Ile Pro Ile Gly Thr Lys Ile Cys Lys 740 745 750 Phe Tyr Asn
Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Ile Ser Tyr 755 760 765 Leu
Gly Tyr Leu Leu Leu Phe Asn Tyr Val Ile Leu Val Arg Met Asp 770 775
780 Gly Trp Pro Ser Leu Gln Glu Trp Ile Val Ile Ser Tyr Ile Val Ser
785 790 795 800 Leu Ala Leu Glu Lys Ile Arg Glu Ile Leu Met Ser Glu
Pro Gly Lys 805 810 815 Leu Ser Gln Lys Ile Lys Val Trp Leu Gln Glu
Tyr Trp Asn Ile Thr 820 825 830 Asp Leu Val Ala Ile Ser Thr Phe Met
Ile Gly Ala Met Ala Thr Arg 835 840 845 Ser Val Met Met Ile Gly Lys
Met Met Ile Asp Met Leu Tyr Phe Val 850 855 860 Val Ile Met Leu Val
Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala 865 870 875 880 Ile Leu
His Pro Glu Glu Lys Pro Ser Trp Lys Leu Ala Arg Asn Ile 885 890 895
Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln 900
905 910 Ile Asp Leu Tyr Ala Met Glu Ile Asn Pro Pro Cys Gly Glu Asn
Leu 915 920 925 Tyr Asp Glu Glu Gly Lys Arg Leu Pro Pro Cys Ile Pro
Gly Ala Trp 930 935 940 Leu Thr Pro Ala Leu Met Ala Cys Tyr Leu Leu
Val Ala Asn Ile Leu 945 950 955 960 Leu Val Asn Leu Leu Ile Ala Val
Phe Asn Asn Thr Phe Phe Glu Val 965 970 975 Lys Ser Ile Ser Asn Gln
Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile 980 985 990 Met Thr Phe His
Asp Arg Pro Val Leu Pro Pro Pro Met Ile Ile Leu 995 1000 1005 Ser
His Ile Tyr Ile Ile Ile Met Arg Leu Ser Gly Arg Cys Arg Lys 1010
1015 1020 Lys Arg Glu Gly Asp Gln Glu Glu Arg Asp Arg Gly Leu Lys
Leu Phe 1025 1030 1035 1040 Leu Ser Asp Glu Glu Leu Lys Arg Leu His
Glu Phe Glu Glu Gln Cys 1045 1050 1055 Val Gln Glu His Phe Arg Glu
Lys Glu Asp Glu Gln Gln Ser Ser Ser 1060 1065 1070 Asp Glu Arg Ile
Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met 1075 1080 1085 Arg
Leu Glu Glu Ile Asn Glu Arg Glu Thr Phe Met Lys Thr Ser Leu 1090
1095 1100 Gln Thr Val Asp Leu Arg Leu Ala Gln Leu Glu Glu Leu Ser
Asn Arg 1105 1110 1115 1120 Met Val Asn Ala Leu Glu Asn Leu Ala Gly
Ile Asp Arg Ser Asp Leu 1125 1130 1135 Ile Gln Ala Arg Ser Arg Ala
Ser Ser Glu Cys Glu Ala Thr Tyr Leu 1140 1145 1150 Leu Arg Gln Ser
Ser Ile Asn Ser Ala Asp Gly Tyr Ser Leu Tyr Arg 1155 1160 1165 Tyr
His Phe Asn Gly Glu Glu Leu Leu Phe Glu Asp Thr Ser Leu Ser 1170
1175 1180 Thr Ser Pro Gly Thr Gly Val Arg Lys Lys Thr Cys Ser Phe
Arg Ile 1185 1190 1195 1200 Lys Glu Glu Lys Asp Val Lys Thr His Leu
Val Pro Glu Cys Gln Asn 1205 1210 1215 Ser Leu His Leu Ser Leu Gly
Thr Ser Thr Ser Ala Thr Pro Asp Gly 1220 1225 1230 Ser His Leu Ala
Val Asp Asp Leu Lys Asn Ala Glu Glu Ser Lys Leu 1235 1240 1245 Gly
Pro Asp Ile Gly Ile Ser Lys Glu Asp Asp Glu Arg Gln Thr Asp 1250
1255 1260 Ser Lys Lys Glu Glu Thr Ile Ser Pro Ser Leu Asn Lys Thr
Asp Val 1265 1270 1275 1280 Ile His Gly Gln Asp Lys Ser Asp Val Gln
Asn Thr Gln Leu Thr Val 1285 1290 1295 Glu Thr Thr Asn Ile Glu Gly
Thr Ile Ser Tyr Pro Leu Glu Glu Thr 1300 1305 1310 Lys Ile Thr Arg
Tyr Phe Pro Asp Glu Thr Ile Asn Ala Cys Lys Thr 1315 1320 1325 Met
Lys Ser Arg Ser Phe Val Tyr Ser Arg Gly Arg Lys Leu Val Gly 1330
1335 1340 Gly Val Asn Gln Asp Val Glu Tyr Ser Ser Ile Thr Asp Gln
Gln Leu 1345 1350 1355 1360 Thr Thr Glu Trp Gln Cys Gln Val Gln Lys
Ile Thr Arg Ser His Ser 1365 1370 1375 Thr Asp Ile Pro Tyr Ile Val
Ser Glu Ala Ala Val Gln Ala Glu Gln 1380 1385 1390 Lys Glu Gln Phe
