U.S. patent application number 11/673521 was filed with the patent office on 2007-07-05 for reagents and methods for identifying gene targets for treating cancer.
This patent application is currently assigned to The Board of Trustees of the University of Illinois. Invention is credited to Bey-Dih Chang, Thomas Priminano, Igor B. Roninson.
Application Number | 20070154933 11/673521 |
Document ID | / |
Family ID | 23186592 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070154933 |
Kind Code |
A1 |
Priminano; Thomas ; et
al. |
July 5, 2007 |
Reagents and Methods for Identifying Gene Targets for Treating
Cancer
Abstract
The invention provides methods and reagents for identifying
mammalian genes necessary for tumor cell growth as targets for
developing drugs that inhibit expression of said genes and inhibit
tumor cell growth thereby.
Inventors: |
Priminano; Thomas; (Chicago,
IL) ; Chang; Bey-Dih; (Lombard, IL) ;
Roninson; Igor B.; (Loudonville, NY) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
The Board of Trustees of the
University of Illinois
Urbana
IL
|
Family ID: |
23186592 |
Appl. No.: |
11/673521 |
Filed: |
February 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10199820 |
Jul 19, 2002 |
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11673521 |
Feb 9, 2007 |
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60306730 |
Jul 20, 2001 |
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Current U.S.
Class: |
435/6.14 ;
435/287.2; 977/924 |
Current CPC
Class: |
G01N 33/5023 20130101;
C07K 7/08 20130101; G01N 2800/52 20130101; G01N 33/5011 20130101;
C12Q 2600/158 20130101; A61K 38/00 20130101; C12Q 1/6886 20130101;
G01N 2500/10 20130101; A61P 43/00 20180101; A61P 35/00 20180101;
C07K 14/47 20130101; G01N 33/57415 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 977/924 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Goverment Interests
[0002] This application was supported by a grant from the National
Institutes of Health, No. R01 CA62099. The government may have
certain rights in this invention.
Claims
1. One or a plurality of target genes for identifying compounds
that inhibit tumor cell growth, wherein inhibition of expression of
at least one of said genes or inhibition of protein activity of its
gene product inhibits growth of the tumor cell, wherein said genes
are selected from the group consisting of the genes set forth in
Table 3.
2. A plurality of target genes according to claim 1, comprising
genes encoding L1CAM, ICAM2, Zinedin, or von Willebrand factor.
3. A pattern of gene expression inhibition or inhibition of protein
activity of the gene product of a plurality of said genes as set
forth in Table 3 wherein detecting said pattern in a tumor cell in
response to contacting the cell with the compound is used to
identify said compound as an inhibitor of tumor cell growth.
4. A pattern according to claim 3, wherein the pattern comprises
genes encoding L1CAM, ICAM2, Zinedin, or von Willebrand factor.
5. A panel of oligonucleotides comprising sequences specific for a
plurality of target genes for identifying compounds that inhibit
tumor cell growth according to claim 1, wherein said genes are
selected from the group consisting of the genes set forth in Table
3.
6. The panel of claim 5, wherein said oligonucleotides comprising
sequences specific for the genes of said panel are immobilized on a
solid substrate.
7. The panel of claim 6, wherein said solid substrate is a
microchip or forms a microarray.
8. The panel of claim 5, comprising oligonucleotides specific for
genes encoding L1CAM, ICAM2, Zinedin, or von Willebrand factor.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/199,820 filed Jul. 19, 2002 which claims priority to
U.S. Provisional Application Ser. No. 60/306,730, filed Jul. 20,
2001.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention is related to methods and reagents for
inhibiting tumor cell growth. Specifically, the invention
identifies genes necessary for tumor cell growth as targets for
developing drugs to inhibit such genes and thereby inhibit tumor
growth. The invention provides methods for screening compounds to
identify inhibitors of said genes, and methods for using said
inhibitors to inhibit tumor cell growth. The invention also
provides peptides encoded by genetic suppressor elements of the
invention and mimetics and analogues thereof for inhibiting tumor
cell growth. Also provided by the invention are normalized random
fragment cDNA libraries prepared from tumor cells of one or a
plurality of tumor cell types wherein the cDNA fragments can be
induced by treating recipient cells with a physiologically-neutral
stimulating agent.
[0005] 2. Summary Of The Related Art
[0006] The completion of the draft sequence of the human genome has
provided the art with a partial list of known and putative human
genes, the total number of which is estimated to be between 30,000
and 45,000 (Venter et al., 2001, Science 291: 1304-1351; Lander et
al., 2001, Nature 409: 860-921). These genes provide many potential
targets for drugs, some of which may be useful in preventing the
growth of cancers. However, the development of clinically useful
gene-targeting anticancer drugs could be greatly facilitated by the
ability to narrow down the list of human genes to those that are
involved in the primary feature of cancer, uncontrolled tumor
growth. It would be especially useful to identify genes necessary
for the growth of tumor cells and to determine which of the genes
play a tumor-specific role and are not required for normal cell
growth. These genes are particularly attractive targets for
developing tumor-specific anticancer agents.
[0007] Most of the effort in tumor-specific drug targeting in the
prior art has focused on oncogenes, the function of which has been
associated with different forms of cancer Perkins and Stern (1997,
in CANCER: PRINCIPLES AND PRACTICE OF ONCOLOGY, DeVita et al.,
eds., (Philadelphia: Lippincott-Raven), pp. 79-102). Oncogene
targets have been viewed in the art as being more "tumor-specific"
than "normal" cellular enzymes that are targeted by the drugs used
in present chemotherapeutic regimens. The tumor specificity of
oncogenes has been suggested primarily by the existence of
oncogene-associated genetic changes, such as mutations or
rearrangements, specific to neoplastic cells. Although oncogenes
are mutated or rearranged in some cases, in other cases they are
merely expressed at elevated levels or at inappropriate stages of
the cell cycle, without changes in the structure of the gene
product (Perkins and Stern, 1997, Id.). Even when mutated, proteins
encoded by oncogenes rarely acquire a qualitatively novel function
relative to the "normal" protooncogene products. Hence, products of
mutated, rearranged or overexpressed oncogenes generally perform
the same biochemical functions as their normal cell counterparts,
except that the functions of the activated oncogene products are
abnormally regulated.
[0008] It is noteworthy that none of the "classical" oncogenes
known in the art have been identified as targets for clinically
useful anticancer drugs discovered by traditional
mechanism-independent screening procedures. Rather the known
cellular targets of chemotherapeutic drugs, such as dihydrofolate
reductase (inhibited by methotrexate and other antifolates),
topoisomerase II ("poisoned" by epipodophyllotoxins, anthracyclines
or acridine drugs), or microtubules that form the mitotic spindle
(the targets of Vinca alkaloids and taxanes) are essential for
growth and proliferation of both normal and neoplastic cells. Tumor
selectivity of anticancer drugs appears to be based not merely on
the fact that their targets function primarily in proliferating
cells, but rather on tumor-specific response to the inhibition of
anticancer drug targets. For example, Scolnick and Halazonetis
(2000, Nature 406 430-435) disclosed that a high fraction of tumor
cell lines are deficient in a gene termed CHFR. In the presence of
antimicrotubular drugs, CHFR appears to arrest the cell cycle in
prophase. CHFR-deficient tumor cells, however, proceed into
drug-impacted abnormal metaphase (Scolnick and Halazonetis, 2000,
Id.), where they die through mitotic catastrophe or apoptosis
(Torres and Horwitz, 1998, Cancer Res. 58: 3620-3626). In addition
to CHFR, tumor cells are frequently deficient in various cell cycle
checkpoint controls, and exploiting these deficiencies is a major
direction in experimental therapeutics (O'Connor, 1997, Cancer
Surv. 29: 151-182; Pihan and Doxsey, 1999, Semin. Cancer Biol. 9:
289-302). In most cases, however, the reasons that inhibition of
anticancer drug targets selectively induces cell death or permanent
growth arrest in tumor cells are unknown. There is therefore need
in the art to identify additional molecular targets in tumor cells,
inhibition of which would arrest tumor cell growth.
[0009] One method known in the art for identifying unknown genes or
unknown functions of known genes is genetic suppressor element
technology, developed by some of the present inventors (in U.S.
Pat. Nos. 5,217,889, 5,665,550, 5,753,432, 5,811,234, 5,866,328,
5,942,389, 6,043,340, 6,060,134, 6,083,745, 6,083,746, 6,197,521,
6,268,134, 6,281,011 and 6,326,488, each of which is incorporated
by reference in its entirety). Genetic suppressor elements (GSEs)
are biologically active cDNA fragments that interfere with the
function of the gene from which they are derived. GSEs may encode
antisense RNA molecules that inhibit gene expression or peptides
corresponding to functional protein domains, which interfere with
protein function as dominant inhibitors. The general strategy for
the isolation of biologically active GSEs involves the preparation
of an expression library containing randomly fragmented DNA of the
target gene or genes. This library is then introduced into
recipient cells, followed by selection for the desired phenotype
and recovery of biologically active GSEs from the selected cells.
By using a single cDNA as the starting material for GSE selection,
one can generate specific inhibitors of the target gene and map
functional domains in the target protein. By using a mixture of
multiple genes or the entire genome as the starting material, GSE
selection allows one to identify genes responsible for a specific
cellular function, since such genes will give rise to GSEs
inhibiting this function. In a variation of this approach, the
vector used for library preparation contains sequences permitting
regulated expression of cDNA fragments cloned therein.
[0010] This method can be used to identify genes required for tumor
cell growth by subjecting the cells to negative growth selection.
One example of this type of selection is known in the art as
bromodeoxyuridine (BrdU) suicide selection, which has long been
used to select conditional-lethal mutants (Stetten et al., 1977,
Exp. Cell Res. 108: 447-452) and growth-inhibitory DNA sequences
(Padmanabhan et al., 1987, Mol. Cell. Biol. 7: 1894-1899). The
basis of BrdU suicide selection is the destruction of cells that
replicate their DNA in the presence of BrdU. BrdU is a photoactive
nucleotide that incorporates into DNA and causes lethal DNA
crosslinking upon illumination with white light in the presence of
Hoechst 33342. The only cells that survive this selection are cells
that do not replicate their DNA while BrdU is present, such as
cells that express growth-inhibitory genes or GSEs. One advantage
of this method is very low background of surviving cells. When used
with GSE libraries under the control of an inducible vector, this
selection method excludes spontaneously arising BrdU-resistant
mutants by the insensitivity of their phenotype to the presence or
absence of the inducing agent. Another major advantage of this
technique is its sensitivity for weak growth-inhibitory GSEs: even
if only a small fraction of GSE-containing cells are
growth-inhibited by GSE induction, such cells will survive BrdU
suicide and will give rise to a recovering clone.
[0011] The applicability of this approach to the isolation of
growth-inhibitory GSEs was first demonstrated by Pestov and Lau
(1994, Proc. Natl. Acad. Sci. USA 91: 12549-12553). These workers
used an IPTG-inducible plasmid expression vector to isolate
cytostatic GSEs from a mixture of cDNA fragments from 19 murine
genes associated with the G.sub.0/G.sub.1 transition. In this work,
three of the genes in the mixture gave rise to growth-inhibitory
GSEs (Pestov and Lau, 1994, Id.). In a subsequent study, Pestov et
al. (1998, Oncogene 17: 13187-3197) used the same approach to
isolate one full-length and one truncated cDNA clone with
growth-inhibitory activity from a 40,000-clone library of nominally
full-length mouse cDNA. However, the method disclosed in the art
cannot be efficiently used for transducing a library of random
fragments representing the total mRNA population from a mammalian
cell such as a tumor cell because the method relies on plasmid
expression vectors for library construction, and only a limited
number of cells can be stably transfected by such libraries.
[0012] There remains a need in the art to discover novel genes and
novel functions of known genes necessary for tumor cell growth,
especially by using methods for identifying genes based on
function. There is also a need in the art to identify targets for
therapeutic drug treatment, particularly targets for inhibiting
tumor cell growth, and to develop compounds that inhibit the
identified targets and thereby inhibit tumor cell growth.
SUMMARY OF THE INVENTION
[0013] The present invention identifies genes that are targets for
developing drugs for the treatment of cancer by inhibiting tumor
cell growth. Such genes are identified as disclosed herein through
expression selection of genetic suppressor elements (GSEs) that
inhibit the growth of tumor cells in vitro. This selection has
revealed multiple genes, some of which have been previously known
to play a role in cell proliferation, whereas others were not known
to be involved in cell proliferation prior to instant invention;
the latter genes constitute novel drug targets and are set forth in
Table 3.
[0014] In a first embodiment, the invention provides a method
identifying a compound that inhibits growth of a mammalian cell,
the method comprising the steps of: [0015] (a) culturing a cell in
the presence or absence of the compound; [0016] (b) assaying the
cell for expression or activity in the sample of one or a plurality
of the genes set forth in Table 3; and [0017] (c) identifying the
compound when expression or activity in the sample of at least one
of the genes set forth in Table 3 is lower in the presence of the
compound than in the absence of the compound.
[0018] In preferred embodiments, the cell is a mammalian cell,
preferably a human cell, and most preferably a human tumor cell. In
further preferred embodiments, gene inhibition is detected by
hybridization with a nucleic acid complementary to the gene,
biochemical assay for an activity of the gene or immunological
reaction with an antibody specific for an antigen comprising the
gene product. In a preferred embodiment, the cell is a recombinant
cell in which a reporter gene is operably linked to a promoter from
a cellular gene in Table 3, to detect decreased expression of the
reporter gene in the presence of the compound than in the absence
of the compound. In further preferred embodiments, the cell is
assayed for cell growth in the presence and absence of the
compound, to identify compounds that inhibit cell growth and a gene
identified in Table 3.
[0019] The invention also provides compounds that inhibit tumor
cell growth that are identified by the methods of the invention,
and pharmaceutical formulations of said compounds. The invention
specifically provides peptides encoded by sense-oriented genetic
suppressor elements of the invention. In addition the invention
provides peptide mimetics comprising all or a portion of any of
said peptides, peptido-, organo- or chemical mimetics thereof.
[0020] In a second embodiment, the invention provides a method for
assessing efficacy of a treatment of a disease or condition
relating to abnormal cell proliferation or tumor cell growth,
comprising the steps of: [0021] (a) obtaining a biological sample
comprising cells from an animal having a disease or condition
relating to abnormal cell proliferation or tumor cell growth before
treatment and after treatment with a compound that inhibits
expression or activity of a gene identified in Table 3; [0022] (b)
comparing expression or activity of at least one gene in Table 3
after treatment with the compound with expression or activity of
said genes before treatment with the compound; and [0023] (c)
determining that said treatment with the compound has efficacy for
treating the disease or condition relating to abnormal cell
proliferation or neoplastic cell growth if expression or activity
of at least one gene in Table 3 is lower after treatment than
before treatment.
[0024] In preferred embodiments, the cell is a mammalian, most
preferably human cell, most preferably a tumor cell.
[0025] In a third aspect, the invention provides a method for
inhibiting tumor cell growth, the method comprising the steps of
contacting a tumor cell with an effective amount of a compound that
inhibits expression of a gene in Table 3.
[0026] In a fourth aspect, the invention provides a method for
treating a disease or condition relating to abnormal cell
proliferation or tumor cell growth, the method comprising the steps
of administering to an animal having said disease or condition a
therapeutically effective amount of a compound that inhibits
expression of a gene in Table 3.
[0027] Pharmaceutically acceptable compositions effective according
to the methods of the invention, comprising a therapeutically
effective amount of a peptide or peptide mimetic of the invention
capable of inhibiting tumor cell growth and a pharmaceutically
acceptable carrier or diluent, are also provided.
[0028] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a schematic diagram illustrating the principles of
genetic suppressor element technology.
[0030] FIG. 2 is a schematic diagram of the structure of the
LNCXCO3 retroviral vector.
[0031] FIG. 3 is a schematic diagram of the BrdU selection
protocol.
[0032] FIG. 4 is a photograph of cell culture plates containing
library-transduced cells subjected to BrdU suicide selection in the
presence or in the absence of IPTG, immediately after G418
selection (top), after one round of BrdU suicide selection in the
presence of IPTG (middle), or after two rounds of BrdU suicide
selection in the presence of IPTG (bottom).
[0033] FIG. 5 is a bar diagram of the results of testing of cell
populations transduced with individual GSEs for IPTG-dependent
resistance to BrdU suicide, measured in triplicates and expressed
as mean and standard deviation of the numbers of colonies surviving
BrdU suicide selection in the presence and in the absence of IPTG.
Sequences for the shown results are GSE (SEQ ID NO):GBC-1 (79),
GBC-3 (94), STAT3 (205), STAT5b (211), PRL31 (192), GBC-11 (85),
L1CAM (125), INTB5 (112), OKCeta (170), VWF (225), ZIN (228), HSPCA
(103), CDC20 (37), PKC zeta (172), CDK10 (39), DAP3 (59), RPA3
(190), NF.kappa.B1 (157), HES6 (99), and MBD1 (142).
[0034] FIG. 6 is a bar diagram of the results of IPTG growth
inhibition assays carried out with clonal cell lines transduced
with individual GSEs, measured in triplicates and expressed as mean
and standard deviation of the cell numbers after 7 days of culture
in the presence and in the absence of IPTG. Sequences for the shown
results are GSE (SEQ ID NO): HNRPF (101), HRMT1L2 (102), STAT5b
(211), CCND1 (57), 28S RNA (17), RPL31 (192), CDK2 (40), AHRG
(183), GBC-1 (79), L1CAM (125), NIN283 (158), MYL6 (155), DAP3
(59), TAF7 (215), STAT3 (205), IF1 (32), GBC-11 (85), LYN (138),
c-KIT (48), GBC-3 (94), eIF-3 (62), PKCeta (170), EFNA1 (67), ATF4
(27), HNRPA2B1(102), GBC-12(86), INTB5 (112), BAM22 (35), FOS (43),
FGFR1 (77), and KIAA1270 (123).
[0035] FIGS. 7A and 7B are photomicrographs illustrating the
morphological effects of an L1CAM-derived GSE (SEQ ID NO 134) in a
clonal IPTG-inhibited cell line. FIG. 7A shows the effects on cell
morphology of four-day treatment with IPTG. FIG. 7B shows evidence
of mitotic catastrophe in IPTG-treated cells.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0036] This invention provides target genes involved in cell
growth, preferably tumor cell growth, methods for identifying
compounds that inhibit expression or activity of these genes and
methods for specifically inhibiting tumor cell growth by inhibiting
expression or activity of these genes. Preferably, the methods of
the invention do not substantially affect normal cell growth.
[0037] This invention provides methods for identifying genes that
are required for tumor cell growth. Such genes, which are potential
targets for new anticancer drugs, are identified through expression
selection of genetic suppressor elements (GSEs). GSEs are
biologically active sense- or antisense-oriented cDNA fragments
that inhibit the function of the gene from which they are derived.
Expression of GSEs derived from genes involved in cell
proliferation is expected to inhibit cell growth. According to the
inventive methods, such GSEs are isolated by so-called "suicide
selection" of cells the growth of which is inhibited under cell
culture conditions in which growing cells are specifically killed.
