U.S. patent application number 10/834375 was filed with the patent office on 2007-08-09 for knockout mouse for the tumor suppressor gene anx7.
This patent application is currently assigned to The Henry M. Jackson Foundation for the Advancement of Military Medicine. Invention is credited to Harvey B. Pollard, Meera Srivastava.
Application Number | 20070186299 10/834375 |
Document ID | / |
Family ID | 32328623 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070186299 |
Kind Code |
A1 |
Srivastava; Meera ; et
al. |
August 9, 2007 |
Knockout mouse for the tumor suppressor gene ANX7
Abstract
A knockout transgenic mouse containing a nonfunctional allele of
the tumor suppressing gene, annexin VII. This mouse is used as a
screening model for potential therapeutic agents useful in the
treatment of tumors resulting from an annexin tumor suppressor
disease.
Inventors: |
Srivastava; Meera; (Potomac,
MD) ; Pollard; Harvey B.; (Potomac, MD) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
The Henry M. Jackson Foundation for
the Advancement of Military Medicine
|
Family ID: |
32328623 |
Appl. No.: |
10/834375 |
Filed: |
April 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09633278 |
Aug 4, 2000 |
6747187 |
|
|
10834375 |
Apr 29, 2004 |
|
|
|
60147255 |
Aug 5, 1999 |
|
|
|
Current U.S.
Class: |
800/18 ;
424/93.2; 435/354; 435/455; 435/7.23 |
Current CPC
Class: |
A01K 2227/105 20130101;
A61K 48/00 20130101; C12N 15/8509 20130101; A01K 2267/0331
20130101; A01K 2217/072 20130101; C12N 2799/022 20130101; C07K
14/4721 20130101; A01K 67/0276 20130101; A01K 67/0275 20130101;
A01K 2217/075 20130101; A01K 2217/05 20130101 |
Class at
Publication: |
800/018 ;
435/354; 435/455; 435/007.23; 424/093.2 |
International
Class: |
A01K 67/027 20060101
A01K067/027; G01N 33/574 20060101 G01N033/574; C12N 5/06 20060101
C12N005/06; A61K 48/00 20060101 A61K048/00 |
Claims
1-26. (canceled)
27. A method of identifying a probability that a patient with
prostate cancer has a severe form of prostate cancer, comprising
assaying ANX7 protein expression in a tissue sample from the
patient's prostate; wherein, if ANX7 protein is not expressed above
a negligible level in the patient's prostate, the patient is
identified as having a high probability of having a severe form of
prostate cancer; and wherein, if ANX7 protein is expressed above a
negligible level in the patient's prostate, the patient is
identified as having a low probability of having a severe form of
prostate cancer.
28. The method of claim 27, wherein ANX7 protein is not expressed
above a negligible level in the patient's prostate and the patient
is identified as having a high probability of having a severe form
of prostate cancer.
29. The method of claim 27, wherein ANX7 protein is expressed above
a negligible level in the patient's prostate and the patient is
identified as having a low probability of having a severe form of
prostate cancer.
30. The method of claim 27, wherein the assaying ANX7 protein
expression in the tissue sample comprises introducing at least one
antibody that can specifically bind to ANX7 protein to the tissue
sample.
31. The method of claim 30, wherein the at least one antibody is a
monoclonal antibody.
32. The method of claim 31, wherein the monoclonal antibody is
labeled.
33. The method of claim 27, wherein the severe prostate cancer is a
metastasized prostate cancer.
34. The method of claim 27, wherein the severe prostate cancer is a
locally recurrent hormone refractory prostate cancer.
35. The method of claim 31, further comprising assaying the
proportion of proliferating cells in the tissue sample from the
patient's prostate.
36. The method of claim 35, wherein assaying the proportion of
proliferating cells in the tissue sample from the patient's
prostate comprises introducing a Ki67 antibody to the tissue
sample.
37. An assay complex comprising at least one prostate tissue sample
or tissue sample extract, at least one antibody that can
specifically bind ANX7, and at least one label.
38. The assay complex of claim 37, in which the prostate tissue
sample or tissue sample extract is bound to a substrate.
39. The assay complex of claim 37, in which the assay complex
comprises an array or microarray of tissue samples or tissue sample
extracts.
40. The assay complex of claim 39, wherein the tissue samples or
tissue sample extracts are bound to a substrate.
Description
[0001] This is a division of application Ser. No. 09/633,278, filed
Aug. 4, 2000, and aims the benefit of U.S. provisional application
No. 60/147,255, filed Aug. 5, 1999, each of which are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention is directed to tumor-susceptible non-human
animals. The invention further pertains to the use of such animals
in the development of anti-cancer agents and therapies.
BACKGROUND
[0003] Cancer is a set of diseases resulting from uncontrolled cell
growth, which causes intractable pain and death for more than
300,000 people per year in the United States alone. Oncogenes and
tumor suppressor genes are at opposite ends of a spectrum of gene
actions that either promote or retard cancer cell growth. The
development of cancer is believed to depend on the activation of
oncogenes and the coincident inactivation of growth suppressor
genes (Park, M., "Oncogenes" in The Genetic Basis of Human Cancer
(B. Vogelstein et al., eds.) pp. 205-228 (1998)). Oncogenes are
mutated, dominant forms of cellular proto-oncogenes that stimulate
cell proliferation, while tumor suppressor genes are recessive and
normally inhibit cell proliferation (Cooper, 1995). The loss or
inactivation of tumor suppressor genes is widely thought to be one
of the contributors to unregulated cancer cell growth. While the
discovery and identification of oncogenes has been relatively
straightforward, identifying tumor suppressor genes has been much
less so (Fearon, The Genetic Basis of Human Cancer (B. Vogelstein
et al., eds.) pp. 229-236 (1998)).
[0004] Both oncogenes and tumor-suppressing genes have a basic
distinguishing feature. The oncogenes identified thus far have
arisen only in somatic cells and thus have been incapable of
transmitting their effects to the germ line of the host animal. In
contrast, mutations in tumor-suppressing genes can be identified in
germ line cells and are thus transmissible to an animal's progeny.
About a dozen such tumor suppressor genes have been identified,
with the hope that knowledge of their mechanism(s) might yield
therapeutically relevant insights.
[0005] Tumor suppressor gene action depends on either mutation or
deletion of both tumor suppressor alleles or on a reduction in the
absolute level of expressed tumor suppressor protein. In their
natural state, tumor suppressor genes act to suppress cell
proliferation. Damage in such genes leads to a loss of this
suppression, and thereby results in tumorigenesis. Knudson's
"two-mutation hypothesis" is a well studied statistical model for
tumor suppressor gene action which is based on the epidemiological
analysis of retinoblastoma. (Knudson, A. G., Proc. Nat. Acad. Sci.
USA. 68:820-823 (1971)). According to this model, the host is
heterozygous for the tumor suppressor gene, and cancer ensues when
the single remaining functional allele also mutates to create a
nullizygous state. An alternative model is the "haplo-insufficient
hypothesis" in which the tumor cell produces abnormally low levels
of wild type tumor suppressor gene product. Thus, in both of these
models the deregulation of cell growth may be mediated by the
inactivation of tumor-suppressing genes (Weinberg, R. A.,
Scientific Amer., September 1988, pp 44-51).
[0006] Tumor suppressor genes are principally known for control of
cell proliferation by their action on the cell cycle. Well-studied
examples include Rb (Weinberg, R. A., Cell, 81:323-330 (1996)), p53
(Greenblatt, M. S. et al., Cancer Res. 54:4855-4878, (1994);
Williams, B. O. et al., Cold Spring Harbor Symp. Quant. Biol.
59:449, (1994)); Levine, A. J., Cell 88:323-331 (1997)), and p16
(Cairns, P., et al., Nat. Genetics 11:210-212, (1995)); Okamoto,
A., et al., Cancer Research 55:1448-1451, (1995)). Another example
of a tumor suppressor gene acting on the cell cycle is the
p27.sup.KIP1 gene, also known simply as p27, which physiologically
inhibits cyclin-dependent kinases, and thereby blocks cell
proliferation (Fero, M. L., et al., Nature 396:177-180 (1998)).
[0007] In understanding how tumor suppressor genes impact the cell
cycle, one must understand that cell cycle transitions are
regulated by specific cyclin dependent kinases that consist of an
activating cyclin subunit and a catalytic Cdk subunit (Polyak, K.,
et al., Cell 78:59-66 (1994)); Hartwell, L., Cell 71:543-546,
(1992)); Nurse, P., Nature 344:503-508, (1990)). The functions of
the respective cyclins and Cdk's in mammalian cells correspond to
the different phases of the cell cycle. For example, during the G1
phase, cyclin D-Cdk4/6 and cyclin E-Cdk2 are catalytically active
and rate limiting for cell cycle progression. Growth factors induce
the synthesis of D-type cyclins to initiate the G1 phase. The
D-type cyclins then associate with Cdk4/Cdk6, and the active Cdk's
then hyperphosphorylate Rb to drive the cell past the restriction
point (Buchkovich, K., et al., Cell 58:1097-1105 (1989)); see
Weinberg, R. A., Cell 81:323-330 (1996)). Tumor suppressor genes
have been found to affect the function of both of these types of
subunits.
[0008] In addition to the cell cycle, tumor suppressor genes can
also control cellular differentiation by acting as transcription
factors and/or by modulating specific downstream DNA repair targets
involved in maintaining genomic integrity. In this class, the tumor
suppressor gene, inactivation of the tumor suppressor gene, p53, is
the most common, resulting in a somatic mutation that causes
malignancy (Nigro, J. M., et al., Nature 342:705-708 (1989); cf.,
review by Nguyen and Jameson, 1998). Of particular note, p53 is a
frequent target for mutation in lung cancer (Takahashi, R., et al.,
Science 246:491-494 (1989)) and bladder cancers (Sidransky, D., et
al., Science 252:706-709 (1991)). A germlne mutation for p53 is the
basis for a familial cancer, the Li-Fraumeni syndrome (Srivastava,
S., et al., Nature 348:747-749 (1990)). At the level of DNA repair,
p53 works in the following manner: When DNA is damaged, a resulting
signal causes stabilization of p53, which in turn causes
transcriptional deregulation of p21, resulting in cell cycle arrest
in the G1 phase (Hunter, T., Cell 75:839-841 (1993)).
[0009] Finally, tumor suppressor genes have also been implicated in
controlling apoptotic cell death (Graeber, T. G., et al., Nature
379:88 (1996)). Again, p53 figures prominently in this process as
well (Basu, A., et al., Mol. Hum. Reprod. 4:1099-1109 (1998)). The
clear message from this brief summary is that the individual tumor
suppressor genes cannot be viewed from a single perspective.
[0010] In order to study these tumor suppressor genes, model
systems must be developed. Recent advances in recombinant DNA and
genetic technologies have made it possible to discover and assess
new tumor suppressor genes. One of the key model systems available
is the transgenic animal. Such animals have been engineered to
contain gene sequences that are not normally or naturally present
in an unaltered animal. The techniques have also been used to
produce animals which exhibit altered expression of naturally
present gene sequences.
[0011] There remains a need for additional transgenic animals and
methods of generating such animals, for the discovery of new
tumor-suppressor genes.
SUMMARY OF THE INVENTION
[0012] The present invention provides a transgenic knockout mammal
having somatic and germline cells comprising a chromosomally
incorporated transgene. At least one allele of a genomic tumor
suppressing annexin gene is disrupted by the transgene such that
the expression of a tumor suppressing annexin gene is inhibited.
This inhibition of the endogenous tumor suppressing annexin gene
results in an increased susceptibility to formation of tumors as
compared to a wild type mammal. The transgenic mammal may be
heterozygous for this disruption. Preferably, the genomic tumor
suppressing annexin gene is annexin VII. The preferred transgenic
mammal is a transgenic rodent, and the more preferred transgenic
mammal is a mouse.
[0013] Another embodiment of this method is the generation of
transgenic embryonic stem cells. The method involves the steps
of:
[0014] (a) constructing a transgene construct containing
[0015] (i) a recombination region having all or a portion of the
endogenous tumor suppressing annexin gene and
[0016] (ii) a marker sequence which provides a detectable signal
for identifying the presence of the transgene in a cell;
[0017] (b) transferring the transgene into embryonic stem cells of
a mammal; and
[0018] (c) selecting embryonic stems cells having a correctly
targeted homologous recombination between the transgene construct
and the tumor suppressing annexin gene.
[0019] Another embodiment of the present invention comprises a
method for generating a transgenic mammal having a functionally
disrupted endogenous tumor suppressing annexin gene. The method
involves the steps of:
[0020] (a) constructing a transgene construct containing
[0021] (i) a recombination region having all or a portion of the
endogenous tumor suppressing annexin gene and
[0022] (ii) a marker sequence which provides a detectable signal
for identifying the presence of the transgene in a cell;
[0023] (b) transferring the transgene into embryonic stem cells of
a mammal;
[0024] (c) selecting embryonic stems cells having a correctly
targeted homologous recombination between the transgene construct
and the tumor suppressing annexin gene;
[0025] (d) transferring the cells of step (c) into a blastocyst and
implanting the resulting chimeric blastocyst into a female mammal,
and
[0026] (e) selecting those offspring harboring an endogenous tumor
suppressing annexin gene allele comprising the correctly targeted
recombination.
[0027] The preferred transgenic mammal for this method is a
transgenic rodent, and the more preferred transgenic mammal is a
transgenic mouse. The most preferred transgenic stem cell is a
transgenic mouse stem cell. The most preferred tumor suppressing
annexin gene is an annexin VII gene.
[0028] Another embodiment of the invention comprises a method for
evaluating the carcinogenic potential of a test agent by contacting
a transgenic mammal containing a disrupted tumor suppressing
annexin gene with a test agent, and comparing the number of
transformed cells in a sample of the treated transgenic mammal with
the number of transformed cells in a sample from an untreated
transgenic mammal. Alternatively, one can compare the number of
transformed cells in a sample of the treated transgenic mammal with
a control agent. The difference in the number of transformed cells
in the treated transgenic mammal, compared to the number of
transformed cells in the absence of treatment or in the presence of
a control agent, indicates the carcinogenic potential of the test
agent.
[0029] Another embodiment comprises a method of treating mammalian
cancer cells lacking endogenous wild-type annexin protein, which
comprises introducing a wild-type annexin tumor suppressor gene
into the mammalian cancer cells, whereby the phenotype of abnormal
proliferation of these mammalian cancer cells' is suppressed by the
expressed annexin protein. Preferably, the mammalian cancer cell
lacks at least one allele of the wild-type annexin tumor suppressor
gene. Preferably, the mammalian cancer cell is an osteosarcoma
cell, lung carcinoma cell, lymphoma cell, leukemia cell,
soft-tissue sarcoma cell, breast carcinoma cell, bladder carcinoma
cell, or prostate carcinoma cell. More preferably, the mammalian
cancer cell has a mutated annexin tumor suppressor gene.
[0030] Another embodiment comprises a method for treating a patient
having a neoplasm characterized by abnormally proliferating cells
in a mammal comprising administering an effective dose of a
recombinant replication deficient virus comprised of a DNA segment
that expresses a protein having the cell growth inhibition activity
of the annexin tumor suppressor gene product. In one embodiment,
the patient has a neoplasm comprised of cells that substantially
lack a functional annexin tumor suppressor gene product. In another
preferred embodiment, the neoplasm is comprised of cells that
substantially lack a functional annexin VII gene product.
Preferably, the replication-deficient virus is selected from the
group consisting of a retrovirus, an adenovirus, a herpes simplex
virus, a vaccinia virus, a papillomavirus, and an adeno-associated
virus. Most preferably, the virus is a recombinant replication
deficient adenovirus expression vector.
[0031] Another embodiment comprises a composition for therapy of a
neoplastic disease characterized by the lack a functional annexin
tumor suppressor gene product. The treatment comprises
administering a therapeutically effective dose of a recombinant
replication deficient adenovirus in a pharmaceutically deliverable
form.
[0032] Another embodiment comprises a method of treating a disease
characterized by abnormally proliferating cells in a mammal,
by:
[0033] (a) administering an expression vector coding for an annexin
protein to the mammal,
[0034] (b) inserting the expression vector into the abnormally
proliferating cells, and
[0035] (c) expressing the tumor suppressor annexin gene in the
abnormally proliferating cells in an amount effective to suppress
proliferation of those cells.
[0036] Another embodiment is a DNA construct containing a
recombination region having all or a portion of the endogenous
tumor suppressing annexin gene and a marker sequence which provides
a detectable signal for identifying the presence of the transgene
in a cell. Preferably, the construct is KSBX.pPNT, as described
below.
[0037] Another embodiment comprises a cell containing the DNA
construct mentioned above. More preferably, the cell is a tumor
cell, and most preferably, the cell is a mammalian cancer cell
lacking endogenous wild-type annexin protein. In another preferable
embodiment, the construct is KSBX.PPNT.