Ala Asp Met Gln Asp Glu His His Val Ala Glu Ala 1395 1400 1405 Ile
Pro Arg Ile Pro Arg Leu Ser Leu Thr Ile Thr Asp Arg Asn Gly 1410
1415 1420 Met Glu Asn Leu Leu Ser Val Lys Pro Asp Gln Thr Leu Gly
Phe Pro 1425 1430 1435 1440 Ser Leu Arg Ser Lys Ser Leu His Gly His
Pro Arg Asn Val Lys Ser 1445 1450 1455 Ile Gln Gly Lys Leu Asp Arg
Ser Gly His Ala Ser Ser Val Ser Ser 1460 1465 1470 Leu Val Ile Val
Ser Gly Met Thr Ala Glu Glu Lys Lys Val Lys Lys 1475 1480 1485 Glu
Lys Ala Ser Thr Glu Thr Glu Cys 1490 1495 8 5055 DNA Homo sapiens
CDS (346)...(4944) 8 actcattata gggntcgagc ggccgcccgg gcaggtttga
gctgtgccct ctccattcca 60 ctgctgtggc agggtcagaa atcttggata
gagaaaacct tttgcaaacg ggaatgtatc 120 tttgtaattc ctagcacgaa
agactctaac aggtgttgct gtggccagtt caccaaccag 180 catatccccc
ctctgccaag tgcaacaccc agcaaaaatg aagaggaaag caaacaggtg 240
gagactcagc ctgagaaatg gtctgttgcc aagcacaccc agagctaccc aacagattcc
300 tatggagttc ttgaattcca gggtggcgga tattccaata aagcc atg tat atc
cgt 357 Met Tyr Ile Arg 1 gta tcc tat gac acc aag cca gac tca ctg
ctc cat ctc atg gtg aaa 405 Val Ser Tyr Asp Thr Lys Pro Asp Ser Leu
Leu His Leu Met Val Lys 5 10 15 20 gat tgg cag ctg gaa ctc ccc aag
ctc tta ata tct gtg cat gga ggc 453 Asp Trp Gln Leu Glu Leu Pro Lys
Leu Leu Ile Ser Val His Gly Gly 25 30 35 ctc cag aac ttt gag atg
cag ccc aag ctg aaa caa gtc ttt ggg aaa 501 Leu Gln Asn Phe Glu Met
Gln Pro Lys Leu Lys Gln Val Phe Gly Lys 40 45 50 ggc ctg atc aag
gct gct atg acc acc ggg gcc tgg atc ttc acc ggg 549 Gly Leu Ile Lys
Ala Ala Met Thr Thr Gly Ala Trp Ile Phe Thr Gly 55 60 65 ggt gtc
agc aca ggt gtt atc agc cac gta ggg gat gcc ttg aaa gac 597 Gly Val
Ser Thr Gly Val Ile Ser His Val Gly Asp Ala Leu Lys Asp 70 75 80
cac tcc tcc aag tcc aga ggc cgg gtt tgt gct ata gga att gct cca 645
His Ser Ser Lys Ser Arg Gly Arg Val Cys Ala Ile Gly Ile Ala Pro 85
90 95 100 tgg ggc atc gtg gag aat aag gaa gac ctg gtt gga aag gat
gta aca 693 Trp Gly Ile Val Glu Asn Lys Glu Asp Leu Val Gly Lys Asp
Val Thr 105 110 115 aga gtg tac cag acc atg tcc aac cct cta agt aag
ctc tct gtg ctc 741 Arg Val Tyr Gln Thr Met Ser Asn Pro Leu Ser Lys
Leu Ser Val Leu 120 125 130 aac aac tcc cac acc cac ttc atc ctg gct
gac aat ggc acc ctg ggc 789 Asn Asn Ser His Thr His Phe Ile Leu Ala
Asp Asn Gly Thr Leu Gly 135 140 145 aag tat ggc gcc gag gtg aag ctg
cga agg ctg ctg gaa aag cac atc 837 Lys Tyr Gly Ala Glu Val Lys Leu
Arg Arg Leu Leu Glu Lys His Ile 150 155 160 tcc ctc cag aag atc aac
aca aga ctg ggg cag ggc gtg ccc ctc gtg 885 Ser Leu Gln Lys Ile Asn
Thr Arg Leu Gly Gln Gly Val Pro Leu Val 165 170 175 180 ggt ctc gtg
gtg gag ggg ggc cct aac gtg gtg tcc atc gtc ttg gaa 933 Gly Leu Val
Val Glu Gly Gly Pro Asn Val Val Ser Ile Val Leu Glu 185 190 195 tac
ctg caa gaa gag cct ccc atc cct gtg gtg att tgt gat ggc agc 981 Tyr
Leu Gln Glu Glu Pro Pro Ile Pro Val Val Ile Cys Asp Gly Ser 200 205
210 gga cgt gcc tcg gac atc ctg tcc ttt gcg cac aag tac tgt gaa gaa
1029 Gly Arg Ala Ser Asp Ile Leu Ser Phe Ala His Lys Tyr Cys Glu
Glu 215 220 225 ggc gga ata ata aat gag tcc ctc agg gag cag ctt cta
gtt acc att 1077 Gly Gly Ile Ile Asn Glu Ser Leu Arg Glu Gln Leu
Leu Val Thr Ile 230 235 240 cag aaa aca ttt aat tat aat aag gca caa
tca cat cag ctg ttt gca 1125 Gln Lys Thr Phe Asn Tyr Asn Lys Ala