In a preferred embodiment the suicide selection protocol is
bromodeoxyuridine (BrdU) suicide selection, in which cells are
incubated with BrdU and then illuminated with bright light. Growing
cells incorporate BrdU into chromosomal DNA, making the DNA
sensitive to illumination with light, which specifically kills
growing cells. GSEs are produced starting from a normalized
(reduced-redundance) library of human cDNA fragments in an
inducible retroviral vector. In preferred embodiments, the
recipient cells are tumor cells, most preferably human tumor cells,
for example breast carcinoma cells.
[0038] For the purposes of this invention, reference to "a cell" or
"cells" is intended to be equivalent, and particularly encompasses
in vitro cultures of mammalian cells grown and maintained as known
in the art.
[0039] For the purposes of this invention, reference to "cellular
genes" in the plural is intended to encompass a single gene as well
as two or more genes. It will also be understood by those with
skill in the art that effects of modulation of cellular gene
expression, or reporter constructs under the transcriptional
control of promoters derived from cellular genes, can be detected
in a first gene and then the effect replicated by testing a second
or any number of additional genes or reporter gene constructs.
Alternatively, expression of two or more genes or reporter gene
constructs can be assayed simultaneously within the scope of this
invention.
[0040] Recombinant expression constructs can be introduced into
appropriate mammalian cells as understood by those with skill in
the art. Preferred embodiments of said constructs are produced in
transmissible vectors, more preferably viral vectors and most
preferably retrovirus vectors, adenovirus vectors, adeno-associated
virus vectors, and vaccinia virus vectors, as known in the art.
See, generally, MAMMALIAN CELL BIOTECHNOLOGY: A PRACTICAL APPROACH,
(Butler, ed.), Oxford University Press: New York, 1991, pp.
57-84.
[0041] In additionally preferred embodiments, the recombinant cells
of the invention contain a construct encoding an inducible
retroviral vector comprising random cDNA fragments from total tumor
cell mRNA, wherein the fragments are each under the transcriptional
control of an inducible promoter. In more preferred embodiments,
the inducible promoter is responsive to a trans-acting factor whose
effects can be modulated by an inducing agent. The inducing agent
can be any factor that can be manipulated experimentally, including
temperature and most preferably the presence or absence of an
inducing agent. Preferably, the inducing agent is a chemical
compound, most preferably a physiologically-neutral compound that
is specific for the trans-acting factor. In the use of constructs
comprising inducible promoters as disclosed herein, expression of
the random cDNA fragments from the recombinant expression construct
is mediated by contacting the recombinant cell with an inducing
agent that induces transcription from the inducible promoter or by
removing an agent that inhibits transcription from such promoter. A
variety of inducible promoters and cognate trans-acting factors are
known in the prior art, including heat shock promoters than can be
activated by increasing the temperature of the cell culture, and
more preferably promoter/factor pairs such as the tet promoter and
fusions thereof with mammalian transcription factors (as are
disclosed in U.S. Pat. Nos. 5,654,168, 5,851,796, and 5,968,773),
and the bacterial lac promoter of the lactose operon and its
cognate lacI repressor protein. In a preferred embodiment, the
recombinant cell expresses the lacI repressor protein and a
recombinant expression construct encoding the random cDNA fragments
under the control of a promoter comprising one or a multiplicity of
lac-responsive elements, wherein expression of the fragments can be
induced by contacting the cells with the physiologically-neutral
inducing agent, isopropylthio-.alpha.-galactoside. In this
preferred embodiment, the lacI repressor is encoded by a
recombinant expression construct identified as 3'SS (commercially
available from Stratagene, LaJolla, Calif.).
[0042] The invention also provides recipient cell lines suitable
for selection of growth-inhibitory GSEs. In preferred embodiments,
the cell lines are human breast, lung, colon and prostate carcinoma
cells, modified to comprise a trans-acting factor such as the lac
repressor and further to express a retroviral receptor cognate to
the tropism of the retroviral vector in which the library is
constructed. In a preferred embodiment, the cells are modified to
express the bacterial lac operon repressor, lacI (to allow for
IPTG-inducible gene expression) and to express the ecotropic mouse
retroviral receptor (to enable high-efficiency infection with
ecotropic recombinant retroviruses). In alternative preferred
embodiments, the cells are telomerase-immortalized normal human
fibroblasts and retinal pigment and mammary epithelial cells that
have been modified to express lacI and the mouse ecotropic
retrovirus receptor.
[0043] The invention utilizes modifications of methods of producing
genetic suppressor elements (GSEs) for identifying tumor cell
growth controlling genes. These DNA fragments are termed "GSE"
herein to designate both sense- and antisense-oriented gene
fragments that can inhibit or modify the function of the target
gene when expressed in a cell. Both types of functional GSEs can be
generated by random fragmentation of the DNA of the target gene and
identified by function-based selection of fragments that confer the
desired cellular phenotype such as cell growth inhibition. Such
function-based GSE selection makers it possible to develop genetic
inhibitors for the selected targets, identify protein functional
domains, and identify genes involved in various complex
phenotypes.
[0044] A generalized scheme of GSE selection is shown in FIG. 1.
Originally developed using a model bacterial system (see U.S. Pat.
No. 5,217,889, incorporated by reference), this method has been
adapted for use in mammalian cells. Because less than 1% of random
fragments derived from a typical cDNA have GSE activity, the size
of expression libraries required for GSE selection is much larger
than the corresponding size of libraries that can be used for
function-based selection of full-length cDNAs. Retroviral vectors
are used to deliver such large libraries into mammalian cells,
because it is a non-stressful delivery system that can be used for
stable transduction into a very high fraction (up to 100%) of
recipient cells. In the preparation of these retroviral-based
libraries, packaging cell lines are used, most preferably human
293-based packaging cell lines, such as BOSC23 (Pear et al., 1993,
Proc. Natl. Acad. Sci. USA 90: 8392-8396), which provide efficient
and uniform retrovirus packaging after transient transfection
(Gudkov and Roninson, 1997, in METHODS IN MOLECULAR BIOLOGY: cDNA
LIBRARY PROTOCOLS, Cowell and Austin, eds. (Totowa, N.J.: Humana
Press), pp. 221-240). Additionally, large-scale expression
selection required modifications in conventional retroviral
vectors. The retroviral vectors used to produce the normalized
tumor libraries of the invention carry one constitutively
expressing and one inducible promoter, which minimizes the problem
of promoter interference under non-inducing conditions. Preferred
embodiments of the modified retroviral vectors of the invention
express the bacterial neomycin resistance gene (neo, selectable in
mammalian cells with G418) from an LTR promoter in the retrovirus.
The vectors also contain a multiple cloning site 3' to the
selectable marker gene and adjacent to a regulatable promoter
comprising promoters from cytomegalovirus (CMV) or Rous sarcoma
virus (RSV) LTR containing 2-4 bacterial lac operator sequences.
The regulatable promoter is cloned in the anti orientation to the
retroviral LTR. A diagram of the topography of one of these
viruses, LNXCO3 is shown in FIG. 2. In alternative embodiments, the
neo gene is exchanged for a gene encoding green fluorescent protein
(Kandel et al., 1997, Somat. Cell Genet. 23: 325-340) or firefly
luciferase (Chang et al., 1999, Oncogene 18: 4808-4818). As a
positive control for growth inhibition an embodiment of LNXCO3 was
used that expressed human p21, a CDK inhibitor know to strongly
inhibit tumor cell growth (see International Patent Application,
Publication No. WO01/38532, incorporated by reference).
[0045] The invention provides a normalized cDNA fragment library
from a mixture of poly(A)+ RNA preparations from one or a
multiplicity of human cell lines, derived from different types of
cancer. This normalized library is prepared in a vector, preferably
a retroviral vector and most preferably a retroviral vector
containing sequences permitting regulated expression of cDNA
fragments cloned therein. In a preferred embodiment, the vector is
the retroviral vector LNXCO3, comprising a promoter inducible by
isopropyl-.beta.-thio-galactoside (IPTG), a physiologically neutral
agent.
[0046] The invention provides methods for isolating
growth-inhibitory GSEs from a normalized cDNA fragment library,
representing most of the expressed genes in a human tumor cell. As
provided herewith, normalized cDNA fragment libraries contain on
the order of 5.times.10.sup.7 clones (Gudkov et al., 1994, Proc.
Natl. Acad. Sci. USA 91: 3644-3748; Levenson et al., 1999, Somat.
Cell Molec. Genet. 25: 9-26), corresponding to >1,000 cDNA
fragments per gene. Selection of individual GSEs from a library of
this size requires a procedure with high sensitivity and low
background, most preferably BrdU suicide selection. The principle
of BrdU suicide selection is illustrated in FIG. 3. In preferred
embodiments, the GSEs are expressed under the control of an
inducible promoter, most preferably a promoter that is inducible by
a physiologically neutral agent (such as IPTG), provided that the
growth inhibitor is induced prior to the addition of BrdU.
Following BrdU selection, the inducer is washed from the culture
and cells infected with growth-inhibitory GSEs begin to
proliferate, thus providing colonies of cells harboring selected
GSEs.
[0047] BrdU suicide is not the only technique that can be used to
select growth-inhibitory genes or GSEs. In one alternative
approach, cells are labeled with a fluorescent dye that integrates
into the cell membrane and is redistributed between daughter cells
with each round of cell division. As a result, cells that have
divided the smallest number of times after labeling show the
highest fluorescence and can be isolated by FACS (Maines et al.,
1995, Cell Growth Differ. 6: 665-671). It is also possible to
isolate cells that die upon the addition of the inducer, by
collecting floating dead cells or isolating apoptotic cells on the
basis of altered staining with DNA-binding fluorescent dyes. These
methods have been used to isolate GSEs from single-gene cDNA
fragment libraries prepared from the MDR1 gene (Zuhn, 1996, Ph.D.
Thesis, Department of Genetics, University of Illinois at Chicago,
Chicago, Ill.) or from BCL2 (U.S. Pat. No. 5,789,389, incorporated
by reference). There are no theoretical problems with any of these
approaches, and all of them work to enrich for growth-inhibitory
elements in low-complexity libraries. The only disadvantage of
these alternatives when compared with BrdU selection is that they
have higher spontaneous background rates that may prevent rare
clones to be selected from an exceedingly complex normalized
library. Thus, BrdU selection is the preferred embodiment of the
inventive methods.
[0048] Prior art methods (Pestov and Lau, 1994, Id.) for adapting
GSE technology to identify growth-inhibitory GSEs were of limited
utility when applied to total tumor cell cDNA. The prior art
methods cannot be efficiently used for transducing a library
representing the total mRNA population from a mammalian cell such
as a tumor cell because the method relies on plasmid expression
vectors for library construction, and only a limited number of
cells can be stably transfected by such libraries. To overcome this
limitation, the invention provides a set of inducible retroviral
vectors that are regulated by IPTG through the bacterial LacI
repressor. This inducible system provides comparable levels of
induction among most of the infected cells. The induced levels of
expression can be finely regulated by using different doses of
IPTG.
[0049] The methods of the invention are exemplified herein by use
of this IPTG-inducible retroviral system to generate a normalized
cDNA library from human breast cancer cells. This library was used
to select GSEs that induce growth arrest in a breast carcinoma cell
line. Using this approach, more than 90 genes were identified that
were enriched by BrdU suicide selection. Many of these GSEs were
shown to have a growth-inhibiting effect when re-introduced into
tumor cells. Included in the genes identified using the inventive
methods are known oncogenes, some of which have been specifically
associated with breast cancer, as well as other genes with a known
role in cell proliferation. Many of the identified genes, however,
had no known function or were not previously known to play a role
in cell cycle progression. The latter genes and their products
represent therefore novel targets for cancer treatment.
Furthermore, some of the genes giving rise to the GSEs that
inhibited the proliferation of breast carcinoma cells appear to be
inessential for normal cell growth, since homozygous knockout of
these genes does not prevent the development of adult mice.
[0050] The invention provides methods for cloning unknown genes
containing GSEs identified using GSE libraries and negative growth
selection methods of the invention. In the practice of this aspect
of the methods of the invention, GSEs with no homology to known
human genes in the NCBI database are used to clone unknown genes by
any technique known in the art.
[0051] In a preferred embodiment, genomic DNA is isolated from the
two-step selected library-transduced cells and used as a template
for PCR, using vector-derived sequences flanking the inserts as
primers. The PCR-amplified mixture of inserts from the selected
cells is recloned into a vector. In further preferred embodiments,
the vector is a TA cloning vector from Invitrogen Life Technologies
that facilitates direct cloning of PCR products. Plasmid clones
from the library of selected fragments are sequenced by
high-throughput DNA sequencing using vector-derived sequences
flanking the inserts as primers. The sequences of growth-inhibitory
GSEs are used as query for the BLAST homology search in the NCBI nr
database to identify genes that gave rise to the selected GSE
fragments.
[0052] In cases where no match can be found in the database, a pair
of oppositely directed primers is designed according to the GSE
sequence. cDNAs from the same human cell lines where the normalized
GSE library is derived is used as template. Rapid Amplification of
cDNA Ends (RACE) is performed using technique known in the art to
capture the missing parts of the cDNA (Frohman et al., 1988, Proc.
Natl. Acad. Sci. USA 85: 8998-9002; also see U.S. Pat. Nos.
5,578,467, and 5,334,515, incorporated by reference). Full-length
cDNA of the unknown gene can be obtained by assembling the RACE
products with the GSE clone. In a preferred embodiment, the GSE is
used to BLAST search the NCBI human EST database. The longest
corresponding EST is obtained from the I.M.A.G.E. Consortium
(distributed by American Type Culture Collection or Research
Genetics) and sequence verified. ORF Finder from NCBI is used to
identify putative open reading frame from the GSE, which helps to
determine if the cDNA fragment lacks the 3' or/and the 5' portion.
The RACE primers are designed according to the extended cDNA
sequence based on the EST sequence to amplify the end segments.
[0053] Alternatively, a GSE with no homology to known human genes
in the NCBI database is PCR-amplified using primers derived from
the end sequences of said GSE. The PCR product is then used as
probe to screen a cDNA library constructed from the same human cell
lines where the GSE library is derived. Positive clones that
hybridize to said probe are sequenced to identify putative open
reading frame. In cases where the cDNA is not full-length, RACE
experiment is performed as described hereinabove.
[0054] The invention provides methods for measuring gene expression
or activity of the gene products corresponding to GSEs identified
using GSE libraries and negative growth selection methods of the
invention. In the practice of this aspect of the methods of the
invention, gene expression or gene product activity is assayed in
cells in the presence or absence of a compound to determine whether
the compound inhibits expression or activity of such a gene or gene
product. In preferred embodiments, gene expression is assayed using
any technique known in the art, such as comparison of northern blot
hybridization to cellular mRNA using a detectably-labeled probe (as
disclosed, for example, in Sambrook et al., 2001, MOLECULAR
CLONING: A LABORATORY MANUAL, 3.sup.rd ed., Cold Strong Harbor
Laboratory Press: N.Y.), or by in vitro amplification methods, such
as quantitative reverse transcription--polymerase chain reaction
(RT-PCR) assays as disclosed by Noonan et al. (1990, Proc. Natl.
Acad. Sci. USA 87: 7160-7164), or by western blotting using
antibodies specific for the gene product (Sambrook et al., 2001,
Id.). Gene product activity is assayed using assays specific for
each gene product, such as immunoassay using antibodies specific
for said gene products or biochemical assay of gene product
function.
[0055] Alternatively, gene expression is assayed using recombinant
expression constructs having a promoter from a gene corresponding
to GSEs identified using GSE libraries and negative growth
selection methods of the invention, wherein the promoter is
operably linked to a reporter gene. The reporter gene is then used
as a sensitive and convenient indicator of the effects of test
compounds on gene expression, and enables compounds that inhibit
expression or activity of genes required for cell, preferably tumor
cell growth to be easily identified. Host cells for these
constructs include any cell expressing the corresponding
growth-promoting gene. Reporter genes useful in the practice of
this aspect of the invention include but are not limited to firefly
luciferase, Renilla luciferase, chloramphenicol acetyltransferase,
beta-galactosidase, green fluorescent protein, and alkaline
phosphatase.
[0056] The invention provides peptides encoded by some of the GSEs
of the invention that have been identified using the GSE-negative
growth selection methods disclosed herein. Such peptides are
presented in Table 5 and in the Sequence Listing as SEQ ID NOS.
229-314. Some of these peptides are derived from proteins that were
previously known to play a role in cell proliferation, and others
from proteins that were first assigned such a role in the instant
inventions. All of the identified peptides, however, are novel
inhibitors of tumor cell proliferation. Also provided are related
compounds within the understanding of those with skill in the art,
such as chemical mimetics, organomimetics or peptidomimetics. As
used herein, the terms "mimetic," "peptide mimetic,"
"peptidomimetic," "organomimetic" and "chemical mimetic" are
intended to encompass peptide derivatives, peptide analogues and
chemical compounds having an arrangement of atoms is a
three-dimensional orientation that is equivalent to that of a
peptide encoded by a GSE of the invention. It will be understood
that the phrase "equivalent to" as used herein is intended to
encompass compounds having substitution of certain atoms or
chemical moieties in said peptide with moieties having bond
lengths, bond angles and arrangements thereof in the mimetic
compound that produce the same or sufficiently similar arrangement
or orientation of said atoms and moieties to have the biological
function of the peptide GSEs of the invention. In the peptide
mimetics of the invention, the three-dimensional arrangement of the
chemical constituents is structurally and/or functionally
equivalent to the three-dimensional arrangement of the peptide
backbone and component amino acid sidechains in the peptide,
resulting in such peptido-, organo- and chemical mimetics of the
peptides of the invention having substantial biological activity.
These terms are used according to the understanding in the art, as
illustrated for example by Fauchere, 1986, Adv. Drug Res. 15: 29;
Veber & Freidinger, 1985, TINS p. 392; and Evans et al., 1987,
J. Med. Chem. 30: 1229, incorporated herein by reference.
[0057] It is understood that a pharmacophore exists for the
biological activity of each peptide GSE of the invention. A
pharmacophore is understood in the art as comprising an idealized,
three-dimensional definition of the structural requirements for
biological activity. Peptido-, organo- and chemical mimetics can be
designed to fit each pharmacophore with current computer modeling
software (computer aided drug design). Said mimetics are produced
by structure-function analysis, based on the positional information
from the substituent atoms in the peptide GSEs of the
invention.
[0058] Peptides as provided by the invention can be advantageously
synthesized by any of the chemical synthesis techniques known in
the art, particularly solid-phase synthesis techniques, for
example, using commercially-available automated peptide
synthesizers. The mimetics of the present invention can be
synthesized by solid phase or solution phase methods conventionally
used for the synthesis of peptides (see, for example, Merrifield,
1963, J. Amer. Chem. Soc. 85: 2149-54; Carpino, 1973, Acc. Chem.
Res. 6: 191-98; Birr, 1978, ASPECTS OF THE MERRIFIELD PEPTIDE
SYNTHESIS, Springer-Verlag: Heidelberg; THE PEPTIDES: ANALYSIS,
SYNTHESIS, BIOLOGY, Vols. 1, 2, 3, 5, (Gross & Meinhofer,
eds.), Academic Press: New York, 1979; Stewart et al., 1984, SOLID
PHASE PEPTIDE SYNTHESIS, 2nd. ed., Pierce Chem. Co.: Rockford,
Ill.; Kent, 1988, Ann. Rev. Biochem. 57: 957-89; and Gregg et al.,
1990, Int. J. Peptide Protein Res. 55: 161-214, which are
incorporated herein by reference in their entirety.)
