Cancer diagnosis and WAF1

Details Cancer diagnosis and WAF1 Cancer diagnosis and WAF1

1995-06-01

2001-10-09

Horlick, Kenneth R. (Department: 1807)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S007230, C436S064000, C436S501000

Reexamination Certificate

active

06300059

ABSTRACT:

TECHNICAL FIELD
The invention relates to the fields of diagnosis and therapy of cancers. More particularly, the invention relates to a protein which can suppress tumor cell growth.
BACKGROUND OF THE INVENTION
Inactivation of p53 is a common event in the development of human neoplasia (Hollstein et al. (1991)
Science
253, 49-53). A variety of mechanisms can lead to such functional inactivation, including p53 point mutations of deletions of p53 (Baker et al. (1989)
Science
244, 217-221; Wolf, D., and Rotter, V. (1985)
Proc. Natl. Acad. Sci. USA
82, 790-794), and interaction with oncogenic viral or cellular proteins (Mietz et al. (1992)
EMBO J.
11, 5013-5020; Momand et al. (1992)
Cell
69, 1237-1245). Wild-type p53 has been shown to be a suppressor of tumor cell growth (for reviews see Mercer, W. E. (1992)
Crit. Rev. Eucar. Gene Exp.
2, 251-263; Oren, M. (1992)
FASEB J.
6, 3169-3176; Lane, D. P. (1992)
Nature
358, 15-16; Perry, M. E., and Levine, A. J. (1993)
Curr. Opin. in Genet. and Devel.
3, 50-54). Inactivation of p53 by any of the above mechanisms thereby leads to a selective growth advantage, generally observed as tumor progression.
The mechanism underlying p53 growth suppression is still undefined. Several biochemical features of p53 have been elucidated, and at least two of these are currently of much interest. First, p53 has been shown to transcriptionally suppress a variety of promoters containing TATA-elements (Ginsberg et al. (1991)
Proc. Natl. Acad. Sci. USA
88, 9979-9983; Santhanam et al. (1991)
Proc. Natl. Acad. Sci. USA
88, 7605-7609; Kley et al. (1992)
Nucl. Acids Res.
20, 4083-4087; Mack et al. (1993)
Nature
363, 281-283). This suppression is apparently sequence independent, and may involve p53 binding to tie TATA-binding protein (TBP) or to other transcription factors (Seto et al. (1992)
Proc. Natl. Acad. Sci. USA
89, 12028-12032; Truant et al. (1993)
J. Biol. Chem.
268, 2284-2287; Ragimov et al. (1993)
Oncogene
8, 1183-1193; Martin et al. (1993)
J. Biol. Chem.
268, 13062-13067; Liu et al. (1993)
Mol. and Cell. Biol.
13, 3291-3300). Second, p53 can bind to DNA in a sequence-specific manner (Kern et al. (1991)
Science
252, 1707-1711). A 20 bp consensus binding site, consisting of two copies of the 10 bp sequence 5′-RRRCWWGYYY-3′, separated by up to 13 bp, has been identified (El-Deiry et al. (1992)
Nature Genet.
1, 45-49; Funk et al. (1992)
Mol. Cell. Biol.
12, 2866-2871). Both copies of the 10 bp sequence are required for efficient binding by p53. p53 contains a strong transcriptional activation sequence near its amino terminus (Fields, S., and Jang, S. K. (1990)
Science
249, 1046-1049; Raycroft et al. (1990)
Science
249, 1049-1051), and can stimulate the expression of genes downstream of its binding site. Such stimulation has been demonstrated in both mammalian (Kern et al. (1992) Science 256, 827-830; Funk et al. (1992)
Mol. Cell. Biol.
12, 2866-2871; Zambetti et al. (1992)
Gen. and Devel.
6, 1143-1152) and yeast cells (Scharer, E., and Iggo, R. (1992)
Nucl. Acids Res.
20, 1539-1545; Kern et al. (1992)
Science
256, 827-830) as well as in an in vitro system (Farmer et al. (1992)
Nature
358, 83-86).
The sequence-specific transcriptional activation by p53 has led to the hypothesis that p53-induced genes may mediate its biological role as a tumor suppressor (Pietenpol et al. (1993)
Cell
(submitted)). To date, several genes containing p53-binding sites have been identified. These include muscle creatine kinase (M C K, Weintraub et al. (1991)
Proc. Natl. Acad. Sci. USA
88, 4570-4574; Zambetti et al. (1992)
Gen. and Devel.
6, 1143-1152), GADD45 (Kastan et al. (1992)
Cell
71, 587-597), MDM2 (Barak et al. (1993)
EMBO
12, 461-468; Wu et al. (1993)
Genes and Devel.
7, 1126-1132), and a GLN retroviral element (Zauberman et al. (1993)
EMBO J.