Gln Ser His Gln Leu Phe Ala 245 250 255 260 att ata atg gag tgc atg
aag aag aaa gaa ctc gtc act gtg ttc aga 1173 Ile Ile Met Glu Cys
Met Lys Lys Lys Glu Leu Val Thr Val Phe Arg 265 270 275 atg ggt tct
gag ggc cag cag gac atc gag atg gca att tta act gcc 1221 Met Gly
Ser Glu Gly Gln Gln Asp Ile Glu Met Ala Ile Leu Thr Ala 280 285 290
ctg ctg aaa gga aca aac gta tct gct cca gat cag ctg agc ttg gca
1269 Leu Leu Lys Gly Thr Asn Val Ser Ala Pro Asp Gln Leu Ser Leu
Ala 295 300 305 ctg gct tgg aac cgc gtg gac ata gca cga agc cag atc
ttt gtc ttt 1317 Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln
Ile Phe Val Phe 310 315 320 ggg ccc cac tgg acg ccc ctg gga agc ctg
gca ccc ccg acg gac agc 1365 Gly Pro His Trp Thr Pro Leu Gly Ser
Leu Ala Pro Pro Thr Asp Ser 325 330 335 340 aaa gcc acg gag aag gag
aag aag cca ccc atg gcc acc acc aag gga 1413 Lys Ala Thr Glu Lys
Glu Lys Lys Pro Pro Met Ala Thr Thr Lys Gly 345 350 355 gga aga gga
aaa ggg aaa ggc aag aag aaa ggg aaa gtg aaa gag gaa 1461 Gly Arg
Gly Lys Gly Lys Gly Lys Lys Lys Gly Lys Val Lys Glu Glu 360 365 370
gtg gag gaa gaa act gac ccc cgg aag ata gag ctg ctg aac tgg gtg
1509 Val Glu Glu Glu Thr Asp Pro Arg Lys Ile Glu Leu Leu Asn Trp
Val 375 380 385 aat gct ttg gag caa gcg atg cta gat gct tta gtc tta
gat cgt gtc 1557 Asn Ala Leu Glu Gln Ala Met Leu Asp Ala Leu Val
Leu Asp Arg Val 390 395 400 gac ttt gtg aag ctc ctg att gaa aac gga
gtg aac atg caa cac ttt 1605 Asp Phe Val Lys Leu Leu Ile Glu Asn
Gly Val Asn Met Gln His Phe 405 410 415 420 ctg acc att ccg agg ctg
gag gag ctt tat aac aca aga ctg ggt cca 1653 Leu Thr Ile Pro Arg
Leu Glu Glu Leu Tyr Asn Thr Arg Leu Gly Pro 425 430 435 cca aac aca
ctt cat ctg ctg gtg agg gat gtg aaa aag agc aac ctt 1701 Pro Asn
Thr Leu His Leu Leu Val Arg Asp Val Lys Lys Ser Asn Leu 440 445 450
ccg cct gat tac cac atc agc ctc ata gac atc ggg ctc gtg ctg gag
1749 Pro Pro Asp Tyr His Ile Ser Leu Ile Asp Ile Gly Leu Val Leu
Glu 455 460 465 tac ctc atg gga gga gcc tac cgc tgc aac tac act cgg
aaa aac ttt 1797 Tyr Leu Met Gly Gly Ala Tyr Arg Cys Asn Tyr Thr
Arg Lys Asn Phe 470 475 480 cgg acc ctt tac aac aac ttg ttt gga cca
aag agg cct aaa gct ctt 1845 Arg Thr Leu Tyr Asn Asn Leu Phe Gly
Pro Lys Arg Pro Lys Ala Leu 485 490 495 500 aaa ctt ctg gga atg gaa
gat gat gag cct cca gct aaa ggg aag aaa 1893 Lys Leu Leu Gly Met
Glu Asp Asp Glu Pro Pro Ala Lys Gly Lys Lys 505 510 515 aaa aaa aaa
aag aaa aag gag gaa gag atc gac att gat gtg gac gac 1941 Lys Lys
Lys Lys Lys Lys Glu Glu Glu Ile Asp Ile Asp Val Asp Asp 520 525 530
cct gcc gtg agt cgg ttc cag tat ccc ttc cac gag ctg atg gtg tgg
1989 Pro Ala Val Ser Arg Phe Gln Tyr Pro Phe His Glu Leu Met Val
Trp 535 540 545 gca gtg ctg atg aaa cgc cag aaa atg gca gtg ttc ctc
tgg cag cga 2037 Ala Val Leu Met Lys Arg Gln Lys Met Ala Val Phe
Leu Trp Gln Arg 550 555 560 ggg gaa gag agc atg gcc aag gcc ctg gtg
gcc tgc aag ctc tac aag 2085 Gly Glu Glu Ser Met Ala Lys Ala Leu
Val Ala Cys Lys Leu Tyr Lys 565 570 575 580 gcc atg gcc cac gag tcc
tcc gag agt gat ctg gtg gat gac atc tcc 2133 Ala Met Ala His Glu
Ser Ser Glu Ser Asp Leu Val Asp Asp Ile Ser 585 590 595 cag gac ttg
gat aac aat tcc aaa gac ttc ggc cag ctt gct ttg gag 2181 Gln Asp
Leu Asp Asn Asn Ser Lys Asp Phe Gly Gln Leu Ala Leu Glu 600 605 610
