U.S. patent application number 09/799946 was filed with the patent office on 2002-07-25 for genes and genetic elements associated with control of neoplastic transformation in mammalian cells.
This patent application is currently assigned to University of Illinois. Invention is credited to Gudkov, Andrei, Kazarov, Alexander, Mazo, Ilya, Roninson, Igor B..
Application Number | 20020099028 09/799946 |
Document ID | / |
Family ID | 22759235 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020099028 |
Kind Code |
A1 |
Gudkov, Andrei ; et
al. |
July 25, 2002 |
Genes and genetic elements associated with control of neoplastic
transformation in mammalian cells
Abstract
The invention provides genetic suppressor elements that confer
the transformed phenotype of malignant mammalian cells upon
untransformed cells, methods for identifying and obtaining such
elements, methods for isolating and identifying genes corresponding
to such elements, and methods of using such elements. The invention
also provides genes corresponding to the GSEs of the invention.
Inventors: |
Gudkov, Andrei; (Glencoe,
IL) ; Kazarov, Alexander; (Baltimore, MD) ;
Mazo, Ilya; (Redwood City, CA) ; Roninson, Igor
B.; (Wilmette, IL) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
University of Illinois
|
Family ID: |
22759235 |
Appl. No.: |
09/799946 |
Filed: |
March 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09799946 |
Mar 6, 2001 |
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09416833 |
Oct 12, 1999 |
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09416833 |
Oct 12, 1999 |
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08204740 |
Mar 2, 1994 |
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08204740 |
Mar 2, 1994 |
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08033086 |
Mar 9, 1993 |
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08033086 |
Mar 9, 1993 |
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PCT/US91/07492 |
Oct 11, 1991 |
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PCT/US91/07492 |
Oct 11, 1991 |
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07599730 |
Oct 19, 1990 |
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Current U.S.
Class: |
514/44R ;
424/93.21 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 2310/18 20130101; C12N 15/1138 20130101; C12N 15/113 20130101;
C12N 2310/321 20130101; C12N 15/1137 20130101; G01N 33/574
20130101; G01N 2800/44 20130101; C07K 14/4703 20130101; C12N 9/90
20130101; C12Y 599/01003 20130101; C12N 15/1034 20130101; C12N
15/67 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/44 ;
424/93.21 |
International
Class: |
A61K 048/00 |
Claims
What is claimed is:
1. A method of inhibiting malignant growth in cancer cells in an
animal, the method comprising the step of expressing in the cancer
cell a complete gene homologous to a nucleotide sequence of a
genetic suppressor element, or its complement, that establishes or
maintains a transformed phenotype in a mammalian cell, wherein the
genetic suppressor element is prepared by a method comprising the
steps of: (a) synthesizing randomly fragmented cDNA prepared from
the total mRNA of a cell to yield DNA fragments; (b) transferring
the DNA fragments to an expression vector to yield a genetic
suppressor element library, wherein the expression vector expresses
the DNA fragments in a living cell that expresses a transformed
phenotype; (c) genetically modifying the living cells by
introducing the genetic suppressor element library into the cells;
(d) isolating or enriching for genetically modified living cells
containing genetic suppressor elements conferring the transformed
phenotype on the cells by selecting the cells under conditions
wherein the transformed cells are identifiable; (e) obtaining the
genetic suppressor element conferring the transformed phenotype
from the surviving genetically modified cells.
2. The method of claim 1 wherein the genetic suppressor element is
selected from the group consisting of SAHH, P120, and the genes
homologous to the GSEs Tr6, Tr19 and Tr22.
Description
[0001] This is a divisional of U.S. patent application Ser. No.
08/204,740, filed Mar. 2, 1994, now U.S. Pat. No. 5,753,432, issued
May 19, 1998, which is a continuation-in-part of U.S. Ser. No.
08/033,086, filed Mar. 9, 1993, which in turn is a
continuation-in-part of International patent application Ser. No.
PCT/US91/07492, filed on Oct. 11, 1991 and which entered the
National stage in the U.S. on Apr. 15, 1993, which is a
continuation-in-part of U.S. Ser. No. 07/599,730, filed Oct. 19,
1990, now U.S. Pat. No. 5, 217,889, issued Jun. 8, 1993.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to genes and genetic suppressor
elements associated with the control of neoplastic transformation
of mammalian cells. More particularly, the invention relates to
methods for identifying such genes and genetic suppressor elements
as well as to uses for such genes and genetic suppressor elements.
The invention specifically provides genetic suppressor elements
derived from genes associated with the transformed phenotype of
mammalian cells, and therapeutic and diagnostic uses related
thereto. The invention also provides genes associated with the
control of neoplastic transformation of mammalian cells.
[0004] 2. Summary of the Related Art
[0005] Cancer remains one of the leading causes of death in the
United States. Clinically, a broad variety of medical approaches,
including surgery, radiation therapy and chemotherapeutic drug
therapy are currently being used in the treatment of human cancer
(see the textbook CANCER: Principles & Practice of Oncology, 2d
Edition, De Vita et al., eds., J.B. Lippincott Company,
Philadelphia, Pa., 1985). However, it is recognized that such
approaches continue to be limited by a fundamental lack of a clear
understanding of the precise cellular bases of malignant
transformation and neoplastic growth.
[0006] The beginnings of such an understanding of the cellular
basis of malignant transformation and neoplastic growth have been
elucidated over the last ten years. Growth of normal cells is now
now understood to be regulated by a balance of growth-promoting and
growth-inhibiting genes, known as proto-oncogenes and tumor
suppressor genes, respectively. Proto-oncogene: are turned into
oncogenes by regulatory or structural mutations that increase their
ability to stimulate uncontrolled cell growth. These mutations are
therefore manifested as dominant (e.g. mutant RAS genes) or
co-dominant (as in the case of amplification of oncogenes such as
N-MYC or HER2/NEU) (see Varmus, 1989, "A historical overview of
oncogenes", in Oncogenes and the Molecular Origin of Cancer,
Weinberg, ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,
pp. 3-44).
[0007] Dominant and co-dominant genes can be effectively identified
and studied using many different techniques based on gene transfer
or on selective isolation of amplified or overexpressed DNA
sequences (Kinzler et al., 1987, Science 236: 70-73; Schwab et al.,
1989, Oncogene 4: 139-144; Nakatani et al., Jpn. J. Cancer Res. 81:
707-710). Expression selection has been successfully used to clone
a number of cellular oncogenes. The dominant nature of the
oncogenes has facilitated the analysis of their function both in
vitro, in cell culture, and in vivo, in transgenic animals. Close
to fifty cellular oncogenes have been identified so far (Hunter,
1991, Cell 64: 249-270).