[0059] The use of solid phase methodology is preferred. Briefly, an
N-protected C-terminal amino acid residue is linked to an insoluble
support such as divinylbenzene cross-linked polystyrene,
polyacrylamide resin, Kieselguhr/polyamide (pepsyn K), controlled
pore glass, cellulose, polypropylene membranes, acrylic acid-coated
polyethylene rods or the like. Cycles of deprotection,
neutralization and coupling of successive protected amino acid
derivatives are used to link the amino acids from the C-terminus
according to the amino acid sequence. For some synthetic peptides,
an FMOC strategy using an acid-sensitive resin may be used.
Preferred solid supports in this regard are divinylbenzene
cross-linked polystyrene resins, which are commercially available
in a variety of functionalized forms, including chloromethyl resin,
hydroxymethyl resin, paraacetamidomethyl resin, benzhydrylamine
(BHA) resin, 4-methylbenzhydrylamine (MBHA) resin, oxime resins,
4-alkoxybenzyl alcohol resin (Wang resin),
4-(2',4'-dimethoxyphenylaminomethyl)-phenoxymethyl resin,
2,4-dimethoxybenzhydryl-amine resin, and
4-(2',4'-dimethoxyphenyl-FMOC-amino-methyl)-phenoxyacetamidonorleucyl-MBH-
A resin (Rink amide MBHA resin). In addition, acid-sensitive resins
also provide C-terminal acids, if desired. A particularly preferred
protecting group for alpha amino acids is base-labile
9-fluorenylmethoxy-carbonyl (FMOC).
[0060] Suitable protecting groups for the side chain
functionalities of amino acids chemically compatible with BOC
(t-butyloxycarbonyl) and FMOC groups are well known in the art.
When using FMOC chemistry, the following protected amino acid
derivatives are preferred: FMOC-Cys(Trit), FMOC-Ser(But),
FMOC-Asn(Trit), FMOC-Leu, FMOC-Thr(Trit), FMOC-Val, FMOC-Gly,
FMOC-Lys(Boc), FMOC-Gln(Trit), FMOC-Glu(OBut), FMOC-His(Trit),
FMOC-Tyr(But), FMOC-Arg(PMC
(2,2,5,7,8-pentamethylchroman-6-sulfonyl)), FMOC-Arg(BOC).sub.2,
FMOC-Pro, and FMOC-Trp(BOC). The amino acid residues can be coupled
by using a variety of coupling agents and chemistries known in the
art, such as direct coupling with DIC (diisopropyl-carbodiimide),
DCC (dicyclohexylcarbodiimide), BOP
(benzotriazolyl-N-oxytrisdimethylaminophosphonium
hexa-fluorophosphate), PyBOP
(benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium
hexafluoro-phosphate), PyBrOP (bromo-tris-pyrrolidinophosphonium
hexafluorophosphate); via performed symmetrical anhydrides; via
active esters such as pentafluorophenyl esters; or via performed
HOBt (1-hydroxybenzotriazole) active esters or by using FMOC-amino
acid fluoride and chlorides or by using FMOC-amino acid-N-carboxy
anhydrides. Activation with HBTU
(2-(1H-benzotriazole-1-yl),1,1,3,3-tetramethyluronium
hexafluorophosphate) or HATU (2-(1H-7-aza-benzotriazole-1-yl),
1,1,3,3-tetramethyluronium hexafluoro-phosphate) in the presence of
HOBt or HOAt (7-azahydroxybenztriazole) is preferred.
[0061] The solid phase method can be carried out manually, although
automated synthesis on a commercially available peptide synthesizer
(e.g., Applied Biosystems 431A or the like; Applied Biosystems,
Foster City, Calif.) is preferred. In a typical synthesis, the
first (C-terminal) amino acid is loaded on the chlorotrityl resin.
Successive deprotection (with 20% piperidine/NMP
(N-methylpyrrolidone)) and coupling cycles according to ABI FastMoc
protocols (ABI user bulletins 32 and 33, Applied Biosystems are
used to build the whole peptide sequence. Double and triple
coupling, with capping by acetic anhydride, may also be used.
[0062] The synthetic mimetic peptide is cleaved from the resin and
deprotected by treatment with TFA (trifluoroacetic acid) containing
appropriate scavengers. Many such cleavage reagents, such as
Reagent K (0.75 g crystalline phenol, 0.25 mL ethanedithiol, 0.5 mL
thioanisole, 0.5 mL deionized water, 10 mL TFA) and others, can be
used. The peptide is separated from the resin by filtration and
isolated by ether precipitation. Further purification may be
achieved by conventional methods, such as gel filtration and
reverse phase HPLC (high performance liquid chromatography).
Synthetic calcitonin mimetics according to the present invention
may be in the form of pharmaceutically acceptable salts, especially
base-addition salts including salts of organic bases and inorganic
bases. The base-addition salts of the acidic amino acid residues
are prepared by treatment of the peptide with the appropriate base
or inorganic base, according to procedures well known to those
skilled in the art, or the desired salt may be obtained directly by
lyophilization out of the appropriate base.
[0063] Generally, those skilled in the art will recognize that
peptides as described herein may be modified by a variety of
chemical techniques to produce compounds having essentially the
same activity as the unmodified peptide, and optionally having
other desirable properties. For example, carboxylic acid groups of
the peptide may be provided in the form of a salt of a
pharmaceutically-acceptable cation. Amino groups within the peptide
may be in the form of a pharmaceutically-acceptable acid addition
salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic,
maleic, tartaric and other organic salts, or may be converted to an
amide. Thiols can be protected with any one of a number of
well-recognized protecting groups, such as acetamide groups. Those
skilled in the art will also recognize methods for introducing
cyclic structures into the peptides of this invention so that the
native binding configuration will be more nearly approximated. For
example, a carboxyl terminal or amino terminal cysteine residue can
be added to the peptide, so that when oxidized the peptide will
contain a disulfide bond, thereby generating a cyclic peptide.
Other peptide cyclizing methods include the formation of thioethers
and carboxyl- and amino-terminal amides and esters.
[0064] Specifically, a variety of techniques are available for
constructing peptide derivatives and analogues with the same or
similar desired biological activity as the corresponding peptide
compound but with more favorable activity than the peptide with
respect to solubility, stability, and susceptibility to hydrolysis
and proteolysis. Such derivatives and analogues include peptides
modified at the N-terminal amino group, the C-terminal carboxyl
group, and/or changing one or more of the amido linkages in the
peptide to a non-amido linkage. It will be understood that two or
more such modifications can be coupled in one peptide mimetic
structure (e.g., modification at the C-terminal carboxyl group and
inclusion of a --CH.sub.2-- carbamate linkage between two amino
acids in the peptide).
[0065] Amino terminus modifications include alkylating,
acetylating, adding a carbobenzoyl group, and forming a succinimide
group. Specifically, the N-terminal amino group can then be reacted
to form an amide group of the formula RC(O)NH-- where R is alkyl,
preferably lower alkyl, and is added by reaction with an acid
halide, RC(O)Cl or acid anhydride. Typically, the reaction can be
conducted by contacting about equimolar or excess amounts (e.g.,
about 5 equivalents) of an acid halide to the peptide in an inert
diluent (e.g., dichloromethane) preferably containing an excess
(e.g., about 10 equivalents) of a tertiary amine, such as
diisopropylethylamine, to scavenge the acid generated during
reaction. Reaction conditions are otherwise conventional (e.g.,
room temperature for 30 minutes). Alkylation of the terminal amino
to provide for a lower alkyl N-substitution followed by reaction
with an acid halide as described above will provide for N-alkyl
amide group of the formula RC(O)NR--. Alternatively, the amino
terminus can be covalently linked to succinimide group by reaction
with succinic anhydride. An approximately equimolar amount or an
excess of succinic anhydride (e.g., about 5 equivalents) are used
and the terminal amino group is converted to the succinimide by
methods well known in the art including the use of an excess (e.g.,
ten equivalents) of a tertiary amine such as diisopropylethylamine
in a suitable inert solvent (e.g., dichloromethane), as described
in Wollenberg et al., U.S. Pat. No. 4,612,132, is incorporated
herein by reference in its entirety. It will also be understood
that the succinic group can be substituted with, for example,
C.sub.2-- through C.sub.6-- alkyl or --SR substituents, which are
prepared in a conventional manner to provide for substituted
succinimide at the N-terminus of the peptide. Such alkyl
substituents are prepared by reaction of a lower olefin (C.sub.2--
through C.sub.6-- alkyl) with maleic anhydride in the manner
described by Wollenberg et al., supra., and --SR substituents are
prepared by reaction of RSH with maleic anhydride where R is as
defined above. In another advantageous embodiments, the amino
terminus is derivatized to form a benzyloxycarbonyl-NH-- or a
substituted benzyloxycarbonyl-NH-- group. This derivative is
produced by reaction with approximately an equivalent amount or an
excess of benzyloxycarbonyl chloride (CBZ-Cl) or a substituted
CBZ-Cl in a suitable inert diluent (e.g., dichloromethane)
preferably containing a tertiary amine to scavenge the acid
generated during the reaction. In yet another derivative, the
N-terminus comprises a sulfonamide group by reaction with an
equivalent amount or an excess (e.g., 5 equivalents) of
R--S(O).sub.2Cl in a suitable inert diluent (dichloromethane) to
convert the terminal amine into a sulfonamide, where R is alkyl and
preferably lower alkyl. Preferably, the inert diluent contains
excess tertiary amine (e.g., ten equivalents) such as
diisopropylethylamine, to scavenge the acid generated during
reaction. Reaction conditions are otherwise conventional (e.g.,
room temperature for 30 minutes). Carbamate groups are produced at
the amino terminus by reaction with an equivalent amount or an
excess (e.g., 5 equivalents) of R--OC(O)Cl or
R--OC(O)OC.sub.6H.sub.4--p--NO.sub.2 in a suitable inert diluent
(e.g., dichloromethane) to convert the terminal amine into a
carbamate, where R is alkyl, preferably lower alkyl. Preferably,
the inert diluent contains an excess (e.g., about 10 equivalents)
of a tertiary amine, such as diisopropylethylamine, to scavenge any
acid generated during reaction. Reaction conditions are otherwise
conventional (e.g., room temperature for 30 minutes). Urea groups
are formed at the amino terminus by reaction with an equivalent
amount or an excess (e.g., 5 equivalents) of R--N.dbd.C.dbd.O in a
suitable inert diluent (e.g., dichloromethane) to convert the
terminal amine into a urea (i.e., RNHC(O)NH--) group where R is as
defined above. preferably, the inert diluent contains an excess
(e.g., about 10 equivalents) of a tertiary amine, such as
diisopropylethylamine. Reaction conditions are otherwise
conventional (e.g., room temperature for about 30 minutes).
[0066] In preparing peptide mimetics wherein the C-terminal
carboxyl group is replaced by an ester (e.g., --C(O)OR where R is
alkyl and preferably lower alkyl), resins used to prepare the
peptide acids are employed, and the side chain protected peptide is
cleaved with base and the appropriate alcohol, e.g., methanol. Side
chain protecting groups are then removed in the usual fashion by
treatment with hydrogen fluoride to obtain the desired ester. In
preparing peptide mimetics wherein the C-terminal carboxyl group is
replaced by the amide --C(O)NR.sub.3R.sub.4, a benzhydrylamine
resin is used as the solid support for peptide synthesis. Upon
completion of the synthesis, hydrogen fluoride treatment to release
the peptide from the support results directly in the free peptide
amide (i.e., the C-terminus is --C(O)NH.sub.2). Alternatively, use
of the chloromethylated resin during peptide synthesis coupled with
reaction with ammonia to cleave the side chain Protected peptide
from the support yields the free peptide amide and reaction with an
alkylamine or a dialkylamine yields a side chain protected
alkylamide or dialkylamide (i.e., the C-terminus is
--C(O)NRR.sub.1, where R and R.sub.1 are alkyl and preferably lower
alkyl). Side chain protection is then removed in the usual fashion
by treatment with hydrogen fluoride to give the free amides,
alkylamides, or dialkylamides.
[0067] In another alternative embodiment, the C-terminal carboxyl
group or a C-terminal ester can be induced to cyclize by
displacement of the --OH or the ester (--OR) of the carboxyl group
or ester respectively with the N-terminal amino group to form a
cyclic peptide. For example, after synthesis and cleavage to give
the peptide acid, the free acid is converted in solution to an
activated ester by an appropriate carboxyl group activator such as
dicyclohexylcarbodiimide (DCC), for example, in methylene chloride
(CH.sub.2Cl.sub.2), dimethyl formamide (DMF), or mixtures thereof.
The cyclic peptide is then formed by displacement of the activated
ester with the N-terminal amine. Cyclization, rather than
polymerization, can be enhanced by use of very dilute solutions
according to methods well known in the art.
[0068] Peptide mimetics as understood in the art and provided by
the invention are structurally similar to the paradigm peptide
encoded by each of the sense-oriented GSEs of the invention, but
have one or more peptide linkages optionally replaced by a linkage
selected from the group consisting of: --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2CH.sub.2--, --CH.dbd.CH-- (in both cis and
trans conformers), --COCH.sub.2--, --CH(OH)CH.sub.2--, and
--CH.sub.2SO--, by methods known in the art and further described
in the following references: Spatola, 1983, in CHEMISTRY AND
BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS, (Weinstein,
ed.), Marcel Dekker: New York, p. 267; Spatola, 1983, Peptide
Backbone Modifications 1: 3; Morley, 1980, Trends Pharm. Sci. pp.
463-468; Hudson et al., 1979, Int. J. Pept. Prot. Res. 14: 177-185;
Spatola et al., 1986, Life Sci. 38: 1243-1249; Hann, 1982, J. Chem.
Soc. Perkin Trans. 1307-314; Almquist et al., 1980, J. Med. Chem.
23: 1392-1398; Jennings-White et al., 1982, Tetrahedron Lett. 23:
2533; Szelke et al., 1982, European Patent Application, Publication
No. EP045665A; Holladay et al., 1983, Tetrahedron Lett. 24:
4401-4404; and Hruby, 1982, Life Sci. 31: 189-199, each of which is
incorporated herein by reference. Such peptide mimetics may have
significant advantages over polypeptide embodiments, including, for
example: being more economical to produce, having greater chemical
stability or enhanced pharmacological properties (such half-life,
absorption, potency, efficacy, etc.), reduced antigenicity, and
other properties.
[0069] Mimetic analogs of the tumor-inhibiting peptides of the
invention may also be obtained using the principles of conventional
or rational drug design (see, Andrews et al., 1990, Proc. Alfred
Benzon Symp. 28: 145-165; McPherson, 1990, Eur. J. Biochem.
189:1-24; Hol et al., 1989a, in MOLECULAR RECOGNITION: CHEMICAL AND
BIOCHEMICAL PROBLEMS, (Roberts, ed.); Royal Society of Chemistry;
pp. 84-93; Hol, 1989b, Arzneim-Forsch. 39:1016-1018; Hol, 1986,
Agnew Chem. Int. Ed. Engl. 25: 767-778, the disclosures of which
are herein incorporated by reference).
[0070] In accordance with the methods of conventional drug design,
the desired mimetic molecules are obtained by randomly testing
molecules whose structures have an attribute in common with the
structure of a "native" peptide. The quantitative contribution that
results from a change in a particular group of a binding molecule
can be determined by measuring the biological activity of the
putative mimetic in comparison with the tumor-inhibiting activity
of the peptide. In a preferred embodiment of rational drug design,
the mimetic is designed to share an attribute of the most stable
three-dimensional conformation of the peptide. Thus, for example,
the mimetic may be designed to possess chemical groups that are
oriented in a way sufficient to cause ionic, hydrophobic, or van
der Waals interactions that are similar to those exhibited by the
tumor-inhibiting peptides of the invention, as disclosed
herein.
[0071] The preferred method for performing rational mimetic design
employs a computer system capable of forming a representation of
the three-dimensional structure of the peptide, such as those
exemplified by Hol, 1989a, ibid.; Hol, 1989b, ibid.; and Hol, 1986,
ibid. Molecular structures of the peptido-, organo- and chemical
mimetics of the peptides of the invention are produced according to
those with skill in the art using computer-assisted design programs
commercially available in the art. Examples of such programs
include SYBYL 6.5.RTM., HQSAR.TM., and ALCHEMY 2000.TM. (Tripos);
GALAXY.TM. and AM2000.TM. (AM Technologies, Inc., San Antonio,
Tex.); CATALYST.TM. and CERIUS.TM. (Molecular Simulations, Inc.,
San Diego, CA); CACHE PRODUCTS.TM., TSAR.TM., AMBER.TM., and
CHEM-X.TM. (Oxford Molecular Products, Oxford, Calif.) and
CHEMBUILDER3D.TM. (Interactive Simulations, Inc., San Diego,
Calif.).
[0072] The peptido-, organo- and chemical mimetics produced using
the peptides disclosed herein using, for example, art-recognized
molecular modeling programs are produced using conventional
chemical synthetic techniques, most preferably designed to
accommodate high throughput screening, including combinatorial
chemistry methods. Combinatorial methods useful in the production
of the peptido-, organo- and chemical mimetics of the invention
include phage display arrays, solid-phase synthesis and
combinatorial chemistry arrays, as provided, for example, by
SIDDCO, Tuscon, Ariz.; Tripos, Inc.; Calbiochem/Novabiochem, San
Diego, Calif.; Symyx Technologies, Inc., Santa Clara, Calif.;
Medichem Research, Inc., Lemont, Ill.; Pharm-Eco Laboratories,
Inc., Bethlehem, Pa.; or N. V. Organon, Oss, Netherlands.
Combinatorial chemistry production of the peptido-, organo- and
chemical mimetics of the invention are produced according to
methods known in the art, including but not limited to techniques
disclosed in Terrett, 1998, COMBINATORIAL CHEMISTRY, Oxford
University Press, London; Gallop et al., 1994, "Applications of
combinatorial technologies to drug discovery. 1. Background and
peptide combinatorial libraries," J. Med. Chem. 37: 1233-51; Gordon
et al., 1994, "Applications of combinatorial technologies to drug
discovery. 2. Combinatorial organic synthesis, library screening
strategies, and future directions," J. Med. Chem. 37: 1385-1401;
Look et al., 1996, Bioorg. Med. Chem. Lett. 6: 707-12; Ruhland et
al., 1996, J. Amer. Chem. Soc. 118: 253-4; Gordon et al., 1996,
Acc. Chem. Res. 29: 144-54; Thompson & Ellman, 1996, Chem. Rev.
96: 555-600; Fruchtel & Jung, 1996, Angew. Chem. Int. Ed. Engl.
35: 17-42; Pavia, 1995, "The Chemical Generation of Molecular
Diversity", Network Science Center, www.netsci.org; Adnan et al.,
1995, "Solid Support Combinatorial Chemistry in Lead Discovery and
SAR Optimization," Id., Davies and Briant, 1995, "Combinatorial
Chemistry Library Design using Pharmacophore Diversity," Id.,
Pavia, 1996, "Chemically Generated Screening Libraries: Present and
Future," Id.; and U.S. Pat. Nos. 5,880,972 to Horlbeck; 5,463,564
to Agrafiotis et al.; 5,331,573 to Balaji et al.; and 5,573,905 to
Lerner et al.