tta tta gac cag tcc tat aag cat gac gag cag atc gct atg aaa ctc
2229 Leu Leu Asp Gln Ser Tyr Lys His Asp Glu Gln Ile Ala Met Lys
Leu 615 620 625 ctg acc tac gag ctg aaa aac tgg agc aac tcg acc tgc
ctc aaa ctg 2277 Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ser Thr
Cys Leu Lys Leu 630 635 640 gcc gtg gca gcc aaa cac cgg gac ttc att
gct cac acc tgc agc cag 2325 Ala Val Ala Ala Lys His Arg Asp Phe
Ile Ala His Thr Cys Ser Gln 645 650 655 660 atg ctg ctg acc gat atg
tgg atg gga aga ctg cgg atg cgg aag aac 2373 Met Leu Leu Thr Asp
Met Trp Met Gly Arg Leu Arg Met Arg Lys Asn 665 670 675 ccc ggc ctg
aag gtt atc atg ggg att ctt cta ccc ccc acc atc ttg 2421 Pro Gly
Leu Lys Val Ile Met Gly Ile Leu Leu Pro Pro Thr Ile Leu 680 685 690
ttt ttg gaa ttt cgc aca tat gat gat ttc tcg tat caa aca tcc aag
2469 Phe Leu Glu Phe Arg Thr Tyr Asp Asp Phe Ser Tyr Gln Thr Ser
Lys 695 700 705 gaa aac gag gat ggc aaa gaa aaa gaa gag gaa aat acg
gat gca aat 2517 Glu Asn Glu Asp Gly Lys Glu Lys Glu Glu Glu Asn
Thr Asp Ala Asn 710 715 720 gca gat gct ggc tca aga aag ggg gat gag
gag aac gag cat aaa aaa 2565 Ala Asp Ala Gly Ser Arg Lys Gly Asp
Glu Glu Asn Glu His Lys Lys 725 730 735 740 cag aga agt att ccc atc
gga aca aag atc tgt gaa ttc tat aac gcg 2613 Gln Arg Ser Ile Pro
Ile Gly Thr Lys Ile Cys Glu Phe Tyr Asn Ala 745 750 755 ccc att gtc
aag ttc tgg ttt tac aca ata tca tac ttg ggc tac ctg 2661 Pro Ile
Val Lys Phe Trp Phe Tyr Thr Ile Ser Tyr Leu Gly Tyr Leu 760 765 770
ctg ctg ttt aac tac gtc atc ctg gtg cgg atg gat ggc tgg ccg tcc
2709 Leu Leu Phe Asn Tyr Val Ile Leu Val Arg Met Asp Gly Trp Pro
Ser 775 780 785 ctc cag gag tgg atc gtc atc tcc tac atc gtg agc ctg
gcg tta gag 2757 Leu Gln Glu Trp Ile Val Ile Ser Tyr Ile Val Ser
Leu Ala Leu Glu 790 795 800 aag ata cga gag atc ctc atg tca gaa cca
ggc aaa ctc agc cag aaa 2805 Lys Ile Arg Glu Ile Leu Met Ser Glu
Pro Gly Lys Leu Ser Gln Lys 805 810 815 820 atc aaa gtt tgg ctt cag
gag tac tgg aac atc aca gat ctc gtg gcc 2853 Ile Lys Val Trp Leu
Gln Glu Tyr Trp Asn Ile Thr Asp Leu Val Ala 825 830 835 att tcc aca
ttc atg att gga gca att ctt cgc cta cag aac cag ccc 2901 Ile Ser
Thr Phe Met Ile Gly Ala Ile Leu Arg Leu Gln Asn Gln Pro 840 845 850
tac atg ggc tat ggc cgg gtg atc tac tgt gtg gat atc atc ttc tgg
2949 Tyr Met Gly Tyr Gly Arg Val Ile Tyr Cys Val Asp Ile Ile Phe
Trp 855 860 865 tac atc cgt gtc ctg gac atc ttt ggt gtc aac aag tat
ctg ggg cca 2997 Tyr Ile Arg Val Leu Asp Ile Phe Gly Val Asn Lys
Tyr Leu Gly Pro 870 875 880 tac gtg atg atg att gga aag atg atg atc
gac atg ctg tac ttt gtg 3045 Tyr Val Met Met Ile Gly Lys Met Met
Ile Asp Met Leu Tyr Phe Val 885 890 895 900 gtc atc atg ctg gtc gtg
ctc atg agt ttc gga gta gcc cgt caa gcc 3093 Val Ile Met Leu Val
Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala 905 910 915 att ctg cat
cca gag gag aag ccc tct tgg aaa ctg gcc cga aac atc 3141 Ile Leu
His Pro Glu Glu Lys Pro Ser Trp Lys Leu Ala Arg Asn Ile 920 925 930
ttc tac atg ccc tac tgg atg atc tat gga gag gtg ttt gca gac cag
3189 Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp
Gln 935 940 945 ata gac ctc tac gcc atg gaa att aat cct cct tgt ggt
gag aac cta 3237 Ile Asp Leu Tyr Ala Met Glu Ile Asn Pro Pro Cys