[0008] It is likely, however, that there are at least as many
cancer-associated genes that are involved in suppression rather
than induction of abnormal cell growth. This class of genes, known
as anti-oncogenes or tumor suppressors, has been defined as
comprising "genetic elements whose loss or inactivation allows a
cell to display one or another phenotype of Aneoplastic growth
deregulations" by Weinberg (1991, Science 254: 1138-1146). Changes
in a tumor suppressor gene that result in the loss of its function
or expression are recessive, because they have no phenotypic
consequences in the presence of the normal allele of the same gene.
The recessive nature of mutations associated with tumor suppressors
makes such genes very difficult to analyze or identify by gene
transfer techniques and explains why oncogene research is far more
advanced than studies of tumor suppressors.
[0009] In normal cells, tumor suppressor genes may participate in
growth inhibition at different levels, from the recognition of a
growth inhibiting signal and its transmission to the nucleus, to
the induction (or inhibition) of secondary response genes that
finally determine the cellular response to the signal. The known
tumor suppressor genes have indeed been associated with different
steps of the regulatory pathway. Thus, the DCC and ErbA genes
encode receptors of two different classes (Fearon et at., 1990,
Science 247: 49-56; Sap et as., 1986, Nature 324: 635-640;
Weinberger et al., 1986, Nature 324 : 641-646). The gene NF-1
encodes a polypeptide that resembles ras-interacting proteins, that
are members of the signaling pathway (Xu et al., 1990, Cell 62 :
599-608; Ballester et al., 1990, Cell 62: 851-859; Buchberg et al.,
1990, Nature 347: 291-294; Barbacid, 1987, Ann. Rev. Biochem. 56:
779-827). p53, RB and WT genes encode nuclear regulatory proteins
(Fields et al., 1990, Science 249: 1046-1049; Raycroft et al.,
1990, Science 249: 1049-1051; Kern et al., 1991, Oncogene 6:
131-136; O'Rourke et al., 1990, Oncogene 5: 1829-1832; Kern et al.,
1991, Science 252: 1708-1711; Lee et al., 1987, Nature 329:
642-645; Friend et al., 1987, Proc. Natl. Acad. Sci. USA 84:
9059-9063; Call et al., 1990, Cell 60: 509-520; Gessler et al.,
1990, Nature 343: 774-778).
[0010] Two approaches have been previously used for cloning tumor
suppressor genes. The first approach is based on isolating the
regions associated with nonrandom genetic deletions or
rearrangements observed in certain types of tumors. This approach
requires the use of extremely laborious linkage analyses and does
not give any direct information concerning the function of the
putative suppressor gene (Friend et al., 1991, Science 251:
1366-1370; Viskochil et al., 1990, Cell 62: 187-192; Vogelstein et
al., 1988, N. Engl. J. Med. 319: 525-532). In fact, among numerous
observations of loss of heterozygosity in certain tumors (Solomon
et al., 1991, Science 245: 1153-1160; LaForgia et al., 1991, Proc.
Natl. Acad. Sci. USA 88: 5036-5040; Trent et al., 1989, Cancer Res.
49: 420-423), there are only a few examples where the function of
the affected gene is understood. In two of these rare cases the
gene function was identified using another method, analysis of
dominant negative mutant proteins (Herskowitz, 1987, Nature 329:
219-222).
[0011] Specifically, the tumor suppressor genes erbA and p53 were
first discovered as altered forms which encoded mutant proteins
(Sap et al., 1986, ibid.; Weinberger et al., 1986, ibid.; Raycroft
et al., 1990, ibid.; Milner et al., 1991, Molec. Cell. Biol. 11:
12-19). These altered genes were initially classified as oncogenes,
since they induced cell transformation when transfected alone or in
combination with other oncogenes (ras in the case of p53 and erbB
in the case of erbA; see Eliyahu et al., 1984, Nature 312: 646-649;
Parada et al., 1984, Nature 312: 649-4651; Graf & Beug, 1983,
Cell 34: 7-9; Damm et al., 1989, Nature 339: 593-597). Later,
however, it was recognized that both of these "oncogenes" acted by
interfering with the normal function of the corresponding wild-type
genes. Thus, the oncogenic mutant ps53 protein forms functionally
inactive complexes with the wild-type protein; such complexes fail
to provide the normal negative regulatory function of the p53
protein (Herskowitz, 1986, ibid.; Milner et al., 1991, ibid.;
Montenarh & Quaiser, 1989, Oncogene 4: 379-382; Finlay et al.,
1988, Molec. Cell. Biol. 8: 531-539). The oncogene erbA, found in
chicken erythroblastosis virus, is a mutant version of the chicken
gene for thyroid hormone receptor, the transcriptional regulatory
protein which participates in the induction of erythroid
differentiation (Damm et al., 1989, ibid.; Damm et al., 1987, EMBO
J. 6: 375-382). The mutant erbA protein blocks the function of the
wild-type receptor by occupying its specific binding sites in the
DNA (Sap et al., 1989, Nature 340: 242-244).
[0012] Thus, naturally arising dominant negative mutants not only
allowed the identification of the corresponding tumor suppressor
genes but also served as tools for their functional analysis. Such
natural tools for recessive gene identification seem to be rare,
however, limiting the utility of this approach for the discovery of
new tumor suppressor genes.
[0013] The discovery and analysis of new recessive genes involved
in neoplastic transformation may be greatly accelerated through the
use of genetic suppressor elements (GSEs), derived from such genes
and capable of selectively suppressing their function. GSEs are
dominant negative factors that confer the recessive-type phenotype
for the gene to which the particular GSE corresponds. Recently,
some developments have been made in the difficult area of isolating
recessive genes using GSE technology. Roninson et al., U.S. Pat.
No. 5,217,889 (issued Jun. 8, 1993) teach a generalized method for
obtaining GSEs (see also Holzmayer et al., 1992, Nucleic Acids Res.
20: 711-717). Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA 90:
3231-3235 teach isolation of GSEs from topoisomerase II cDNA that
induce resistance to topoisomerase II-interactive drugs. Co-pending
U.S. patent applications Ser. No. 08/033,986, filed Mar. 9, 1993,
and Ser. No. 08/177,571, filed Jan. 5, 1994, disclosed the
discovery by the present inventors of the novel and unexpected
result that GSEs isolated from RNA of cells resistant to the
anticancer DNA damaging agent, etoposide, include a GSE encoding an
antisense RNA homologous to a portion of a linesin heavy chain
gene. Additionally, co-pending U.S. patent application Ser. No.
08,033,986 disclosed two other GSEs from previously-unknown genes,
the expression of said GSEs conferring etoposide resistance on
mammalian cells. Co-pending U.S. patent application Ser. No.
08/______ , filed Feb. 22, 1994, disclosed GSEs from
previously-unknown genes, the expression of said GSEs conferring
cisplatin resistance on mammalian cells.