[0073] The invention also provides methods for using the genes
identified herein (particularly the genes set forth in Table 3) to
screen compounds to identify inhibitors of expression or activity
of said genes. In the practice of this aspect of the methods of the
invention, cells expressing a gene required for cell growth,
particularly a gene identified in Table 3, are assayed in the
presence and absence of a test compound, and test compounds that
reduce expression or activity of the gene or gene product
identified thereby. Additionally, the assays can be performed under
suicide selection conditions, wherein compounds that inhibit cell
growth by inhibiting expression or activity of the gene select for
survival of the cells. In alternative embodiments, reporter gene
constructs of the invention are used, wherein expression of the
reporter gene is reduced in the presence but not the absence of the
test compound.
[0074] The methods of the invention are useful for identifying
compounds that inhibit the growth of tumor cells, most preferably
human tumor cells. The invention also provides the identified
compounds and methods for using the identified compounds to inhibit
tumor cell, most preferably human tumor cell growth. Exemplary
compounds include neutralizing antibodies that interfere with gene
product activity; antisense oligonucleotides, developed either as
GSEs according to the methods of the invention or identified by
other methods known in the art; ribozymes; triple-helix
oligonucleotides; and "small molecule" inhibitors of gene
expression or activity, preferably said small molecules that
specifically bind to the gene product or to regulatory elements
responsible for mediating expression of a gene in Table 3. It is
recognized by one skilled in the art that a gene of the present
invention can be used to identify biological pathways that contain
the protein encoded by such. Any member of such pathways may be
used to identify compounds that inhibit the growth of tumor
cells.
[0075] The invention also provides embodiments of the compounds
identified by the methods disclosed herein as pharmaceutical
compositions. The pharmaceutical compositions of the present
invention can be manufactured in a manner that is itself known,
e.g., by means of a conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0076] Pharmaceutical compositions for use in accordance with the
present invention thus can be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries that facilitate processing of the active
compounds into preparations that can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen.
[0077] Non-toxic pharmaceutical salts include salts of acids such
as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic,
formic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric,
tartaric, maleic, hydroiodic, alkanoic such as acetic,
HOOC--(CH.sub.2).sub.n--CH.sub.3 where n is 0-4, and the like.
Non-toxic pharmaceutical base addition salts include salts of bases
such as sodium, potassium, calcium, ammonium, and the like. Those
skilled in the art will recognize a wide variety of non-toxic
pharmaceutically acceptable addition salts.
[0078] For injection, tumor cell growth-inhibiting compounds
identified according to the methods of the invention can be
formulated in appropriate aqueous solutions, such as
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological saline buffer. For transmucosal
and transcutaneous administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0079] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained with solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents can
be added, such as the cross-linked polyvinyl pyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate.
[0080] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions can be used, which can
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments can be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0081] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds can
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers can be added. All formulations for oral administration
should be in dosages suitable for such administration. For buccal
administration, the compositions can take the form of tablets or
lozenges formulated in conventional manner.
[0082] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator
can be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0083] The compounds can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0084] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds can be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions can
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension can also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions. Alternatively,
the active ingredient can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0085] The compounds can also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0086] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0087] A pharmaceutical carrier for the hydrophobic compounds of
the invention is a cosolvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. The cosolvent system can be the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
can be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components can be varied: for example, other
low-toxicity nonpolar surfactants can be used instead of
polysorbate 80; the fraction size of polyethylene glycol can be
varied; other biocompatible polymers can replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or
polysaccharides can substitute for dextrose.
[0088] Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds can be employed. Liposomes and emulsions
are well known examples of delivery vehicles or carriers for
hydrophobic drugs. Certain organic solvents such as
dimethylsulfoxide also can be employed, although usually at the
cost of greater toxicity. Additionally, the compounds can be
delivered using a sustained-release system, such as semipermeable
matrices of solid hydrophobic polymers containing the therapeutic
agent. Various sustained-release materials have been established
and are well known by those skilled in the art. Sustained-release
capsules can, depending on their chemical nature, release the
compounds for a few weeks up to over 100 days. Depending on the
chemical nature and the biological stability of the therapeutic
reagent, additional strategies for protein and nucleic acid
stabilization can be employed.
[0089] The pharmaceutical compositions also can comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0090] The compounds of the invention can be provided as salts with
pharmaceutically compatible counterions. Pharmaceutically
compatible salts can be formed with many acids, including but not
limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic,
succinic, phosphoric, hydrobromic, sulfinic, formic,
toluenesulfonic, methanesulfonic, nitic, benzoic, citric, tartaric,
maleic, hydroiodic, alkanoic such as acetic,
HOOC--(CH.sub.2).sub.n--CH.sub.3 where n is 0-4, and the like.
Salts tend to be more soluble in aqueous or other protonic solvents
that are the corresponding free base forms. Non-toxic
pharmaceutical base addition salts include salts of bases such as
sodium, potassium, calcium, ammonium, and the like. Those skilled
in the art will recognize a wide variety of non-toxic
pharmaceutically acceptable addition salts.
[0091] Pharmaceutical compositions of the compounds of the present
invention can be formulated and administered through a variety of
means, including systemic, localized, or topical administration.
Techniques for formulation and administration can be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa. The mode of administration can be selected to maximize delivery
to a desired target site in the body. Suitable routes of
administration can, for example, include oral, rectal,
transmucosal, transcutaneous, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0092] Alternatively, one can administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a specific tissue, often in a depot or
sustained release formulation.
[0093] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amounts is well within the capability of those
skilled in the art, especially in light of the detailed disclosure
provided herein.
[0094] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays, as disclosed herein. For example, a dose can be
formulated in animal models to achieve a circulating concentration
range that includes the EC.sub.50 (effective dose for 50% increase)
as determined in cell culture, i.e., the concentration of the test
compound which achieves a half-maximal inhibition of bacterial cell
growth. Such information can be used to more accurately determine
useful doses in humans.
[0095] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination,
the severity of the particular disease undergoing therapy and the
judgment of the prescribing physician.
[0096] Preferred compounds of the invention will have certain
pharmacological properties. Such properties include, but are not
limited to oral bioavailability, low toxicity, low serum protein
binding and desirable in vitro and in vivo half-lives. Assays may
be used to predict these desirable pharmacological properties.
Assays used to predict bioavailability include transport across
human intestinal cell monolayers, including Caco-2 cell monolayers.
Serum protein binding may be predicted from albumin binding assays.
Such assays are described in a review by Oravcova et al. (1996, J.
Chromat. B 677: 1-27). Compound half-life is inversely proportional
to the frequency of dosage of a compound. In vitro half-lives of
compounds may be predicted from assays of microsomal half-life as
described by Kuhnz and Gieschen (1998, DRUG METABOLISM AND
DISPOSITION, Vol. 26, pp. 1120-1127).
[0097] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio between LD.sub.50 and ED.sub.50.
Compounds that exhibit high therapeutic indices are preferred. The
data obtained from these cell culture assays and animal studies can
be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage can vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
(See, e.g. Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1, p. 1).
[0098] Dosage amount and interval can be adjusted individually to
provide plasma levels of the active moiety that are sufficient to
maintain tumor cell growth-inhibitory effects. Usual patient
dosages for systemic administration range from 100-2000 mg/day.
Stated in terms of patient body surface areas, usual dosages range
from 50-910 mg/m.sup.2/day. Usual average plasma levels should be
maintained within 0.1-1000 .mu.M. In cases of local administration
or selective uptake, the effective local concentration of the
compound cannot be related to plasma concentration.
[0099] The following Examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature.
EXAMPLES
1. Production of Normalized Tumor Library from MCF-7 Human Breast
Cancer Cells
[0100] A normalized cDNA fragment library was generated from MCF-7
breast carcinoma cell line (estrogen receptor positive, wild-type
for p53; ATCC Accession No. HTB22, American Type Culture
Collection, Manassas, Va.). Poly(A)+ RNA from MCF-7 cells was used
to prepare a population of normalized cDNA fragments through a
modification of the procedure described in Gudkov and Roninson
(1997). Briefly, RNA was fragmented by heating at 100.degree. C.
for 9 minutes. Double-stranded cDNA was generated from this
heat-fragmented RNA using the Gibco Superscript kit with a
reverse-transcription primer (5'-GGATCCTCACTCACTCANNNNNN-3'; SEQ ID
NO. 1). This primer contains a random octamer sequence at its 3'
end for random priming, and it carries a tag (termed "stop adaptor"
in its double-stranded form) that provides TGA stop codons in all
three open reading frames, together with BamHI restriction site.
PCR assays were used to establish the presence of
.beta.2-microglobulin, .beta.-actin and estrogen receptor mRNA
sequences in this cDNA preparation. Double-stranded cDNA fragments
were ligated to the following adaptor:
5'GTACCTGAGTTATAGGATCCCTGCCATGCCATGCCATG3'(SEQ ID NO. 2)
3'CCTAGGGACGGTACGGTACGGTAC 5'(SEQ ID NO. 3) The latter adaptor
("start adaptor") contains translation start sites in all three
frames, together with a BamHI site. The double-stranded cDNA was
amplified by PCR with primers that anneal to the start and stop
adaptors. Although the start adaptor is initially ligated at both
ends of cDNA fragments, the PCR products were generated
predominantly by the two different primers and contain the start
adaptor only at the 5' but not at 3' end. This desirable outcome is
explained by the "PCR suppression effect", due to PCR inhibition by
panhandle-like structures formed upon renaturation of sequences
flanked by an inverted repeat (Siebert et al., 1995, Nucleic Acids
Res. 23: 1087-1088). Furthermore, any residual start adaptors at
the 3' ends were subsequently removed by BamHI digestion prior to
cloning. The amplified cDNA fragment population was again tested
for the presence of .beta.2-microglobulin, .beta.-actin and
estrogen receptor sequences. This procedure produced a population
of randomly initiating and terminating double-stranded cDNA
fragments (100-400 bp size), which are tagged by different adaptors
at the ends corresponding to the 5' and 3' direction of the
original mRNA. The 5' adaptor contains translation initiation
codons in three open reading frames, and the 3' adaptor contains
stop codons in all three reading frames. Such fragments direct the
synthesis of peptides derived from the parental protein when cloned
in sense orientation, or give rise to antisense RNA molecules when
cloned in antisense orientation.
[0101] The cDNA fragment mixture was subjected to normalization,
through a modification of the procedure of Patanjali et al. (1991,
Proc. Natl. Acad. Sci. USA 88: 1943-1947), based on C.sub.ot
fractionation. Normalization was achieved by reannealing portions
of denatured cDNA for 24, 48, 72, or 96 hours. Single-stranded
products were separated from re-annealed double stranded DNA by
hydroxyapatite chromatography. Normalization of cDNA fragments was
tested by Southern hybridization with probes corresponding to genes
expressed to different levels in MCF-7 cells and performed with
each single-stranded fraction. This analysis indicated that the
content of .beta.-actin, an abundant mRNA species, decreased over
normalization time, with the lowest content found at the 96 hr time
point. Conversely, a moderately-abundant cDNA sequence, c-MYC and a
low-abundant cDNA sequence, MDR1 (which was undetectable in MCF-7
cDNA prior to normalization) increased their levels to those
comparable with .beta.-actin by 96 hr, suggesting that the 96 hr
fraction was the best-normalized. To confirm the normalization of
the 96 hr fraction, this DNA was digested (on a small scale) with
BamHI, ligated into a plasmid vector and transformed into E. coli
(Top10) by electroporation. Colony hybridization analysis was
performed on nitrocellulose filters to which 10,000 colonies were
plated, using radiolabeled probes for different genes. The
following signal numbers per filter were obtained: .beta.-actin, 3
signals; MDR1, 3 signals; C-MYC, 2 signals; C--FOS, 2 signals.
These results indicated that the sequences from the tested genes
are found on average in 1 of 3,000-5,000 clones of this library,
and also confirmed that the 96 hr fraction was normalized.
[0102] The normalized cDNA fraction was amplified by PCR and
ligated into IPTG-inducible retroviral vector LNXCO3 (Chang and
Roninson, 1996, Gene 183: 137-142). The ligation produced a library
of approximately 50 million clones. Percent recombination in this
library was assessed by PCR of the DNA from bacterial colonies,
using primers that flank the insertion site of LNXCO3. The number
of clones containing an insert was 131/150 or 87%. Most of the
inserts ranged in size from 100 to 300 bp. For further
characterization of the library, a fraction of the inserts were
recloned into the pcDNA3 vector. The insert sequences of 69
randomly picked clones in pcDNA3 were determined using a
high-throughput DNA sequencer, and analyzed for homology to known
gene sequences in the public-domain database. Fifty-two of the
inserts matched no known genes, 16 corresponded to different human
genes, and one sequence was found to be of bacterial origin. This
normalized MCF-7 cDNA fragment library was used to select
growth-inhibitory GSEs in breast carcinoma cells.
2. Production of Breast Cancer Recipient Cells
[0103] The normalized tumor library described in Example 1 was
prepared from MCF-7 human breast carcinoma cells. As recipient
cells for GSE selection, a different breast carcinoma cell line,
MDA-MB-231 (ATCC Accession No. HTB26) was chosen. This line
represents a more malignant class of breast cancers relative to
MCF-7: it is estrogen receptor-negative and p53-deficient. The
choice of different cell lines as the source of RNA and as the
recipient was aimed at isolating growth-inhibitory GSEs that are
more likely to be effective against different types of breast
cancer.
[0104] MDA-MB231 cells were first rendered susceptible to infection
with ecotropic retroviruses, which can be readily generated at a
high titer using convenient packaging cell lines, and are not
infectious to humans or unmodified human cells. MDA-MB-231 cells
were infected with amphotropic recombinant virus that carries the
gene for the murine ecotropic receptor in retroviral vector LXIHis
(Levenson et al., 1998, Hum. Gene Ther. 9: 1233-1236), and the
infected cell population was selected with histidinol. The
susceptibility of the selected cells to infection with ecotropic
retroviruses was determined by infecting such cells with an
ecotropic retrovirus LXSE (Kandel et al., 1997, Id.) that carries
the gene for the Green Fluorescent Protein (GFP). Over 86% of
LXSE-infected cells were positive for GFP fluorescence (as
determined by flow cytometry), indicating a correspondingly high
infection rate. These cells were next transfected with the 3'SS
plasmid (Stratagene) that carries the LacI repressor (Fieck et al.,
1992, Nucleic Acids Res. 20: 1785-1791) and the hygromycin
resistance marker, and stable transfectants were selected with
hygromycin. The selected transfectants were subcloned, and 33
single-cell clones were individually tested for IPTG-regulated
expression of a LacI-inhibited promoter. This testing was carried
out by transient transfection of the cell clones with pCMVI3luc
plasmid (Stratagene) that expresses luciferase from the
LacI-regulated CMV promoter. As a positive control, the same assay
was carried out on a previously characterized well-regulated
fibrosarcoma cell line HT1080 3'SS6 (Chang and Roninson, 1996, Id.;
Chang et al., 1999, Id.). Three of the tested clones showed the
induction of luciferase expression in the presence of IPTG at a
level similar to that of HT1080 3'SS6.
[0105] These clones were further tested by the following assays.
The first assay was infection with LXSE ecotropic retrovirus,
followed by FACS analysis of GFP fluorescence, to determine the
susceptibility to ecotropic infection. The second assay was
ecotropic retroviral transduction with IPTG-regulated retrovirus
LNLucCO3 (Chang and Roninson, 1996), followed by G418 selection and
testing for IPTG inducibility of luciferase expression. The third
assay was the infection with IPTG-regulated ecotropic retrovirus
LNp21CO3 (Chang et al., 1999, Id.), which carries the cell cycle
inhibitor p21 (a positive control for an IPTG-inducible genetic
inhibitor), followed by BrdU suicide selection (described below) in
the presence and in the absence of IPTG. Based on the results of
these assays, a cell line called MDA-MB231 3'SS31 was selected as
being optimal for growth-inhibitory GSE selection. This cell line
showed about 80% infectability with ecotropic retroviruses,
approximately 10-fold inducibility by IPTG (which is higher than
the concurrently determined value for HT1080 3'SS6) and over
20-fold increase in clonogenic survival of BrdU suicide upon
infection with LNp21 CO3.
3. Isolation of Tumor Cell Growth Inhibiting Genetic Suppressor
Elements
[0106] The MCF-7 derived normalized tumor library in the LNXCO3
vector was transduced into MDA-MB231 3'SS31 cell line by ecotropic
retroviral transduction using the BOSC23 packaging cell line (Pear
et al., 1993, Id.), as described in Roninson et al. (1998, Methods
Enzymol. 292: 225-248). Two hundred million (2.times.10.sup.8)
recipient cells were infected and selected with G418. The infection
rate (as determined by the frequency of G418-resistant colonies)
was 36%. Eighty million (8.times.10.sup.7) G418-selected infectants
were subjected to selection for IPTG-dependent resistance to BrdU
suicide, as follows. Cells were plated at 10.sup.6 cells per P150
and treated with 50 .mu.M IPTG for 36 hrs, then with 50 .mu.M IPTG
and 50 .mu.M BrdU for 48 hrs. Cells were thereafter incubated with
10 .mu.M Hoechst 33342 for 3 hrs and illuminated with fluorescent
white light for 15 min on a light box, to destroy the cells that
grew and incorporated BrdU in the presence of IPTG. Cells were then
washed twice with phosphate-buffered saline and allowed to recover
in G418-containing medium without IPTG or BrdU for 7-10 days. The
surviving cells were then subjected to a second step of BrdU
selection under the same conditions. Control plates were selected
in the absence of IPTG, and representative plates were stained to
count the colonies; these results are shown in FIG. 4. The number
of surviving colonies after the second step of selection in the
presence of IPTG was approximately three times higher than the
corresponding number in the absence of IPTG. In contrast, control
cells infected with an insert-free LNXCO3 vector showed no
difference in BrdU survival in the presence or in the absence of
IPTG. As a positive control, cells were infected with
p21-expressing LNp21CO3, but the number of survivors in the
presence of IPTG was too high to count. These results demonstrated
that the frequency of library-infected cells that survived BrdU
suicide selection increased in IPTG-dependent manner, consistent
with successful selection of IPTG-inducible growth-inhibitory
GSEs.
[0107] Genomic DNA was isolated from the two-step selected
library-transduced cells and used as a template for PCR, using
vector-derived sequences flanking the inserts as primers. The
PCR-amplified mixture of inserts from the selected cells was
recloned into LNXCO3 vector and close to 3,000 randomly picked
plasmid clones from the library of selected fragments were
sequenced by high-throughput DNA sequencing by PPD Discovery, Inc.,
Menlo Park, Calif. 1482 clones containing human cDNA fragments were
identified among these sequences by BLAST homology search using the
NCBI database and analyzed to identify genes that gave rise to the
selected cDNA fragments. Ninety-three genes were found to give rise
to two or more of the sequenced clones, indicating the enrichment
for such genes in the selected library, with 67 genes represented
by three or more clones. Forty-nine of the enriched genes were
represented by two or more non-identical sequences. The sequences
of the enriched clones are provided in Table 4 and the Sequence
Listing. Many of these clones encode peptides derived from the
corresponding gene products. The sequences of these
growth-inhibitory peptides are provided in Table 5 and in the
Sequence Listing as SEQ ID NOS. 229-314. The enriched genes with
the corresponding accession numbers, as well as the numbers of
selected clones and different sequences derived from each genes are
listed in Table 1. Table 2 lists enriched genes previously known to
be involved in cell proliferation, and Table 3 lists enriched genes
that were not previously known to be involved in cell
proliferation.