Gly Glu Asn Leu 950 955 960 tat gat gag gag ggc aag cgg ctt cct ccc
tgt atc ccc ggc gcc tgg 3285 Tyr Asp Glu Glu Gly Lys Arg Leu Pro
Pro Cys Ile Pro Gly Ala Trp 965 970
975 980 ctc act cca gca ctc atg gcg tgc tat cta ctg gtc gcc aac atc
ctg 3333 Leu Thr Pro Ala Leu Met Ala Cys Tyr Leu Leu Val Ala Asn
Ile Leu 985 990 995 ctg gtg aac ctg ctg att gct gtg ttc aac aat act
ttc ttt gaa gta 3381 Leu Val Asn Leu Leu Ile Ala Val Phe Asn Asn
Thr Phe Phe Glu Val 1000 1005 1010 aaa tca ata tcc aac cag gtg tgg
aag ttc cag cga tat cag ctg att 3429 Lys Ser Ile Ser Asn Gln Val
Trp Lys Phe Gln Arg Tyr Gln Leu Ile 1015 1020 1025 atg aca ttt cat
gac agg cca gtc ctg ccc cca ccg atg atc att tta 3477 Met Thr Phe
His Asp Arg Pro Val Leu Pro Pro Pro Met Ile Ile Leu 1030 1035 1040
agc cac atc tac atc atc att atg cgt ctc agc ggc cgc tgc agg aaa
3525 Ser His Ile Tyr Ile Ile Ile Met Arg Leu Ser Gly Arg Cys Arg
Lys 1045 1050 1055 1060 aag aga gaa ggg gac caa gag gaa cgg gat cgt
gga ttg aag ctc ttc 3573 Lys Arg Glu Gly Asp Gln Glu Glu Arg Asp
Arg Gly Leu Lys Leu Phe 1065 1070 1075 ctt agc gac gag gag cta aag
agg ctg cat gag ttc gag gag cag tgc 3621 Leu Ser Asp Glu Glu Leu
Lys Arg Leu His Glu Phe Glu Glu Gln Cys 1080 1085 1090 gtg cag gag
cac ttc cgg gag aag gag gat gag cag cag tcg tcc agc 3669 Val Gln
Glu His Phe Arg Glu Lys Glu Asp Glu Gln Gln Ser Ser Ser 1095 1100
1105 gac gag cgc atc cgg gtc act tct gaa aga gtt gaa aat atg tca
atg 3717 Asp Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met
Ser Met 1110 1115 1120 agg ttg gaa gaa atc aat gaa aga gaa act ttt
atg aaa act tcc ctg 3765 Arg Leu Glu Glu Ile Asn Glu Arg Glu Thr
Phe Met Lys Thr Ser Leu 1125 1130 1135 1140 cag act gtt gac ctt cga
ctt gct cag cta gaa gaa tta tct aac aga 3813 Gln Thr Val Asp Leu
Arg Leu Ala Gln Leu Glu Glu Leu Ser Asn Arg 1145 1150 1155 atg gtg
aat gct ctt gaa aat ctt gcg gga atc gac agg tct gac ctg 3861 Met
Val Asn Ala Leu Glu Asn Leu Ala Gly Ile Asp Arg Ser Asp Leu 1160
1165 1170 atc cag gca cgg tcc cgg gct tct tct gaa tgt gag gca acg
tat ctt 3909 Ile Gln Ala Arg Ser Arg Ala Ser Ser Glu Cys Glu Ala
Thr Tyr Leu 1175 1180 1185 ctc cgg caa agc agc atc aat agc gct gat
ggc tac agc ttg tat cga 3957 Leu Arg Gln Ser Ser Ile Asn Ser Ala
Asp Gly Tyr Ser Leu Tyr Arg 1190 1195 1200 tat cat ttt aac gga gaa
gag tta tta ttt gag gat aca tct ctc tcc 4005 Tyr His Phe Asn Gly
Glu Glu Leu Leu Phe Glu Asp Thr Ser Leu Ser 1205 1210 1215 1220 acg
tca cca ggg aca gga gtc agg aaa aaa acc tgt tcc ttc cgt ata 4053
Thr Ser Pro Gly Thr Gly Val Arg Lys Lys Thr Cys Ser Phe Arg Ile
1225 1230 1235 aag gaa gag aag gac gtg aaa acg cac cta gtc cca gaa
tgt cag aac 4101 Lys Glu Glu Lys Asp Val Lys Thr His Leu Val Pro
Glu Cys Gln Asn 1240 1245 1250 agt ctt cac ctt tca ctg ggc aca agc
aca tca gca acc cca gat ggc 4149 Ser Leu His Leu Ser Leu Gly Thr
Ser Thr Ser Ala Thr Pro Asp Gly 1255 1260 1265 agt cac ctt gca gta
gat gac tta aag aac gct gaa gag tca aaa tta 4197 Ser His Leu Ala
Val Asp Asp Leu Lys Asn Ala Glu Glu Ser Lys Leu 1270 1275 1280 ggt
cca gat att ggg att tca aag gaa gat gat gaa aga cag aca gac 4245
Gly Pro Asp Ile Gly Ile Ser Lys Glu Asp Asp Glu Arg Gln Thr Asp
1285 1290 1295 1300 tct aaa aaa gaa gaa act att tcc cca agt tta aat
aaa aca gat gtg 4293 Ser Lys Lys Glu Glu Thr Ile Ser Pro Ser Leu