[0038] Another embodiment is an expression vector comprising an
isolated polynucleotide sequence, which hybridizes to an annexin
sequence under standard hybridization conditions and encodes a
protein having the cell growth inhibition activity of an annexin
protein. Preferably, the expression vector is selected from the
group consisting of a retrovirus, an adenovirus, a herpes simplex
virus, a vaccinia virus, a papillomavirus, and an adeno-associated
virus. More preferably, the expression vector is a recombinant
replication deficient adenovirus, and the polynucleotide sequence
corresponds to the annexin VII gene.
[0039] Another embodiment comprises a cell transformed by the
expression vector mentioned above.
[0040] Another embodiment comprises a method for identifying a
polymorphism or a mutation in an exon of a human or animal tumor
suppressor annexin VII gene. This method involves:
[0041] (a) incubating, under amplification conditions, a sample of
genomic DNA comprising an exon of a human or animal tumor
suppressor annexin gene with a primer pair comprising:
[0042] (i) a first primer which hybridizes to a promoter region or
to an intron upstream of the exon, and
[0043] (ii) a second primer which hybridizes to the 3'-noncoding
region or to an intron downstream of the exon,
[0044] such that at least one primer of the primer pair hybridizes
to an intron;
[0045] (b) producing an amplification product;
[0046] (c) determining the nucleotide sequence of the amplification
product of the exon; and
[0047] (d) comparing the sequence of the exon obtained in step (b)
to the sequence of a corresponding wild type exon.
[0048] A polymorphism or mutation is identified as a difference
between these two sequences. Preferably, the exon is selected from
the group consisting of exon 4, exon 5, exon 6, exon 7, and exon
8.
[0049] Another embodiment comprises a pharmaceutical preparation
comprising an expression vector comprising an isolated
polynucleotide sequence, which hybridizes to an annexin sequence
under standard hybridization conditions and that encodes a protein
having the cell growth inhibition activity of annexin VII, and a
physiologically tolerable diluent.
BRIEF DESCRIPTION OF THE FIGURES
[0050] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawings will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0051] FIG. 1A depicts the targeting vector, KSBX.pPNT.
[0052] FIG. 1B depicts the restriction map of the mouse anx7
gene.
[0053] FIG. 2 depicts the southern blot analysis of the targeting
construct.
[0054] FIG. 3A depicts the PCR analysis of ES cell clones
transfected with KSBX.pPNT.
[0055] FIG. 3B depicts the southern blot analysis of genomic DNA
digested with Xba 1, blotted and hybridized with KXX probe.
[0056] FIG. 4 illustrates a schematic sketch of the generation of
chimera.
[0057] FIG. 5A illustrates a recipient nonagouti (a/a) (black
female) C57B1/6 blastocyst.
[0058] FIG. 5B illustrates the synexin transgenic chimeric mouse
generated by injecting ES cells (with targeting construct) derived
from an agouti (A/A) mouse (brown).
[0059] FIG. 5C illustrates the all agouti progeny from this
chimeric mouse, when bred to a C57B1/6 black male or C57B1/6 black
female.
[0060] FIG. 6 illustrates the PCR analysis of genomic DNA from yolk
sac of anx7 (+/+), anx7 (+/-), and anx7 (-/-) embryos.
[0061] FIG. 7A illustrates the increased growth of an anx7 (+/-)
mouse compared with the control mouse.
[0062] FIG. 7B. Representative growth curve of thirty anx7 (+/+)
and anx7 (+/-) littermates as a function of age.
[0063] FIG. 8. The percent increase in organ weights of control
mice compared with anx7 (+/-) transgenic mice.
[0064] FIG. 9A. Metastatic Lymphosarcoma of the thymus stained with
hematoxylin and eosin (H and E) from a control littermate of an
anx7 (+/-) mouse.
[0065] FIG. 9B. Metastatic Lymphosarcoma of the thymus stained with
hematoxylin and eosin (H and E) from an anx7 (+/-) mouse.
[0066] FIG. 10A. Lymphosarcoma of the thymus metastatic to the lung
from a control littermate of an anx7 (+/-) mouse.
[0067] FIG. 10B. Lymphosarcoma of the thymus metastatic to the lung
from anx7 (+/-) mouse.
[0068] FIG. 11A. Hepatocellular carcinoma in liver tissue from a
control littermate of an anx7 (+/-) mouse.
[0069] FIG. 11B. Hepatocellular carcinoma in liver tissue from an
anx7 (+/-) mouse.
[0070] FIG. 12A. Growth suppression of tumor cells by anx7 and p53
in DU145, a prostate tumor cell line, transfected with pcDNA3.1
alone (vector) or vector expressing anx7 (+anx7) or p53 (+p53).
[0071] FIG. 12B. Growth suppression of tumor cells by anx7 and p53
in LNCaP, a prostate tumor cell line, transfected with pcDNA3.1
alone (vector) or vector expressing anx7 (+anx7) or p53 (+p53).
[0072] FIG. 12C. Growth suppression of tumor cells by anx7 and p53
in MCF-7, a breast cancer cell line, transfected with pcDNA3.1
alone (vector) or vector expressing anx7 (+anx7) or p53 (+p53).
[0073] FIG. 12D. Growth suppression of tumor cells by anx7 and p53
in Saos, an osteosarcoma cell line, transfected with pcDNA3.1 alone
(vector) or vector expressing anx7 (+anx7) or p53 (+p53).
[0074] FIG. 13 depicts EM pictures showing pancreatic .beta.-cells
from anx7 (+/-) and control (+/+) littermates.
[0075] FIG. 14 depicts the dominant negative activity of ANX7J
mutant when mixed with wild typ ANX7 in an in vitro membrane fusion
assay.
[0076] FIG. 15 depicts the phoshorylation of ANX7 by different
protein kinase subunits.
[0077] FIG. 16A depicts the frequency of ANX7 expression in a stage
specific manner in a prostate tissue microarray containing 301
specimens from all stages of human prostate tumor progression.
[0078] FIG. 16B depicts H&E stained sections (left side) and
brown diaminobenzidine (DAB) stain from an anti-ANX-7 monoclonal
antibody immunostaining (right side) of typical examples taken from
samples of the human tumor microarray shown in FIG. 16A.
(BPH--benign prostatic hypertrophy; PIN--primary intraepithelial
neoplasms.)
[0079] FIG. 17A depicts the immunostaining of human prostate cancer
cells by Ki67 antibody.
[0080] FIG. 17B depicts example histological images stained by
antibody Ki67 (left column) or consecutive sections stained for
ANX7 protein (right column). (BPH--benign prostatic hypertrophy;
PIN--primary intraepithelial neoplasms; MET--metastatic prostate
cancer.)
[0081] FIG. 18 depicts data from a breast cancer tissue microarray;
normal breast tissue expresses virtually no ANX7 (see FIG. 26). The
percent of samples that are positive for ANX7 become progressively
greater as the diagnostic category gets "worse". Fractions are
number of cases that are positive/total cases in this diagnostic
category. Abbreviations: dcis (ductal carcinoma in situ).
[0082] FIG. 19. Survival curves for patients in the pathological
stage pT:1. ANX7=3, 2, and 1 are descending relative expression
levels of ANX7 protein. Relative expression is assessed in terms of
percent positive cells in the sample. Samples were taken for
analysis at time=0. Note that for pT:1, ANX7 levels do not seem to
affect or be affected by survival.
[0083] FIG. 20. Survival curves for patients in the pathological
stage pT:2. ANX7=3, 2 and 1,0 are descending relative expression
levels of ANX7 protein. Relative expression is assessed in terms of
percent positive cells in the sample. Samples were taken for
analysis at time=0. Note that for this slightly worse pathological
stage, survival seems to be less likely as ANX7 levels rise.
[0084] FIG. 21. Survival curves for patients in the pathological
stage pT:3. ANX7=3, 2 and 1,0 are descending relative expression
levels of ANX7 protein. Relative expression is assessed in terms of
percent positive cells in the sample. Samples were taken for
analysis at time=0. Note that for this even worse pathological
stage, survival is less likely with higher ANX7 levels.
[0085] FIG. 22. Survival curves for patients in the pathological
stage pT:4. ANX7=3, 2 and 1,0 are descending relative expression
levels of ANX7 protein. Relative expression is assessed in terms of
percent positive cells in the sample. Samples were taken for
analysis at time=0. Note that for this even worse pathological
stage, survival is less likely with higher ANX7 levels. The values
for separate ANX7 levels are more clearly delineated for the
different levels of ANX7.
[0086] FIG. 23. Survival curves for patients in the clinical stage
BRE:1. ANX7 =3, 2 and 1 are descending relative expression levels
of ANX7 protein. Relative expression is assessed in terms of
percent positive cells in the sample. Samples were taken for
analysis at time=0. At this mild stage, the survival frequency is
not apparently affected by the level of ANX7 protein.
[0087] FIG. 24. Survival curves for patients in the clinical stage
BRE:2. ANX7 =3, 2 and 1 are descending relative expression levels
of ANX7 protein. Relative expression is assessed in terms of
percent positive cells in the sample. Samples were taken for
analysis at time=0. At this more aggressive stage, the survival
frequency is profoundly affected by the highest level of ANX7
protein, ANX7=3.
[0088] FIG. 25. Survival curves for patients in the clinical stage
BRE:3. ANX7 =3, 2 and 1 are descending relative expression levels
of ANX7 protein. Relative expression is assessed in terms of
percent positive cells in the sample. Samples were taken for
analysis at time=0. At this even more aggressive stage, the
survival frequency is profoundly affected by the highest level of
ANX7 protein, ANX7=3. However, the general level of survival is not
good in general.
[0089] FIG. 26. Tumor types in which the normal tissue is low in
ANX7, and where some of the tumors tend to be higher. Data are
given as percent of tumor cells positive for ANX7 protein. Upper
left panel: Breast cancer; see FIG. 18 for these data without
control. Upper right panel: Sarcoma's. Lower left panel: lung
cancer; note that normal adult lung is virtually deficient in ANX7,
while fetal lung is 25% positive. Carcinoid, small, and large cell
lung cancers are profoundly distinct from ANX7 levels found in
normal tissue. Lower right panel: testes; ledig tumor seems to be
the most distinct from normal tissue.
[0090] FIG. 27. Tumor types in which the normal tissue is high in
ANX7, and where some of the tumors tend to be low. Upper left
panel: skin; melanomas appear to be the most distinct. Upper right
panel: lymphoid tissue; the three types of tumors studied appear to
be distinct from normal lymph node tissue. Lower left panel:
prostate; see earlier parts of this description for detailed
studies on the prostate. Lower right panel: nerve. Another type of
tumor with as aspect of this pattern of behavior is gynecological
(see FIG. 28).
[0091] FIG. 28. Tumor types in which the normal levels of ANX7
protein can be ca. 50%, and where tumors also vary in the same
range. Upper left panel: salivary gland tumors; note that
adenocarcinoma is completely positive. Upper right panel: renal.
Lower left panel: gynecological; while the normal uterine cervix is
completely positive, the normal placenta is intermediate. Lower
right panel: thyroid.
[0092] FIG. 29. Other tumors for which controls are not necessarily
obvious.
[0093] FIG. 30. Brain; levels of ANX7 are generally low in this
tissue, and in derived tumors.
[0094] FIG. 31. GI tumors vary in level of ANX7. Normal exocrine
pancreas is 100% positive, while normal colon is in the range of
80%. Note that for the progression of colon adenoma G1 (grade 1),
colon adenoma G2 (grade 2), and colon cancer, there is the
appearance of a steady downward projection in ANX7 positive
cells.
[0095] FIG. 32. Endocrine tumors for which normal tissue is
available for comparison, but where variation by the tumors is not
dramatic. Normal endocrine tissues tend to be high in ANX7
protein.
DETAILED DESCRIPTION
[0096] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.
Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New
York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol.
185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Immunochemical Methods In
Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,
London, 1987); Handbook Of Experimental Immunology, Volumes I-IV
(D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986).
I. Definitions
[0097] As is well known, the cells of humans and animals
(especially, rodents (i.e. mouse, rat, hamster, etc.), rabbits,
sheep, goats, fish, pigs, cattle and non-human primates) are
"diploid" cells, and thus naturally contain two copies ("alleles")
of each and every gene of their genome. A cell's "genome" consists
of all of its heritable DNA (either chromosomal or non-chromosomal
(i.e. episomal, viral, etc.). One of the two alleles of a gene is
provided by the animal's or cell's maternal parent; the other set
is provided by its paternal parent. The diploid nature of human and
animal cells is described by Lewin, B. (Genes V, Oxford Univ.
Press, New York (1994)), and in other similar treatises.
[0098] When a cell has two identical or substantially similar
alleles of a gene, it is said to be "homozygous." In contrast, when
the cell has two substantially different alleles it is said to be
"heterozygous" for that gene. If both alleles are nonfunctional,
then the cell is said to be "nullizygous."
[0099] An allele may be capable of being expressed by the natural
processes operating in a cell. The expression of an allele results
in the production of a gene product. The term "allele" as used
herein is intended to denote any nucleotide sequence that affects
the expression of a particular gene. It thus is intended to refer
to any enhancer, promoter, processing, intervening, coding or
termination sequence or region of the gene, or any sequence that
stabilizes the gene product, or its mRNA, etc.
[0100] An allele of a gene is said to be "mutated" if (1) it is not
expressed in a cell or animal, (2) the expression of the allele is
altered with respect to the expression of the normal allele of the
gene, or (3) the allele expresses a gene product, but that gene
product has altered structure, activity, or characteristics
relative to the gene product of a normal allele of that gene.
[0101] Thus, the terms "mutation" or "mutated" as used herein are
intended to denote an alteration in the "normal" or "wild-type"
nucleotide sequence of any nucleotide sequence or region of the
allele. As used herein, the terms "normal" and "wild-type" are
intended to be synonymous, and to denote any nucleotide sequence
typically found in nature. The terms "mutated" and "normal" are
thus defined relative to one another; where a cell has two
chromosomal alleles of a gene that differ in nucleotide sequence,
at least one of these alleles is a "mutant" allele as that term is
used herein. Based on these definitions, an "endogenous tumor
suppressing gene" is the "wild-type" tumor suppressing gene that
exists normally in a cell, and a "mutated annexin tumor suppressor
gene" defines a gene that differs in nucleotide sequence from the
wild-type gene.
[0102] Mutations may have one of three effects. One effect is that
a mutation may detectably alter the expression of an allele. This
denotes any change in nucleotide sequence affecting the extent to
which the allele is transcribed, processed or translated. Such
alterations may be, for example, in (1) an enhancer, (2) a
promoter, (3) a coding or termination region of the allele, (4) a
mutation which stabilizes the gene product, or its mRNA, etc.
[0103] A second effect is that a mutation may detectably alter the
activity of an allele. This denotes any change in nucleotide
sequence that alters the capacity of the expressed gene product to
mediate a function of the gene product. Such mutations include
changes that diminish or inactivate one or more functions of the
expressed product. Significantly, such mutations also include
changes that result in an increase in the capacity of the gene
product to mediate any function (for example, a catalytic or
binding activity) of that gene product.
[0104] Third, a mutation may detectably alter the function of an
allele. This denotes any change in nucleotide sequence that alters
the capacity of a binding molecule (such as a binding protein) to
specifically bind to the allele.
[0105] The mutations that cause these effects in a tumor
suppressing annexin gene can be readily identified by sequencing,
tumorigenicity, resilience to tumorigenicity, binding activity,
etc. (see, for example, Eliyahu et al., Nature 312:646-649 (1984);
Finlay et al., Molec. Cell. Biol. 8:531-539 (1988); Nigro, J. M. et
al., Nature 342:705-708 (1989), all herein incorporated by
reference).
[0106] An allele is said to be "chromosomal" if it either is, or
replaces, one of the two alleles of a gene which a cell inherits
from its ancestors, or which an animal inherits from its parents.
An allele is not "chromosomal," as that term is used herein, if the
allele increases the copy number of the total number of alleles of
a particular gene which are present in a cell.
[0107] The cells that can be produced in accordance with the
present invention include both "germ-line" and "somatic" cells. A
"germ-line" cell is a sperm cell or egg cell, or a precursor or
progenitor of either; such cells have the potential of transmitting
their genome (including the altered tumor-suppressor allele) in the
formation of progeny animals. A "somatic" cell is a cell that is
not a germ-line cell.
[0108] As used herein, the term "transgene" refers to a nucleic
acid sequence which is partly or entirely heterologous, i.e.,
foreign, to the transgenic animal or cell into which it is
introduced, or, is homologous to an endogenous gene of the
transgenic animal or cell into which it is introduced, but which is
designed to be inserted, or is inserted, into the animal's genome
in such a way as to alter the genome of the cell into which it is
inserted (e.g., it is inserted at a location which differs from
that of the natural gene or its insertion results in a knockout). A
transgene can be operably linked to one or more transcriptional
regulatory sequences and any other nucleic acid, such as introns,
that may be necessary for optimal expression of a selected nucleic
acid. Exemplary transgenes of the present invention encode, for
instance an annexin polypeptide, preferably an ANX7-polypeptide.