[0014] These results further underscored the power of the GSE
technology developed by these inventors to elucidate recessive
gene-mediated biological phenomenon involving unexpected
mechanisms, including drug resistance in cancer cells, thereby
providing the opportunity and the means for overcoming drug
resistance in cancer patients. This technology has now been applied
to isolating and identifying GSEs that confer the transformed
phenotype of malignant mammalian cells in previously untransformed
cells expressing such GSES, and for isolating and identifying genes
associated with the transformed phenotype.
BRIEF SUMMARY OF THE INVENTION
[0015] The invention provides genetic suppressor elements (GSEs)
that are random fragments derived from genes associated with the
transformed phenotype of malignant mammalian cells, and that confer
the transformed phenotype upon cells expressing such GSEs. The
invention is based in part on the discoveries disclosed in
co-pending U.S. patent applications, Ser. No. 08/033,086, filed
Mar. 9, 1993, Ser. No. 08/177,157, filed Jan. 5, 1994, and Ser. No.
08/______ , filed Feb. 22, 1994, incorporated by reference,
providing a method for identifying and isolating GSEs that confer
resistance to chemotherapeutic drugs upon cells expressing such
GSEs.
[0016] In a first aspect, the invention provides a method for
identifying GSEs that confer the transformed phenotype on cells
expressing the GSEs. This method utilizes selection of cells that
harbor clones from a random fragment expression library derived
from total cDNA derived from normal cells, preferably normal mouse
or human fibroblasts, and subsequent rescue of library inserts from
immortalized, morphologically-transformed or frankly tumorigenic
cells. In a second aspect, the invention provides a method for
identifying and cloning genes that are associated with the
transformed phenotype of malignant mammalian cells, and also
provides the genes themselves. This method comprises the steps of
screening a full length cDNA library with a GSE that confers the
transformed phenotype upon cells (or, alternatively, with an
oligonucleotide or polynucleotide constituting a portion of such a
GSE) and determining the nucleotide sequence of the cDNA insert of
any positive clones obtained. Alternatively, the technique of
"anchored PCR" (see Example 3 below) can be used to isolate cDNAs
corresponding to transformed phenotype-conferring GSEs. Also
embodied in this aspect of the invention is isolation of genomic
DNA encoding genes associated with the transformed phenotype, for
example from genomic DNA libraries. In a third aspect, the
invention provides a diagnostic assay for characterizing
transformed cells, particularly human tumor cells, that express the
transformed phenotype due to the absence of expression or
underexpression of a particular gene. This diagnostic assay
comprises measuring, preferably quantitatively, the level of
expression of the particular gene product by a particular tumor
cell sample to be tested, compared with the level of expression in
normal, untransformed cells. One feature of this aspect of the
invention is the development of antibodies specific for proteins
whose underexpression or absence of expression is associated with
the transformed phenotype in malignant mammalian, most preferably
malignant human, cells. Such antibodies have utility as diagnostic
agents for detecting tumor cells in biopsy or other tissue samples,
and in characterizing the nature and degree of expression of the
transformed phenotype in such cells. In a fourth, the invention
provides a starting point for in vitro drug screening and rational
design of pharmaceutical products that are useful against tumor
cells, i.e., are anticancer agents. By examining the structure,
function, localization and pattern of expression of genes
associated with the transformed phenotype, strategies can be
developed for creating pharmaceutical products that will
selectively kill or inhibit the growth of such cells, in which such
genes are either not expressed or underexpressed. Also provided by
the invention are cultures of mammalian cells which express the
transformed phenotype-conferring GSEs of the invention and are
transformed thereby. Such cells are useful for determining the
physiological and biochemical basis for malignant mammalian cell
transformation. Such cells also have utility in the development of
pharmaceutical and chemotherapeutic agents for selectively killing
or inhibiting the growth of such cells, and thus are ultimately
useful in establishing improved chemotherapeutic protocols to more
effectively treat neoplastic disease.
[0017] 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
[0018] FIG. 1 shows the structure of the adaptor used in cDNA
cloning. The nucleotide sequences are shown for the ATG-sense
[SEQ.ID.No.:1] and ATG-antisense [SEQ.ID.No.:2] strands of the
adaptor.
[0019] FIG. 2 shows the structure of the pLNCX vector used in cDNA
cloning.
[0020] FIG. 3 shows a scheme for selection of immortalizing GSEs in
MEF cells from a random fragment expression library (RFEL) from
mouse NIH 3T3 cell cDNA: Panel A illustrates selection of such GSEs
via one round of selection for cells that survive crisis; Panel B
shows a scheme for re-selection and enrichment of immortalizing
GSEs from populations of immortalized MEFs produced according to
the scheme shown in Panel A.
[0021] FIG. 4 shows polyacrylamide gel electrophoretic analysis of
PCR fragments comprising MEF immortalizing GSE.
[0022] FIG. 5 shows the nucleotide sequence of the Tr6-GSE (SEQ ID
No.:3).
[0023] FIG. 6 shows the results of an experiment demonstrating that
Tr6GSE (SEQ ID No.:3) is capable of conferring the morphologically
transformed phenotype on both Swiss 3T3 cells and MEF cells (Panel
A), and is also capable of immortalizing MEF cells in which
spontaneous immortalization is suppressed by expression of an
exogenously-introduced p53 gene (Panel B).
[0024] FIG. 7 shows a scheme for selecting morphological
transformation-conferring GSEs.
[0025] FIG. 8 shows the results of an experiment in which rescued
transforming GSE-carrying retroviruses were used to re-infect fresh
NIH 3T3 cells: Panel A shows the results of selection for G418
resistance (as a measure of infection efficiency) and morphological
transformation in media supplemented with 5% FCS; Panel B shows the
results of PCR analysis of retroviral inserts from genomic DNA of
morphologically transformed foci.
[0026] FIG. 9 shows the nucleotide sequence of the SAHH-GSE (SEQ ID
No.:4).
[0027] FIG. 10 shows a comparison between the nucleotide sequence
of SAHH-GSE (SEQ ID No.:4) and the SAHH gene sequence (SEQ ID
No.:5).
[0028] FIG. 11 shows a comparison between the amino acid sequence
of the peptide encoded by the SAHH-GSE (SEQ ID No.:6) and the SAHH
protein amino acid sequence (SEQ ID No.:7).
[0029] FIG. 12 shows the nucleotide sequence of the Tr19-GSE (SEQ
ID No.:8).
[0030] FIG. 13 shows the, results of an experiment demonstrating:
Panel A, that SAHH-GSE was capable of conferring both
immortalization and morphological transformation on MEF cells;
Panel B, that Tr19-GSE is capable of immortalizing MEF cells; and
Panel C that both the SAHH-GSE and an anti-khcs GSE could
immortalize MEF cells, but only the SAHH-GSE could morphologically
transform MEF cells.
[0031] FIG. 14 shows a scheme for selecting tumorigenic GSEs.
[0032] FIG. 15 polyacrylamide gel electrophoretic analysis of PCR
fragments comprising tumorigenic GSEs.