Asn Lys Thr Asp Val 1305 1310 1315 ata cat gga cag gac aaa tca gat
gtt caa aac act cag cta aca gtg 4341 Ile His Gly Gln Asp Lys Ser
Asp Val Gln Asn Thr Gln Leu Thr Val 1320 1325 1330 gaa acg aca aat
ata gaa ggc act att tcc tat ccc ctg gaa gaa acc 4389 Glu Thr Thr
Asn Ile Glu Gly Thr Ile Ser Tyr Pro Leu Glu Glu Thr 1335 1340 1345
aaa att aca cgc tat ttc ccc gat gaa acg atc aat gct tgt aaa aca
4437 Lys Ile Thr Arg Tyr Phe Pro Asp Glu Thr Ile Asn Ala Cys Lys
Thr 1350 1355 1360 atg aag tcc aga agc ttc gtc tat tcc cgg gga aga
aag ctg gtc ggt 4485 Met Lys Ser Arg Ser Phe Val Tyr Ser Arg Gly
Arg Lys Leu Val Gly 1365 1370 1375 1380 ggg gtt aac cag gat gta gag
tac agt tca atc acg gac cag caa ttg 4533 Gly Val Asn Gln Asp Val
Glu Tyr Ser Ser Ile Thr Asp Gln Gln Leu 1385 1390 1395 acg acg gaa
tgg caa tgc caa gtt caa aag atc acg cgc tct cat agc 4581 Thr Thr
Glu Trp Gln Cys Gln Val Gln Lys Ile Thr Arg Ser His Ser 1400 1405
1410 aca gat att cct tac att gtg tcg gaa gct gca gtg caa gct gag
caa 4629 Thr Asp Ile Pro Tyr Ile Val Ser Glu Ala Ala Val Gln Ala
Glu Gln 1415 1420 1425 aaa gag cag ttt gca gat atg caa gat gaa cac
cat gtc gct gaa gca 4677 Lys Glu Gln Phe Ala Asp Met Gln Asp Glu
His His Val Ala Glu Ala 1430 1435 1440 att cct cga atc cct cgc ttg
tcc cta acc att act gac aga aat ggg 4725 Ile Pro Arg Ile Pro Arg
Leu Ser Leu Thr Ile Thr Asp Arg Asn Gly 1445 1450 1455 1460 atg gaa
aac tta ctg tct gtg aag cca gat caa act ttg gga ttc cca 4773 Met
Glu Asn Leu Leu Ser Val Lys Pro Asp Gln Thr Leu Gly Phe Pro 1465
1470 1475 tct ctc agg tca aaa agt tta cat gga cat cct agg aat gtg
aaa tcc 4821 Ser Leu Arg Ser Lys Ser Leu His Gly His Pro Arg Asn
Val Lys Ser 1480 1485 1490 att cag gga aag tta gac aga tct gga cat
gcc agt agt gta agc agc 4869 Ile Gln Gly Lys Leu Asp Arg Ser Gly
His Ala Ser Ser Val Ser Ser 1495 1500 1505 tta gta att gtg tct gga
atg aca gca gaa gaa aaa aag gtt aag aaa 4917 Leu Val Ile Val Ser
Gly Met Thr Ala Glu Glu Lys Lys Val Lys Lys 1510 1515 1520 gag aaa
gct tcc aca gaa act gaa tgc tagtctgttt tgtttcttta 4964 Glu Lys Ala
Ser Thr Glu Thr Glu Cys 1525 1530 attttttttt ttaacagtca gaaacccact
aatgggtgtc atcttggccc atcctaaaca 5024 catmtccaat ttcctaaaaa
cattttccct t 5055 9 1533 PRT Homo sapiens 9 Met Tyr Ile Arg Val Ser
Tyr Asp Thr Lys Pro Asp Ser Leu Leu His 1 5 10 15 Leu Met Val Lys
Asp Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val His
Gly Gly Leu Gln Asn Phe Glu Met Gln Pro Lys Leu Lys Gln 35 40 45
Val Phe Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50
55 60 Ile Phe Thr Gly Gly Val Ser Thr Gly Val Ile Ser His Val Gly
Asp 65 70 75 80 Ala Leu Lys Asp His Ser Ser Lys Ser Arg Gly Arg Val
Cys Ala Ile 85 90 95 Gly Ile Ala Pro Trp Gly Ile Val Glu Asn Lys
Glu Asp Leu Val Gly 100 105 110 Lys Asp Val Thr Arg Val Tyr Gln Thr
Met Ser Asn Pro Leu Ser Lys 115 120 125 Leu Ser Val Leu Asn Asn Ser
His Thr His Phe Ile Leu Ala Asp Asn 130 135 140 Gly Thr Leu Gly Lys
Tyr Gly Ala Glu Val Lys Leu Arg Arg Leu Leu 145 150 155 160 Glu Lys
His Ile Ser Leu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly 165 170 175
Val Pro Leu Val Gly Leu Val Val Glu Gly Gly Pro Asn Val Val Ser 180
185 190 Ile Val Leu Glu Tyr Leu Gln Glu Glu Pro Pro Ile Pro Val Val
Ile 195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu Ser Phe