Other exemplary transgenes are directed to disrupting one or more
genomic annexin genes by homologous recombination with genomic
sequences of an annexin gene, preferably an anx7 gene.
[0109] The transgenic animals of the present invention all include
within a plurality of their cells a transgene of the present
invention, which transgene alters the phenotype of the "host cell"
with respect to regulation of cell growth, death and/or
differentiation. Since it is possible to produce transgenic
organisms of the invention utilizing one or more of the transgene
constructs described herein, a general description will be given of
the production of transgenic organisms by referring generally to
exogenous genetic material. This general description can be adapted
by those skilled in the art in order to incorporate specific
transgene sequences into organisms utilizing the methods and
materials described below.
[0110] In an exemplary embodiment, the "transgenic non-human
animals" of the invention are produced by introducing transgenes
into the germlne of the non-human animal. Embryonal target cells at
various developmental stages can be used to introduce transgenes.
Different methods are used depending on the stage of development of
the embryonal target cell. The specific line(s) of any animal used
to practice this invention are selected for general good health,
good embryo yields, good pronuclear visibility in the embryo, and
good reproductive fitness. In addition, the haplotype is a
significant factor. For example, when transgenic mice are to be
produced, strains such as C57BL/6 or FVB lines are often used
(Jackson Laboratory, Bar Harbor, Me.). The line(s) used to practice
this invention may themselves be transgenics, and/or may be
knockouts (i.e., obtained from animals which have one or more genes
partially or completely suppressed).
[0111] The transgene construct may be introduced into a single
stage embryo. The zygote is the best target for micro-injection.
The use of zygotes as a target for gene transfer has a major
advantage in that in most cases the injected DNA will be
incorporated into the host gene before the first cleavage (Brinster
et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of
the transgenic animal will carry the incorporated transgene. This
will in general also be reflected in the efficient transmission of
the transgene to offspring of the founder since 50% of the germ
cells will harbor the transgene.
[0112] Normally, fertilized embryos are incubated in suitable media
until the pronuclei appear. At about this time, the nucleotide
sequence comprising the transgene is introduced into the female or
male pronucleus as described below. In some species such as mice,
the male pronucleus is preferred. It is most preferred that the
exogenous genetic material be added to the male DNA complement of
the zygote prior to its being processed by the ovum nucleus or the
zygote female pronucleus. It is thought that the ovum nucleus or
female pronucleus release molecules which affect the male DNA
complement, perhaps by replacing the protamines of the male DNA
with histones, thereby facilitating the combination of the female
and male DNA complements to form the diploid zygote.
[0113] Thus, the exogenous genetic material should be added to the
male complement of DNA or any other complement of DNA prior to its
being affected by the female pronucleus. For example, the exogenous
genetic material is added to the early male pronucleus, as soon as
possible after the formation of the male pronucleus, which is when
the male and female pronuclei are well separated and both are
located close to the cell membrane. Alternatively, the exogenous
genetic material could be added to the nucleus of the sperm after
it has been induced to undergo decondensation. Sperm containing the
exogenous genetic material can then be added to the ovum or the
decondensed sperm could be added to the ovum with the transgene
constructs being added as soon as possible thereafter.
[0114] Any technique which allows for the addition of the exogenous
genetic material into nucleic genetic material can be utilized so
long as it is not destructive to the cell, nuclear membrane, or
other existing cellular or genetic structures. Introduction of the
transgene nucleotide sequence into the embryo may be accomplished
by any means known in the art such as, for example, microinjection,
electroporation, or lipofection. The exogenous genetic material is
preferentially inserted into the nucleic genetic material by
microinjection. Microinjection of cells and cellular structures is
known and is used in the art. In the mouse, the male pronucleus
reaches the size of approximately 20 micrometers in diameter which
allows reproducible injection of 1-2 pl of DNA solution. Following
introduction of the transgene nucleotide sequence into the embryo,
the embryo may be incubated in vitro for varying amounts of time,
or reimplanted into the surrogate host, or both. In vitro
incubation to maturity is within the scope of this invention. One
common method in to incubate the embryos in vitro for about 1-7
days, depending on the species, and then reimplant them into the
surrogate host.
[0115] The number of copies of the transgene constructs which are
added to the zygote is dependent upon the total amount of exogenous
genetic material added and will be the amount which enables the
genetic transformation to occur. Theoretically only one copy is
required; however, generally, numerous copies are utilized, for
example, 1,000-20,000 copies of the transgene construct, in order
to insure that one copy is functional. As regards the present
invention, there will often be an advantage to having more than one
functioning copy of each of the inserted exogenous DNA sequences to
enhance the phenotypic expression of the exogenous DNA
sequences.
[0116] Transgenic offspring of the surrogate host may be screened
for the presence and/or expression of the transgene by any suitable
method. Screening is often accomplished by Southern blot or
Northern blot analysis, using a probe that is complementary to at
least a portion of the transgene. Western blot analysis using an
antibody against the protein encoded by the transgene may be
employed as an alternative or additional method for screening for
the presence of the transgene product. Typically, DNA is prepared
from tail tissue and analyzed by Southern analysis or PCR for the
transgene. Alternatively, the tissues or cells believed to express
the transgene at the highest levels are tested for the presence and
expression of the transgene using Southern analysis or PCR,
although any tissues or cell types may be used for this
analysis.
[0117] Alternative or additional methods for evaluating the
presence of the transgene include, without limitation, suitable
biochemical assays such as enzyme and/or immunological assays,
histological stains for particular marker or enzyme activities,
flow cytometric analysis, and the like. Analysis of the blood may
also be useful to detect the presence of the transgene product in
the blood, as well as to evaluate the effect of the transgene on
the levels of various types of blood cells and other blood
constituents.
[0118] Progeny of the transgenic animals may be obtained by mating
the transgenic animal with a suitable partner, or by in vitro
fertilization of eggs and/or sperm obtained from the transgenic
animal. Where mating with a partner is to be performed, the partner
may or may not be transgenic and/or a knockout; where it is
transgenic, it may contain the same or a different transgene, or
both. Alternatively, the partner may be a parental line. Where in
vitro fertilization is used, the fertilized embryo may be implanted
into a surrogate host or incubated in vitro, or both. Using either
method, the progeny may be evaluated for the presence of the
transgene using methods described above, or other appropriate
methods.
[0119] The transgenic animals produced in accordance with the
present invention will include exogenous genetic material. As set
out above, the exogenous genetic material will, in certain
embodiments, be a DNA sequence which results in the production of
an
[0120] ANX7 protein (either agonistic or antagonistic), the
sequence will be attached to a transcriptional control element,
e.g., a promoter, which preferably allows the expression of the
transgene product in a specific type of cell.
[0121] Retroviral infection can also be used to introduce transgene
into a non-human animal. The developing non-human embryo can be
cultured in vitro to the blastocyst stage. During this time, the
blastomeres can be targets for retroviral infection (Jaenich, R.
(1976) PNAS 73:1260-1264). Efficient infection of the blastomeres
is obtained by enzymatic treatment to remove the zona pellucida
(Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1986). The viral vector
system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et
al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS
82:6148-6152). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells
(Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al. (1982) Nature 298:623-628). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic non-human animal.
Further, the founder may contain various retroviral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line by intrauterine
retroviral infection of the midgestation embryo (Jahner et al.
(1982) supra).
[0122] A third type of target cell for transgene introduction is
the embryonal stem cell (ES) and is the preferred method of this
invention. ES cells are obtained from pre-implantation embryos
cultured in vitro and fused with embryos (Evans et al. (1981)
Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258;
Gossler et al. (1986) PNAS 83:9065-9069; and Robertson et al.
(1986) Nature 322:445-448). Transgenes can be efficiently
introduced into the ES cells by DNA transfection or by
retrovirus-mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a non-human animal.
The ES cells thereafter colonize the embryo and contribute to the
germ line of the resulting chimeric animal. For review see
Jaenisch, R. (1988) Science 240:1468-1474.
II. The Invention
[0123] Using any of the methods described above, the present
invention relates to the production of non-human transgenic and
chimeric animals and cells which contain at least one mutated
chromosomal allele of a tumor suppressor gene. The present
invention encompasses the formation of such cells and non-human
animals for any annexin tumor suppressor gene. The invention is
illustrated below with reference to the annexin VII tumor
suppressor gene, but this example is not meant to limit the scope
of the invention. The ability to manipulate this gene and to
produce non-human transgenic animals which carry such mutated
alleles is illustrated with respect to a particular disrupted
allele. It is to be understood, however, that the invention and the
methods disclosed herein can be used to produce any possible
mutation in the anx7 gene. In particular, the invention includes
the production of animal cells and non-human transgenic or chimeric
animals which carry the particular mutations of the anx7 gene that
are responsible for the lethal nullizygous state discussed
below.
[0124] The nullizygous anx7 (-/-) transgenic mouse mutant has a
lethal phenotype during early embryogenesis. However, the
heterozygous anx7 (+/-) transgenic mouse exhibits a phenotype of
gender dimorphic gigantism, generalized organomegaly, focal
hyperplasia and dysplasia, and increased incidence of disparate
spontaneous tumors. The combination of dysplasia and increased
incidence of tumors was the first hint that the anx7 gene may be a
tumor suppressor gene. As a preliminary direct test of tumor
suppressor activity, the wild type human anx7 gene was transfected
into two human prostate tumor cell lines, a breast cancer cell
line, and an osteosarcoma cell line. The experiments with the anx7
gene systematically result in tumor cell growth arrest, as did the
positive controls with the wild type p53 gene. It was therefore
concluded that the anx7 is a tumor suppressor gene. The loss
(either by mutation or deletion) of both anx7 alleles has been
found to be an embryonic lethal event.
III. The Annexin Genes and Specifically, the Annexin VII Gene (anx
7)
[0125] The present invention concerns a non-human animal or an
animal (including human) cell in which one of the two naturally
present copies of an annexin gene, preferably the anx7 gene, of
such non-human animal or animal cell has been rendered
non-functional through a mutation (such as a deletion, insertion,
or substitution in the naturally occurring annexin gene
sequence).
[0126] A. General Properties of Annexins
[0127] Annexins are a family of structurally related proteins that
all have the ability to bind Ca.sup.2+ and phospholipid. These
genes have been described in many organisms from mammals to molds
to plants. (Raynal and Pollard, BBA 1197:63-93 (1994)). In the
presence of Ca.sup.2+, the annexins bind to acidic phospholipids
with very high affinity (K.sub.d in the nM range for annexin V.)
The Ca.sup.2+ binding similarities of all the annexins is due to
their common primary structure, a unique N-terminal domain (the
`tail`) and a conserved C-terminal domain (the `core`). With the
exception of annexin VI, the conserved C-terminal domain is always
composed of 4 repeats (annexin VI having 8) of .about.70 amino
acids containing an increased homology region called the "endonexin
fold". Due to this conserved primary structure, all annexins have a
high degree of identity with each other. Within mammals, annexins
have between 40% and 60% identity with any other member of the
family. (Hauptmann, R. et al. Eur. J. Biochem. 185:63-71
(1989)).
[0128] B. Molecular Biology of Annexins
[0129] Genomic analysis performed on annexins I, II, III, and VII
showed striking similarities in the organization of these annexin
genes. For annexins I, II, and III, the location of exon-intron
boundaries is very well conserved in the core domain. However,
comparison of the genomic structure of the anx7 gene with other
annexins showed that only five of the 10 splice junctions in the
core domain were conserved. These findings suggest that annexin
genes may derive from a common ancestor gene, but that a precursor
underwent divergent remodeling during its evolution towards
annexins I, II, and III, on the one hand, and anx7 on the other.
There is no apparent relationship between the exon-intron
organization of annexin genes and the primary structure of the
their respective proteins.
[0130] C. Annexins as Tumor Suppressor Genes
[0131] Recently attention has been directed towards the family of
annexin genes, particularly the anx7 gene (a.k.a. synexin), as
tumor suppressor gene candidates. Early work on the anx7 gene has
shown that it is expressed in small amounts in nearly every cell
(Creutz, E. C., et al., J. Biol. Chem. 254:553-558 (1978); ibid,
1979; Raynal and Pollard, BBA Biomembranes 1197:63-93 (1994)). In
fact, anx7 is found throughout phylogeny as a single copy gene in
organisms as diverse as man (Shirvan, A., et al., Biochemistry
33:6888-6901 (1994)), mouse (Zhang-Keck, Z-Y., et al., Biochem. J.
289: 735-741 (1993), Zhang-Keck, Z-Y., et al., Biochemical J.
301:835-845 (1994), Xenopus (Srivastava, S., et al., Biochemical J.
316:729-736 (1996)), and Dictyostelium (Greenwood, M., et al.,
Biochim Biophys Acta 1088(3):429-32 (1991); Doring, V., et al., J.
Biol. Chem. 266:17509-17515 (1991); Gierke, V., et al., J. Biol.
Chem. 226:1697-1700 (1991)).
[0132] D. Annexin VII
[0133] In man, the anx7 gene is found on chromosome 10q21. Other
potential tumor suppressor genes have been hypothesized to exist on
chromosome 10q in the same vicinity as the anx7 gene. Examples
include myxoid chondrosarcoma at 10q21.1 (Shen, W. P., et al.,
Cancer Genet. Cytogenet. 45:207-215 (1990)); sporadic nonmedullary
thyroid carcinoma at 10q21.1 (Jenkins, R. B., et al., Cancer
66:1213-1220 (1990)); renal cell carcinoma at 10q21-23 (Morita, R.,
et al., Cancer Res. 51:5817-5820 (1991)); chronic myelogenous
leukemia at 10q21 (Shah, N. K., et al., Cancer Genet Cytogenet.,
61;183-192 (1992)); glioma at 10q21-26 (Oberstrass, J., et al.,
Verh Dtsch. Ges. Pathol. 78:413-417, (1994)); gliobiastoma, two
independent regions at 10pter-q11 and 10q24-q26 (Steck, P. A., et
al., Genes Chromosomes Cancer 12:255-261 (1995)); colonic
denocarcinoma, an inverted, non-ret duplication of 10q11 to 10q21
(Solic, N., et al., Int. J. Cancer 62:48-57, (1995)); lung
carcinoma at 10q21-10qter (Petersen, S., et al., Br. J. Cancer
77:270-276 (1998)); hepatocellular carcinoma at 10q (Piao, Z., et
al., Int. J. Cancer 75:29-33 (1996)); and prostate cancer, two
independent loci at 10q2I and 10q23-24 (Lacombe, L., et al., Int. J
Cancer 69:110-113 (1996)). A frequently deleted locus on chromosome
10q24-25 has recently been shown to harbor the PTEN tumor
suppressor gene (Li J., et al., Science 275:1943-1947 (1997)), thus
supporting the concept of multiple candidate tumor suppressor genes
in this region. Finally, Ford, S., et. al. have shown that the long
arm of chromosome 10 is rearranged in the prostate adenocarcinoma
cell line LNCaP (Cancer Genet. Cytogenet. 102:6-11 (1998)).
[0134] The subcellular distribution of ANX7 protein is
predominantly in membranes and to a lesser extent in the nucleus
(Cardenas, A. M., et al., Biochim. Biophys. Acta. 1:234, 255-260
(1994); Kuijpers, G. A. J., et al., Cell Tissue Res. 269:323-330
(1992)). The ANX7 protein has Ca.sup.2+-dependent membrane fusion
activity (Creutz, C. E., et al., J. Biol. Chem. 253:2858-2866
(1978); Creutz, C. E., et al., J. Biol. Chem. 254:553-558 (1979)),
which is profoundly potentiated by GTP (Caohuy, H., et al., Proc.
Nat. Acad. Sci. (USA), 93:10797-10802, (1996)). The action of GTP
on ANX7 function is regulated by an intrinsic Ca.sup.2+-activated
GTPase. ANX7 GTPase activity is sensitive to such critical
modulators of conventional G-proteins as Al.sub.2F.sub.6 and
mastoparan (Caohuy, H., et al., Secretory Systems and Toxins (eds.,
Linial, M., et al.) 2:439-449 (1998)). In studies with cultured
cells, ANX7 can be shown to bind and hydrolyze GTP. ANX7 protein
also forms Ca.sup.2+ channels in membranes (Pollard, H. B., et al.,
Proc. Natl. Acad. Sci. (USA) 85:2974-2978 (1988)), which can be
stabilized in long open states by GTP.