[0033] FIG. 16 shows the nucleotide sequence of the Tr22-GSE (SEQ
ID No.:9).
[0034] FIG. 17 shows the nucleotide sequence of the 1bb1-GSE (SEQ
ID No.:10).
[0035] FIG. 18 shows a comparison between the nucleotide sequence
of the 1bb1-GSE (SEQ ID No.:10) and the P120 human nucleolar
antigen gene sequence (SEQ ID No.:11).
[0036] FIG. 19 shows a comparison between the amino acid sequence
of the peptide encoded by the 1bb1-GSE (SEQ ID No.:12) and a
portion of the P120 human nucleolar antigen protein amino acid
sequence (SEQ ID No.:13).
[0037] FIG. 20 shows the results of a focus-formation assay using
infection of Swiss 3T3 cells with retrovirus carrying the 1bb1-GSE
(SEQ ID No.:10).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] The invention relates to means for identifying specific gene
functions that are associated with the transformed phenotype of
malignant mammalian cells. The invention provides genetic
suppressor elements (GSEs), the expression of such GSEs conferring
the transformed phenotype on untransformed fibroblast cells. The
invention further provides methods for identifying such GSES, as
well as methods for their use. For purposes of this invention, the
terms "the transformed phenotype of malignant mammalian cells" and
"the transformed phenotype" are intended to encompass, but not be
limited to, any of the following phenotypic traits associated with
cellular transformation of mammalian cells: immortalization,
morphological or growth transformation, and tumorigenicity, as
detected by prolonged growth in cell culture, growth in semi-solid
media, or tumorigenic growth in immuno-incompetent or syngeneic
animals.
[0039] In a first aspect, the invention provides a method for
identifying GSEs that confer upon untransformed cells the
transformed phenotype of malignant mammalian cells. The GSEs
identified by this method will be homologous to a gene that is
associated with the transformed phenotype of malignant mammalian
cells. For purposes of the invention, the term "homologous to a
gene" has two different meanings, depending on whether the GSE acts
through an antisense or antigene mechanism, or through a mechanism
of interference at the protein level. In the former case, a GSE
that is an antisense or antigene oligonucleotide or polynucleotide
is homologous to a gene if it has a nucleotide sequence that
hybridizes under physiological is conditions to the gene or its
mRNA transcript by Hoogsteen or Watson-Crick base-pairing. In the
latter case, a GSE that interferes with a protein molecule is
homologous to the gene encoding that protein molecule if it has an
amino acid sequence that is the same as that encoded by a portion
of the gene encoding the protein, or that would be the same, but
for conservative amino acid substitutions. In either case, as a
practical matter, whether the GSE is homologous to a gene is
determined by assessing whether the GSE is capable of inhibiting or
reducing the function of the gene.
[0040] The method according to this aspect of the invention
comprises the step of screening a total cDNA or genomic DNA random
fragment expression library phenotypically to identify clones that
confer the transformed phenotype on untransformed recipient cells.
Preferably, the library of random fragments of total cDNA or
genomic DNA is cloned into a retroviral expression vector. In this
preferred embodiment, retrovirus particles containing the library
are used to infect cells and the infected cells are tested for
their ability to exhibit the transformed phenotype, for example, by
exhibiting the ability to grow past "crisis" in Witro culture, or
to grow in a manner that is recognized as being
morphologically-transformed, or to grow in semisolid media, such as
soft agar or agarose, or in methylcellulose, or by frankly
tumorigenic growth In vivo in an animal. Preferably, the inserts in
the library will range from about 100 bp to about 700 bp and more
preferably, from about 200 bp to about 500 bp in size. Most
preferably, the random fragment library will be a normalized
library containing roughly equal numbers of clones corresponding to
each gene expressed in the cell type from which it was made,
without regard for the level of expression of any gene. However,
normalization of the library is unnecessary for the isolation of
GSEs that are homologous to abundantly or moderately expressed
genes. Once a clonal population of cells that exhibit the
transformed phenotype has been isolated, the library clone encoding
the GSE is rescued from the cells. At this stage, the insert of the
expression library may be tested for its nucleotide sequence.
Alternatively, and preferably, the rescued library clone may be
further tested for its ability to confer the transformed phenotype
in additional transfection or infection and selection assays, prior
to nucleotide sequence determination. Determination of the
nucleotide sequence, of course, results in the identification of
the GSE. This method is further illustrated in Examples 1 and
2.
[0041] In a second aspect, the invention provides a method for
identifying and cloning genes that are associated with control of
neoplastic growth in mammalian cells, as well as the genes derived
by this method. This is because GSEs, or portions thereof, can be
used as probes to screen full length cDNA or genomic libraries to
identify their gene of origin. Alternatively, the technique of
"anchored PCR" (see Example 3 below) can be used to isolate cDNAs
corresponding to transformed phenotype-conferring GSEs. It will be
recognized that the genes associated with control of neoplastic
transformation in mammalian cells are sufficiently evolutionarily
conserved that the GSEs provided by the invention, or the genes
corresponding to such GSEs, can be used as probes to isolate genes
corresponding to such neoplastic growth-associated GSEs from any
mammalian species, including man.
[0042] In some cases, genes that are associated with the
transformed phenotype will turn out to be quite surprising. For
example, GSEs that have been found to be capable of conferring the
transformed phenotype upon untransformed cells include GSEs derived
from the mouse homolog of the human P120 nucleolar antigen gene,
and the gene for S-adenosyl homocysteine hydrolase, as well as from
three GSEs from previously unidentified genes. In addition, a GSE
derived from a mouse kinesin gene and associated with etoposide
resistance has been previously discovered to be capable of
conferring cell culture growth immortalization on mouse embryo
fibroblasts (MEF) and normal human fibroblasts, as disclosed in
co-pending U.S. patent applications, Ser. No. 08/177,154, filed
Jan. 5, 1994, and Ser. No. 08/033,086, filed Mar. 9, 1993. The
method according to this aspect of the invention therefore also
provides valuable information about the genetic basis for
senescence. The method according to this aspect of the invention
and its use for studying genes identified thereby and their
cellular effects are further illustrated in Example 3.