12, 2799-2808). Each of these genes contains a 20 bp sequence with high homology to the p53 consensus binding site (Prives, C., and Manfredi, J. J. (1993)
Gen. and Devel.
7, 529-534). The p53-binding sites in GADD45 and MDM2 are located within introns, the MCK site is 3 kb upstream of the transcription start site, and the GLN element is located within an LTR. The relationship of any of these genes to suppression of cell growth by p53 remains unclear. It has been suggested that MDM2 may be a feedback regulator of p53 action, by being transcriptionally induced (Barak et al. (1993)
EMBO
12, 461-468; Wu et al. (1993)
Genes and Devel.
7, 1126-1132), then inhibiting p53 function (Momand et al. (1992)
Cell
69, 1237-1245; Oliner et al. (1993)
Nature
362, 857-860; Wu et al. (1993)
Genes and Devel.
7, 1126-1132). In this regard, MDM2 functions as an oncogene rather than as a tumor suppressor gene (Fakharzadeh et al. (1991)
EMBO J.
10, 1565-1569; Finlay, C. A. (1993)
Mol. and Cell. Biol.
13, 301-306).
There is a need in the art for elucidation of the pathway by which p53 exerts its tumor suppressive effects. There is also a need in the art for new diagnostic and therapeutic tools for evaluating and ameliorating human cancers.
SUMMARY OF THE INVENTION
It is an object of the invention to provide DNA molecules useful for diagnosing and treating human tumors.
It is another object of the invention to provide proteins useful for treating human tumors and for raising diagnostically useful antibodies.
It is still another object of the invention to provide antibodies which are useful for diagnosing human cancer.
It is yet another object of the invention to provide methods of suppressing growth of tumor cells.
It is an object of the invention to provide a method for screening potential therapeutic agents for treating cancer.
It is another object of the invention to provide methods for diagnosing cancer.
It is yet another object of the invention to provide a reporter construct, useful for screening potential antineoplastic agents.
It is an additional object of the invention to provide an antisense construct for inhibiting expression of a tumor suppressor gene.
It is still another object of the invention to provide antisense oligonucleotides for inhibiting expression of a tumor suppressor gene.
It is yet another object of the invention to provide methods for promoting growth of cells in which a tumor suppressor gene's expression is inhibited.
It is another object of the invention to provide a method for assessing susceptibility to cancers.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention an isolated and purified subchromosomal DNA molecule is provided. The molecule encodes WAF1 protein as shown in SEQ ID NO: 2, and contains three exons of 168 bp, 450 bp and 1600 bp. The sequence of said exons is shown in SEQ ID NO: 1.
In another embodiment of the invention an isolated and purified WAF1 protein is provided. The protein has a sequence as shown in SEQ ID NO: 2.
In yet another embodiment of the invention an antibody is provided. The antibody is specifically reactive with human WAF1 protein.
In still another embodiment of the invention a method of suppressing growth of tumor cells is provided. The method comprises administration of a WAF1 protein having a sequence as shown in SEQ ID NO: 2 to said cells.
In an additional embodiment of the invention a method of suppressing growth of tumor cells is provided. The method comprises administration to said cells of a DNA molecule which causes said cells to express WAF1, said DNA molecule having a sequence as shown in SEQ ID NO: 1.
According to another embodiment of the invention a method for screening potential therapeutic agents for the ability to suppress the growth of tumor cells by activating the expression of WAF1 is provided. The method comprises incubation of a potential therapeutic agent with a cell which contains a WAF1 reporter construct, said reporter construct comprising a WAF1 transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product. Further, the method compris

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