[0135] Protein kinase C phosphorylates ANX7 with a 2:1 P/Protein
molar ratio, both in vitro and in vivo. This is of possible
relevance to ANX7 function in the cell cycle since many isoforms of
PKC have been directly implicated in activating intracellular
signaling (Nishizuka, Y., Science 258:607-614 (1992), and in
specifically activating mitosis (Kolch, W., et al., Nature
364:426-428 (1993); Berra, E., et al., Cell 74:555-563 (1993);
Cacace, A., et al., Oncogene 8:2094-2104 (1993); Morrisson, D. K.,
et al., Proc. Nat. Acad. Sci. (USA) 85:8855-8859 (1998); and
tumorigenicity (Housey, G. M., et al., Cell, 52:343-354 (1988);
Mischak, H., et al., J. Biol. Chem. 268:6090-6096 (1993); Persons,
D. A., et al., Cell 52:447-458 (1988)). Quantitative phospho-ANX7
adducts have also been prepared in vitro with EGF (epidermal growth
factor) receptor and pp603.sup.src. In vivo, cells treated with
tyrosine kinase activators such as epidermal growth factor (EGF)
and platelet derived growth factor (PGDF) also support
phosphorylation of endogenous ANX7. These reactions are of as yet
unknown biological significance. However, the relevance of such
reactivity to tumor suppressor gene activity is manifest by reports
that splice variants of the breast and ovarian cancer
susceptibility gene BRCA1 contain phosphotyrosine and play a role
in cell cycle regulation (Cui, J. Q., et al., Oncol. Rep. 5:585-589
(1998); Wang, H., et al., Oncogene 15:143-157 (1997); Zhang, H. T.,
et al., Oncogene 14:2863-2869 (1997)).
IV. The Interaction of Mutant and Normal Annexin Gene Products
[0136] Studies of the clonal nature of tumor formation have
suggested that tumors have a monoclonal composition, and hence
arise by the clonal propagation of a single progenitor cell
(Fearson, E. R. et al., Science 247:193-197 (1987)).
[0137] The simplest model to explain the mechanism of action of a
tumor-suppressing gene is that malignancy requires two separate
genetic events (e.g., loss by deletion or mutation of both
functional anx7 alleles in a cell). Inactivation of only one of the
two natural anx7 alleles causes the animal to be more susceptible
to cancerous growths.
[0138] Transgenic animals may be used to investigate the biological
implications of tumor-suppressing genes (Capecchi, M. R., Science
244:1288-1292 (1989)). Lavigueur, A. et al. constructed a
transgenic mouse which had a single added mutant p53 gene in
addition to the endogenous two wild-type p53 alleles. The mouse and
its progeny overexpressed the added p53 gene. The mice were found
to have a high incidence of lung, bone, and lymphoid tumors
(Lavigueur, A. et al., Molec. Cell. Biol. 9:3982-3991 (1989)).
[0139] Thus, this invention provides a transgenic animal whose
genome possesses one normal and functional anx7 allele and one
non-functional (mutant) anx7 allele. Such animals could be used to
study the consequences resulting from the loss of one anx7 allele,
and thus would more clearly aid in elucidating the processes of
oncogenesis and tumorigenesis. Such animals would also be useful in
screening potential carcinogens, in developing novel antineoplastic
therapeutics, and in gene therapy.
V. Homologous Recombination
[0140] The present invention uses the process of homologous
recombination to introduce a specific mutation into the naturally
present anx7 sequence of an animal cell, most preferably an
embryonic stem (ES) cell. The mutated ES cells of non-human animals
can then be either cultured in suitable cell culture medium or
introduced into the uterus of a suitable recipient and permitted to
develop into a non-human animal. Alternatively, the methods of the
present invention may be used to alter the somatic cells of a
non-human animal to produce a chimeric non-human animal.
[0141] An understanding of the process of homologous recombination
(Watson, J. D., In: Molecular Biology of the Gene, 3rd Ed., W. A.
Benjamin, Inc., Menlo Park, Calif. (1977)) is thus desirable in
order to fully appreciate the present invention.
[0142] In brief, homologous recombination is a well-studied natural
cellular process which results in the scission of two nucleic acid
molecules having identical or substantially similar sequences
(i.e., "homologous"), and the ligation of the two molecules such
that one region of each initially present molecule is now ligated
to a region of the other initially present molecule (Sedivy, J. M.,
Bio-Technol. 6:1192-1196 (1988)).
[0143] Homologous recombination is, thus, a sequence specific
process by which cells can transfer a "region" of DNA from one DNA
molecule to another. As used herein, a "region" of DNA is intended
to generally refer to any nucleic acid molecule. The region may be
of any length from a single base to a substantial fragment of a
chromosome. For homologous recombination to occur between two DNA
molecules, the molecules must possess a "region of homology" with
respect to one another. Such a region of homology must be at least
two base pairs long and having a substantially similar nucleic acid
sequence.
[0144] Recombination is catalyzed by enzymes which are naturally
present in both prokaryotic and eukaryotic cells. The transfer of a
region of DNA may be envisioned as occurring through a multi-step
process.
[0145] If either of the two participant molecules is a circular
molecule, then the recombination event results in the integration
of the circular molecule into the other participant. Importantly,
if a particular region is flanked by regions of homology (which may
be the same, but are preferably different), then two
recombinational events may occur, and result in the exchange of a
region of DNA between two DNA molecules. Recombination may be
"reciprocal," and thus results in an exchange of DNA regions
between two recombining DNA molecules. Alternatively, it may be
"non-reciprocal," (also referred to as "gene conversion") and
result in both recombining nucleic acid molecules having the same
nucleotide sequence. There are no constraints regarding the size or
sequence of the region which is exchanged in a two-event
recombinational exchange.
[0146] The frequency of recombination between two DNA molecules may
be enhanced by treating the introduced DNA with agents which
stimulate recombination. Examples of such agents include
trimethylpsoralen, UV light, etc.
VII. Production of Chimeric and Transgenic Animals: Gene Targeting
Methods
[0147] One approach to producing animals having defined and
specific genetic alterations has used homologous recombination to
control the site of integration of an introduced marker gene
sequence in tumor cells and in fusions between diploid human
fibroblast and tetraploid mouse erythroleukemia cells (Smithies, O.
et al., Nature 317:230-234 (1985)).
[0148] This approach was further exploited by Thomas, K. R., and
co-workers, who described a general method, known as "gene
targeting," for targeting mutations to a preselected, desired gene
sequence of an ES cell in order to produce a transgenic animal
(Mansour, S. L. et al., Nature 336:348-352 (1988); Capecchi, M. R.,
Trends Genet. 5:70-76 (1989); Capecchi, M. R., Science
244:1288-1292 (1989); Capecchi, M. R. et al., In: Current
Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 45-52;
Frohman, M. A. et al., Cell 56:145-147 (1989)).
[0149] It may now be feasible to deliberately alter any gene in a
mouse (Capecchi, M. R., Trends Genet. 5:70-76 (1989); Frohman, M.
A. et al., Cell 56:145-147 (1989)). Gene targeting involves the use
of standard recombinant DNA techniques to introduce a desired
mutation into a cloned DNA sequence of a chosen locus. That
mutation is then transferred through homologous recombination to
the genome of a pluripotent, embryo-derived stem (ES) cell. The
altered stem cells are microinjected into mouse blastocysts and are
incorporated into the developing mouse embryo to ultimately develop
into chimeric animals. In some cases, germ line cells of the
chimeric animals will be derived from the genetically altered ES
cells, and the mutant genotypes can be transmitted through
breeding.
[0150] Gene targeting has been used to produce chimeric and
transgenic mice in which an nptII gene has been inserted into the
.beta..sub.2-microglobulin locus (Koller, B. H. et al., Proc. Natl.
Acad. Sci. (U.S.A.) 86:8932-8935 (1989); Zijlstra, M. et al.,
Nature 342:435-438 (1989); Zijistra, M. et al., Nature 344:742-746
(1989); DeChiaba et al., Nature 345:78-80 (1990)). Similar
experiments have enabled the production of chimeric and transgenic
animals having a c-abl gene which has been disrupted by the
insertion of an nptil gene (Schwartzberg, P. L. et al., Science
246:799-803 (1989)). The technique has been used to produce
chimeric mice in which the en-2 gene has been disrupted by the
insertion of an nptll gene (Joyner, A. L. et al., Nature
338:153-155 (1989)).
[0151] In order to utilize the "gene targeting" method, the gene of
interest must have been previously cloned, and the intron-exon
boundaries determined. The method results in the insertion of a
marker gene (i.e. the nptII gene) into a translated region of a
particular gene of interest. Thus, use of the gene targeting method
results in the gross destruction of the gene of interest.
[0152] Significantly, the use of gene targeting to alter a gene of
a cell results in the formation of a gross alteration in the
sequence of that gene. The efficiency of gene targeting depends
upon a number of variables, and is different from construct to
construct.
VII. The Production of Chimeric and Transgenic Animals
[0153] The chimeric or transgenic animal cells of the present
invention are prepared by introducing one or more DNA molecules
into a precursor pluripotent cell, most preferably an ES cell, or
equivalent (Robertson, E. J., In: Current Communications in
Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1989), pp. 39-44, which reference is
incorporated herein by reference). The term "precursor" is intended
to denote only that the pluripotent cell is a precursor to the
desired ("transfected") pluripotent cell which is prepared in
accordance with the teachings of the present invention. The
pluripotent (precursor or transfected) cell may be cultured in
vivo, in a manner known in the art (Evans, M. J. et al., Nature
292:154-156 (1981)) to form a chimeric or transgenic animal.
[0154] Any ES cell may be used in accordance with the present
invention. It is, however, preferred to use primary isolates of ES
cells. Such isolates may be obtained directly from embryos such as
the CCE cell line disclosed by Robertson, E. J., In: Current
Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 39-44),
or from the clonal isolation of ES cells from the CCE cell line
(Schwartzberg, P. A. et al., Science 246:799-803 (1989), which
reference is incorporated herein by reference). Such clonal
isolation may be accomplished according to the method of E. J.
Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, (E. J. Robertson, Ed.), IRL Press, Oxford,
1987) which reference and method are incorporated herein by
reference. The purpose of such clonal propagation is to obtain ES
cells which have a greater efficiency for differentiating into an
animal. Clonally selected ES cells are approximately 10-fold more
effective in producing transgenic animals than the progenitor cell
line CCE. For the purposes of the recombination methods of the
present invention, clonal selection provides no advantage.
[0155] An example of ES cell lines which have been clonally derived
from embryos are the ES cell lines, AB1 (hprt.sup.+) or AB2.1
(hprt.sup.-). The ES cells are preferably cultured on stromal cells
(such as STO cells (especially SNC4 STO cells) and/or primary
embryonic fibroblast cells) as described by E. J. Robertson (In:
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,
(E. J. Robertson, Ed., IRL Press, Oxford, 1987, pp 71-112), which
reference is incorporated herein by reference. Methods for the
production and analysis of chimeric mice are disclosed by Bradley,
A. (In: Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, (E. J. Robertson, Ed.), IRL Press, Oxford, 1987, pp
113-151), which reference is incorporated herein by reference. The
stromal (and/or fibroblast) cells serve to eliminate the clonal
overgrowth of abnormal ES cells. Most preferably, the cells are
cultured in the presence of leukocyte inhibitory factor ("lif")
(Gough, N. M. et al., Reprod. Fertil. Dev. 1:281-288 (1989);
Yamamori, Y. et al., Science 246:1412-1416 (1989), both of which
references are incorporated herein by reference). Since the gene
encoding lif has been cloned (Gough, N. M. et al., Reprod. Fertil.
Dev. 1:281-288 (1989)), it is especially preferred to transform
stromal cells with this gene, by means known in the art, and to
then culture the ES cells on transformed stromal cells that secrete
lif into the culture medium.
[0156] ES cell lines may be derived or isolated from any species
(for example, chicken, etc.), although cells derived or isolated
from mammals such as rodents (i.e. mouse, rat, hamster, etc.),
rabbits, sheep, goats, fish, pigs, cattle, primates and humans are
preferred.
IX. Uses of the Present Invention
[0157] The present invention provides human or animal cells which
contain a desired gene sequence in one of the two annexin gene
alleles of the cell's genome. In a first embodiment, the invention
also provides a means for producing non-human chimeric or
transgenic animals whose cells contain such a sequence. The animals
which may be produced through application of the described method
include chicken, non-human mammals (especially, rodents (i.e.
mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs,
cattle and non-human primates).
[0158] The cells and non-human animals of the present invention
have both diagnostic and therapeutic utility.
[0159] A. Diagnostic Utility
[0160] Since the invention provides a cell, or a transgenic or
chimeric non-human animal, that contains a single functional allele
of the anx7 gene, and since such cells will become tumor cells upon
the mutation of the functional allele to a non-functional form, the
present invention can be used to identify an agent that is capable
of affecting a characteristic of an animal cell that is
attributable to the presence or expression of a tumor-suppressing
gene. A characteristic of an animal cell is said to be
"attributable to the presence or expression of a tumor-suppressing
gene," if the characteristic is altered by the absence or lack of
expression of the tumor-suppressing gene. Examples of such
characteristics include tumorigenesis, resilience to tumorigenesis,
the extent, distribution, incidence, location, grade, etc. of
tumors, etc.
[0161] In one embodiment, such agents can decrease the tumorigenic
(or neoplastic) potential of the cells or animals. Such agents are
discussed below with regard to the therapeutic potential of the
invention.
[0162] In a second embodiment, such agent can increase the
tumorigenic (or neoplastic) potential of the cells or animals.
Thus, the cells and non-human animals of the present invention have
utility in testing potential or suspected carcinogens for
tumorigenic activity. They may be used to identify and assess the
tumorigenic effects of agents that may be present, for example, in
the environment (such as environmental pollutants in air, water or
soil), or resulting from environmental exposures to chemicals,
radioisotopes, etc. They may also be used to facilitate studies of
the effects of diet on oncogenesis. They may be used to determine
whether potential or present food additives, chemical waste
products, chemical process by-products, water sources, proposed or
presently used pharmaceuticals, cosmetics, etc., have tumorigenic
activity. They may also be used to determine the tumorigenic
potential of various energy forms (such as UV rays, X-rays,
ionizing radiation, gamma rays of elemental isotopes, etc.).
[0163] The frequency at which a mutational event occurs is
dependent upon the concentration of a mutagenic chemical agent, or
the intensity of a mutagenic radiation. Thus, since the frequency
of a single cell receiving two mutational events is the square of
the frequency at which a single mutational event will occur, the
cells and non-human animals of the present invention shall be able
to identify neoplastic (mutagenic) agents at concentrations far
below those needed to induce neoplastic changes in natural cells or
animals. This is because one allele of the tumor suppressing gene
anx7 has already been mutated in the transgenic mouse of the
present invention.
[0164] One especially preferred cell is a non-human cell in which
one of the natural anx7 alleles has been replaced with a functional
human anx7 allele and the other of the natural anx7 alleles has
been mutated to a non-functional form. Alternatively, one may
employ a non-human cell in which the two natural anx7 alleles have
been replaced with a functional and a non-functional allele of the
human anx7 gene.
[0165] Such cells may be used, in accordance with the methods
described above, to assess the neoplastic potential of agents in
cells containing the human anx7 allele. More preferably, such cells
are used to produce non-human animals which do not contain any
natural functional anx7 alleles, but which contain only one
functional human anx7 allele. Such non-human animals can be used to
assess the tumorigenicity of an agent in a non-human animal
expressing the human anx7 gene product.
[0166] 1. In Vitro Assays
[0167] In one embodiment, one may employ the cells of the present
invention, in in vitro cell culture, and incubate such cells in the
presence of an amount of the agent whose tumorigenic potential is
to be measured. This embodiment therefore comprises an in vitro
assay of tumorigenic activity.
[0168] Although many carcinogenic agents may directly mediate their
tumorigenic effects, some agents will not exhibit tumorigenic
potential until metabolized, or until presented to a susceptible
cell along with one or more "co-carcinogenic" factors. The present
invention permits the identification of such "latent" carcinogenic
and "co-carcinogenic" agents. In accordance with this embodiment of
the invention, the presence of a "latent" carcinogen can be
identified by merely maintaining cell or animal exposure to a
candidate agent. Alternatively, the cells of the present invention
can be incubated in "conditioned" culture medium (i.e. medium
containing the candidate agent that was used to culture other cells
before being used to culture the cells of the present
invention).
[0169] The present invention permits the identification of
co-carcinogenic factors capable of inducing neoplastic effects in
the presence of a second agent. Such factors can be identified by
culturing the cells of the present invention in the presence of two
or more candidate agents simultaneously, and then assaying for
neoplasia. The transformation of the cells to a neoplastic state
would be indicative of tumorigenic (or neoplastic) activity of the
assayed agent. Such a neoplastic state may be evidenced by a change
in cellular morphology, by a loss of contact inhibition, by the
acquisition of the capacity to grow in soft agar, or most
preferably, by the initiation of expression of tumor antigens.
[0170] The use of tumor antigens as a means of detecting neoplastic
activity is preferred since such antigens may be readily detected.
As is well known in the art, antibodies, or fragments of
antibodies, may be used to quantitatively or qualitatively detect
the presence tumor of antigens on cell surfaces. Since any cell
type (i.e. lung, kidney, colon, etc.) may be employed to form the
anx7-mutated cells of the present invention, it is possible to
determine whether an agent has a tissue specific tumorigenic
potential. To accomplish this goal, one would incubate a candidate
agent in the presence of anx7-mutated cells derived from any of a
variety of tissue types. Since tumors have tumor-specific antigens,
and since antibodies capable of binding to such antigens have been
isolated, it is possible to use such antibodies to characterize any
tumor antigens which may be expressed by the anx7-mutated cells.