Ala His Lys 210 215 220 Tyr Cys Glu Glu Gly Gly Ile Ile Asn Glu Ser
Leu Arg Glu Gln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys Thr Phe
Asn Tyr Asn Lys Ala Gln Ser His 245 250 255 Gln Leu Phe Ala Ile Ile
Met Glu Cys Met Lys Lys Lys Glu Leu Val 260 265 270 Thr Val Phe Arg
Met Gly Ser Glu Gly Gln Gln Asp Ile Glu Met Ala 275 280 285 Ile Leu
Thr Ala Leu Leu Lys Gly Thr Asn Val Ser Ala Pro Asp Gln 290 295 300
Leu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln 305
310 315 320 Ile Phe Val Phe Gly Pro His Trp Thr Pro Leu Gly Ser Leu
Ala Pro 325 330 335 Pro Thr Asp Ser Lys Ala Thr Glu Lys Glu Lys Lys
Pro Pro Met Ala 340 345 350 Thr Thr Lys Gly Gly Arg Gly Lys Gly Lys
Gly Lys Lys Lys Gly Lys 355 360 365 Val Lys Glu Glu Val Glu Glu Glu
Thr Asp Pro Arg Lys Ile Glu Leu 370 375 380 Leu Asn Trp Val Asn Ala
Leu Glu Gln Ala Met Leu Asp Ala Leu Val 385 390 395 400 Leu Asp Arg
Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val Asn 405 410 415 Met
Gln His Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr 420 425
430 Arg Leu Gly Pro Pro Asn Thr Leu His Leu Leu Val Arg Asp Val Lys
435 440 445 Lys Ser Asn Leu Pro Pro Asp Tyr His Ile Ser Leu Ile Asp
Ile Gly 450 455 460 Leu Val Leu Glu Tyr Leu Met Gly Gly Ala Tyr Arg
Cys Asn Tyr Thr 465 470 475 480 Arg Lys Asn Phe Arg Thr Leu Tyr Asn
Asn Leu Phe Gly Pro Lys Arg 485 490 495 Pro Lys Ala Leu Lys Leu Leu
Gly Met Glu Asp Asp Glu Pro Pro Ala 500 505 510 Lys Gly Lys Lys Lys
Lys Lys Lys Lys Lys Glu Glu Glu Ile Asp Ile 515 520 525 Asp Val Asp
Asp Pro Ala Val Ser Arg Phe Gln Tyr Pro Phe His Glu 530 535 540 Leu
Met Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Val Phe 545 550
555 560 Leu Trp Gln Arg Gly Glu Glu Ser Met Ala Lys Ala Leu Val Ala
Cys 565 570 575 Lys Leu Tyr Lys Ala Met Ala His Glu Ser Ser Glu Ser
Asp Leu Val 580 585 590 Asp Asp Ile Ser Gln Asp Leu Asp Asn Asn Ser
Lys Asp Phe Gly Gln 595 600 605 Leu Ala Leu Glu Leu Leu Asp Gln Ser
Tyr Lys His Asp Glu Gln Ile 610 615 620 Ala Met Lys Leu Leu Thr Tyr
Glu Leu Lys Asn Trp Ser Asn Ser Thr 625 630 635 640 Cys Leu Lys Leu
Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His 645 650 655 Thr Cys
Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg 660 665 670
Met Arg Lys Asn Pro Gly Leu Lys Val Ile Met Gly Ile Leu Leu Pro 675
680 685 Pro Thr Ile Leu Phe Leu Glu Phe Arg Thr Tyr Asp Asp Phe Ser
Tyr 690 695 700 Gln Thr Ser Lys Glu Asn Glu Asp Gly Lys Glu Lys Glu
Glu Glu Asn 705 710 715 720 Thr Asp Ala Asn Ala Asp Ala Gly Ser Arg
Lys Gly Asp Glu Glu Asn 725 730 735 Glu His Lys Lys Gln Arg Ser Ile
Pro Ile Gly Thr Lys Ile Cys Glu 740 745 750 Phe Tyr Asn Ala Pro Ile
Val Lys Phe Trp Phe Tyr Thr Ile Ser Tyr 755 760 765 Leu Gly Tyr Leu
Leu Leu Phe Asn Tyr Val Ile Leu Val Arg Met Asp 770 775 780 Gly Trp
Pro Ser Leu Gln Glu Trp Ile Val Ile Ser Tyr Ile Val Ser 785 790 795
800 Leu Ala Leu Glu Lys Ile Arg Glu Ile Leu Met Ser Glu Pro Gly Lys
805 810 815 Leu Ser Gln Lys Ile Lys Val Trp Leu Gln Glu Tyr Trp Asn
Ile Thr 820 825 830 Asp Leu Val Ala Ile Ser Thr Phe Met Ile Gly Ala
Ile Leu Arg Leu 835 840 845 Gln Asn Gln Pro Tyr Met Gly Tyr Gly Arg
Val Ile Tyr Cys Val Asp 850 855 860 Ile Ile Phe Trp Tyr Ile Arg Val
Leu Asp Ile Phe Gly Val Asn Lys 865 870 875 880 Tyr Leu Gly Pro Tyr