[0108] The following criteria were used for assigning genes to
Table 2 or Table 3. The function of each gene was first confirmed
according to the corresponding entry in the LocusLink database of
NCBI. On the basis of this information, genes that are essential
for basic cell functions (such as general transcription or
translation), and genes known to play a role in cell cycle
progression or carcinogenesis were excluded from Table 3 and
assigned to Table 2. The functions of the other genes were then
investigated through a database search of the art, using all the
common names of the gene listed in LocusLink as keywords for the
search. Through this analysis, additional genes were assigned to
Table 2 by the following criteria (i) if overexpression of the
gene, alone or in combinations, was shown to promote neoplastic
transformation or cell immortalization; (ii) if inhibition of the
gene function or expression was shown to produce cell growth
inhibition or cell death; (iii) if homozygous knockout of the gene
was shown to be embryonic lethal in mammals; or (iv) if the gene
was found to be activated through genetic changes (such as gene
amplification, rearrangement or point mutations) in a substantive
fraction of any type of cancers. Genes that did not satisfy any of
the above criteria were then assigned to Table 3.
4. Analysis of Tumor Cell Growth Inhibiting Genetic Suppressor
Elements
[0109] Individual selected clones representative of enriched genes
have been analyzed by functional testing for GSE activity. Results
of these assays are summarized in Table 1. The principal assay
involves the transduction of individual putative GSE clones (in the
LNXCO3 vector) into MDA-MB-231-3'SS31 cells, followed by G418
selection of infected populations (for the neo gene of LNXCO3) and
testing the transduced populations for IPTG-dependent survival of
BrdU suicide. The latter assay was carried out as follows. Infected
cells (200,000 per P100, in triplicate) were treated with 50 .mu.M
IPTG for 72 hrs, then with 50 .mu.M IPTG and 50 .mu.M BrdU for 48
hrs. A parallel set of cells was treated in the same way but
without IPTG (in triplicate). Cells were then illuminated with
white light and allowed to recover in the absence of BrdU and IPTG
for 12-14 days. Results are expressed as the average number of
colonies per P100, with standard deviations. In each set of assays,
insert-free LNXCO3 vector was used as a negative control. As a
positive control, LNXCO3 vector expressing CDK inhibitor p21 was
used, but this control consistently gave excessively positive
values of surviving colonies. Alternative positive controls
comprised a GSE derived from a proliferation-associated
transcription factor Stat3, which produced moderate but
reproducibly positive results in multiple assays. Table 1 lists the
results of this assay (IPTG-dependent survival of BrdU suicide) as
positive ("A" in Functional Assays column) if t-test analysis of
the difference in the number of colonies surviving in the presence
and in the absence of IPTG provides a significance value of
P<0.05. Results of this analysis on a subset of positive GSEs
are shown in FIG. 5.
[0110] The assay for IPTG-dependent survival of BrdU suicide was
performed for GSEs derived from 38 genes with positive results.
Several infected cell populations that scored positive in this
assay were also tested by a more stringent assay for direct growth
inhibition by IPTG. None of the tested populations, however, showed
significant growth inhibition by IPTG. A similar result (positivity
in BrdU selection but not in the growth inhibition assay) was
reported by Pestov et al. (1998, Id.) for a weak growth-inhibitory
cDNA clone encoding a ubiquitin-conjugation enzyme. To determine
whether increased BrdU survival in such cell populations reflects
the heterogeneity of GSE expression and function among the infected
cells, multiple (10 or more) clonal cell lines were generated from
a subset of infected populations and tested for the ability to be
growth-inhibited by IPTG. Through this process, IPTG-inhibited cell
lines containing GSEs from 19 of the enriched genes were produced.
The genes that scored positive by this assay are indicated in Table
1 ("B" in Functional Assays column). In contrast to these
GSE-containing cell lines, cells transduced with an insert-free
LNXCO3 vector showed no growth inhibition in the presence of IPTG.
Results of IPTG growth inhibition assays with positive cell lines
are shown in FIG. 6.
[0111] Putative GSEs from 7 of the tested genes gave a greatly
diminished yield of G418-resistant infectants, relative to cells
infected with the control LNXCO3 virus or with other tested clones.
When the resulting small populations of G418-resistant cells
infected with these clones were expanded and tested for
IPTG-dependent survival of BrdU suicide, almost all of these
populations produced negative results. Remarkably, most of the
genes in this category ("C" in Functional Assays column of Table 1)
are known to be important positive regulators of cell growth (JUN
B, INT-2, MCM-3 replication protein, delta and eta isoforms of
protein kinase C) and therefore are expected to give rise to
growth-inhibitory GSEs. Since LNXCO3 vector is known to provide
substantial basal expression in the absence of IPTG (Chang and
Roninson, 1996), it seems likely that this group may include the
strongest functional GSEs, which inhibit cell growth even in the
absence of IPTG. Altogether, GSEs from a total of 51 genes have so
far been confirmed by functional assays (IPTG-dependent survival of
BrdU suicide or IPTG-dependent growth inhibition) or a putative
positive criterion (decreased apparent infection rate).
[0112] The genes shown in Table 2 are known to be positive
regulators of the cell growth or neoplastic transformation. These
include genes directly involved in cell cycle progression (such as
CCN D1 and CDK2) or DNA replication (e.g. PCNA, RPA3 or MCM-3),
growth factors (e.g. INT-2/FGF-3 and TDGF1) and growth factor
receptors (e.g. FGFR1, C-KIT), transcription factors known to be
positive regulators of cell proliferation (e.g. STAT3, c-FOS,
NF.kappa.B-1), several proliferation-associated signal transduction
proteins, such as three isoforms of PKC (the primary target of
tumor promoters) and three integrin proteins, as well as several
ribosomal components required for protein synthesis. The enriched
genes include many known protooncogenes, such as JunB and c-FOS
(which gave rise to two of three growth-inhibitory GSEs isolated by
Pestov and Lau (1994, Id.) from a 19-gene library in NIH 3T3
cells), a FOS-related gene, INT-2, c-KIT, LYN B (YES
protooncogene), MET, RAN (a member of RAS family), several
growth-promoting genes that are known to be amplified in cancers
(CCN D1, CDK2, FGFR1), and several genes reported to be
overexpressed in cancers. Some of the enriched genes have specific
associations with breast cancer, including INT-2, originally
identified as a mammary oncogene (Peters et al., 1984, Nature 309:
273-275), CCN D1 and FGFR1 found to be amplified in a substantial
minority of breast cancers (Barnes and Gillett, 1998, Breast Cancer
Res Treat. 52: 1-15; Jacquemier et al., 1994, Int. J. Cancer 59:
373-378), and HSPCA, which was shown to be expressed in all the
tested breast cancers (143 total) at a higher level than in
non-malignant breast tissue (Jameel et al., 1992, Int. J. Cancer
50: 409-415). The abundance of such genes among the selected
sequences provides strong validation of this approach to the
elucidation of positive growth regulators in breast carcinoma
cells.
[0113] The genes in Table 3 have no known function in growth
regulation. These genes encode several transcription factors,
proteins involved in signal transduction or cell adhesion, a number
of proteins involved in RNA transport or protein trafficking and
processing, a group of genes with miscellaneous other functions
that are not related to cell growth, and 10 genes, the functions of
which are presently unknown.
[0114] Of special interest, at least three of the genes in Table 3
appear to be inessential for growth of normal cells, since
homozygous knockout of these genes in mice does not prevent the
development of adult animals (except for some limited developmental
abnormalities). These genes include L1CAM (Dahme et al., 1997, Nat.
Genet. 17 346-349), ICAM2 (Gerwin et al., 1999, Immunity 10: 9-19),
and von Willebrand factor (Denis et al., 1998, Proc Natl Acad Sci
USA 95: 9524-9529). The effect of GSEs derived from these genes on
breast carcinoma cells suggests that inhibition of such
"inessential" genes may have a desirable tumor-specific or
tissue-specific antiproliferative effect.
[0115] A striking example of an apparently inessential gene
enriched in the selected library, which has been independently
identified as a highly promising target for breast cancer
treatment, is provided by HSPCA (included in Table 2). The basic
function of this gene, which belongs to of a heat shock responsive
family of chaperone proteins, which play a role in refolding of
mature proteins, does not indicate that it should be required for
cell growth. HSPCA, however, was found to play a role in
stabilizing several proteins that are involved in oncogenic
pathways, including Raf, Met, steroid receptors, and members of the
HER kinase family, and to serve as the target of an antitumor
antibiotic geldanamycin (Stebbins et al., 1997, Cell 89: 239-250).
The HSPCA-inhibiting geldanamycin analog 17-AAG has been shown to
arrest the growth of breast carcinoma cell lines (including
MDA-MB-231; Munster et al, 2001, Cancer Res. 61: 2945-2952) and to
sensitize such cells to chemotherapy-induced apoptosis (Munster et
al., 2001, Clin Cancer Res 7: 2228-2236); 17-AAG is currently in
clinical trial. The example of HSPCA suggests that other apparently
inessential genes identified by GSE selection are likely to provide
similarly promising targets for cancer treatment. Some of these
potential novel targets are described in more detail in the next
section.
5. Potential Novel Drug Targets.
[0116] Several of the selected genes warrant consideration as
potential novel targets for cancer drug development. Non-limiting
examples are as follows.
[0117] L1CAM. L1 cell adhesion molecule (L1CAM) is represented in
the set of growth-inhibiting GSEs by eight sense-oriented and four
antisense-oriented GSEs. L1CAM is a 200-220 kDa type I membrane
glycoprotein of the immunoglobulin superfamily expressed in neural,
hematopoietic and certain epithelial cells. The non-neuronal
(shortened) form of L1CAM is expressed highly in melanoma,
neuroblastoma, and other tumor cell types, including breast. L1CAM
is found not only in membrane-bound form but also in the
extracellular matrix of brain and tumor cells. Soluble L1CAM
directs the migration of glioma cells, and one of anti-L1CAM
antibodies was found to inhibit this migration (Izumoto et al.,
1996, Cancer Res. 56: 1440-1444). Such an antibody might be useful
as an initial prototype agent to validate L1CAM as a cancer drug
target.
[0118] As a cell surface molecule, L1CAM should be easily
accessible to different types of drugs. FIGS. 7A and 7B illustrate
morphological effects of an L1CAM-derived GSE in a clonal
IPTG-inhibited cell line. Four-day treatment with IPTG drastically
altered cell morphology, with the cells developing lamellipodia and
apparent focal adhesion plaques (FIG. 7A). This effect suggests
that the IPTG-induced GSE affects cell adhesion, as would have been
expected from targeting L1CAM. GSE induction not only arrested cell
growth but also induced mitotic catastrophe in 15-20% of
IPTG-treated cells. Mitotic catastrophe is a major form of tumor
cell death (Chang et al., 1999, Id.), which is characterized by
abnormal mitotic figures and formation of cells with multiple
micronuclei (FIG. 7B). The ability of a GSE to induce mitotic
catastrophe is a good general indication for the potential promise
of a GSE-inhibited target.
[0119] Human L1CAM gene is mutated in patients with a severe
X-linked neurological syndrome (CRASH: corpus callosum hypoplasia,
retardation, aphasia, spastic paraplegia and hydrocephalus). L1CAM
"knockout" (-/-) mice develop to adulthood and appear superficially
normal (slightly smaller than adults), but they have a shortened
lifespan due to CRASH-like neurological deficits, which may be
related to a decrease in neurite outgrowth (Dahme et al., 1997,
Id.). These observations suggest that targeting L1CAM in an adult
cancer patient should not have major toxicity outside of the
nervous system, where most drugs will not penetrate due to the
blood-brain barrier. Furthermore, it is quite likely that the
neurological effects result only from a lack of L1CAM during
embryonic development and would not develop from L1CAM inhibition
in an adult.
[0120] ICAM2. The intercellular cell adhesion molecule-2 (ICAM2) is
represented in the set of growth-inhibiting GSEs by two
sense-oriented and one antisense-oriented GSE. ICAM2 has many
similarities to L1CAM and is also inessential for the growth of
normal cells (Gerwin et al., 1999, Id.). Anti-ICAM2 antibodies, for
example, are attractive possibilities for prototype drugs.
[0121] NIN283. This gene has recently been described (Araki et al.,
2001, J. Biol. Chem. 276: 34131-34141) as being induced in Schwann
cells upon nerve injury and termed NIN283. Induction of NIN283 is a
part of injury response of Schwann cells, which then act to promote
the growth of the injured nerve. NIN283 is also induced by nerve
growth factor (NGF). Like L1CAM, NIN283 is expressed primarily in
the brain. It is localized to lysosomes, is highly conserved in
evolution (with identifiable homologs in Drosophila and C.
elegans), and contains a unique combination of a single zinc finger
and a RING finger motif. Based on these structural features and
localization, Araki et al. (2001, Id.) speculated that NIN283 may
be involved in ubiquitin-mediated protein modification and
degradation. With this putative function in protein modification,
stress inducibility and evolutionary conservation, NIN283 appears
analogous to the above-discussed HSPCA.
[0122] Here, this gene was found to give rise to one of the
strongest functionally active GSEs in breast carcinoma
growth-inhibition assays. The available information on functional
domains of NIN283 should be useful in structure-based rational
design of small molecule inhibitors of this interesting
protein.
[0123] ATF4. Activating transcription factor 4 gave rise to the
most highly enriched antisense GSE in these selection assays.
Homozygous knockout of ATF4 results in only minor developmental
abnormalities (in the eye lens; Tanaka et al., 1998, Genes Cells 3:
801-810; Hettmann et al., 2000, Dev. Biol. 222: 110-123),
indicating that this factor is not essential for normal cell
growth. The results disclosed herein implicate ATF4 in breast
cancer cell proliferation and are strengthened by reports in the
art that ATF4 expression and function are augmented by heregulin
.beta.1, a factor that stimulates the growth of breast cancer cells
(Talukder et al., 2000, Cancer Res. 60: 276-281).
[0124] Zinedin. Zinedin is a recently described calmodulin-binding
protein with a WD repeat domain, which is preferentially expressed
in the brain (Castets et al., 2000, J. Biol. Chem. 275:
19970-19977). This expression pattern suggests that
zinedin-targeting drugs are unlikely to have an effect on any
normal proliferating cells. An antisense-oriented GSE derived from
zinedin, however, was found herein to inhibit breast carcinoma cell
growth, both by the IPTG-dependent BrdU suicide assay and by the
ability to give rise to an IPTG-inhibited cell line. Structural
analysis of zinedin indicates specific domains that apparently
mediate its interactions with calmodulin and caveolin (Castets et
al., Id.). Structure-based targeting of these domains, as well as
screening based on the interference with zinedin-calmodulin
interactions, can be used as strategies for developing
zinedin-targeting drugs.
[0125] Novel genes. Several genes identified by this selection have
no known function, no significant homologies with known genes or
identifiable functional domains. These results provide the first
functional evidence for such genes. One of the most highly enriched
and functionally active GSEs is designated GBC-1 (Growth of Breast
Carcinoma 1). Translated protein sequence of GBC-1 matches a
partial sense-oriented sequence of a hypothetical unnamed protein
(accession No. XP.sub.--031920). GBC-1 GSE encodes a helical-repeat
peptide. The strong growth-inhibitory activity of this GSE suggests
that molecules derived from or mimicking this peptide are likely to
have antitumor activity. The GBC-1 peptide disclosed herein can be
regarded as a prototype drug, the structure of which can be used to
direct rational design of a synthetic compound.
[0126] Among other novel genes identified in the instant invention,
two genes, designated herein GBC-3 (Growth of Breast Carcinoma 3)
and GBC-11 (Growth of Breast Carcinoma 11) are the most highly
enriched, and their GSEs show strong functional activity. Cell
lines that comprise these GSE and that are efficiently
growth-inhibited by treatment with IPTG are useful for
characterizing the cellular effects of GBC-3 or GBC-11 inhibition.
GBC-3 matches an otherwise uncharacterized EST AA443027 and maps to
chromosome 3q29, GBC-11 maps to chromosome 14 and does not match
any known cDNA sequences. GBC-3 appears according to "Virtual
Northern" analysis carried out using the NCBI SAGE database
(http://www.ncbi.nlm.nih.gov/SAGE/sagevn.cgi) to be expressed at a
very low level in all cell types, suggesting that it may be an easy
target to inhibit.
6. In Vivo Testing of Test Compounds
[0127] The efficacy of inhibiting expression or activity of the
genes set forth in Table 3 is tested in vivo as follows.
[0128] Cells (1-2.times.10.sup.6) expressing an IPTG-inducible GSE
of the invention that inhibits expression or activity of a gene in
Table 3 are injected into a mouse as a xenograft, most preferably
in one flank of the mouse so that tumor growth can be visually
monitored. IPTG-regulated gene expression in mouse xenografts of
MDA-MB-231 breast carcinoma has been demonstrated in the art, for
example by Lee et al. (1997, Biotechniques 23: 1062-1068) and the
experiments described herein can be performed substantially as
described by Lee et al. but using the GSE-containing tumor cells of
the invention. Conveniently, GSE-naive tumor cells are injected in
the opposite flank in each mouse. Two sets of injected mice are
housed and maintained in parallel, with one set of mice having feed
supplemented with IPTG at a concentration as taught by Lee et al.
and the other set of mice not receiving IPTG supplemented food.
Emergent tumors are observed on the mice under humane animal care
conditions until the extent of tumor cell growth is
life-threatening or inhumane. Biopsy samples are taken and the
tumors measured and weighed after animal sacrifice to determine
differences between the GSE-expressing and non-GSE-expressing
tumors in each mouse and between mice fed IPTG and mice without
IPTG supplementation.
[0129] IPTG-fed mice will bear one tumor of naive xenograft cells
whose growth is unaffected by IPTG. These tumors will be
substantially identical to the size of both naive xenograft cell
and GSE-containing xenograft cell tumor in mice not fed IPTG. In
contrast, the tumor produced from the GSE-containing xenograft
cells in mice fed IPTG will be substantially smaller than the other
tumors. Biopsy will show proliferating tumor cells in both naive
xenograft cell and GSE-containing xenograft cell tumor in mice not
fed IPTG and naive xenograft cells from IPTG-fed mice, and
quiescent or dying cells in the GSE-containing xenograft tumor.
[0130] These results demonstrate that inhibition of expression or
activity of genes set forth in Table 3 inhibits tumor cell growth
in vivo.