Such detection may be accomplished using any of a variety of
immunoassays. For example, by radioactively labeling the antibodies
or antibody fragments, it is possible to detect the antigen through
the use of radioimmune assays.
[0171] A good description of a radioimmune assay (RIA) may be found
in Laboratory Techniques and Biochemistry in Molecular Biology, by
Work, T. S., et al., North Holland Publishing Company, NY (1978),
with particular reference to the chapter entitled "An Introduction
to Radioimmune Assay and Related Techniques" by Chard, T.,
incorporated by reference herein. Examples of suitable
radioisotopic labels include .sup.3H, .sup.111In, .sup.125I,
.sup.131I, .sup.32P, .sup.35S, .sup.14C, .sup.51Cr, .sup.57To,
.sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu, .sup.90Y, .sup.67CU,
.sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc, .sup.109Pd, etc.
[0172] Alternatively, enzyme labels, non-radioactive isotopic
labels, fluorescent labels, chemiluminescent labels or other
suitable labels can be employed. Examples of suitable enzyme labels
include malate dehydrogenase, staphylococcal nuclease,
delta-5-steroid isomerase, yeast-alcohol dehydrogenase,
alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,
peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine
esterase, etc. Examples of suitable non-radioactive isotopic labels
include .sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, .sup.56Fe,
etc.
[0173] Examples of suitable fluorescent labels include an
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, a fluorescamine
label, etc.
[0174] Examples of chemiluminescent labels include a luminal label,
an isoluminal label, an aromatic acridinium ester label, an
imidazole label, an acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, an aequorin label, etc.
Those of ordinary skill in the art will know of other suitable
labels which may be employed in accordance with the present
invention. The binding of these labels to antibodies or fragments
thereof can be accomplished using standard techniques commonly
known to those of ordinary skill in the art. Typical techniques are
described by Kennedy, J. H., et al. (Clin. Chim. Acta 70:1-31
(1976)), and Schurs, A. H. W. M., et al. (Clin. Chim. Acta 81:1-40
(1977)). Coupling techniques mentioned in the latter are the
glutaraldehyde method, the periodate method, the dimaleimide
method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method,
all of which methods are incorporated by reference herein.
[0175] The above-described in vitro assay has the advantageous
features of potentially lower cost than presently used assays, and
the capacity to readily screen large numbers of agents. Use of this
embodiment facilitates comparisons of test results obtained at
different times and conditions. Moreover, because it is possible to
use very large numbers of cells in such assays, it is possible to
detect the tumorigenic activity of tumorigenic agents even at very
low concentrations. Lastly, since this embodiment can be performed
using human cells, it provides a means for determining the
tumorigenic (or neoplastic) potential of a test compound on human
cells.
[0176] 2. In Vivo Assays
[0177] In a second embodiment, one may employ the non-human animals
of the present invention, and provide to such animals (by, for
example, inhalation, ingestion, injection, implantation, etc.) an
amount of the agent whose tumorigenic potential is to be measured.
The formation of tumors in such animals (as evidenced by direct
visualization by eye, or by biopsy, imaging, detection of tumor
antigens, etc.) would be indicative of tumorigenic activity of the
assayed agent.
[0178] The use of the non-human animals of the present invention is
preferred over naturally occurring non-human animals because
natural animals contain two functional anx7 alleles and thus
require two mutational events in order to lead to loss of
functional anx7 activity. In contrast, since the non-human animals
of the present invention have only one functional anx7 allele, only
one mutational event is needed to cause total loss of anx7
function.
[0179] The detection of tumors in such animals can be accomplished
by biopsy, imaging, or by assaying the animals for the presence of
cells which express tumor antigens. For example, such detection may
be accomplished by removing a sample of tissue from a subject and
then treating the isolated sample with any suitably labeled
antibodies (or antibody fragments) as discussed above. Preferably,
such in situ detection is accomplished by removing a histological
specimen from the subject, and providing the labeled antibody to
such specimen. The antibody (or fragment) is preferably provided by
applying or by overlaying the labeled antibody (or fragment) to a
sample of tissue. Through the use of such a procedure, it is
possible to determine not only the presence of antigen, but also
the distribution of the antigen on 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.
[0180] Alternatively, the detection of tumor cells may be
accomplished by in vivo imaging techniques, in which the labeled
antibodies (or fragments thereof) are provided to the subject, and
the presence of the tumor is detected without the prior removal of
any tissue sample. Such in vivo detection procedures have the
advantage of being less invasive than other detection methods, and
are, moreover, capable of detecting the presence of
antigen-expressing cells in tissue which cannot be easily removed
from the patient. Additionally, it is possible to assay for the
presence of tumor antigens in body fluids (such as blood, lymph,
etc.), stools, or cellular extracts. In such immunoassays, the
antibodies (or antibody fragments) may be utilized in liquid phase
or bound to a solid-phase carrier, as described below.
[0181] The use of an in vivo assay has several advantageous
features. The in vivo assay permits one not only to identify
tumorigenic agents, but also to assess the kind(s) of tumors
induced by the agent, the number and location (i.e. whether organ
or tissue specific) of any elicited tumors, and the grade (clinical
significance) of such elicited tumors. It further permits an
assessment of tumorigenicity which inherently considers the
possible natural metabolism of the introduced agent, the
possibility that the introduced agent (or its metabolic
by-products) might selectively accumulate in specific tissues or
organs of the recipient animal, the possibility that the recipient
animal might recognize and repair or prevent tumor formation. In
short, such an assay provides a true biological model for studying
and evaluating the tumorigenic potential of an agent in a living
non-human animal.
[0182] 3. Immunoassays of Tumor Antigens
[0183] The in vitro, in situ, or in vivo detection of tumor
antigens using antibodies (or fragments of antibodies) can be
improved through the use of carriers. Well-known carriers include
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
agaroses, 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 may have virtually any possible
structural configuration so long as the coupled molecule is capable
of binding to an antigen. Thus, the support configuration may 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 may be flat such as a sheet, test strip, etc. Those
skilled in the art will note many other suitable carriers for
binding monoclonal antibody, or will be able to ascertain the same
by use of routine experimentation.
[0184] The binding molecules of the present invention may also be
adapted for utilization in an immunometric assay, also known as a
"two-site" or "sandwich" assay. In a typical immunometric assay, a
quantity of unlabeled antibody (or fragment of antibody) is bound
to a solid support that is insoluble in the fluid being tested
(i.e., blood, lymph, liquified stools, tissue homogenate, etc.) and
a quantity of detectably labeled soluble antibody is added to
permit detection and/or quantitation of the ternary complex formed
between solid-phase antibody, antigen, and labeled antibody.
[0185] Typical immunometric assays include "forward" assays in
which the antibody bound to the solid phase is first contacted with
the sample being tested to extract the antigen from the sample by
formation of a binary solid phase antibody-antigen complex. After a
suitable incubation period, the solid support is washed to remove
the residue of the fluid sample, including unreacted antigen, if
any, and then contacted with the solution containing an unknown
quantity of labeled antibody (which functions as a "reporter
molecule"). After a second incubation period to permit the labeled
antibody to complex with the antigen bound to the solid support
through the unlabeled antibody, the solid support is washed a
second time to remove the unreacted labeled antibody. This type of
forward sandwich assay may be a simple "yes/no" assay to determine
whether antigen is present or may be made quantitative by comparing
the measure of labeled antibody with that obtained for a standard
sample containing known quantities of antigen. Such "two-site" or
"sandwich" assays are described by Wide at pages 199-206 of
Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S.
Livingstone, Edinburgh, 1970.
[0186] In another type of "sandwich" assay, which may also be
useful to identify tumor antigens, the so-called "simultaneous" and
"reverse" assays are used. A simultaneous assay involves a single
incubation step as the antibody bound to the solid support and
labeled antibody are both added to the sample being tested at the
same time. After the incubation is completed, the solid support is
washed to remove the residue of fluid sample and uncomplexed
labeled antibody. The presence of labeled antibody associated with
the solid support is then determined as it would be in a
conventional "forward" sandwich assay.
[0187] In the "reverse" assay, stepwise addition first of a
solution of labeled antibody to the fluid sample followed by the
addition of unlabeled antibody bound to a solid support after a
suitable incubation period is utilized. After a second incubation,
the solid phase is washed in conventional fashion to free it of the
residue of the sample being tested and the solution of unreacted
labeled antibody. The determination of labeled antibody associated
with a solid support is then determined as in the "simultaneous"
and "forward" assays.
[0188] The immunometric assays for antigen require that the
particular binding molecule be labeled with a "reporter molecule."
These reporter molecules or labels, as identified above, are
conventional and well-known to the art. In the practice of the
present invention, enzyme labels are a preferred embodiment. No
single enzyme is ideal for use as a label in every conceivable
immunometric assay. Instead, one must determine which enzyme is
suitable for a particular assay system. Criteria important for the
choice of enzymes are turnover number of the pure enzyme (the
number of substrate molecules converted to product per enzyme site
per unit of time), purity of the enzyme preparation, sensitivity of
detection of its product, ease and speed of detection of the enzyme
reaction, absence of interfering factors or of enzyme-like activity
in the test fluid, stability of the enzyme and its conjugate,
availability and cost of the enzyme and its conjugate, and the
like. Included among the enzymes used as preferred labels in the
immunometric assays of the present invention are peroxidase,
alkaline phosphatase, beta-galactosidase, urease, glucose oxidase,
glycoamylase, malate dehydrogenase, and glucose-6-phosphate
dehydrogenase. Urease is among the more preferred enzyme labels,
particularly because of chromogenic pH indicators which make its
activity readily visible to the naked eye.
[0189] B. Therapeutic Utility
[0190] Significantly, the cells and animals of the present
invention can be used to identify agents that decrease the
tumorigenic (or neoplastic) potential of the cells or animals. Such
agents can be "anti-tumor agents" and/or "chemopreventative
agents." "Anti-tumor agents" act to decrease the proliferation of
the cells (or the growth, dissemination, or metastasis of tumors in
the chimeric or transgenic animals). "Chemopreventative agents" act
to inhibit the formation of new tumors. Such agents may have
general activity (inhibiting all new tumor formation), or may have
a specific activity (inhibiting the distribution, frequency, grade,
etc.) of specific types of tumors in specific organs and tissue.
Thus, the present invention permits the identification of novel
antineoplastic therapeutics. Any of the assays in section A. above
may be used for determining tumor-suppressing activity.
[0191] The transgenic cells and non-human animals of the present
invention can be used to study human gene regulation of the anx7
gene. For example, such cells and animals can be used to
investigate the interactions of the anx7 gene with oncogenes or
other tumor suppressor genes. Thus, they may be used to identify
therapeutic agents which have the ability to impair or prevent
neoplastic or tumorigenic development. Such agents have utility in
the treatment and cure of cancer in humans and animals.
Significantly, potential therapeutic agents are frequently found to
induce toxic effects in one animal model but not in another animal
model. To resolve the potential of such agents, it is often
necessary to determine the metabolic patterns in various species,
and to then determine the toxicities of the metabolites. The
present invention permits one to produce transgenic cells or
animals which could facilitate such determinations.
[0192] When providing the therapeutic agents of the present
invention to the cells of an animal, pharmaceutically acceptable
carriers (i.e. liposomes, etc.) are preferably employed. Such
agents can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby these materials, or
their functional derivatives, are combined in admixture with a
pharmaceutically acceptable carrier vehicle. Suitable vehicles and
their formulation, are described, for example, in Nicolau, C. et
al. (Crit. Rev. Ther. Drug Carrier Syst. 6:239-271 (1989)), which
reference is incorporated herein by reference.
[0193] In order to form a pharmaceutically acceptable composition
suitable for effective administration, such compositions will
contain an effective amount of the desired gene sequence together
with a suitable amount of carrier vehicle.
[0194] Additional pharmaceutical methods may be employed to control
the duration of action. Control release preparations may be
achieved through the use of polymers to complex or absorb the
desired gene sequence (either with or without any associated
carrier). The controlled delivery may be exercised by selecting
appropriate macromolecules (for example polyesters, polyamino
acids, polyvinyl, pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine, sulfate) and
the concentration of macromolecules as well as the methods of
incorporation in order to control release. Another possible method
to control the duration of action by controlled release
preparations is to incorporate the agent into particles of a
polymeric material such as polyesters, polyamino acids, hydrogels,
poly(lactic acid) or ethylene vinylacetate copolymers.
Alternatively, instead of incorporating these agents into polymeric
particles, it is possible to entrap these materials in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatine-microcapsules and poly(methylmethacylate)
microcapsules, respectively, or in colloidal drug delivery systems,
for example, liposomes, albumin microspheres, microemulsions,
nanoparticles, and nanocapsules or in macroemulsions.
[0195] C. Use in Research and in Gene Therapy
[0196] The cells and non-human animals of the present invention may
be used to investigate gene regulation, expression and organization
in animals. The methods of the present invention may be used to
produce alterations in a regulatory region of the native anx7 gene
sequence. Thus, the invention provides a means for altering the
nature or control of transcription or translation of the anx7 gene,
and of altering the anx7 gene itself. For example, the invention
enables one to introduce mutations which result in increased or
decreased gene expression. Similarly, it enables one to impair or
enhance the transcriptional capacity of the natural anx7 gene in
order to decrease or increase its expression. Thus, the present
invention permits the manipulation and dissection of the anx7 gene.
Such abilities are especially valuable in gene therapy protocols
and in the development of improved animal models of cancer.
[0197] The principles of gene therapy are disclosed by Oldham, R.
K. (In: Principles of Biotherapy, Raven Press, N.Y., 1987), and
similar texts. Disclosures of the methods and uses for gene therapy
are provided by Boggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990));
Karson, E. M. (Biol. Reprod. 42.39-49 (1990)); Ledley, F. D., In:
Biotechnology, A Comprehensive Treatise, volume 7B, Gene
Technology, VCH Publishers, Inc. NY, pp 399-458 (1989)), all of
which references are incorporated herein by reference.
[0198] In one embodiment of the present invention, DNA encoding
either a functional anx7 gene, variants of that gene, or other
genes which influence the activity of the anx7 gene, may be
introduced into the somatic cells of an animal (particularly
mammals including humans) in order to provide a treatment for
cancer (i.e. "gene therapy"). Most preferably, viral or retroviral
vectors are employed for this purpose.
[0199] Retroviral vectors are a common mode of delivery and in this
context are retroviruses from which all viral genes have been
removed or altered so that no viral proteins are made in cells
infected with the vector. Viral replication functions are provided
by the use of retrovirus "packaging" cells that produce all of the
viral proteins but that do not produce infectious virus.
[0200] Introduction of the retroviral vector DNA into packaging
cells results in production of virions that carry vector RNA and
can infect target cells, but such that no further virus spread
occurs after infection. To distinguish this process from a natural
virus infection where the virus continues to replicate and spread,
the term transduction rather than infection is often used.
[0201] Non-retroviral vectors have been used in genetic therapy.
One such alternative is the adenovirus (Rosenfeld, M. A., et al.,
Cell 68:143155 (1992); Jaffe, H. A. et al., Nature Genetics
1:372-378 (1992); Lemarchand, P. et al., Proc. Natl. Acad. Sci. USA
89:6482-6486 (1992)). Major advantages of adenovirus vectors are
their potential to carry large segments of DNA (36 Kb genome), a
very high titre (10.sup.11/ml), ability to infect non-replicating
cells, and suitability for infecting tissues in situ, especially in
the lung. The most striking use of this vector so far is to deliver
a human cystic fibrosis transmembrane conductance regulator (CFTR)
gene by intratracheal instillation to airway epithelium in cotton
rats (Rosenfeld, M. A., et al., Cell 63:143-155 (1992)). Similarly,
herpes viruses may also prove valuable for human gene therapy
(Wolfe, J. H. et al., Nature Genetics 1:379-384 (1992)). Of course,
any other suitable viral vector may be used for genetic therapy
with the present invention.
[0202] Another gene transfer method for use in humans is the
transfer of plasmid DNA in liposomes directly to human cells in
situ (Nabel, E. G., et al., Science 249:1285-1288 (1990)). Plasmid
DNA should be easy to certify for use in human gene therapy
because, unlike retroviral vectors, it can be purified to
homogeneity. In addition to liposome-mediated DNA transfer, several
other physical DNA transfer methods, such as those targeting the
DNA to receptors on cells by conjugating the plasmid DNA to
proteins, have shown promise in human gene therapy (Wu, G. Y., et
al., J. Biol. Chem. 266:14338-14342 (1991); Curiel, D. T., et al.,
Proc. Natl. Acad. Sci. USA, 88:8850-8854 (1991)).
[0203] In applying these methods of therapy, it has been observed
that certain tumor cells return to normal function when fused with
normal cells, suggesting that replacement of a missing factor, such
as a wild-type tumor suppressor gene expression product may serve
to restore a tumor cell to a normal state (reviewed by Weinberg, R.