Val Met Met Ile Gly Lys Met Met Ile Asp Met 885 890 895 Leu Tyr Phe
Val Val Ile Met Leu Val Val Leu Met Ser Phe Gly Val 900 905 910 Ala
Arg Gln Ala Ile Leu His Pro Glu Glu Lys Pro Ser Trp Lys Leu 915 920
925 Ala Arg Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val
930 935 940 Phe Ala Asp Gln Ile Asp Leu Tyr Ala Met Glu Ile Asn Pro
Pro Cys 945 950 955 960 Gly Glu Asn Leu Tyr Asp Glu Glu Gly Lys Arg
Leu Pro Pro Cys Ile 965 970 975 Pro Gly Ala Trp Leu Thr Pro Ala Leu
Met Ala Cys Tyr Leu Leu Val 980 985 990 Ala Asn Ile Leu Leu Val Asn
Leu Leu Ile Ala Val Phe Asn Asn Thr 995 1000 1005 Phe Phe Glu Val
Lys Ser Ile Ser Asn Gln Val Trp Lys Phe Gln Arg 1010 1015 1020 Tyr
Gln Leu Ile Met Thr Phe His Asp Arg Pro Val Leu Pro Pro Pro 1025
1030 1035 1040 Met Ile Ile Leu Ser His Ile Tyr Ile Ile Ile Met Arg
Leu Ser Gly 1045 1050 1055 Arg Cys Arg Lys Lys Arg Glu Gly Asp Gln
Glu Glu Arg Asp Arg Gly 1060 1065 1070 Leu Lys Leu Phe Leu Ser Asp
Glu Glu Leu Lys Arg Leu His Glu Phe 1075 1080 1085 Glu Glu Gln Cys
Val Gln Glu His Phe Arg Glu Lys Glu Asp Glu Gln 1090 1095 1100 Gln
Ser Ser Ser Asp Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu 1105
1110 1115 1120 Asn Met Ser Met Arg Leu Glu Glu Ile Asn Glu Arg Glu
Thr Phe Met 1125 1130 1135 Lys Thr Ser Leu Gln Thr Val Asp Leu Arg
Leu Ala Gln Leu Glu Glu 1140 1145 1150 Leu Ser Asn Arg Met Val Asn
Ala Leu Glu Asn Leu Ala Gly Ile Asp 1155 1160 1165 Arg Ser Asp Leu
Ile Gln Ala Arg Ser Arg Ala Ser Ser Glu Cys Glu 1170 1175 1180 Ala
Thr Tyr Leu Leu Arg Gln Ser Ser Ile Asn Ser Ala Asp Gly Tyr 1185
1190 1195 1200 Ser Leu Tyr Arg Tyr His Phe Asn Gly Glu Glu Leu Leu
Phe Glu Asp 1205 1210 1215 Thr Ser Leu Ser Thr Ser Pro Gly Thr Gly
Val Arg Lys Lys Thr Cys 1220 1225 1230 Ser Phe Arg Ile Lys Glu Glu
Lys Asp Val Lys Thr His Leu Val Pro 1235 1240 1245 Glu Cys Gln Asn
Ser Leu His Leu Ser Leu Gly Thr Ser Thr Ser Ala 1250 1255 1260 Thr
Pro Asp Gly Ser His Leu Ala Val Asp Asp Leu Lys Asn Ala Glu 1265
1270 1275 1280 Glu Ser Lys Leu Gly Pro Asp Ile Gly Ile Ser Lys Glu
Asp Asp Glu 1285 1290 1295 Arg Gln Thr Asp Ser Lys Lys Glu Glu Thr
Ile Ser Pro Ser Leu Asn 1300 1305 1310 Lys Thr Asp Val Ile His Gly
Gln Asp Lys Ser Asp Val Gln Asn Thr 1315 1320 1325 Gln Leu Thr Val
Glu Thr Thr Asn Ile Glu Gly Thr Ile Ser Tyr Pro 1330 1335 1340 Leu
Glu Glu Thr Lys Ile Thr Arg Tyr Phe Pro Asp Glu Thr Ile Asn 1345
1350 1355 1360 Ala Cys Lys Thr Met Lys Ser Arg Ser Phe Val Tyr Ser
Arg Gly Arg 1365 1370 1375 Lys Leu Val Gly Gly Val Asn Gln Asp Val
Glu Tyr Ser Ser Ile Thr 1380 1385 1390 Asp Gln Gln Leu Thr Thr Glu
Trp Gln Cys Gln Val Gln Lys Ile Thr 1395 1400 1405 Arg Ser His Ser
Thr Asp Ile Pro Tyr Ile Val Ser Glu Ala Ala Val 1410 1415 1420 Gln
Ala Glu Gln Lys Glu Gln Phe Ala Asp Met Gln Asp Glu His His 1425
1430 1435 1440 Val Ala Glu Ala Ile Pro Arg Ile Pro Arg Leu Ser Leu
Thr Ile Thr 1445 1450 1455 Asp Arg Asn Gly Met Glu Asn Leu Leu Ser
Val Lys Pro Asp Gln Thr 1460 1465 1470 Leu Gly Phe Pro Ser Leu Arg
Ser Lys Ser Leu His Gly His Pro Arg 1475 1480 1485 Asn Val Lys Ser
Ile Gln Gly Lys Leu Asp Arg Ser Gly His Ala Ser 1490 1495 1500 Ser
Val Ser Ser Leu Val Ile Val Ser Gly Met Thr Ala Glu Glu Lys 1505
1510 1515 1520 Lys Val Lys
Lys Glu Lys Ala Ser Thr Glu Thr Glu Cys 1525 1530
* * * * *