[0043] In a third aspect, the invention provides a diagnostic assay
for characterizing transformed cells, particularly human tumor
cells, that express the transformed phenotype due to the absence of
expression or underexpression of a particular gene. By using the
methods according to the first and second aspects of the invention
such a gene is identified and cloned. To determine whether absence
of expression or underexpression of such a gene is a naturally
occurring, and thus medically significant basis for neoplastic
growth and cancer, human tumor cells are assessed for their level
of expression of the particular gene of interest. Absence of
expression or significantly reduced expression, relative to
expression in normal tissues that give rise to the tumor, would
then be correlated with the natural history of the particular
cancer, including cell and tissue type, incidence, invasiveness,
capacity to metastasize, and other relevant properties of the
particular tumor. Accordingly, such reduced or absent expression
can be the basis for a diagnostic assay for the presence and extent
of tumorigenic cells in a tissue sample. Malignant transformation
and neoplastic growth as the result of over-expression of a gene is
also detectable using similar diagnostic assays provided by the
invention. A first embodiment of a diagnostic assay according to
this aspect of the invention utilizes an oligonucleotide or
oligonucleotides that is/are homologous to the sequence of the gene
for which expression is to be measured. In this embodiment, RNA is
extracted from a tissue or tumor sample, and RNA specific for the
gene of interest is quantitated by standard filter hybridization
procedures, an RNase protection assay, or by quantitative cDNA-PCR
(see Noonan et al., 1990, Proc. Natl. Acad. Sci. USA 87:
7160-7164). In a second embodiment of a diagnostic assay according
to this aspect of the invention, antibodies are raised against a
synthetic peptide having an amino acid sequence that is identical
to a portion of the protein that is encoded by the gene of
interest. These antibodies are then used in a conventional
quantitative immunoassay (e.g., RIA or immunohistochemical assays)
to determine the amount of the gene product of interest present in
a sample of proteins extracted from the tumor cells to be tested,
or on the surface or at locations within the tumor cells to be
tested. In a third embodiment, an enzymatic activity that is a
property of a gene associated with neoplastic transformation of
cancer cells can be used to measure whether the gene encoding said
protein is over- or under-expressed in the cancer cells.
[0044] In a fourth aspect, the invention provides a starting point
for in vitro drug screening and rational design of pharmaceutical
products that can counteract tumorigenicity and neoplastic growth
by tumor cells in vivo. In this regard, the invention provides
cultures of mammalian cells which express the transformed
phenotype-conferring GSEs of the invention and are immortalized
and/or transformed thereby. Included within this aspect of the
invention are cell cultures that are representative of almost any
tissue or cell type. Such cells are useful for determining the
physiological and biochemical basis for malignant transformation of
mammalian cells, as well as for screening pharmaceutical and
chemotherapeutic agents for killing or selectively inhibiting the
growth of such transformed cells. Identification of such agents
would lead to the development of improved chemotherapeutic
protocols to more effectively treat neoplastic disease.
[0045] The protein sequence encoded by genes from which the GSEs
were derived can be deduced from the cDNA sequence, and the
function of the corresponding proteins may be determined by
searching for homology with known genes or by searching for known
functional motives in the protein sequence. If these assays do not
indicate the protein function, it can be deduced through the
phenotypic effects of the GSEs suppressing the gene. Such effects
can be investigated at the cellular level, by analyzing various
growth-related, morphological, biochemical or antigenic changes
associated with GSE expression. The GSE effects at the organism
level can also be studied by introducing the corresponding GSEs as
transgenes in transgenic animals (e.g. mice) and analyzing
developmental abnormalities associated with GSE expression. The
gene function can also be studied by expressing the full-length
cDNA of the corresponding gene, rather than a GSE, from a strong
promoter in cells or transgenic animals, and studying the changes
associated wit overexpression of the gene.
[0046] Full-length or partial cDNA sequences can also be used to
direct protein synthesis in a convenient prokaryotic or eukaryotic
expression system, and the produced proteins can be used as
immunogens to obtain polyclonal or monoclonal antibodies. These
antibodies can be used to investigate the protein localization and
as specific inhibitors of the protein function, as well as for
diagnostic purposes. In particular, antibodies raised against a
synthetic peptide encoded by the sequence of the GSEs Tr6, Tr19 and
Tr22, or the corresponding region of the P120 nucleolar antigen
gene or the SAHH gene should be particularly useful (see Examples 2
and 3 and FIGS. 5, 9-11, & 15-18).
[0047] Understanding the biochemical function of a gene involved in
malignant transformation of mammalian cells is also likely to
suggest pharmaceutical means to stimulate or mimic the function of
such a gene and thus augment the cytotoxic response to anticancer
drugs. For example, if the gene encodes an enzyme producing a
certain compound, such a compound can be synthesized chemically and
administered in combination with cytotoxic drugs. If a
pharmaceutical approach is not apparent from the protein function,
one may be able to upmodulate gene expression at the level of
transcription. This can be done by cloning the promoter region of
the corresponding gene and analyzing the promoter sequence for the
presence of cis elements known to provide the response to specific
biological stimulators. Such an approach is useful to replace the
function of tumor-suppressor genes, for example, to restore the
tumor-suppressing function of such genes that has been lost through
mutation or other biological insult, resulting in neoplastic
disease.
[0048] The most straightforward way to increase the expression of
gene identified through the GSE approach, the loss of which results
in malignant transformation of a cell no longer functionally
expressing the gene, would be to insert a full-length cDNA for such
a gene into a gene therapy expression vector, for example, a
retroviral vector. Such a vector, in the form of a recombinant
retrovirus, will be delivered to tumor cells in vivo, and, upon
integration, would act to reduce or eliminate neoplastic growth of
such cells. The selective delivery to tumor cells can be
accomplished on the basis of the selectivity of retrovirus-mediated
transduction for dividing cells. Alternatively, the selectivity can
be achieved by driving the expression of the gene from a tissue- or
tumor-specific promoter, such as, for example, the promoter of the
carcinoembryonic antigen gene.
[0049] The protein structure deduced from the cDNA sequence can
also be used for computer-assisted drug design, to develop new
drugs that affect this protein in the same manner as the known
anticancer drugs. The purified protein, produced in a convenient
expression system, can also be used as the critical component of in
vitro biochemical screen systems for new compounds with anticancer
activity. In addition, mammalian cells that express tranformed
phenotype-conferring GSEs according to the invention are useful for
screening compounds for the ability to selectively kill or inhibit
the neoplastic growth associated with down-regulation of the
corresponding gene.
[0050] The following Examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature.
EXAMPLE 1
Generation of a Normalized Random Fragment cDNA Library in a
Retroviral Vector and Introduction Into Virus-Packaging Cell
Lines
[0051] A normalized cDNA population was prepared as described in
co-pending U.S. patent application Ser. No. 08/033,086, filed Mar.
9, 1993, which is incorporated by reference. Briefly, poly(A).sup.+
RNA was purified from total RNA extracted in equal amounts from
exponentially-growing and quiescent, confluent monolayer cultures
of mouse NIH 3T3 cells (Accession No. ______, American Type Culture
Collection, Rockville, Md.), an immortalized mouse cell line known
to be useful in cellular transformation assays (see Shih et al.,
1979, Proc. Natl. Acad. Sci. USA 76: 5714-5718). To avoid
over-representation of the 5'-end sequences in a randomly primed
cDNA population, RNA was fragmented by boiling for 5 minutes to an
average size of 600-1000 nucleotides. These RNA fragments were then
used for preparing randomly primed double-stranded cDNA. This
randomly primed cDNA was then ligated to a synthetic adaptor
providing ATG codons in all three possible reading frames and in a
proper context for translation initiation (see FIG. 1). The
structure of the adaptor determined its ligation to the blunt-ended
fragments of the cDNA in such a way that each fragment started from
initiation codons independently from its orientation. The ligated
mixture was amplified by PCR, using the "sense" strand of the
adaptor as a PCR primer, in twelve separate reactions that were
subsequently combined, in order to minimize random over-or
under-amplification of specific sequences and to increase the yield
of the product. The PCR-amplified mixture was then
size-fractionated by electrophoresis in a 6% polyacrylamide gel,
and fragments ranging in size from approximately 200-500 basepairs
(bps) were selected for further manipulations.