A., Cancer Research 49:3713-3721, at 3717 (1989)).
[0204] These observations have led to research aimed at providing
genetic treatment of tumor cells having defective tumor suppressor
genes. The proposed method of treatment requires identification of
the damaged tumor suppressor gene, and introduction of the
corresponding undamaged gene (including a promoter and a complete
encoding sequence) into the affected tumor cells by means of a
vector such as an adenovirus vector able to express the gene
product. It is proposed that the incorporated functional gene will
convert the target cell to a non-malignant state.
[0205] For example, The Regents of the University of California, in
Patent Cooperation Treaty patent application (by Lee et al., number
WO 90/05180, having an international filing date of Oct. 30, 1989
and published May 17, 1990), disclose a scheme for identifying an
inactive or defective tumor suppressor gene and then replacing such
a defective gene with its functional equivalent. In particular, the
WO 90/05180 application proposes, based on in vitro studies, to
insert a functional RB.sup.110 gene into an
[0206] RB-minus tumor cell by means of a retroviral vector in order
to render such cells non-malignant.
[0207] Although, as indicated above, such gene therapy can be
provided to a recipient in order to treat (i.e. suppress,
attenuate, or cause regression) an existing neoplastic state, the
principles of the present invention can also be used to provide a
prophylactic gene therapy to individuals who, due to inherited
genetic mutations, or somatic cell mutation, contain cells having
impaired anx7 gene expression (for example, only a single
functional allele of the anx7 gene). Such therapy could be
administered in advance of the detection of cancer in order to
lessen the individual's predisposition to the disease.
[0208] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE I
Production and Characterization of a Transgenic Mouse for the anx7
Gene
[0209] Before beginning the actual production of transgenic mice,
the genomic locus for annexin VII (anx7) was characterized. Then, a
transgene construct ("targeting vector") was prepared based on this
characterization. This construct carries the necessary elements to
facilitate the transgenic animal construction.
[0210] The anx7 genomic locus from a 129SV/CPJ mouse genomic
library (Stratagene), which contained 14 exons of the anx7 gene
spanning about 34 kb, was screened with mouse anx7 cDNA probe
(Zhang-Keck, et al., Biochem. J. 301:835-845.)
[0211] In order to assess which segments of the genomic locus were
most suitable for use in the targeting vector, several restriction
fragments including three Xba I fragments (1.9, 3.6 and 3.1 kb) and
one Xho I fragment (2.0 kb) encompassing this region were
subcloned, labeled with .sup.32[P], and tested for the presence of
repetitive sequences. Repetitive sequences are undesirable because
they can cause random insertion of the anx7 gene into any part of
the chromosome. Therefore, these regions were removed. A Southern
blot analysis revealed that only the 3.1 kb Xba I genomic DNA
fragment and the 2.0 kb Xho I genomic DNA fragment of the four
fragments tested, when used as a probe, gave sharp bands on a
genomic Southern of mouse ES cell DNA. In FIG. 2, the genomic DNA
derived from ES cells was digested with Hind III, blotted and
hybridized with .sup.32P-labeled genomic fragments. A single band
was seen with probes from the 3.1 kb Xba I fragment (lane 1) and
the 2.0 kb Xho I fragment (lane 2) of mouse anx7. A smear was noted
with the 1.9 kb and 3.6 kb Xba I fragments (lanes 3, 4 and 5, 6,
respectively). Thus, the conclusion drawn is that the single band
fragments do not contain repetitive sequences and therefore, were
used in the targeting vector.
[0212] A. Construction of the Targeting Vector
[0213] To construct the anx7 gene targeting vector, the 2.0 kb Xho
I genomic DNA fragment (containing exons 4 and 5) and the 3.1 kb
Xba I genomic DNA fragment (containing exons 7 and 8) were inserted
into the Xho I site and the XbaI site of pPNT, respectively, to
generate the replacement type targeting vector termed KSBX.pPNT
(See FIG. 1A). The vector, PPNT (obtained from Dr. Heiner
Westphal's laboratory at NICHD, NIH) contained PGKneo and PGKtk
cassettes, separated and flanked by a number of unique cloning
sites. The neo gene was determined to be in the same orientation as
the anx7 gene. A herpes simplex virus thymidine kinase (TK) gene
was also added to the targeting vector as a marker sequence which
would be deleted in the event of homologous recombination between
the targeting vector and the wild type allele. This allowed
selection against cells that had undergone nonhomologous
integration.
[0214] B. Transfection and Selection of ES Cells
[0215] Pluripotent embryonic stem cells (referred to as "ES" cells)
are cells which may be obtained from embryos until the early
post-implantation stage of embryogenesis. The cells may be
propagated in culture, and are able to differentiate either in
vitro or in vivo upon implantation into a mouse as a tumor. ES
cells have a normal karyotype (Evans, M. J. et al., Nature
292:154-156 (1981); Martin, G. R. et al., Proc. Natl. Acad. Sci.
(U.S.A.) 78:7634-7638 (1981)).
[0216] Upon injection into a blastocyst of a developing embryo, ES
cells will proliferate and differentiate, thus resulting in the
production of a chimeric animal. ES cells are capable of colonizing
both the somatic and germ-line lineages of such a chimeric animal
(Robertson, E. et al., Cold Spring Harb. Conf. Cell Prolif.
10:647-663 (1983); Bradley A. et al., Nature 309:255-256 (1984);
Bradley, A. et al., Curr. Top. Devel. Biol. 20:357-371 (1986);
Wagner, E. F. et al., Cold Spring Harb. Symp. Quant. Biol.
50:691-700 (1985); (all of which references are incorporated herein
by reference).
[0217] Because ES cells may be propagated in vitro, it is possible
to manipulate such cells using the techniques of somatic cell
genetics. Thus, it is possible to select ES cells which carry
mutations (such as in the hprt gene (encoding hypoxanthine
phosphoribosyl transferase) (Hooper, M. et al., Nature 326:292-295
(1987); Kuehn, M. R. et al., Nature 326:295-298 (1987)). Such
selected cells can then be used to produce chimeric or transgenic
mice which fail to express an active enzyme, and thus provide
animal models for diseases.
[0218] The ES cells used here were derived from mouse strain 129SvJ
and maintained in culture on primary mouse embryo fibroblast (PMEF)
feeder cells carrying a neomycin gene. The culture medium was
supplemented with leukemia inhibitory factor (1500 units/ml). The
targeting vector was linearized by the restriction endonuclease Not
I and transfected into the J1 cell line (Li et al. 1992) by
electroporation of 3.times.10.sup.6 ES cells. The genetically
altered ES cells containing the targeted allele were selected with
G418 (Gibco) at 350 .mu.g/ml and Gancyclovir (Bristol Myers) at 0.2
.mu.M.
[0219] To screen for homologous recombinant ES clones, genomic DNA
was isolated from those clones exhibiting dual resistance (selected
against neomycin and Gancyclovir). Genomic DNA was isolated from
cultured cells by digestion overnight at 55.degree. C. in lysis
buffer (10 mM Tris-HCl, pH 7.5/100 mM NaCl/1 mM EDTA/100 .mu.g/ml
proteinase K) followed by precipitation with iso-propanol. The
pellet was washed with 70% ethanol and dissolved in 100 .mu.l of
1.times.TE, pH 8.0 at 55.degree. C. overnight.
[0220] The genomic DNA from each clone was used as a template for
PCR amplifications with a anx7 specific flanking primer and a
PGK-neo-specific primer (5'-CGGATCGATCCCCTCAGAAGAAC-3'). Three out
of 250 clones yielded a PCR band of the correct size. FIG. 3A
depicts the PCR analysis of ES cell clones transfected with
KSBX.pPNT. To verify the results of the PCR screening, DNA from
PCR-positive ES clones was digested with Xba I and hybridized with
a genomic DNA probe, KXX, which is external to the 5'-flank
introduced into the targeting vector (See FIG. 3B). The probe
detected the predicted 3.6 kb wild-type and 4.5 kb mutant fragments
representing the normal and altered alleles of anx7, respectively.
Thus, the data indicated that the targeting vector had been
successfully generated and that genetically altered heterozygous ES
cells had been isolated which had undergone a single targeted
integration event at the anx7 locus.
[0221] C. Preparation of Chimeras
[0222] The strategy for generating the chimeras containing the
desired targeted mutation is shown in FIG. 4. In this example, the
altered ES cells were microinjected into the blastocoel cavity of a
4.5 day preimplantation mouse embryo from a C57B1/6J mouse. Then,
the embryos were transferred surgically into the uterine horn of a
pseudopregnant mouse and development was allowed to progress to
birth.
[0223] Resulting chimeric animals were backcrossed to C57BL6/J
mice, and germline transmission was scored by coat color. All
agouti (A/A) mice (i.e., brown) offspring were tested for the
presence of the mutated anx7 allele by PCR amplification using the
same conditions described above for the detection of homologous
recombination events in the ES cells. Normally, the ES cells are
derived from mice with distinguishable coat color alleles (brown,
shown in FIG. 4 as black) compared to recipient blastocyst (black,
shown in FIG. 4 as white).
[0224] More specifically, ES cells (with targeting construct)
derived from an agouti (A/A) mouse (brown) were injected into a
recipient nonagouti (a/a) (black female) C57B1/6 blastocyst (See
FIG. 5A). Chimeric males and females were mated to non-agouti
(black, a/a) females and males, respectively. Any progeny having
black coats were excluded immediately. One of the chimeric females
gave birth to a brown male mouse and so was a candidate for
harboring the chosen mutation in one of the two copies of the anx7
gene in every cell. This chimeric female is illustrated in FIG. 5B:
an anx7 transgenic chimeric mouse having an almost entirely agouti
brown coat and thus, greater than 95% of the hair follicle cells
were derived from the ES cells. Further, since the progeny from
this chimeric mouse, when bred to a C57B1/6 black male or black
female, were all agouti (brown), it was concluded that most, if not
all, of the germline cells produced by this mouse were also derived
from the ES cells (FIG. 5C).
[0225] Breeding of the chimeras with C57B1/6J strain mice resulted
in thirty germ line heterozygotes for the anx7 gene. No anx7 (-/-)
mutants were found out of 140 pups screened, implying that anx7
deficient mutants die in utero. To investigate the timing of
embryonic lethality, mice from embryonic days E8 to E17 were
genotyped. Of the viable 120 embryos analyzed, 25% were anx7 (-/-)
at E10, but by E11 none of them had survived. The Yolk sac DNA of
these embryos was used as a template for PCR analyses as described
above. Polymerase chain reaction analysis showed the absence of
anx7 transcripts in anx7 (-/-) mutants. (See FIG. 6.)
[0226] D. Anatomical and Histological Studies of the Chimeras
[0227] Thirty F2 generation hybrids from anx7 (+/-) and anx7 (+/+)
mice were weighed at regular intervals. Nine month old anx7 (+/-)
and anx7 (+/+) mice were sacrificed and their internal organs were
weighed. The following tissues were fixed in 10% buffered formalin:
brain, pituitary, heart, lung, liver, pancreas, adrenals, kidney,
spleen and thymus. For pathological studies, these tissues were
embedded in paraffin, cut in 5 .mu.m sections, and stained with
hematoxylin and eosin. The results are shown in FIG. 8 and
discussed below.
[0228] At birth, the anx7 (+/-) heterozygotes are indistinguishable
in size or behavior from wild type littermates or founder mates.
However, as shown in FIG. 7A, by 6-8 weeks, the larger male anx7
(+/-) heterozygotes are clearly distinguishable in their relative
gigantism from smaller normal. To measure this difference
quantitatively, the weights of a set of animals systematically
followed (see FIG. 7B). By the 9th month of life, male
heterozygotes weighed 40.7.+-.4.4 (SEM, n=5) grams, compared to
controls, which were 33.2.+-.2.2 (SEM, n=5) grams. This relative
weight increment for males of ca. 25% was statistically significant
and not due to obesity. By contrast, no changes were noted in the
weight gains with age for females.
[0229] Male anx7 (+/-) mice begin to grow at a greater rate than
normal littermate controls by about the fourth week after birth. By
contrast, female anx7 (+/-) mice do not vary from their controls.
Male mutant growth thereafter does not appear to abate. The data in
FIG. 7 show growth up to six months of age. However, when these
same animals were weighed at 13 months of age, evidence of
continued growth over this subsequent time period was noted.
Weights as high as 60 grams were noted for some of these
heterozygous animals. Postmortem examination has systematically
shown that the animals are not fat, but merely large. Organ weight
studies performed at 6 months of age showed that many internal
organs in anx7 (+/-) males weighed much more than normal, but were
of grossly normal structure. It is remarkable that, in light of our
subsequent focus on the hyperplastic Islets of Langerhans, the
pancreas was one of several organs that were not larger than
normal. However, the islets do make up less than 2% of the islet by
volume.
[0230] The growth phenotype of gender-specific gigantism and
organomegaly of the anx7 (+/-) mouse is fundamentally different
from that of other reported mouse knockouts. With the exception of
the p27.sup.kip1(-/-) mouse (Fero et al, 1996; Nakayama, K., et
al., Cell 85:707-720 (1996)), the reported instances of
mutation-based gigantism are mostly endocrine in origin, and are
due to increases of either growth hormone (Palmiter, R., et al.,
Nature 300:611-615 (1982)), IGF-1 (Mathews, L., et al.,
Endocrinology 123:2827-2833 (1988)), or IGF-2 (Wolf, E., et al.,
Endocrinology 135:1877-1886 (1994)). However, the levels of serum
IGF-1 in the anx7 (+/-) mouse are within normal limits. In
addition, since the levels of IGF-1 integrate the pulsatile levels
of growth hormone, it was concluded that average GH levels were
probably normal as well in anx7 (+/-) mice. GH levels measured in
overnight-fasted animals showed no change in males. One qualitative
parallel between the growth kinetics of male anx7 (+/-) mice and
those mice transgenic for GH or IGF-1, is a postpartum delay in the
onset of enhanced growth. The anx7 (+/-) male mice and mice
transgenic for GH begin to grow at 3-4 weeks, while those
transgenic for IGF-1 begin to grow only after 6-8 weeks. Mice
overproducing IGF-2 are heavier than control mice at birth, but do
not sustain the increase in weight into adulthood. Finally,
pituitary gland histology in male and female anx7 (+/-) mutants
cannot be distinguished from wildtype histologies (data not shown).
Consistently, the selective distribution of organomegaly noted for
the anx7 (+/-) male mutant is distinct from that associated with
high levels of GH, IGF-1 and IGF-2 (Palmiter, R., et al., Science
222:809-814 (1983); Mathews, L., et al., Endocrinology
123:2827-2833 (1988); Quaife, C., et al., Endocrinology 114:40-48
(1989); Wolf, E., et al., Endocrinology 135:1877-1886 (1994); Ward,
A., et al., Proc. Nat. Acad. Sci. (USA) 91:10365-10369 (1994)), or
with the generalized, gender-independent organomegaly reported for
the p27kip1(-/-) mouse. Finally, blood insulin levels in fasting or
fed anx7 (+/-) mice were not profoundly different from levels in
control animals, indicating that hyperinsulinism is not a viable
explanation either. Together, these data thus further validate the
conclusion that the documented growth anomalies in the anx7 (+/-)
mouse are probably not related to pituitary hyperfunction. The fact
that unique growth anomalies in the anx7 (+/-) mouse are
gender-specific constitute a further distinct internal genetic
control for the anx7 (+/-) mouse mutation.
[0231] As further shown in FIG. 8, the increased weight of the anx7
male (+/-) mice was found to occur coincidentally with enlargement
of many internal organs. Furthermore, in this separate study, many
of the major organs appear to be disproportionately larger than the
ca. 25% increment in whole body weights. The most evident example
of this situation is the heart, which is nearly 80% heavier than
hearts from normal male littermate controls. The brain is 8%
heavier on average than brains in normal controls, but the
difference is not statistically significant. Grossly, the male
mutants were just large, not fat.
[0232] Finally, in an effort to determine whether sexually
dimorphic anomalies of growth hormone or other hormones of
pituitary origin might explain the male gigantism, we examined the
concentrations of IGF-1 and corticosterone in plasma from wildtype
and heterozygous animals. However, neither IGF-1 nor corticosterone
levels varied between wildtype or mutant when comparing like
genders. In addition, the pituitaries of males and females, mutant
and normal littermate controls were not found to be histologically
different when comparing like genders (n=6, each; data not
shown).
EXAMPLE II
Spontaneous Tumors in anx7 (+/-) Mice
[0233] A total of 50 heterozygous animals, aged 100-200 days, were
subjected to a complete post mortem examination, and 10 proved to
have histologically verifiable, macroscopic tumors. These tumors
occurred in both male and female animals. The tumors found
principally included lymphosarcoma of the thymus, insulinoma, and
hepatocellular carcinoma. No instance of defined tumors was
detected in the control animals.