[0131] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims. TABLE-US-00001 TABLE 1 Genes Enriched among 1482 Sequences
of Clones Containing cDNA Inserts in the Selected Library
#Sequences Functional Gene Accession # (s/as) # clones Assays* ATF4
NM_001675.1 5(as) 369 A STAT5b NM_012448.1 4(s), 4(as) 152 A, B
GBC-1 NM_031221.1 2 (s) 70 A, B ARHG NM_001665.1 5(s), 1(as) 43 A
VWF NM_000552.2 6(s), 5(as) 39 B MCM3 NM_002388.2 3(s), 4(as) 38 C
18S RNA K03432.1 8(s), 4(as) 33 A ITGB5 NM_002213.1 4(s), 1(as) 30
A, B HSPCA NM_005348.1 2(s) 27 B STAT3 NM_003150.1 4(s), 3(as) 25
A, B L1CAM NM_000425.2 8(s), 4(as) 20 A, B 28S RNA M27830.1 3 (s)
17 A C-FOS NM_005252.2 3(s), 3(as) 17 A C-KIT NM_021099.2 4(s),
2(as) 12 A FEN1 NM_004111.3 2(s), 2(as) 12 A GBC-3 AA443027 1(s) 12
A, B NIN283 NM_032268 1(s) 11 A ADPRT NM_001618 1(s), 1(as) 10 CCN
D1 NM_001758.1 2(s), 2(as) 9 A CDC20 NM_001255 1(as) 9 B EFNA1
NM_004428 1(s), 3(as) 9 A KIAA1270 XM_044835 1(as) 9 A RPL31
NM_013403.1 2(s) 9 A, B 7SL X04248.1 4(s), 1(as) 8 C ENO1 NM_001428
2(s) 8 GSTP NM_000852 2(s) 8 ICAM2 NM_000873 2(s), 1(as) 8
INT-2/FGF3 NM_005247 2(s) 8 C LYN NM_002350 2(as) 8 A RPS24
NM_001026 1(s), 1(as) 8 FGFR1 NM_000604.2 2(s), 1(as) 6 A HES6
XM_043579 1(s) 6 B PKC zeta NM_002744 2(s), 1(as) 6 B RAN NM_006325
1(s) 6 RPA3 NM_002947.1 1(s) 6 A ZIN NM_013403.1 1(as) 6 A, B TAF7
NM_005642 1(s) 6 A AP1B1/BAM22 NM_001127.1 2(s) 5 A HNRPF NM_004966
1(s) 5 A HNRPMT AF222689 1(s) 5 A NFkB-1 NM_003998.1 1(as) 5 A, B
NR3C1 NM_000176 1(s) 5 A PKC delta NM_006254.1 2(s), 1(as) 5 C
BAG-1 NM_004323.2 2(s) 4 A GBC-11 W84777 1(s) 4 A, B HNRPA2B1
NM_002137 1(s) 4 A IF1 NM_016311.1 1(s) 4 A ITGA4 NM_000885 1(s),
1(as) 4 JunB NM_002229.1 1(s) 4 C GRP58 NM_005313.1 1(s), 1(as) 4
PKC eta NM_006255.1 3(s), 1(as) 4 A, B, C PSMB7 NM_002799 1(s) 4
RAB2L NM_004761 1(s) 4 RPL35 NM_004632.1 2(as) 4 C CDK2 NM_001798.1
2(s) 3 A DAP-3 NM_004632.1 2(as) 3 A, B EIF-3 NM_003750 3(s) 3 A
GBC-12 1(s) 3 A IGF2R NM_000876 2(s) 3 KIFC1 XM_042626 1(as) 3 MET
NM_031517 2(s), 1(as) 3 PCNA NM_002592 1(s) 3 PPP2R1B NM_002716
2(as) 3 RAB5B NM_002868.1 1(s), 1(as) 3 TDGF1 NM_003212 1(as) 3
ARFAPTIN1 NM_014447 1(as) 2 CDK10 NM_003674 2(s) 2 B CREB1
NM_004379 1(s) 2 EDF-1 NM_003792 1(s) 2 FLJ10006 XM_041928 1(as) 2
ELJ13052 NM_023018 1(s) 2 FOSL2 NM_005253.1 1(s), 1(as) 2 GBC-13
1(s) 2 GBC-14 AL557138 1(s) 2 GBC-15 BE079876 1(s) 2 GBC-16 1(s) 2
GBC-17 1(s) 2 GBC-18 1(s) 2 GNAS M21139 1(as) 2 IL4R NM_000418
1(as) 2 ITGA3 NM_002204 1(as) 2 MAP2K2 NM_002755 2(as) 2 MBD-1
NM_015847 1(s), 1(as) 2 B MCM-6 NM_005915 1(s) 2 MYL6 NM_021019
2(s) 2 A NUMA1 NM_006185 1(s) 2 PC4 NM_006713 1(s) 2 RAD23A
NM_005053 1(s) 2 REL NM_002908 1(s) 2 RPA1 NM_002945 1(as) 2 RPL12
NM_000976 1(s) 2 RPS29 NM_001032 1(s) 2 SQSTM1 NM_003900 1(s) 2 *A,
confirmed by BrdU suicide assay; B, gave rise to cell line
inhibited by IPTG; C, low infection rate
[0132] TABLE-US-00002 TABLE 2 Enriched Genes Previously Implicated
in Cell Proliferation #Sequences # Gene Accession No. (s/as) clones
Description Association with cancer CCN D1 NM_001758 2(s), 2(as) 9
Cyclin, G1/S transition Amplified in cancers CDK2 NM_001798 2(s) 3
Cyclin-dependent kinase, S- Amplified in cancers phase PCNA
NM_002592 1(s) 3 DNA replication Upregulated in cancers RPA3
NM_002947 1(s) 6 DNA replication, excision repair RPA1 NM_002945
1(as) 2 DNA replication MCM3 NM_002388 3(s), 4(as) 38 DNA
replication MCM6 NM_005915 1(s) 2 DNA replication FEN1 NM_004111
2(s), 2(as) 12 DNA replication and repair CDC20 NM_001255 1(as) 9
CDC2-related kinase, mitosis NUMA1 NM_006185 1(s) 2 Nuclear
reassembly in late mitosis RAN NM_006325 1(s) 6 Small GTPase,
mitosis Ras family CDK10 NM_003674 2(s) 2 Cell cycle, G2/M C-KIT
NM_021099 4(s), 2(as) 12 Growth factor receptor, Protooncogene
oncogene EFN A1 NM_004428 1(s), 3(as) 9 Receptor tyrosine kinase
ligand RAS pathway regulator LYN NM_002350 2(as) 8 Tyrosine kinase
YES protooncogene INT-2/FGF-3 NM_005247 2(s) 8 Fibroblast growth
factor Mammary oncogene FGFR1 NM_000604 2(s), 1(as) 6 Fibroblast
growth factor Amplified in breast cancers receptor, tyrosine kinase
IGF2R NM_000876 2(s) 3 Insulin-like growth factor 2 Mutated in
breast cancers receptor TDGF1 NM_003212 1(as) 3 Teratocarcinoma
derived growth Overexpressed in teratocarcinomas factor 1 (EGF
family) MET NM_031517 2(s), 1(as) 3 Hepatocyte growth factor
Protooncogene receptor IL4R NM_000418 1(as) 2 Interleukin-4
receptor STAT3 NM_003150 4(s), 3(as) 25 Transcription factor
Upregulated in breast ca (proliferation) STAT5b NM_012448 4(s),
4(as) 152 Transcription factor (proliferation) C-FOS NM_005252
3(s), 3(as) 17 AP-1 component Protooncogene NF.kappa.B-1 NM_003998
1(as) 5 Stress, apoptosis, paracrine activities TAF7 NM_005642 1(s)
6 Transcription initiation factor PC4 NM_006713 1(s) 2 General
positive coactivator of transcription CREB1 NM_004379 1(s) 2
Transcription factor, regulates expression of cAMP-inducible genes
including Cyclin A JUNB NM_002229 1(s) 4 AP-1 component
Protooncogene FOSL2 NM_005253 1(s), 1(as) 2 AP-1 component
FOS-related REL NM_002908 1(s) 2 Transcription factor Protooncogene
ADPRT NM_001618 1(s), 1(as) 10 Poly (ADP ribosyl) transferase PKC
zeta NM_002744 2(s), 1(as) 6 Serine/threonine protein kinase
Stimulated by tumor promoters PKC delta NM_006254 2(s), 1(as) 5
Serine/threonine protein kinase Stimulated by tumor promoters PKC
eta NM_006255 3(s), 1(as) 4 Serine/threonine protein kinase
Stimulated by tumor promoters MAP2K2 NM_002755 2(as) 2 MAP kinase
kinase Implicated in medulloblastoma metastasis GRP58 NM_005313
1(s), 1(as) 4 Membrane signal transduction PPP2R1B NM_002716 2(as)
3 Protein phosphatase 2 regulatory subunit .beta. BAG1 NM_004323
2(s) 4 Apoptosis inhibitor (Bcl-2 Overexpressed in cancers family)
DAP3 NM_004632 2(as) 3 Positive/negative apoptosis Overexpressed in
gliomas regulator ITGA4 NM_000885 1(s), 1(as) 4 Cell adhesion,
signal Involved in Src pathway transduction ITGA3 NM_002204 1(as) 2
Cell adhesion, signal Involved in colorectal cancer growth
transduction ITGB5 NM_002213 4(s), 1(as) 30 Cell adhesion, signal
Correlates with invasiveness in transduction gastric ca AHRG
NM_001665. 5(s), 1(as) 43 Small GTPase, cytoskeletal Ras family,
contributes to Ras reorganization transforming activity GNAS
complex M21139 1(as) 2 G-protein alpha subunit s, knockout is
embryonic lethal HSPCA NM_005348 2(s) 27 Chaperone, protein folding
Overexpressed in breast ca, activates tyrosine kinases EIF-3
NM_003750 3(s) 3 Translation initiation factor RPL31 NM_013403 2(s)
9 Ribosomal protein L31 RPL35 NM_004632 2(as) 4 Ribosomal protein
L35 RPL12 NM_000976 1(s) 2 Ribosomal protein L12 RPS29 NM_001032
1(s) 2 Ribosomal protein S29 RPS24 NM_001026 1(s), 1(as) 8
Ribosomal protein S24 18S RNA K03432.1 8(s), 4(as) 33 Ribosomal RNA
28S RNA M27830 3(s) 17 Ribosomal RNA 7SL X04248 4(s), 1(as) 8 RNA
component of signal recognition particle
[0133] TABLE-US-00003 TABLE 3 Enriched Genes That Have Not Been
Previously Implicated in Cell Proliferation #Sequences #
Association with Gene Accession No. (s/as) clones Description
cancer Transcription factors ATF4 NM_001675 5(as) 369 Activating
transcription factor Induced in breast ca by heregulin HES6
XM_043579 1(s) 6 Transcription co-factor, differentiation inducer
NR3C1 NM_000176 1(s) 5 Glucocorticoid receptor EDF1 NM_003792 1(s)
2 Transcription factor, stimulates endothelial cell growth,
represses endothelial cell differentiation MBD1 NM_015847 1(s),
1(as) 2 Methylated DNA binding protein, transcription inhibitor RNA
transport HRPMT1L2 NM_001536 1(s) 5 Hnrp arginine methyltransferase
HNRPF NM_004966 1(s) 5 Heterogeneous nuclear ribonucleoprotein F
HNRPA2B1 NM_002137 1(s) 4 Heterogeneous nuclear ribonucleoprotein
A2/B1 Signal transduction and cell adhesion ZIN NM_013403 1(as) 6
Calmodulin-binding WD repeat protein Arfaptin1 NM_014447 1(as) 2
Similar to POR1 GTP-binding protein; may act in cellular membrane
ruffling and formation of lamellipodia L1CAM NM_000425 8(s), 4(as)
20 Cell adhesion, neural ICAM2 NM_000873 2(s), 1(as) 8 Cell
adhesion, intercellular Intracellular transport AP1B1/BAM22
NM_001127 2(s) 5 Clathrin-associated adaptor protein RAB2L
NM_004761 1(s) 4 Small GTPase, intracellular transport Ras family
KIFC1 XM_042626 1(as) 3 Intracellular trafficking Rab5B NM_002868
1(s), 1(as) 3 Small GTPase, vesicle transport Ras family Protein
processing NIN283 NM_032268 1(s) 11 ubiquitin-mediated protein
modification PSMB7 NM_002799 1(s) 4 Proteasome subunit .beta.7
SQSTM1 NM_003900 1(s) 2 Sequestosome 1; ubiquitin-mediated protein
degradation RAD23A NM_005053 1(s) 2 Nucleotide excision repair,
ubiquitin-mediated protein degradation Other VWF NM_000552 6(s),
5(as) 39 Blood clotting GSTP NM_000852 2(s) 8 Xenobiotic metabolism
ENO1 NM_001428 2(s) 8 Glycolysis IF1 NM_016311 1(s) 4 Inhibitor of
Fo/F1 mitochondrial ATPase MYL6 NM_021019 2(s) 2 Contractility
FLJ13052 NM_023018 1(s) 2 NAD kinase (predicted) GBC-14 AL557138
1(s) 2 similar to tyrosine 3- monooxygenase/tryptophan 5-
monooxygenase activation protein, zeta polypeptide KIAA1270
XM_044835 1(as) 9 Alanyl-tRNA synthetase homolog IGF2R NM_000876
2(s) 3 Insulin-like growth factor 2 receptor Mutated in breast
cancers Unknown function GBC-1 NM_031221 2(s) 70 Contains helical
repeat peptide FLJ10006 XM_041928 1(as) 2 GBC-3 AA443027 1(s) 12 HC
3q29 GBC-11 1(s) 4 HC 14 GBC-12 1(s) 3 HC 1 GBC-13 1(s) 2 GBC-15
BE079876 1(s) 2 GBC-16 1(s) 2 GBC-17 1(s) 2 GBC-18 1(s) 2
[0134] TABLE-US-00004 TABLE 4 Nucleotide Sequences of GSEs SEQ
Gene/Acces- No. of Orien- ID sion No. Clones tation NO Sequence 18S
RNA 1 AS 4 1089
gccgctagaggtgaaattccttggaccggcgcaagacggaccagagcgaaagcatttgccaagaatgtt
K03432.1
ttcattaatcaagaacgaaagtcggaggttcgaagacgatcagataccgtcgtagttccgaccataaac
gatgccgaccggcgatgcggcggcgttattcccatgacccgccgg 1271 2 AS 5 1413
ccggacacggacaggattgacagattgatagctctttctcgattccgtgggtggtggtgcatggccgtt
cttagttggtggagcgatttgtctggttaattccgataacgaacgaga 1529 6 S 6 177
caaagattaagccatgcatgtctaagtacgcacggccggtacagtgaaactgcgaatggctca-
ttaaat cagttatggttcctttggtcgct 268 7 S 7 1414
cggacacggacaggattgacagattgatagctctttctcgattccgtgggtggtggtgcatggccgttc
1482 4 AS 8 154
ctgccagtagcatatgcttgtctcaaagattaagccatgcatgtctaagtacgcacggccggtac
218 1 AS 9 199
taagtacgcacggccggtacagtgaaactgcgaatggctcattaaatcagttatggt 255 2 S
10 570 cggagagggagcctgagaaacggctaccacatccaaggaaggca 613 3 S 11 177
caaagattaagccatgcatgtctaagtacgcacggccggta 217 1 S 12 1040
cggaactgaggccatgattaagagggacggccggg 1074 1 S 13 1433
cagattgatagctctttctcgattccgtgggtggt 1467 1 S 14 224
aactgcgaatggctcattaaatcagttatggttcctttggtcgct 268 4 S 15 185
aagccatgcatgtctaagtacgcacggccg 214 28S RNA 10 S 16 83
ccctactgatgatgtgttgttgccatggtaatcctgctcagtacgagaggaaccgcaggttcagacatt
M27830.1 tggtgtatgtgcttggctgaggagccaatggggcgaacgtaccatctgt 200 4 S
17 1 gaattcaccaagcgttggattgttcacccactaatagggaacgtgagct 49 3 S 18
136 cgcaggttcagacatttggtgtatgtg 162 7SL RNA 3 S 19 29
cccagctactcgggaggctgaggctggaggatcgcttgagtccaggagttctgggctgtagtgcgctat
X04248.1 gccgatcgggtgtccgcactaagttcggcatcaatatgg 136 1 S 20 70
ccaggagttctgggctgtagtgcgctatgccgatcgggtgtccgcactaagttcggcatcaat-
atggt 137 3 S 21 144 ccgggagcgggggaccaccaggttgcctaaggaggggtga 183 9
AS 22 24 gtagtcccagctactcgggaggctgaggctggaggatcgcttga 67 3 S 23 153
ggggaccaccaggttgcctaaggaggggtga 183 ADPRT 9 S 24 2736
gctgtggcacgggtctaggaccaccaactttgctgggatcctgtcccagggtcttcggatagccc
NM_001618 cgcctgaagcgcccgtgacag 2821 1 AS 25 2422
gaccctcccctgagcagactgtaggccacctcgatgtccagcaggttgtcaagcatttcc
accttggcctgcacactgtctgc 2504 ARFAPTIN1 2 AS 26 26
ttcacactgaccaaccgccgaggacagtcggaccggcgacctctcaacccagcc 79 NM_014447
ATF4 359 AS 27 833
acaccttcgaattaagcacattcctcgattccagcaaagcaccgcaacatgaccgaaatgagcttcctg
NM_001675.1 agcagcg 909 6 AS 28 833
gacaccttcgaattaagcacattcctcgattccagcaaagcaccgcaaca 883 2 AS 29 838
----ccttagaattaagcacattcctcgattccagcaaagcgccgcaacatgacggaaa 893 1
AS 30 843
---------gaattaagcactttcctcgagtccagcaaagccccgca------------ 880 1
AS 31 864 cgctgctcagcaagctctgttcggtcatgttgcggtgctttgctgg 909 IF1 4
S 32 13 ccagcagcaatggcagtgacggcgttggcggcgcggacgtggcttggcgtgtggggc
69 NM_016311.1 BAG1 3 S 33 434
ccgggacgaggagtcgacccggagcgaggaggtgaccagggaggaaatggcggcagctgggctcaccgt