[0052] For normalization, the cDNA preparation was denatured and
reannealed, using the following time-points for reannealing: 0, 24,
48, 72, 96 and 120 hours. The single-stranded and double-stranded
DNAs from each reannealed mixture were then separated by
hydroxyapatite chromatography. These DNA fractions from each time
point of reannealing were PCR-amplified using adaptor-derived
primers and analyzed by slot blot hybridization with probes
corresponding to genes expressed at different levels in human
cells. .alpha.-tubulin and c-myc probes were used to represent
highly-expressed genes, adenosine deaminase and topoisomerase-II
(using separate probes for the 5' and 3' ends of the latter cDNA)
probes were used to represent intermediately-expressed genes, and a
c-fos probe was used to represent low-level expressed genes. The
fraction that contained similar proportions of high-, medium- and
low-expressed genes was used for the library preparation.
[0053] The normalized cDNA preparation was cloned into a ClaI site
of the MoMLV-based retroviral vector pLNCX, which carries the neo
(G418 resistance) gene, expressed under the transcriptional control
of the promoter contained in the retroviral long terminal repeat
(LTR), and which expresses the cDNA insert sequences from a
cytomegalovirus (CMV)-derived promoter (see FIG. 2 and Miller and
Rosman, 1989, Biotechniques 7: 980-986). pLNCX contains translation
termination codons in all three reading frames within 20 bp
downstream of the cloning site. To generate a representative-size
library for GSE selection, this ligation mixture was divided into
five portions and used to transform E. coli in 5 separate
electroporation experiments, using conventional techniques and
standard conditions for electroporation (see Sambrook et al., 1992,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The transformed
bacteria were plated on a total of 500 agar plates (150 mm in
diameter) and the plasmid produced (18 mg total) was isolated from
the colonies washed off the agar. A total of approximately
5.times.10.sup.7 clones were obtained, more than 60% of which
carried inserts of normalized cDNA, as estimated by PCR
amplification of 50 randomly-picked colonies.
[0054] Plasmid DNA was used for in vivo selection of GSEs capable
of conferring a transformed phenotype of appropriate cells as
discussed in Example 2 below. The plasmid library prepared as
described above was converted into a mixture of retroviral
particles by transfection into twenty P150 culture plates
containing a 1:1 mixture of ecotropic and amphotropic packaging
cells (derived from NIH 3T3 cells; see Markowitz et al., 1988,
Virology 167: 400-406), the cells having been seeded the day before
transfection at a density of 1.5.times.10.sup.6 cells per plate. 15
.mu.g of random fragment retroviral library (RFRL) plasmid DNA were
transfected per P150 plate. The retrovirus-containing cell culture
supernatant was collected every 12 hours over three days
post-transfection and purified by filtration through 0.22 .mu.m
membranes.
EXAMPLE 2
Introduction Of A Retroviral Random Fragment Library Into Mouse
Fibroblast Cells
[0055] The purified retrovirus-containing supernatant prepared
according to Example 1 was used in each of three assays chosen to
detect three distinct aspects of the transformed phenotype in
mammalian cells. Selection of transforming GSEs required the use of
suitable indicator cells capable of undergoing identifiable and
selectable transformation-associated changes. Three different
selection protocols for GSEs that induce phenotypic traits
associated with neoplastic transformation were used. First, for
selection of GSEs capable of immortalizing senescent cells, mouse
embryonic fibroblasts were used as the indicator cell system. The
other two selection protocols utilized three different types of
immortalized mouse fibroblasts, each of which differ in
transformation-associated traits, in order to select GSEs specific
for different stages of neoplastic transformation. Two of these
cell lines are subvariants of NIH 3T3 cells, and the third type of
cells comprise several populations of Swiss 3T3 cells, newly
established from spontaneously-transformed MEF cells. These latter
cells were expected to contain multiple phenotypic variants which
would be differentially susceptible to the effects of different
GSEs, thereby increasing the number of different types of GSEs that
could be detected. Some characteristic properties of each of the
three types of immortalized cells are shown in Table I.
1 TABLE I Rate of Spontaneous Plating Tumorigenicity.sup.a Cell
Type Focus Formation Efficiency 3 Weeks 6 weeks NIH 3T3-HF 2-5
.times. 10.sup.-6 20-30% 0/6 5/6 NIH 3T3-LF <1 .times. 10.sup.-7
20-30% 0/6 0/6 Swiss 3T3 <1 .times. 10.sup.-7 <0.1% N.T. N.T.
.sup.a = Number of mice with tumors/Number of mice tested N.T = not
tested
[0056] A. Selection of GSEs Capable of Immortalizing Mouse Embryo
Fibroblasts
[0057] GSE selection for the ability to immortalize senescent cells
was carried out on cultures of mouse embryo fibroblast (MEF) cells
infected with retroviral particles comprising the RFRL of Example
1, using a protocol depicted in FIG. 3. Primary MEF cultures were
prepared from 11-day old Swiss Webster mouse embryos using a
conventional trypsinization procedure. Cells were split every
three-four days, with 2.5.times.10.sup.6 cells plated per P150
culture plate at each passage, grown in Dulbecco's Modified Eagle's
medium (DMEM) supplemented with 10% (v/v) fetal calf serum.
Additionally, about 5.times.10.sup.6 cells were preserved after
every second passage until the culture underwent senescence and
"crisis", by freezing in a cryogenic protective solution at
-70.degree. C. For retroviral infection experiments, cells frozen 4
passages before crisis were thawed and grown in culture on 10 P150
plates at a density of 1.times.10.sup.6 cells/plate. The thawed
cells were infected with RFRL-derived retrovirus over 3 days, at 12
hour intervals, and MEFs were repeatedly infected with each
collected supernatant. Each P150 plate was processed independently
beginning with infection with the RFRL-derived retrovirus. The
efficiency of infection was estimated by plating equal numbers of
infected cells in the presence and absence of G418 for 5 days, at
which time relative cell viability was measured using the MTT assay
(see Pauwels et al., 1988, J. Virol. Meth. 20: 309-321. Typical
infections efficiencies obtained in such assays indicated that
about 70% of the MEFs were infected with RFRL-derived
retroviruses.