[0234] In a second set of 50 heterozygotes, aged ca. 1 year old, a
vastly increased tumor incidence of ca. 50% was detected. In the
older animals the principal tumors were also lymphosarcoma of the
thymus and hepatocellular carcinoma. In some instances more than
one type of tumor was detected in the same animal. In addition,
there were several instances of dysplastic thymic organization in
otherwise "normal" mutant animals.
[0235] Lymphosarcoma of the thymus is a frequently occurring tumor
in these mutants. One particularly interestingly example of this
tumor is shown in FIG. 9. This tumor was found as an
unencapsulated, 1-cm.sup.3 tan fleshy mass occupying the anterior
thoracic cavity, which surrounded the heart and compressed the
lungs. The tumor mass is composed of sheets of monomorphic cells
supported by a fine fibrovascular stroma. As shown in FIG. 9B, the
cells are small, round, and non-adherent, with well-demarcated
borders, scant lightly basophilic cytoplasm, single central round
deeply basophilic nuclei, and, generally, a single central
prominent nucleolus. There is a moderate mitotic rate, averaging
1/high powered field, and there are numerous large `tingible body`
macrophages which are scattered among the neoplastic cells. At the
organismic level the neoplastic cells were found to infiltrate and
expand the mediastinum, and to extend into the lung along branches
of the pulmonary artery (see FIG. 10B). The tumor effaced the
bronchial lymph nodes and was also seen to disseminate to the
kidneys (not shown).
[0236] Although less frequently found than the lymphosarcoma, the
hepatocellular carcinomas are remarkable by their size. The
hepatocellular carcinoma shown in FIG. 11 is an unencapsulated mass
(1.times.0.5.times.0.5 cm) composed of large polygonal cells
arranged in cords and trabeculae. The mitotic rate is less than
1/10 high power fields. Cells have discrete cytoplasmic borders,
abundant granular to finely vacuolated eosinophilic cytoplasm and a
large centralized round vesicular nucleus. In most cells there is a
single prominent magenta nucleolus, although occasional nuclei
contain multiple nucleoli. Neoplastic cells are observed to
infiltrate adjacent hepatic parenchyma.
[0237] (1) Example of Lymphosarcoma of the Thymus:
[0238] A section from a lymphosarcoma of the thymus, taken at 50-X
magnification, is shown in FIG. 9B, with a sample of normal thymus
shown in FIG. 9A for comparison. The board certified veterinary
pathologist's description is as follows: [0239] Description of
thymic mass in mouse, MS9801634: There is a 1 cm.sup.3 tan fleshy
mass occupying the anterior thoracic cavity, surrounding the heart
and compressing the lungs. The mass is composed of sheets of
monomorphic cells supported by a fine fibrovascular stroma. The
cells are small, round, and non-adherent, with well demarcated
borders, scant lightly basophilic cytoplasm, single central round
deeply basophilic nuclei and generally a single central prominent
nucleolus. There is a moderate mitotic rate, averaging 1/high
powered field. Numerous large `tingible body` macrophages are
scattered among the neoplastic cells. The mass is unencapsulated.
Neoplastic cells infiltrate and expand the mediastinum, extend into
the lung along branches of the pulmonary artery, efface the
bronchial lymph nodes, and disseminate to the kidneys. Cell
morphology is consistent with lymphosarcoma.
[0240] A section is shown of tumor cell infiltration into the lung
in FIG. 10B, in which extensions along branches of the pulmonary
artery are prominent. For comparison, control lung from an anx7
(+/+) mouse is shown in FIG. 10A. In many other examples of
lymphosarcoma of the thymus, metastases to the pancreas have been
frequently noted.
[0241] (2) Example of Hepatocellular Carcinoma:
[0242] A section from a hepatocellular carcinoma, taken at 100-X
magnification, is shown in FIG. 11B. For comparison a sample of
normal liver from an anx7 (+/+) mouse shown in FIG. 11A. The board
certified veterinary pathologist's description is as follows.
[0243] Description of mass (1.times.0.5.times.0.5 cm) from (region
around liver of) mouse MS9901058: . . . is composed of large
polygonal cells arranged in cords and trabeculae. Cells have
discrete cytoplasmic borders, abundant granular to finely
vacuolated eosinophilic cytoplasm and a large centralized round
vesicular nucleus. In most (cells) there is a single prominent
magenta nucleolus; occasional nuclei contain multiple nucleoli. The
mass is unencapsulated and neoplastic cells infiltrate adjacent
hepatic parenchyma. The mitotic rate is less than 1/10 high power
fields.
[0244] Cell morphologies are consistent with lymphosarcoma of the
thymus and hepatocellular carcinoma, respectively. Since other
types of tumors have also been detected, albeit with lesser
frequencies, it would appear that the anx7 (+/-) phenotype is not
expressed as an obvious preference for one tumor type to the
exclusion of others. The wild type human anx7 gene suppresses
growth of a variety of human tumor cell lines.
EXAMPLE III
[0245] Determination of Levels of ANX7 Protein in Tissues from anx7
(+/-) Mouse.
[0246] As noted above, certain organs (e.g., heart and pancreatic
islets) in the anx7 (+/-) mouse exhibit organomegaly. Tissues from
anx7 (+/-) and control mice were harvested, frozen on dry ice, and
then homogenized in boiling SDS buffer, and then assessed for ANX7
protein. Aliquots containing identical amounts of protein were
separated by SDS-PAGE, transblotted to nitrocellulose, and ANX7
visualized using rabbit anti-ANX7 primary antibody, HRP-conjugated
secondary antibody, and ECL detection on X-Ray film.
[0247] As shown in FIG. 13, ANX7 levels in the pancreas of male
anx7 (+/-) mice contain 20-30% of the ANX7 levels in pancreatic
tissue from control animals. Heart tissue was also run in parallel,
with similar results. ANX7 from heart has a tissue-specific
cassette exon edited into the higher molecular weight edited
product.
[0248] ANX7 levels appear to be much lower in mutant than in
control pancreas, heart, and other tissues. Tissue specific editing
processes do not appear to influence the lower expression levels in
mutant mice. Thus the remaining intact copy of the anx7 gene in the
anx7 (+/-) mouse appears to be unable to compensate for the loss of
function of the knocked out allele.
EXAMPLE IV
Production of Recombinant Adenovirus Expressing Wild Type and
Mutant anx7 for Gene Therapy.
[0249] Compared with the chemically based gene transfer systems,
the adenovirus system is more efficient and quantitative for
introducing specific genes into cells. Adenovirus recombinants of
anx7 sense, anti-sense, and mutations are constructed by
cotransfection into human embryonic kidney cells (HEK293) with a
replication-deficient adenovirus vector, QBI-Ad5 (Quantum
Biotechnologies, Inc., Laval, Quebec, Canada). In the HEK293 cells
recombinant adenoviral vectors containing the anx7 cDNA sequence
are formed by homologous recombination. HEK293 cell lysates from
approximately 20 plaques per construct are analyzed for recombinant
virus by PCR, using primers from the anx7 cDNA. Cell lysates from
plaques that are positive by PCR analysis are then further
characterized for expression of ANX7 protein by Western blot
analysis. Plaques that express ANX7 robustly are further
plaque-purified and isolated in higher titer from HEK293 cells for
further experiments.
[0250] Wild type and mutant anx7 genes are engineered into
replication-deficient adenoviral vectors, and adenoviral particles
prepared, purified, titered, and systematically tested by
administration to HEK293 cells. A variety of wild type and mutant
anx7 genes have been prepared in the adenovirus vector, and many
have been expressed as recombinant adenoviral particles. The anx7
mutations, not shown here, include 16 combinations of the mutated
calcium binding sites in the four repeats; two site directed
mutations against protein kinase C sites; five mutations directed
against GTP binding sites; and an antisense anx7 construct.
EXAMPLE V
Production of Dominant Negative Mutants of ANX7
[0251] Dominant negative mutants of tumor suppressor genes have
been useful for investigating the mechanism of action of tumor
suppressor genes. An alternative approach to study the role of the
tumor suppressing anx7 gene is to use mutated anx7 constructs with
dominant negative activity to suppress the function of endogenous
anx7 gene. "Dominant negative" genes encode abnormal proteins that
repress the function of their normal counterparts in a dominant
manner. Thus, one way to examine the role of anx7 is to utilize
"dominant negative" mutant constructs that would suppress normal
ANX7 function in wild type cells and then determine if the
expression of these constructs would alter the growth and
differentiation, especially under Ca.sup.2+ limiting
conditions.
[0252] In order to construct these dominant negative mutants,
mutations at some or all of the four Ca.sup.2+ binding sites on
ANX7 were chosen as the sites of mutational events. Using standard
techniques, site directed mutations were introduced into the
calcium binding sites in combinations of all four
crystallographically defined endonexin fold motifs. All four have
the consensus sequence [GXGTDE] and the mutations were engineered
to generate the amidated analogues of the charged residues (viz
[GXGTNQ]). Thus, 16 different combinations were prepared, including
the wild type ANX7. The combinations were single mutations (e.g.,
1, 2, 3 & 4); mutations at two sites (e.g., 1 & 2, 1 &
3, etc.) mutations at three sites (e.g., 1 &2&3,
2&3&4, etc.) and all four sites (e.g., 1
&2&3&4).
[0253] All the mutants were prepared and tested in the
phosphatidylserine liposome fusion assay (Couhay, et al., Proc.
Nat. Acad. Sci. (USA) 93:10797-10802 (1996)). Some were as active
as the wild type, while others were much less active. As shown in
FIG. 14, one mutant, ANX7J, was both intrinsically inactive, and
profoundly inhibitory when mixed with equi-molar amounts of wild
type ANX7 (viz., 1 .mu.g each of ANX7 proteins). Thus, ANX7J
behaves as a dominant negative mutant in the in vitro test.
EXAMPLE VI
Human ANX7 as a Target for Protein Kinases, In Vitro and In
Vivo
[0254] Threonine/serine protein kinases such as Protein Kinase C
(PKC) and tyrosine kinases are known to phosphorylate tumor
suppressor genes such as p53 or BRCA1, respectively. For in vitro
tests, purified recombinant ANX7 is mixed with purified protein
kinases, and assays performed. For in vivo tests, agonists for
specific receptors are mixed with cells, and endogenous ANX7
labeling is detected.
[0255] A series of purified protein kinases were tested for in
vitro activity on recombinant ANX7. These included protein kinase C
(PKC), cAMP-dependent protein Kinase (PKA), cGMP-dependent protein
kinase (PKG), Casein kinase I and casein Kinase II,
Ca.sup.2+/calmodulin Kinase II, and the tyrosine kinases,
p60.sup.src and epidermal growth factor receptor kinase
(EGFR-kinase). The assays determined the molar ratio of
.sup.32[P]/ANX7 protein after a 30-minute incubation under the best
experimental conditions. As partially summarized in FIG. 15, of the
enzymes tested, only five were active. These were PKC (molar
ratio=2.0), PKG (molar ratio=1.0), PKA (molar ratio=1.0),
p60.sup.src (molar ratio=1.0), and EGFR kinase (molar ratio not
determined). ANX7 activity in a Ca.sup.2+-dependent membrane fusion
assay was vastly potentiated by PKC treatment of the ANX7. One of
two candidate PKC sites on ANX7 were selected and mutated from
serine (S) to alanine (A), with substantial loss of activity. By
contrast, no activity was detected when recombinant ANX7 was
exposed to casein kinase I, casein kinase II, or
Ca.sup.2+/calmodulin kinase II.
[0256] To test for PKC phosphorylation under in vivo conditions,
chromaffin cells were equilibrated with .sup.32[p] to label
endogenous ATP, and exposed to phorbol-12-myristate-13-acetate
(PMA) to activate PKC. Substantial .sup.32[P]-labeled endogenous
ANX7 was detected by immunoprecipitation, which was blocked by
specific PKC inhibitors.
[0257] In addition, at the in vivo cellular level, we also asked
whether endogenous ANX7 might be labeled when human adenocarcinoma
A431 cells were exposed to either epidermal growth factor (EGF) or
platelet derived growth factor (PDGF). In both cases substantial
levels of .sup.32[P]-labeled endogenous ANX7 were detected by
immunoprecipitation.
[0258] Thus, ANX7 can be labeled by a broad spectrum of protein
kinases, both in vitro and in vivo. The exceptions of casein
kinases I and II serve to distinguish ANX7 from p53 types of target
molecules.
EXAMPLE VII
GTP-binding Site Mutations in Human anx7
[0259] ANX7 is a Ca.sup.2+-activated GTPase, which contains the
five putative RAS-type canonical GTP binding sites. Since it was
not known prior to these experiments which mutations in these
GTPase domains might be important for ANX7 activity, mutant ANX7's
containing discrete site-directed RAS-like mutations were
constructed and expressed. These mutations were G-2 (QinT); G-4
(NRsN); and G-5 (EiSG). Binding of 8-azido-GTP could then be used
to assess GTP binding.
[0260] The wild type and mutant ANX7 proteins were expressed in the
pTrc99A expression system in E. coli, and purified to ca. 90% by
differential ammonium sulfate precipitation and column
chromatography on Ultragel AcA54 (see Cauhuy et al, 1996 for more
details). Specific ANX7 protein content of the 47 KDa or the 51 KDa
bands were estimated by using the .sup.125[I] anti-mouse IgG
secondary antibody to label transblotted samples on nitrocellulose
that had been bound by primary monoclonal antibody 10E7. ANX7 and
ANX7 mutants were photolabeled by 8-N.sub.3-.sup.32[P]-GTP in the
presence of 2 mM glutathione to block non-specific binding.
[0261] Western blots and protein blots showed that substantial
amounts of mutant proteins could be prepared. The Phospholmager
data reveal that the LI mutation entirely blocks GTP binding, while
NI, TA1 and FL mutants are approximately 60% active. By contrast,
the TA2 mutation is approximately 50% activated. "FLS" represents
recombinant full-length anx7, or ANX7. The TA1 and TA2 mutations
are in a higher molecular weight. ANX7 isoform containing the
cassette exon #6, and for that reason run slower on the SDS gel. In
RAS, the equivalent LI mutation prevents GTP from binding, just as
it does in ANX7.
[0262] These data serve to validate the structural basis of the
intrinsic GTPase activity of ANX7.
EXAMPLE VIII
Culture and Assay of Tumor Suppressor Gene Activity in Tumor Cell
Lines: Suppression of Human Tumor Cell Proliferation by Human anx7
Gene
[0263] Tumor cell lines can be grown in vitro, and this growth is
suppressed when wild type tumor suppressor genes are transfected
into the tumor cells (e.g., Greenblatt, M. S., et al., Mutations in
the p53 Tumor Suppressor Gene: Clues to Cancer Etiology and
Molecular Pathogenesis, Cancer Res., 54:4855-4878 (1994)). For
example, certain human prostate tumor cell lines can be suppressed
when a mutated Rb gene is supplanted by a wild type Rb gene (Huang,
H. J-S., et al., Science 242:1563-1566, (1988); Bookstein, R., et
al., Science 247:712-715, (1990)). Equivalent results have been
reported for a human bladder carcinoma cell line (Takahashi, R., et
al., Proc. Nat. Acad. Sci. (USA) 88:5257-5261 (1991)). Similar
reports have also been made for the p53 gene (e.g., Eliyahu, D., et
al., Proc. Nat. Acad. Sci. (USA), 86:8763-8767 (1989); Finlay, C.
A., et al., Cell 57:1083-1093 (1989); Isaacs, W. B., et al., Cancer
Res., 51:4716-4720 (1991)). Specific examples include suppression
of growth of human colorectal cancer cells (Baker, S. J., et al.,
Science 249:912-915 (1990)) and human prostate cancer cells lines
such as LNCaP and DU145 (Srivastava, S., et al., Nature 348:747-749
(1998)). Although many susceptible tumor cells contain p53
mutations, it is even possible to suppress the growth of cancer
cells with transfected p53 which contain endogenous wild type p53
(Clayman, G. L., et al., Cancer Res. 55:1-6 (1995); Katayose, D.,
et al., Clin. Cancer Res. 1:889-897 (1995)). With the transfection
paradigm, it is possible to raise wild type p53 levels as high as
100-fold over control expression levels. [0264] Cells were obtained
from the ATCC and handled as follows: [0265] Prostate cancer cell
line DU145 was cultured in Eagle's Minimum Essential Medium with 2
mM L-glutamine and Earle's BSS, adjusted to contain 1.5 g/L sodium
bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium
pyruvate (Sigma Chemical Co., St. Louis, Mo.), and 10% fetal bovine
serum (Intergen Co., Purchase, N.Y.). [0266] Prostate cancer cell
line LNCaP was cultured in RPMI 1640 medium with 2 mM L-glutamine
adjusted to contain 1.5% sodium bicarbonate, 4.5 g/L glucose, 10 mM
HEPES, 1.0 mM pyruvate (Sigma Chemical Co.), and 10% fetal bovine
serum (Intergen Co., Purchase, N.Y.). [0267] Osteosarcoma cell line
Saos-2 was cultured in McCoy's 5a medium with 1.5 mM L-glutamine
(Sigma Chemical Co.), and 15% fetal bovine serum (Intergen Co.,
Purchase, N.Y.). [0268] Breast cancer cell line MCF7 was cultured
in Eagle's Minimum Essential Medium with 2 mM L-glutamine and
Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM
non-essential amino acids, 1.0 mM sodium pyruvate (Sigma Chemical
Co., St. Louis, Mo.), 0.01 mg/ml bovine insulin (Life Technologies
Inc.), and 10% fetal bovine serum (Intergen Co., Purchase,
N.Y.).