NM_004323.2 gactgtcacccacagc 518 1 S 34 461
ggaggtgaccagggaggaaatggcggcagctgggctcaccgtgactgtcacccacagc 518
AP1B1 5 S 35 275
gccaagagtcagcctgacatggccattatggccgtcaacacctttgtgaaggactgtgagga 336
NM_001127.1 1 S 36 286
gcctgacatggccattatggccgtcaacacctttgtgaaggactgtgag 334 CDC20 4 AS 37
1001
gccagggacaccatgctacggccttgacagccccttgatgctgggtgaatgtctgcagaggaa
NM_001255
cccagccaccctctccaggagcactgggccacacattgaccaagttatcattaccaccactggccaaat
gtcgtccatctggggcccagcgcagcccacacacttcctggctgtggccactcagtgtggccacatggt
gttctgct 1209 CDK10 1 S 38 1159
gccccagccacctccgagggccagagcatgcgctgtaaacc 1199 NM_003674 1 S 39
1734
ctaccaggagagccctgggctggaggctgagctgcatccctgctccccacatggaggacccaa
caggaggccgtggctctgatgctgagcgaagct 1829 CDK-2 2 S 40 322
agatctctctgcttaaggagcttaaccatcctaatattgtcaagctg 368 NM_001798.1 1 S
41 645 tacacccatgaggtggtgaccctgtggtaccgagctcctgaaatcctcctgggctgca
702 c-FOS 1 AS 42 347
cactgccatctcgaccagtccggacctgcagtggctggtgcagcccgccctcgtctcctctgtggcccc
NM_005252.2 atcgcagaccagagcccctcaccctttcggagtccccgccccc 458 1 AS 43
246
cactcacccgcagactccttctccagcatgggctcgcctgtcaacgcgcaggacttctgcacggacctg
gcc 317 12 S 44 57
agcgaacgagcagtgaccgtgctcctacccagctctgcttcacagcgcccacctgtctccgcccct
122 1 S 45 1342
gcccgagctggtgcattacagagaggagaaacacatcttccctagagggttcctgtagacc taggg
1407 1 AS 46 717
gaggcagggtgaaggcctcctcagactccggggtggcaacctctggcaggcccccagtcagatca
agggaagccacagacatctcttctgggaagcccaggtcatcagggatcttgcaggcgggtcggtgagct
gccaggatgaactctagtttttccttctcctt 882 1 S 47 596
taagatggctgcagccaaatgccgcaaccggagga 630 c-KIT 2 AS 48 2448
gcgatttcgggctagccagagacatcaggaatgattcgaattacgtggtcaaaggaaatgcacgactgc
NM_021099.2 ccgtgaagtggatggcaccagagagcattttcagctgcg 2555 4 AS 49
2632
cccagggatgccggtcgactccaagttctacaagatgatcaaggaaggcttccggatggtcagcccgga
gcacgcgcctgccgaaatgtatgacgtcatgaagacttgctgggacg 2747 2 S 50 3466
aacggggcatcggaagtctggtcacgctaagaagaccgaggctgagaaggaacaagccaggggaagcgt
ga 3536 1 S 51 4650
gctggtttggaggtcctgtggtcatgtacgagactgtcaccagttaccgcgctctgtttgaaacatgtc
4718 2 S 52 3508
tgagaaggaacaagccaggggaagcgtgaacaatgatgctctgctctgggctgccgctcgggcttct
gtacaactgacctggttt 3592 1 S 53 3595
gaacaagccagggaagcgtgaacaatgatgctctgctctgggctgccgctcgggcttctgtacaac
tgacctggtttctc 3515 CREB1 2 S 54 199
aagcccagccacagattgccacattagcccaggtatctatgccagcagctcatgcaacatcatctg
NM_004379 264 CCND1 6 S 55 311
tgcggaagatcgtcgccacctggatgctggaggtctgcgaggaacagaagtgcgaggaggaggtcttcc
NM_001758.1 cgctggccatgaactacctggaccgcttcctgtcgctgg 418 1 S 56 935
agaacatggaccccaaggccgcc 957 2 AS 57 331
tggatgctggaggtctgcgaggaacagaagtgcgaggaggaggtcttc
ccgctggccatgaactacctggaccgcttcctg 411 1 58 406
cacagcttctcggccgtcagggggatggtctccttcatcttagaggccacgaacatgcaagtg-
gccc ccagcagctgcaggcggctctttttcacgggctccagcgacaggaa 518 DAP3 2 AS
59 1249
gcggcactgtgcctacctctaagccaagatcacagcatgtgaggaagacagtggacatctgctttatgc
NM_004632.1
tggacccagtaagatgaggaagtcgggcagtacacaggaagaggagccaggcccttgtacctatgggat
tggacaggactgcagttggctctggacctgc 1417 1 AS 60 1259
gcctacctctaagccaagatcacagcatgtgaggaagacagtggacatctgctttatgctggacccagt
aagatgaggaagtcgggcagtacacaggaagaggagccaggcccttgtacctatgggattggacaggac
tgcagttggctctggacctgc 1417 EDF1 2 S 61 97
ggccaaatccaagcaggctatcttagcggcacagagacgaggaggagat 145 NM_003792
eIF-3 1 S 62 3259
ggcgaggaggcgctgatgatgagcgatcatcctggcgtaatgctgatgatgaccggggtcccaggcgag
NM_003750 ggttggatga 3337 1 S 63 40 gcagcgttgggcccatgcaggacgc 64 1
S 64 269 cagcttcaggcagaaacagaaccaa 293 ENO-1 7 S 65 5
agatctcgccggctttacgttcacctcggtgtctgcagcaccctccgcttcctctcctaggcgacg
70 NM_001428 1 S 66 11
cgccggctttacgttcacctcggtgtctgcagcaccctccgcttcct 57 EFNA1 5 S 67 228
cgcactatgaagatcactctgtggcagacgctgccatggagcagtacatactgtacctggtggagca
NM_004428 tgaggagtaccagctgt 311 2 AS 68 517
tgctgcaagtctcttctcctgtggattgacatgggcctgaggactgtgagtgattttgcca 577 1
AS 69 1183
tggcacagcccccctgctggcacagctctggggagtgctgccccaggatgggagagaatgcagtacctg
gctacaaacttctctgtggcagctccacagatgaggtctt 1291 1 AS 70 467
gacagtcaccttcaacctcaagcagcggtcttcatgctggtggatggg 514 FEN1 5 S 71
634
gccacagctcaagtcaggcgagctggccaaacgcagtgagcggcgggctgaggcagagaagcagctgca
NM_004111.3 gcaggctcaggctgctgg 720 4 AS 72 841
ggcagaggccagctgtgctgccctggtgaaggctggcaaagtctatgctgcggctaccga 900 2
AS 73 634 gccacagctcaagtcaggcgagctggccaaacgcagtgagcggc 677 1 S 74
651
gcgacctggacaaacgcattgagcggcggcctgaggcagagaagcagctgtatcatgctcaagctgctg
g 720 FGFR1 1 S 76 2004
ggtaacagtgtctgctgactccagtgcatccatgaactctggggttcttctggttcggccatcacggct
NM_000604.2
ctcctccagtgggactcccatgctagcaggggtctctgagtatgagcttcccgaagaccctcgc
gggagctgcctcgggacagactggtcttaggc 2169
1 AS 77 2844
ggaggaacttttcaagctgctgaaggagggtcaccgcatggacaagcccagtaactgcaccaacgagct
gtacatgatgatgcgggactgctggcatgcagtgccctcacagagacccaccttcaagcagctggt
2978 4 S 78 1930
ggtaccaagaagagtgacttccacagccagatggctgtgcacaagctggccaagagcatcctctgcgca
gacaggtaacagtgtctgctgactccagtg 2029 GBC-1 68 S 79 876
tcctcacatcccagacgatgggcggccaggcagagacgctcctcacttcccagacggggtagcggccg
XM_031920 943 2 S 80 876
tcctcacatcccagacgatgggcggccaggcagagacgctcctcacttcccag 928 FLJ10006
2 AS 81 1010
agaaagtgaggaccctcaggaggctgcaggccagtgagtcagcaaatgaagagattcccgaaccccgaa
XM_041928 tcagtgattcggaaagtgaggatcc 1102 FLJ13052 2 S 82 2508
ctaacacagcgagggactcaacacgctgattctcctcctgcctctcccg 2556 NM_023018
FOSL2 1 S 83 708
ggcggggctggacaatgcccagcgctctgtctcaagcccatcagcattgctgggggcttctacggtgag
NM_005253.1 gatcccc 784 1 AS 84 881
ggtgactcctgctccaggacgctaggataggtga 848 GBC-11 4 AS 85 437
cagagccccaaaacgctgggcagagttgacaggacccaaatgctaaagttgtggaggg 378
W84777 GBC-12 4 S 86
tggggagacccggagacggtggctggggtgtcctcagcccgggagagctgagtcagccgcgccccgcac
acagcatacttaggagccaaggacttggacctcgcttctcgccggtacgcga GBC-13 3 S 87
acccctggnaacatggnaaatataaaacaacttggtgtttttgaaaaaccgcaaagcgttatggtgtgg
atgtaacacaggggtgtggtgt GBC-14 2 S 88 176
tggaggaaaccccgtgtctgcggagcggctgtagcctgtgagcagcgagatccagggacag 236
AL557138 GBC-15 2 S 89 107
cagctacccagaagtctgaggcaggagaaatgctggaacccgggaggcagagg 159 BE079876
GBC-16 2 S 90
Cagcgatccgtccagcagatgacgaatatcgacggccatttccggcataccgagctgttgcataatgcc
cgcagactgtgct GBC-17 2 S 91
Cggaagagctcacaatgctcatttcgcgtctcgctcgggtgttgtgctgttctttaatactgtgggcaa
ttcaggtgtgtcgcttagaaaacggaggtactcaatggagtcctcaacaatgaggggccctgttcatgg
ctttgtgttggccgttcgttccacatgttctt GBC-18 2 S 92
Cgatgattattttcttggcaaagtttttagcagaacgtcaaaaattgattacatcttttaaacgtggtt
tattaccggc GBC-2 1 93 1
agagcgaggcgtgaagtccacacgcccagccccgtcgcagtgtggttgccgagcaaggctacgtctgcg
gcgcgtgcggta 81 GBC-3 12 5 94 4
ccgggatgaagtgacccagcagaaataccagagaccggagacggaatggcccagggtcagcctccaccc
AA443027 ggaaccggaggatgcagcgaagacgtctc 101 GBC-4 1 AS 95 87
cctcgctcaggattgcttcccgcggtgcctcccgcggctgcacggaaggccacgaaccgacaacttgca
AV710590 cagcagccatcttttct 1 GNAS 2 AS 96 44
cgcgcgcagctccccgcccctcgagccgaggccgagggggctgatggccgccgccgggccgag 106
NM_000516 GSTP 7 S 97 275
ggaccagcaggaggcagccctggtggacatggtgaatgacggcgtggaggacctccgctgca 336
NM_000852 1 S 98 670
tgcctggctgcgtttcccctgctctcagcatatgtggggcgcctcagcgcccggcccaagctca
aggccttcctggcctcccctgagtacgtgaacttccccatcaatggcaacgggaaacagtgagg
gttg gg 537 HES6 6 S 99 935
gcagggcagcccctggtaaccagcccagtcaggccccagccccgtttcttaagaaacttttaggg
XM_043579 accctgcagctctg 1013 HNRPA2B1 4 S 100 826
cggaccaggaccaggaagtaactttagaggaggatctgatggatatggcagtggacgtggattt
NM_002137 ggggatggctata 902 HNRPF 5 S 101 1000
caggcctggaaaggatgaggcctggtgcctacagcacaggctacgggggctacgaggagtacagt
NM_004966
ggcctcagtgatggctacggcttcaccaccgacctgttcgggagagacctcagctactgtctctccgga
atgtatgaccacagatacgccgac 1157 HRMT1L2 5 S 102 2707
ggtgcgggtgaagatggcggcagccgaggccgcgaactgcat 2748 NM_001536 HSPCA 24
S 103 1554 caaggaccaggtagctaactcagcctttgtggaacgtcttcggaaacatggc
1605 NM_005348.1 3 S 104 1553 ccaaggaccaggtagctaactcagcctttgtggaac
1588 ICAM2 5 S 105 12
ggcagcccttggctggtccctgcgagcccgtggagactgccagagatgtcctctttcggtta
NM_000873 caggaccctgactgtggccctcttcaccctgatctgctg 112 2 S 106 705
gagcctgtgtcggacagccagatggtcatcatagtcacggtggtgtcggtgttgctgtccctgt
768 1 AS 107 745
gccgctcactccccgtaggtgcccatccgctgctggcgcaagtgctggccgaagatgaagcaga
gcaggacagatgtcacgaacagggacagcaacaccgacacca 850 IGF2R 2 S 108 903
gaagctggtgcgcaaggacaggcttgtcctgagttacgt 941 NM_000876 1 S 109 1571
gcggtgccaccgacgggnaagaagcgctatgacctgtccgcgctggtccgccatgcagaacc 1631
IL4R 2 AS 110 1178
ctcctcctcctcacactccaccgggngcctcaaacaactccacacatcgcaccacgctgatgctct
NM_000418
ctggccagaggactgtcttgctgatctccactgggcaccatgctgattttccagagcc 1300
INTB5 25 S 111 67
tggggctctgcgcgctcctgccccggctcgcaggtctcaacatatgcactagtggaagtgccacctcat
NM_002213.1
gtgaagaatgtctgctaatccacccaaaatgtgcctggtgctccaaagaggacttcggaagcc 198
2 S 112 2088
ccaaggactgcgtcatgatgttcacctatgtggagctccccagtgggaagtccaacctgaccgtcctc
agggagccagagtgtggaaacacccccaacgccatgaccatcctcct 2203 1 S 113 1722
ggccatggcgagtgtcactgcggggaatgcaagtgccatgcaggttacatcggggacaactgtaactgc
tcgacagacatcagcaca 1808 1 S 114 2118
gtggagctccccagtgggaagtccaacctgaccgtcctcagggagccagagtgtggaaacacccccaa
cgccatgaccatcctcctggctg 2208 1 AS 115 2047
tgaaagatgaccaggaggctgtgctatgtttctaca 2082 ITGA3 2 AS 118 1993
tgggcgtcctccccggagcgctccgaggtccgggtgttcgtcacgttgatgctcaggagcaattt
NM_002204 ccggacgtctctgctgtactggagcctg 2085 ITGA4 2 S 119 1188
ggcgcgaacccggcccccgaaggccgccgtccgggagacggtgatgctgttgctgtgcctgggggt
NM_000885 cccgaccggccgcccctacaacgt 1276 1 AS 120 2797
tgtgttctacagttagcttctctgctggacacctgtatgcttcnctgtaatca 2848 JunB 1 S
121 306 cgggatacggccgggcccctggtggcctctctctacacgactacaaac 353
NM_002229.1 1 S 122 322 ccctggtggcctctctctacacgactac 349 KIAA1270 9
AS 123 1591
cctgtccaagaggaggccacagcgctggcctttccccacggaggccactgctgtcccgtcctctgt
XM_044835 atacagttgcaacacctgggcctcacaggt 1683 KIFC1 3 AS 124 2193
tctggatccgtcttcacttcctgttggcctgagcagtaccaataacacactggttcaccttggaggcaa
XM_042626 2125 L1CAM 1 AS 125 4465
ttggggacccaggagacgacacttggatgttgtgtggtgggtaccgaaggcagcgtgtgtatggagctc
NM_000425.2 ctgaaagccggccatggggtgggc 4392 1 AS 126 2457
caggcaatccctgagctggaaggcattgaaatcctcaactcaagtgccgtgctggtcaagtggcggccg
gtggacctggcccaggtcaagggccacctccgcggatacaatg 2568 2 S 127 1389
agtgttcagtggctggacgaggatgggacaacagtgcttcaggacgaacgcttcttcccctatgccaat
gggaccctgggcattcgagacctccaggccaatgacac 1495 2 AS 128 1518
gccaatgaccaaaacaatgttaccatcatggctaacctgaaggttaaagatgcaactcagatcactcag
gggccccgcagcacaatcgagaagaaaggttccaggg 1623 1 S 129 666
accaggaccatcattcagaaggaacccattgacctccgggtcaaggccaccaacagcatgattgacagg
aagccgcgcctgctcttccccaccaactccagcagccacc 774 1 S 130 591
ggcaacctctactttgccaatgtgctcacctccgacaaccactcagactacatctgccacgcccacttc
ccaggcaccaggaccatcatt 680 1 S 131 253
ccaaggaagagctgggtgtgaccgtgtaccagtcgccccact 294 1 S 132 1367
ggccttcggagcgcctgtgcccagtgttcagtggctggacgaggatgggacaacagtgctt 1427
1 S 133 729 gacaggaagccgcgcctgctcttccccaccaactccagcagccacctggtg 779
12 S 134 94 aatatgaaggacaccatgtgatggagccacctgtcatcac 133 1 AS 135
2889 cccctggatgaggggggcaaggggcaact 2917 7 S 136 94
aatatgaaggacaccatgtgatggagc 120 LYN 1 AS 137 1243
tacatcatcaccgagttcatggctaagggtagtttgctggatttcctcaagagtgatgaaggtggcaag
NM_002350 gtgctgctgcccaagctcattgacttctcggcccagattgca 1353 4 AS 138
1208 ggctgtacgctgtggtcaccaaggaggagcccatctacatcatcaccg 1255 PSMB7 4
S 139 595 caagaatctggtgagcgaagccatcgcagctggcatcttcaacgacctgggc 647
NM_002799 MAP2K2 1 AS 140 435
tcatcgtctttgagttcgccgaccttggctttctgggtgag 475 NM_030662.1 1 AS 141
881 ccgctccggagccatgtaggagcgcgtgcccacgaaggagttggccatggagtctatgagct
ggccgctcaccccgaagtcacacagcttgatctcc 977 MBD1 1 S 142 2829
cctcgtgccgaattcttggcctcgagggccaaattccctatagtgagtcgtattaaattcg 2889
NM_015847 1 AS 143 2846 tttaatacgactcactatagggaatttggccctcgaggcc
2885 MCM3 3 AS 144 2207
cactccaaagacggcagactcacaggagaccaaggaatcccagaaagtggagttgagtgaatccaggtt
NM_002388.2
gaaggcattcaaggtggccctcttggatgtgttccgggaagctcatgcgcagtcaatcggcatgaatcg
cctcacagaatccatcaaccgggacagcgaagagcccttctcttcagttg 2394 6 S 145
1597
tgcccttgggtagtgctgtggatatcctggccacagatgatcccaactttagccaggaagatcagcagg
acacccagat 1675 14 AS 146 1707
accaagaagaaaaaggagaagatggtgagtgcagcattcatgaagaagtacatccatgtggccaaaatc
atcaagcc 1783 4 AS 147 1597
tgcccttgggtagtgctgtggatatcctggccacagatgatcccaactttagccaggaagatcagcagg
acacccagat 1675 6 S 148 2410
tgagcaagatgcaggatgacaatcaggtcatggtgtctgag 2450
1 AS 149 2400
acccaagttcggagacgaggcctcctcagatgaggaagatgatgccctcagacaccatgacctgatt
gtcatcctgcatcttgctcagagcaacctg 2496 1 S 150 2799
agcagtggctcatccgccctacttcccatcccacacaaacccaattgtaaataacatatgacttcgt
gagtacttttggg 2721 MCM6 2 S 151 2127
gccctgctcctgtgaacgggatcaatggctacaatgaagacataaatcaagagtctgctcccaaagcc
NM_005915 2194 MYL6 1 S 155 35