[0058] After the cell cultures overcame senescenc and crisis, the
surviving cells from each plate were fused with ecotropic packaging
cells to rescue the virus, using polyethylene glycol a previously
described in co-pending U.S. patent application Ser. No. 08/______
, filed on Feb. 22, 1994. The complexity of the rescued virus
population was estimated by PCR amplification of proviral inserts,
using the oligonucleotide corresponding to the sense strand of the
cloning adaptor as PCR primer (as shown in FIG. 4). The PCR
products from RFRL-derived retrovirus infected MEF cells initially
formed a continuous smear of fragments 200-500 bps in length. As
the cells proceeded through crisis, the complexity of the cDNA
inserts decreased, and separate bands became visible (FIG. 2).
[0059] The rescued viral preparations from post-crisis cells,
containing the virus at relatively low titre (.about.10.sup.4/mL),
were used to infect fresh populations of pre-crisis MEF cells,
which were then allowed to go through crisis. The efficiency of
these secondarily-infected cells was estimated by G418 selection
before and after crisis; in several secondary selection
experiments, the proportion of infected cells increased after
crisis, suggesting enrichment for GSE-carrying cells. PCR analysis
performed on cellular DNA from immortalized cells surviving this
second round selection indicated the selection of several cDNA
inserts, containing putative immortalization-conferring GSEs.
[0060] These inserts are each individually subcloned into the pLNCX
retroviral vector and tested for the ability to immortalize MEF
cells as shown in FIG. 3B. MEFs that are two passages before crisis
are infected by GSE-carrying viruses and then plated at low density
(e.g., 3.times.10.sup.4 cells/100 mm culture plate) and then fixed
and stained two weeks after plating. The number of surviving
colonies reflects the proportion of immortalized cells in the
infected population.
[0061] B. Isolation of GSEs that Can Morphologically Transform
Mouse Fibroblasts
[0062] To isolate GSEs capable of inducing morphological
transformation of immortalized MEFs, immortalized MEF cells as
described in subsection A above were used. Cells were plated into
10 P100 plates at a density of 2.5.times.10.sup.6 cells/plate and
maintained in DMEM/10% FCS for three weeks. 2-20 foci of
morphologically-transformed cells appeared in each plate. Two foci
were isolated and expanded by growth in culture. Cells from these
expanded foci were then fused with packaging cells and the hybrid
cells selected with G418 and used to rescue retroviral populations
as described above. Viruses isolated in this way from the expanded
foci were used to infect fresh Swiss 3T3 cells, and the infected
cells were maintained in DMEM/5% FCS.
[0063] Viruses rescued from each of these two foci, isolated from
one of the original plates of immortalized MEF cells, induced
morphological transformation of Swiss 3T3 cells in two separate
experiments. PCR analysis of the cDNA insert present in the
transforming virus (termed Tr6-GSE), performed on genomic DNA
isolated from four independent foci of transformed Swiss 3T3 cells,
revealed a single insert band. DNA from this band was re-cloned
into the pLNCX vector and the nucleotide sequence determined using
conventional techniques (see Sambrook et al., ibid.). This clone
was found to contain a 285 bp insert (shown in FIG. 5), which
showed no significant homology with known nucleic acid and protein
sequences present in the National Center for Biotechnology
Information database. The recloned Tr6-GSE-carrying retrovirus was
efficient in inducing morphological transformation of NIH 3T3 cells
and immortalized MEF (shown in FIG. 6A). Infection of senescent MEF
cells with this virus produced no significant increase in the
number of immortalized cells, relative to background.
[0064] Tr6, however, was found to have an effect on MEF
immortalization by a different assay. In this assay, MEF cells 2
passages from senescence were infected with LNCX, or LNCX carrying
Tr6-GSE, or a retroviral construct carrying a full-length cDNA
encoding the cellular tumor suppressor gene p53, or a combination
of the p53 retrovirus and Tr6-GSE carrying retrovirus. MEF cells
infected with the LNCX vector retrovirus produced a low background
spontaneously-immortalized cells (FIG. 6B). In contrast, MEF cells
infected with the recombinant retrovirus carrying a full-length
cDNA of the p53 tumor suppressor gene under conditions where all
the cells were infected, failed to give rise to any immortalized
colonies. However, when the same cells were infected under the same
conditions with retroviruses carrying Tr6 and p53, immortalized
colonies were formed (FIG. 6B).
[0065] GSEs were also selected for the ability to induce
morphological transformation of NIH 3T3 cells (shown in FIG. 7). In
these experiments, RFRL plasmid DNA was transfected into a 1:1
mixture of ecotropic and amphotropic virus-packaging cells.
Retroviral particle-containing tissue culture media supernatant was
collected at 24, 48 and 72 h after infection and used for repeat
infection of NIH 3T3 cells. The total amount of virus used for
infection was estimated to be >10.sup.7 infectious units.
Recipient NIH 3T3 cells were plated in ten P150 plates at a density
of 1.times.10.sup.6 cells/plate and incubated in DMEM10% FCS. Four
plates were infected with control virus containing no GSE insert,
produced by transient transfection of packaging cells with the
vector plasmid pLNCX, to estimate the rate of spontaneous (i.e.,
non-GSE mediated) transformation in these cells.
[0066] The day after the last infection, a portion of the infected
NIH 3T3 cells were frozen as described above, and another portion
was split into 10 P150 culture plates at a density of
2.times.10.sup.6 cells/plate and cultured in DMEM/5% FCS for two
weeks. The efficiency of infection was evaluated by G418 selection;
typically, at least 50% of the cells were found to be infected.
Similar numbers of apparently transformed cells were observed in
both the experimental and control plates (5-15 foci/plate,
corresponding to 2.5-7.5.times.10.sup.-6 foci/cell). Individual
foci were picked and expanded as described above, and virus rescued
from each focus by fusion with ecotropic packaging cells. Fresh NIH
3T3 cells were infected with rescued retrovirus, and cells infected
with 2/50 rescued virus populations were found to produce cell
populations which showed altered growth properties, including
reaching a much higher density in 5% serum (shown in FIG. 8). PCR
analysis of genomic DNA from these populations showed that each of
the two virus preparations inducing such altered cellular growth
properties carried a single cDNA insert.
[0067] The two cDNA inserts carried by the transforming
retroviruses isolated in this manner were sequenced and analyzed
for homology with known nucleic acid and protein sequences present
in the NCBI database. This analysis showed that one of the
transforming viruses carried a 285 bp fragment corresponding to the
beginning of the coding region of the cDNA encoding the enzyme
S-adenosyl homocysteine hydrolase (SAHH), cloned in the sense
orientation (shown in FIGS. 9-11). SAHH is known to be involved in
many biochemical pathways, including methionine, cysteine and
S-adenosylmethionine synthesis, the latter compound being the major
source of methyl groups in methylation reactions. Abnormal SAHH
expression may cause general alterations in cellular DNA
methylation patterns and is known to alter various cellular
characteristics (see Wolos et al., 1993, J. Immunol. 150:
3264-3273; Liu et al., 1992, Antvir. Res. 19: 247-265; Duerre et
al., 1992, Biochim. Biolog. Cellulaire 70: 703-711). The
SAHH-derived cDNA insert from this experiment was recloned into the
pLNCX vector in the same orientation as in the original provirus
(i.e., in the sense orientation) and used for further testing as
described below.