[0269] Cells were plated in 6-well plates (35 mm wells) and grown
in appropriate media to approximately 70% confluency for
transfection in media appropriate to the cell type. Transfection
parameters were initially optimized using a plasmid expressing
.beta.-galactosidase. These studies suggested that 2-4 ug plasmid
DNA and 6 ul lipofectamine would produce maximum transfection
efficiency. Cells were therefore transfected for 5 hrs with various
amounts (1-6 ug) of several plasmids (pcDNA3.1 alone obtained from
or containing/expressing cDNA encoding human anx7, p53 or NMDA
receptor subunit 2C) and lipofectamine (6 ul; Life Technologies
Inc., Grand Island, N.Y.) in reduced serum medium (Optimem 1, Life
Technologies Inc.) essentially as recommended by the supplier.
[0270] Approximately 36 hrs later, selection with G418 (Geneticin,
Life Technologies Inc.) at 800 ug per ml medium was initiated.
Cells were then maintained with medium changes every 3-4 days,
always containing G418. After approximately 1 week of G418
selection, most non-transfected cells had died. After approximately
2 weeks of selection, when many macroscopic colonies could be seen
in wells transfected with pcDNA3, the cells were rinsed with
phosphate-buffered saline (PBS), fixed with 2% formaldehyde in PBS
for 15 min, stained with 0.5% crystal violet in PBS for 15 min and
rinsed 1-2 times with distilled H.sub.2O, dried and stored for
subsequent quantification of colonies. Colonies visible in each
well without magnification were counted and average values
(mean+standard error of the mean) were determined for wells
transfected with each concentration of each plasmid.
[0271] As shown in FIG. 12, all four tumor cell lines are
suppressed in a DNA-dose dependent manner by both anx7 and p53, but
not by the vector controls. Two prostate tumor cell lines, DU145
(FIG. 12A) and LNCaP (FIG. 12B), a breast cancer cell line MCF-7
(FIG. 12C), and an osteosarcoma cell line (Saos-2) (FIG. 12D), were
transfected with the vector alone or the vector expressing anx7
(+anx7) or a vector expressing p53 (+p53).
EXAMPLE IX
State of anx7 mRNA and ANX 7 Protein During Cell Cycle in Human
Fibroblasts
[0272] Tumor suppressor genes are principally known for control of
cell proliferation by their action on different aspects of the cell
cycle. To determine whether anx7 plays a role in this process, it
is crucial to know the state of anx7 mRNA and ANX7 protein as a
function of position in the cell cycle of untransformed cells. The
state of anx7 mRNA and ANX7 protein in human IMR-90 fibroblasts can
be studied by the serum-deprivation/addition method to synchronize
the cell cycles, and changes occurring during this period can then
be studied.
[0273] (1) Cells, Cell Cycle Assays, and Immunodetection:
[0274] IMR-90 cells, obtained from the ATCC, were cultured and
synchronized by serum deprivation to arrest the cells in G.sub.o,
and then activation by serum addition, as described by Raynal et
al. (1997) (See above). Progression through the cell cycle was
followed by .sup.3[H]-thymidine incorporation (5 .mu.Ci/ml) over a
4 hour period and by monoclonal immunodetection of cdc-2 (Zymed).
ANX7 was detected with a polyclonal rabbit anti-ANX7 antibody
against a conserved internal ANX7 peptide, (RDLEKDI RSDTSG).
Detection was on the basis of .sup.125[I]-second antibody and
Molecular Dynamics Phospholmager quantification. Our own
recombinant ANX7 was used to standardize the assays.
[0275] (2) Analysis of mRNA:
[0276] For construction of an anx7 RNAse protection assay,
templates were constructed using a PCR amplification kit
(Perkin-Elmer Cetus, Norwalk, Conn.), with a primer set as
described by Raynal et al (1997). RNAse protection assays were
analyzed by separating the products by denaturing PAGE and
autoradiography.
[0277] Confluent human IMR-90 fibroblasts are incubated for 72
hours in a serum-free medium, and 10% serum is added at time zero
to activate the cell cycle. As shown in the Raynal, et al (Biochem.
J. 322:365-371 (1997), incorporated by reference), cdc2 (a.k.a.,
cdk2) synthesis is followed by immunoblotting to mark the
G2/M-phase, while DNA synthesis to mark the S-phase is followed by
incorporation of .sup.3[H]-thymidine. The relative expression of
ANX7 over the cycle period is determined by Western blot analysis.
Other annexins serve as controls for the experiment of Raynal et
al.
[0278] There is a small but significant reduction of ANX7 protein
levels at the transition between S and G2/M. However, anx7 mRNA
levels do not vary appreciably over the entire cell cycle.
EXAMPLE X
Methods for Determining Polymorphism or Mutation in the anx7
Gene
[0279] (1) Analysis of RNA Transcripts:
[0280] Matched tumor and adjacent normal tissues from mice and
human are obtained and immediately embedded in OCT (Miles Inc.
Diagnostics Division Elkhart, Ind.) and frozen at -70.degree. C.
With LCM (laser-gene capture microdissection) of tumor and normal
cells are obtained from a heterozygous mouse and human specimens.
Using a cryotome, 1.0-micron sections are cut from frozen tissues
and stained by hematoxylin (H) and eosin (E). The H & E slides
are read by the pathologist to ensure the presence of >70% tumor
cells. The neoplastic area are outlined on each slide. The
unstained frozen sections on the slides are stored at -70.degree.
C. until DNA was extracted. The H&E stained slides are used as
a template and corresponding frozen sections will be superimposed
on it. The normal and tumor cells dissected by LCM are used for RNA
extraction and purification by Ransom B reagent (Tel-Test, Inc.,
Friendwood, Tex.). Tumor and normal tissue RNAs are
reverse-transcribed using random hexamers and Superscript (Life
Technology, Gaithersburg, Md.). Five cDNA fragments representing
the complete anx7 protein coding sequence are amplified using pfu
DNA polymerase. Since pfu DNA polymerase has proof-reading
activity, it is less error prone as compared to Taq DNA polymerase.
The PCR fragments are subjected to "Cold SSCP" with temperature
optimized for each fragment. Aberrant bands from SSCP gels are
reamplified and sequenced.
[0281] (2) Analysis of Allelic Loss at the anx7 Gene Locus in the
Tumor Samples:
[0282] Defined areas of the tumor cells are scraped with a fresh
razor blade, taking care not to scrape adjacent normal tissues as
described above. The scraped tissue are digested with proteinase K,
extracted with phenol/chloroform, followed by ethanol
precipitation. The integrity and concentration of genomic DNA from
frozen tissue is determined on agarose gels. The matching normal
DNA is extracted from
[0283] histologically normal tissue sections that did not contain
tumor cells. To detect polymorphism and deletions in the anx7
locus, Southern blot analysis is carried out using high molecular
weight. DNA digested with restriction enzymes. The bands are
fractionated by electrophoresis on 0.6% agarose gel and transferred
onto nylon membranes. The nylon membranes are hybridized with nick
translated cDNA anx7 probe. The polymorphic changes in the disease
samples are then analyzed.
[0284] (3) PCR and Polymorphism Analysis:
[0285] Primers flanking the dinucleotide repeat sequences have been
identified in the anx7 gene (Shirvan et al., 1994). The PCR is
performed on the genomic DNA samples using the following
conditions: 5 nanograms (ng) of DNA template, 50 ng of each primer,
0.5 unit of AmpliTaq Gold (Perkin Elmer Emeryville, Calif.), Ix PCR
buffer, 200 .mu.l dNTP mix in a 50 .mu.l final volume. PCR
conditions are identical for all primers used. PCR cycles includes
1 cycle of 95.degree. C. for 10 min. followed by 25 cycles of
95.degree. C. for 30 sec., 55.degree. C. for 45 sec. and 72.degree.
C. for 1 min. One of the primers is end labeled using .sup.32P-ATP
and T4 polynucleotide kinase kit (Life Technology, Gaithersburg,
Md.). Each locus exhibiting allelic loss/gain is co-amplified with
.beta.-actin to ascertain that we have used similar amounts of the
input DNA in PCR reactions. Human placental DNA is used as a
positive control for PCR reactions. For samples which will be
analyzed by radioactive methods, PCR products are subjected to
electrophoresis on a 7% acrylamide/urea/formamide gel. The gel is
dried and processed for autoradiography.
[0286] (4) Mutational Analysis of anx7 by "Cold SSCP"/DNA
Sequencing:
[0287] PCR products representing anx7 cDNA are denatured with
methyl mercury hydroxide and electrophoresed through pre-made 20%
polyacrylamide minigels (Novex) at a high voltage and a constant
temperature. Temperature of the buffer and gel is accurately
maintained by constant temperature water circulation through a
specially designed cooling system. It is also important to point
out that SSCP is more sensitive than direct DNA sequencing in
detecting mutant anx7 alleles in the presence of wt anx7 sequences.
These wt anx7 sequences are unavoidably present due to the presence
of normal cells in tumor tissue architecture, or due to tumor cell
heterogeneity where all the cells in tumor tissue may not contain
anx7 mutations. It is difficult to make a call for mutation by
direct DNA sequence analysis of PCR products containing 30% or less
of the mutant allele in the presence of wt sequence. However, due
to mobility shifts of the mutant alleles in SSCP, .about.10% mutant
alleles are confidently detected in the large excess of the wt
allele. Once the mutant conformation is identified it can be
isolated from the SSCP gel and selectively amplified for DNA
sequencing. These issues are especially relevant for tumor DNAs
from primary samples of cancer, where tumor heterogeneity is well
recognized. Silver staining or SYBR green staining of the gels, are
responsible for the increased sensitivity of detection of SSCP
bands on gel. The aberrant SSCP bands identified by "Cold SSCF"
procedure are cut from the gel and reamplified using the same
primers. PCR products are purified and sequenced using the
(Rhodamine-terminator cycle sequencing kit (PE Applied Biosystem)
following the supplier's recommended methods. DNA sequences are
analyzed on an automated DNA sequencer (310 Genetic Analyzer,
Applied Biosystem).
[0288] (5) Assessment of LOH:
[0289] After PCR, LOH "Loss of Heterozysosity" of tumor samples
will be initially determined visually by comparing the intensity of
bands representing the alleles between normal DNA and tumor DNA. A
decrease of >50% signal intensity in tumor DNA, as compared to
normal, on more than one of the alleles in tumor DNA is scored as
LOH. Quantitation is undertaken by exposing the dried gels to
phosphor storage screens for 4-5 hours and images are collected on
a Molecular Dynamics Phosphorimager and analyzed with Image Quant
software (Sunnyvale, Calif.). Quantitation is done by subtracting
the background and by the volume integration method within
equal-sized rectangular regions that are placed manually over
bands.
EXAMPLE XI
Levels of ANX7 Protein Expression in Human Prostate Tumor Tissue
Micrarrays.
[0290] We determined the level of ANX7 protein expression in a
prostate tissue microarray containing 301 specimens taken from all
stages of human prostate tumor progression. As shown in FIG. 16A,
significant reductions in ANX7 expression were found to occur in a
stage-specific manner. ANX7 expression was completely lost in a
high proportion of metastases (57%) and in local recurrences of
hormonal refractory prostate cancer (63%). By contrast, ANX7 occurs
at close to normal levels in benign prostate glands, high grade
prostatic intraepithelial neoplasms (PIN), and stage T2 and T3/4
primary tumors (all in the range of 89-96%). In FIG. 16B, typical
examples taken from samples used in the human tumor microarray were
stained with H&E (left side) and brown diaminobenzidine (DAB)
using an anti-ANX-7 monoclonal antibody (right side). (BPH--benign
prostatic hypertrophy.) The top three sections are heavily stained,
while the bottom two sections, representing metastatic and locally
recurrent tumors, respectively, are negative. The p value for
stage-specific loss is p=0.0001. This visual comparison reinforces
the statistically significant lack of ANX7 in the two worst
prognostic situations.
EXAMPLE XII
Association Between Ki67 (Growth Fraction) and ANX7 Expression in
Prostate Cancers
[0291] Using Ki67 immunostaining as an index of tumor cell
proliferation, we found a positive correlation between a high Ki67
labeling index and a lack of ANX7 expression, as well as a
correlation with advanced stage prostate cancer and high Gleason
score. As seen in FIG. 17A, ANX7 positive human prostate cancer
cells have significantly fewer cells with high levels of
immunostaining by Ki67 antibody (red bar), as compared to the
percentage of cells with a low level of immunostaining (purple
bar). In contrast, ANX7 negative human prostate cancer cells have a
higher percentage of cells with high levels of immunostaining by
Ki67 antibody (red bar). These data are based on the analysis of
301 tumors and are statistically significant (p=0.0003).
[0292] FIG. 17B shows example histological images taken from the
samples used in FIG. 17A. The samples in the left column were
stained by Ki67, which is indicative of the proliferative state.
The samples in the right column are consecutive sections stained
for ANX7 protein. Samples exhibiting benign prostatic hypertrophy
(BPH) and primary intraepithelial neoplasms (PIN) were low in Ki67,
but high in ANX7 protein. In contrast, samples exhibiting
metastatic prostate cancer (MET) had high levels of Ki67, but
virtually no ANX7 staining.
EXAMPLE XIII
Levels of ANX7 Protein Expression in Human Breast Cancer Tissue
Micrarrays
[0293] Different normal tissues are characterized by either "high"
or "low" levels of ANX7 gene expression. As seen above, in the case
of prostate cancer, the normal prostate has high levels of ANX7,
while metastatic and hormone insensitive local recurrences have
very low levels of ANX7. By contrast, normal breast tissue has
quite low levels (see FIG. 26), while cancerous forms have
increasingly more ANX7 levels (see FIG. 19). We also show in FIGS.
19-25 that increasing levels of ANX7 protein expression in breast
cancer strongly correlates with lower likelihood of survival.
EXAMPLE XIV
Levels of ANX7 Protein Expression in Other Tumor Types
[0294] We determined the level of ANX7 protein expression in a
variety of other tumor types in which the normal tissue was found
to have a low level of ANX7, as well as tumor types that tend to
have higher levels. Data are given as percent of tumor cells
positive for ANX7 protein. These tumor types include sarcoma, lung
cancer, and testes, and the results are depicted in FIG. 26. Normal
adult lung was found to be virtually deficient in ANX7, while fetal
lung was 25% positive. Carcinoid, small and large cell lung cancers
are profoundly distinct from normal tissue ANX7 levels.
[0295] For the tumor types depicted in FIG. 27, normal tissue gave
high levels of ANX7, but some of the tumors tend to have low
levels. The tissues represented in FIG. 27 include skin, lymphoid
tissue, prostate (see earlier parts of this description for
detailed studies on the prostate), and nerve tissue.
[0296] In FIG. 28, we found that the tumor types had normal levels
of ANX7 protein of ca. 50%. These included salivary gland tumors
(adenocarcinoma is completely positive), renal, gynecological, and
thyroid.
[0297] Brain tissue had generally low levels of ANX7, as did
derived tumors from this tissue. (See FIG. 30).
[0298] GI tumors vary in level of ANX7 (See FIG. 31.) Normal
exocrine pancreas is 100% positive, while normal colon is in the
range of 80%. Note that for the progression of colon adenoma G1
(grade 1), colon adenoma G2 (grade 2), and colon cancer, there is
the appearance of a steady downward projection in ANX7 positive
cells.
[0299] Variation in endocrine tumors is not dramatic. Normal
endocrine tissues tend to be high in ANX7 protein. (See FIG.
32.)
[0300] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification
which are hereby incorporated by reference. The embodiments within
the specification provide an illustration of embodiments of the
invention and should not be construed to limit the scope of the
invention. The skilled artisan readily recognizes that many other
embodiments are encompassed by the invention.
Sequence CWU 1
1
4 1 23 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 cggatcgatc ccctcagaag aac 23 2 6 PRT Artificial Sequence
Description of Artificial Sequence Consensus sequence MOD_RES (2)
Variable amino acid 2 Gly Xaa Gly Thr Asp Glu 1 5 3 6 PRT
Artificial Sequence Description of Artificial Sequence Consensus
sequence MOD_RES (2) Variable amino acid 3 Gly Xaa Gly Thr Asn Gln
1 5 4 13 PRT Homo sapiens 4 Arg Asp Leu Glu Lys Asp Ile Arg Ser Asp
Thr Ser Gly 1 5 10
* * * * *