gtcaagatgtgtgacttcaccgaagaccagaccgcagagttcaaggaggccttccagctgtttgaccga
NM_021019 acag 107 1 S 156 54
ccgaagaccagaccgcagagttcaaggaggccttccagctgtttgaccgaacaggtgatggc
aagatcctgtacagccagtg 135 NFkB1 5 AS 157 1
ggccaccggagcggcccggcgacgatcgctgacagcttcccctgcc 46 NM_003998.1
NIN283 11 S 158 1116
ggcaccccttctgcactgacttccagatatggttctcccttcctccctgaggacaccaaattg
NM_032268 gatgagagcaagtttgagagaag 1202 NR3C1 5 S 159 511
gcaaacctcatatgtcgaccagtgttccagagaaccccaagagttcagcatccactgctgtgt
NM_000176 ctgctgcccccacagagaaggagtt 599 NUMA1 2 S 160 4197
ggagctgacctcacaggctgagcgtgcggaggagctgggccaagaattgaaggcgtggc 4255
NM_006185 GRP58 3 S 161 1166
caatctgaagagatacctgaagtctgaacctatcccagagagcaatgatgggcctgtgaaggtagtggt
NM_005313.1 agc 1237 1 AS 162 1084
ttagcagttctgatagcaacaacaggaatctctccagcagtgctctccaagtgagtgagcggccgc
1034 PC4 2 S 163 93
tgctccagaaaaacctgtaaagagacaaaagacaggtgagacttcgagagccctg 147
NM_006713 PCNA 3 S 164 1
ccgctacaggcaggcgggaaggaggaaagtctagctggtttcggcttcaggagcctcaga
NM_002592 gcgagcgggcgaacgtcgcgacgacgggctgagacct 97 PKC delta 1 S
165 897
gcggcatcaaccagaagcttttggctgaggccttgaaccaagtcacccagagagcctccc
NM_006254.1
ggagatcagactcagcctcctcagagcctgttgggatatatcagggtttcgagaagaagaccggagtt
1024 1 S 166 667
gatcatcggcagatgcactggcaccgcggccaacagccgggacactatattccagaaaga
acgcttcaacatcgacatgccgcaccgcttcaaggttcacaactacatg 775 3 AS 167 1935
cacccagagactacagtaactttgaccaggagttcctgaacgagaaggcgcgcctctcctacagcg
2000 PKC eta 1 S 168 327
tgggccagaccagcaccaagcagaagaccaacaaacccacgtacaacgaggagttttgcgctaacgtca
NM_006255.1 ccgacggcggccacctcgagttg 418 1 S 169 383
tgcgctaacgtcaccgacggcggccacctcgagttggccgtcttccacgagacccccctgggctacgac
cacttcgtggccaactgcaccctgcagttccaggagct 486 1 AS 170 371
aacgaggagttttgcgctaacgtcaccgacggcggccacctcgagttggccgtcttccacgagaccccc
ctgggc 445 1 S 171 362 cccacgtacaacgaggagttttgcgctaa 390 PKCZETA 4
S 172 386
acggccacctcttccaagccaagcgctttaacaggagagcgtactgcggtcagtgcagcg 445
NM_002744 1 S 173 163
ccgctcaccctcaagtgggtggacagcgaaggtgacccttgcacggtgtcctcccagatgg
agctggaagaggctttccgcctggcccgtcagtgcagggatgaaggcctcatcattcatg 283 1
AS 174 842
gacgtactcaatgaccaggaacaaccgacttgtcgtctggaagcaggagtgtaatccgacca
ggaaggggttgctggatgcctgctcaaacacgtgcttctctgtctgtacccagtcaatatcctcgccat
catgcaccagctctttcttcaccactt 999 PPP2R1B 2 AS 175 504
acggaattgctgtctgatttctgctttaacagcatttgatgccctgggatagcaaacgctg
aacaaac NM_002716 cacatgc 578 1 AS 176 805
aggacccatggctttctggagctctgaaaatctgtcagccaccatatagcgaacgcgcca
agatttatcttctgctgcttgtcgaagtg 893 RAB2L 4 S 177 871
gtcacacagtttaacaaggtggcaggggcagtggttagttctgtcctgggggctacttcc
NM_004761 actggagagggacctggggaggtgaccatacggcc 965 RAB5B 2 S 178 834
aacaccaggcagctgttccgactggcctcct 864 NM_002868.1 1 AS 179 1345
gggcggaggtggaggtgcagggtcaactgtggctctgta 1383 RAD23A 2 S 180 1351
gcctgctcanagaagctggcaggactgggaggcgacagatgggcccctcttggcctctgtcccagctct
NM_005053 1419 RAN 6 S 181 750
ggatggtgacctgtgagaatgaagctggagcccagcgtcagaagtctagttttataggcagctgtcc
NM_006325 816 REL 2 S 182 1727
tgaatcttgaaaacccctcatgtaattcagtgttagacccaagagacttgagacagctccatc
NM_002908
agatgtcctcttccagtatgtcagcaggcgccaattccaatactactgcccattgtttcacaatcagat
gcatttgagggatctgacttcagttgtgcagataacagcatgataaatg 1906 AHRG 36 S
183 518
aggagcagagccaggcgcccatcacaccgcagcagggccaggcactcgcgaaacagatcc
NM_001665.1
acgctgtgcgctacctcgaatgctcagccctgcaacaggatggtgtcaaggaagtgttcgccgag
gctgtccgggctgtgctc 660 2 AS 184 377
ccattgccagtccgccgtcctatgagaacgtgcggcacaagtggcatccagaggtgtgccacca
ctgccctgatgtgcccatcctgctggtgggcaccaagaaggacctgagagcccagcctgacaccctacg
gc 511 1 S 185 518 aggagcagagccaggcgcccatcacaccgcagcagggccaggcact
563 2 S 186 273
ggcaatggagaaacagatgacgaaaacgttggtctgagggtaggagagtgtacggaggcggtcatact
cctcctggcccgcagtgtcccacaggttcaggttcactttgcgc 384 1 S 187 516
caccatcctgttgcagggctgagcattcgaggtagcgcacagcgtggatctgcttggccagtgcctggc
cctgctgcggtgtgatgggcgcctggccctgctccttg 622 1 S 188 541
gagcacagcccggacagcctcggcgagctattccttggctccatcgtgttgcaggggtggcgtcctagg
tagcgcgcagcgtggatatgctcggccagtgcatggccctgatgcggtgt 660 RPA1 2 AS
189 2163
tggagaagcaaaaacctagttacataatttacttcatggtctgcagttagggtcagtgactta
NM_002945
cgacataattcctgcttgatgataatgaaattgacagaagcctgaaggctgagtgagtga 2285
RPA3 6 S 190 8 agccgcagtcttggaccataatcatgg 34 NM_002947.1 RPL12 2 S
191 24
ggccaaggtgcaacttccttcggtcgtcccgaatccgggttcatccgacaccagccgcctcca
NM_000976 ccatgccgccgaagttcgaccccaacga 114 RPL31 9 S 192 28
tggcgagaagaaaaagggccgttctgccatcaacgaagtggtaacccgagaat 80
NM_013403.1 1 S 193 44 ggccgttctgccatcaacgaagtggtaacccgagaat 80
RPL35 2 AS 194 12 ggcggcttgtgcagcaatggccaagatcaaggctcgagatct 53
NM_004632.1 1 AS 195 12 ggcggcttgtgcagcaatggccaagatcaaggc 44 RPS24
4 AS 196 351
gccagcaccaacattggcctttgcagtccccctgactttcttcattctgttcttgcgttcct
ttcgtt NM_001026 gct 421 4 s 197 373
cagaatgaagaaagtcagggggactgcaaaggccaatgttggtgctggcaaaaag 427 RPS29 2
S 198 4 ttacctcgttgcactgctgagagcaagatgggtcaccagcagctgtactggagcca 59
NM_001032 SQSTM1 2 S 199 1278
ggcagcaaaacaagtgacatgaagggagggtccctgtgtgtgtgtgc 1324 NM_003900
STAT3 11 5 200 2288
gagagccaggagcatcctgaagctgacccaggtagcgctgccccatacctgaagaccaagttta
NM_003150.1
tctgtgtgacaccaacgacctgcagcaataccattgacctgccgatgtccccccgc 2407 7 AS
201 2111
aagacccagatccagtccgtggaaccatacacaaagcagcagctgaacaacatgtcatttgctgaaatc
atcatgggctataagatcatggatgctaccaatatcctg 2218 2 S 202 667
ggatgtccggaagagagtgcaggatctagaacagaaaatgaaagtggtagagaatctcca
ggatgactttgatttcaactataaaaccctcaagagtc 764 2 S 203 431
ttcctgcaagagtcgaatgttctctatcagcacaatctacgaagaatcaagcagtttcttcagagcagg
tatcttgagaagccaatggagattgcccggattgtggcccggtgcc 545 1 AS 204 834
agatgctcactgcgctggaccagatgcggagaagcatcgtgagtgagctggcggggcttttgtcagcga
tggagtacgtgcagaa 918 1 S 205 413
gaccagcagtatagccgcttcctgcaagagtcgaatgttctctatca 459 1 AS 206 935
gagctggctgactggaagaggcggcaacagatggagtacgtgcagaa 980 STAT5b 102 AS
207 287 tcttgataatccacaggagaacattaaggccacccagctcctggagggcctggtgcag
NM_012448.1 gagctgcagaagaaggcagaacaccaggtgggggaagatgggttttt 391 1
AS 208 303
gagaacattaaggccacccagctcctggagggcctggcgcaggagctgcagaacaaggcacaacaccag
gagggggaagatg 384 3 S 209 1941
aacaagcagcaggcccacgacctgctcatcaacaagccagatgggaccttcctgctgcgcttcagcgac
tcggaaatcgggggcatcaccattgcttggaagtttga 2047 36 S 210 1409
aaacgaatcaagaggtctgaccgccgtggtgcagagtcggtcacggaagagaagttcacaatcttgttt
gactcacagttcagtgttggtggaaatgagctggt 1513 3 AS 211 287
tcttgataatcctcaggaggccattaagcccacccagctcatgaagggcatggtgcagtagctgcagaa
gaagagcagaactccaggtgggggaagatgggttt 389 1 AS 212 287
tcttgataatccacaggagaacattaaggccacccagctcctggaggg 334 2 S 213 1467
acaatcctgtttgaatcccagttcagtgttggtggaaatgagctggt 1513 1 S 214 1484
ccagttcagtgttggtggaaatgagctggt 1513 TAF7 6 S 215 65
cgagctgcgcctctcggcaagatttcgcgctgaccatcccgggccctttcatcactaatcggt 127
(TFIID) NM_005642 TDGF1 3 AS 216 57
ggtcgtagcagaagcaggagcaaggcgtccaggggaaactggagggctt 105 NM_003212 VWF
8 S 217 3646
ccagcatggcaaggtggtgacctggaggacggccacattgtgcccccagagctgcgaggagaggaatct
NM_000552.2 ccgggagaacgggtatgagtgtgagtggcgctataacagctgtgcacctgcctg
3768 3 AS 218 4687
ccttgcccctgaagcccctcctcctactctgcccccccacatggcacaagtcactgtgggcccggggct
cttgggggtttcgaccctggggcccaagaggaactccatggttctggatgtggcgttc 4813 3 S
219 1124
gcccggacctgtgcccaggagggaatggtgctgtacggctggaccgaccacagcgcgtgcagcccagtg
tgccctgctggtatg 1207
2 S 220 7776
agtgctgtggaaggtgcctgccatctgcctgtgaggtggtgactggctcaccgcggggggactcccagt
cttcctg 7851 2 S 221 5082
tggtcagccagggtgaccgggagcaggcgcccaacctggtctacatggtcaccggaaatcctg
5144 3 S 222 6003
agtgccacaccgtgacttgccagccagatggccagaccttgctgaagagtcatcgggtcaactgt
6067 1 AS 223 4725
acatggcacaagtcactgtgggcccggggctcttgggggtttcgaccctggggcccaagaggaactcca
tggttctggatg 4805 2 S 224 4376
tccaccagcgaggtcttgaaatacacactgttccaaatcttcagcaagatcgaccgccctgaagc
4440 1 AS 225 7818
ctggctcaccgcggggggactcccagtcttcctggaagagtgtcggctcccagtggg 7874 1 AS
226 1380 accctcccggcacctccctctctcgagactgcaacacctgcatttgccgaaacagcc
1436 2 AS 227 8762 agctgcatgggtgcctgctgctgcc 8786 ZIN 6 AS 228 1782
ctcagtggccttcaccagcaccgagcctgcccacatcgtggcctccttccgctctggcgacaccgtctt
NM_013403.1
gtatgacatggaggttggcagtgccctcctcacgctggagtcccggggcagcagcggtccaaccca
1916
[0135] TABLE-US-00005 TABLE 5 Peptides encoded by sense-oriented
GSEs Location in Parent GSE Peptide Protein SEQ ID SEQ ID (AA Gene
NO NO Residues) Sequence ADPRT 24 229 860-887
LWHGSRTTNFAGILSQGLRIAPPEAPVT IF1 32 230 1-16 MAVTALAARTWLGVWG BAG1
33 231 53-80 RDEESTRSEEVTREEMAAAGLTVTVTHS BAG1 34 232 62-80
EVTREEMAAAGLTVTVTHS AP1B1 35 233 76-97 YAKSQPDMAIMAVNTFVKDCED AP1B1
36 234 81-96 PDMAIMAVNTFVKDCE CDK10 38 235 347-360 APATSEGQSKRCKP
CDK2 40 236 51-66 EISLLKELNHPNIVKL CDK2 41 237 159-177
YTHEVVTLWYRAPEILLGC c-FOS 45 238 362-378 PELVHYREEKHVFPQRF c-FOS 47
239 148-158 KMAAAKCRNRR CREB1 54 240 27-49 VQAQPQIATLAQVSMPAAHATSS
CCND1 55 241 56-91 MRKIVATWMLEVCEEQKCEEEVFPLAMN YLDRFLSL EDF1 61
242 22-37 AKSKQAILAAQRRGGD EIF1 62 243 1050-1063 RGGADDERSSWRNA
EFNA1 67 244 53-79 HYEDHSVADAAMEQYILYLVEHEEYQL FEN1 71 245 90-101
PQLKSGELAKRS FGFR1 76 246 427-470
VTVSADSSASMNSGVLLVRPSRLSSSGTPMLAGVSEYELPEDPR FGFR1 78 247 402-421
GTKKSDFHSQMAVHKLAKSI GBC1 79 248 36-54 LTSQTMGGQAETLLTSQKG FOS2L 83
249 246-261 IKPISIAGGFYGEEPL GSTP 97 250 83-102
DQQEAALVDMVNDGVEDLRC GSTP 98 251 170-210
CLDAFPLLSAYVGRLSARPKLKAFLASP EYVNLPINGNGKQ GBC-3 94 252
WMDGRDEVTQQKYQRPETEWPRVSLH PEPEDAAKTSLSE HES6 99 253 874-948
RAAPGNQPSQAPAPFLKKLLGTLQL HNRPA2B1 100 254 786-866
ISDQDQEVTLEEDLMDMAVDVDLGMAI HNRPF 101 255 226-278
AGLERMRPGAYSTGYGGYEEYSGLSDGYGFTTDLFGRDLSYCL SGMYDHRYGD HRMT1L2 102
256 2701-2748 GVGAGEDGGSRGRELH HSPCA 103 257 499-515
KDQVANSAFVERLRKHG ICAM2 105 258 1-19 MSSFGYRTLTVALFTLICC ICAM2 106
259 216-229 YEPVSDSQMVIIVT IGF2R 108 260 253-265 KLVRKDRLVLSYV
IGF2R 109 261 481-496 KKRYDLSALVRHAEPE INTB5 111 262 12-56
LLGLCALLPRLAGLNICTSGSATSCEECLLIHPKCAWCSKEDFGS INTB5 112 263 688-724
KDCVMMFTYVELPSGKSNLTVLREPECGNTPNAMTIL INTB5 113 264 457-485
GHGECHCGECKCHAGYIGDNCNCSTDIST INTB5 114 265 697-726
VELPSGKSNLTVLREPECGNTPNAMTILLA ITGA4 119 266 18-41
PEAAVRETVMLLLCLGVPTGRPYN JUNB 121 267 19-34 GYGRAPGGLSLHDYKL JUNB
122 268 24-32 PGGLSLHDY L1CAM 127 269 457-491
SVQWLDEDGTTVLQDERFFPYANGTLGIRDLQAND L1CAM 129 270 216-251
TRTIIQKEPIDLRVKATNSMIDRKPRLLFPTNSSSH L1CAM 130 271 191-220
GNLYFANVLTSDNHSDYICHAHFPGTRTII L1CAM 132 272 450-469
AFGAPVPSVQWLDEDGTTVL L1CAM 134 273 25-39 EYEGHHVMEPPVITE L1CAM 131
274 79-91 KEELGVTVYQSPH L1CAM 133 275 237-253 DRKPRLLFPTNSSSHLV
PSMB7 139 276 193-211 EAKNLVSEAIAAGIFNDLG MCM3 145 277 519-543
PLGSAVDILATDDPNFSQEDQQDTQ MCM3 148 278 789-802 LSKMQDDNQVMVSE MCM6
151 279 690-711 PAPVNGINGYNEDINQESAPKA MET 154 280 1253-1317
YSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWS FGVVLWELMTRGAPP YPDVNTFDITVYLLQG
MYL6 155 281 2-23 MCDFTEDQTTEFKEAFQLFDRT MYL6 156 282 7-32
EDQTTEFKEAFQLFDRTGDGKILYNQ NR3C1 159 283 132-155
STSVPENPKSSASTAVSAAPTEKE NUMA1 160 284 1314-1332
ELTSQAERAEELGQELKAW GRP58 161 285 360-382 NLKRYLKSEPIPESNDGPVKVVV
PC4 163 286 32-49 APEKPVKKQKTGETSRAL PKC delta 165 287 281-322
GINQKLLAEALNQVTQRASRRSDSASSEPVGIYQGFEKKTGV PKC delta 166 288
204-239 IIGRCTGTAANSRDTIFQKERFNIDMPHRFKVHNYM PKC eta 168 289 55-84
GQTSTKQKTNKPTYNEEFCANVTDGGHLEL PKC eta 169 290 73-106
CANVTDGGHLELAVFHETPLGYDHFVANCTLQFQE PKC zeta 172 291 130-148
GHLFQAKRFNRRAYCGQCS PKC zeta 173 292 55-94
PLTLKWVDSEGDPCTVSSQMELEEAFRLARQCRDEGLIIH RAB2L 177 293 291-321
VTQFNKVAGAVVSSVLGATSTGEGPGEVTIR REL 182 294 518-553
NLENPSCNSVLDPRDLRQLHQMSSSSMSAGANSNTT AHRG 183 295 131-177
EQSQAPITPQQGQALAKQIHAVRYLECSALQQDGVKEVFAEAVRAVL AHRG 186 296 49-85
GWMEEQSQAPITPQQGQALE AHRG 187 297 130-164
KEQSQAPITPQQGQALAKQIHAVRYLECSALQQDG AHRG 188 298 138-155
TPQQGQALAKQIHAVRYL RPL12 191 299 209-228 SRIRVHLTPAASTMLPKFNP RPL31
192 300 8-24 GEKKKGRSAINEVVTRE RPS24 197 301 113-130
WMDGRMKKVRGTAKANVGAGKK STAT3 200 302 6150-729
ESQEHPEADPGSAAPYLKTKFICVTPTTCSNTIDLPMSPR STAT3 202 303 90-181
DVRKRVQDLEQKMKVVENLQDDFDFNYKTLKS STAT3 203 304 71-108
FLQESNVLYQHNLRRIKQFLQSRYLEKPMEIARIVARC STAT3 205 305 65-79
DQQYSRFLQESNVLY STAT5 209 306 599-633
NKQQAHDLLINKPDGTFLLRFSDSEIGGITIAWKF STAT5 210 307 422-455
KRIKRSDRRGAESVTEEKFTILFESQFSVGGNEL STAT5 213 308 441-455
TILFESQFSVGGNEL VWF 217 309 1113-1152
QHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPA VWF 219 310 272-299
ARTCAQEGMVLYGWTDHSACSPVCPAGM VWF 220 311 2490-2513
CCGRCLPSACEVVTGSPRGDSQSS VWF 221 312 1592-1611 VSQGDREQAPNLVYMVTGNP
VWF 222 313 1899-1919 CHTVTCQPDGQTLLKSHRVNC VWF 224 314 1356-1376
STSEVLKYTLFQIFSKIDRPE
* * * * *
References