[0068] The insert from the second transforming virus preparation
was found to contain two different linked cDNA fragments, connected
on one another by the adaptor. One of these fragments was derived
from a cDNA encoding a structural protein, filamin. The sequence of
the other fragment, termed Tr19GSE (shown in FIG. 12) had no
significant homology with any known genes in the NCBI database.
These two fragments were recloned separately into the pLNCX
retroviral vector for further testing.
[0069] Each of the re-cloned cDNA fragments were tested by
transfection into ecotropic packaging cells and the resulting virus
used to infect NIH 3T3 cells (to test for morphological
transformation capacity for each cDNA insert) and MEF cells (to
test for both immortalization and morphological transformation
capacities). The NIH 3T3 cell experiments produced highly variable
results. The MEF cell experiments, on the other hand, were more
efficient and reproducible, and the results of these experiments
are shown in FIG. 13. Infection with virus carrying SAHH cDNA
sequences (SAHH-GSE) resulted in both immortalization and
morphological transformation of MEF cells. Infection with virus
carrying the filamin cDNA fragment had no effect on MEF cells, but
the Tr19-GSE-carrying virus was found to be capable of inducing
immortalization of MEF cells, although at a lower efficiency than
the SAHH-GSE. These results confirmed that the strategy disclosed
herein had resulted in the isolation of two transforming GSES, one
of which was previously unknown (Tr19) and the other derived from a
gene which, although known, had not been implicated in neoplastic
transformation until now.
[0070] C. Selection of GSEs Enabling Tumorigenic Growth in Nude
Mice
[0071] The following experiments were performed to isolate GSEs
capable of enabling tumorigenic growth of NIH 3T3 cells in
immuno-incompetent, nude (nu/nu) mice. The scheme for these
experiments is shown in FIG. 14. For this selection, RFRL-infected
NIH 3T3 cells, prepared as described above, were inoculated
subcutaneously into the flank of nude mice (Balb/c strain), at
5.times.10.sup.5 cells per mouse. NIH 3T3 cells infected with
pLNCX-vector derived virus were used as a control. Mice were
examined weekly for tumor formation for up to six weeks
post-inoculation. The results of these experiments are summarized
in Table II.
2TABLE II Number of Tumor-bearing Mice Cell Type Week 2 Week 3 Week
4 Week 5 Week 6 Control 0/3 0/3 0/3 1/3 1/3 RFRL 0/9 6/9 7/9 9/9
9/9
[0072] These results, showing a higher frequency of tumorigenic
variants among the NIH 3T3 cells infected with the RFRL-derived
retrovirus than the LNCX-derived retrovirus, indicated the
existence of tumorigenic GSEs in the population of RFRL-derived
retroviruses. When the tumor size reached 5 mm in diameter, each
tumor was explanted and established in culture. PCR analysis
performed using genomic DNA from three of these tumor-derived
cultures showed the presence of several proviruses carrying
different cDNA inserts. Virus was then rescued from these tumor
cells by fusion of the tumor cells with ecotropic packaging cells,
as described above, infection of fresh NIH 3T3 cells and selection
in nude mice for tumorigenicity. Two mice were used per each
transduced cell population, and proviral inserts from tumors formed
in these mice were characterized by PCR analysis (shown in FIG.
15). In two of the three populations tested, a single insert was
found to be enriched in the secondary tumors of both
independently-injected mice. A different insert was detected in the
secondary tumors of mice injected with cells infected with virus
derived from the third original NIH 3T3 cell population.
[0073] Both of these putative tumorigenic GSEs were characterize by
nucleotide sequencing and the sequences compared with known nucleic
acid and protein sequences present in the NCBI database. One of the
cDNA inserts, termed Tr22-GSE, was found to share no significant
homology with any of the sequences in the database, and hence
represents a fragment of a novel gene (this sequence is shown in
FIG. 16). The other cDNA insert, termed 1bb1-GSE, is a
sense-oriented GSE that encodes 87 amino acids from the internal
region of the mouse homolog of the human P120 nucleolar antigen of
proliferating cells. The nucleotide sequence of this GSE is shown
in FIG. 17, and nucleic acid and amino acid sequence comparisons
between the P120 sequence and the GSE sequence are shown in FIGS.
18 and 19, respectively.
[0074] The 1bb1 fragment was re-cloned intro the pLNCX vector,
transfected into ecotropic packaging cells, and the resulting virus
used to infect Swiss 3T3 cells. Infection with the 1bb1-carrying
virus resulted in the formation of morphologically-transformed foci
in these cells (FIG. 20). These results are consistent with a
recent report that a full-length cDNA of P120 is capable of acting
as a dominant oncogene in NIH 3r3 cells (Peraky et al., 1992,
Cancer Res. 52: 428-436). The results disclosed herein indicate
that the portion of the P120 cDNA comprising the 1bb1GSE encodes a
functional oncogenic domain representing about 10% of the P120
protein. This result is the first demonstration that such a small
portion of an oncogenic protein is oncogenically functional.
EXAMPLE 3
Cloning And Analysis Of The Genes From Which Each Transforming GSE
Was Derived
[0075] The results described in Example 2 above discloses the
isolation of three newly-identified genes implicated in cellular
transformation in tumor cells. Each of the genes corresponding to
these three GSEs are isolated as follows. Each GSE is used as a
hybridization probe to screen a mouse or human cDNA library
prepared from normal cells. Interspecific DNA hybriudization at the
appropriate stringency is expected to enable the isolation of genes
corresponding to GSEs from any mammalian species, using nucleic
acid probes that are homologous to GSEs or genes corresponding to
such GSEs isolated as described in Example 2 above. The nucleotide
sequence of the longest cDNA clone isolated in this way for each
GSE is then determined, and the sequence analyzed to identify the
longest open reading frame (ORF) encoding the putative gene product
from each strand. Sequence homology analysis, as described above,
is then performed on the sequence of the longest ORF to determine
whether a related protein has been previously identified. If
necessary, any additional nucleotides encoding amino acids from the
amino terminus are then determined from 5'-specific cDNA isolated
using the "anchored PCR" technique, as described by Ohara et al.
(1989, Proc. Natl. Acad. Sci. USA 86: 5763-5677). Additional
missing 3' terminal sequences are also isolated using this
technique. The "anchored PCR" technique can also be used to isolate
full-length cDNA starting directly from the GSE sequence without
library screening.
[0076] 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.
Sequence CWU 1
1
* * * * *