Apoptosis gene EI24, compositions, and methods of use

Details Apoptosis gene EI24, compositions, and methods of use Apoptosis gene EI24, compositions, and methods of use

1998-09-11

2003-07-01

Mckelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S375000, C435S455000, C530S350000, C536S023100

Reexamination Certificate

active

06586204

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the field of cell physiology, and more particularly, to apoptosis. Even more particularly, the present invention is related to a novel apoptosis associated protein EI24 and its corresponding gene EI24; to nucleotide sequences encoding EI24; to products and processes involved in the cloning, preparation and expression of genes and nucleotide sequences encoding EI24; to antibodies with specificity to EI24; and to diagnostic and therapeutic uses of the above.
BACKGROUND OF THE INVENTION
“Apoptosis” refers to cell suicide that proceeds by an active, physiological process (Kerr, J. F., et al.,
Br. J. Cancer
26:239-257 (1972); Wyllie, A. H.,
Nature
284:555-556 (1980)). Apoptosis plays an important role in developmental processes, including morphogenesis, maturation of the immune system, and tissue homeostasis whereby cell numbers are limited in tissues that ale continually renewed by cell division (Ellis, R. E., et al.,
Annu. Rev. Cell. Biol.
7:663-698 (1991); Oppenheim, R. W., et al.,
Neurosci.
14:453-501 (1991); Cohen, J. J., et al.,
Annu. Rev. Immunol.
10:267-293 (1992); Raff, M. C.,
Nature
356:397-400 (1992)).
In addition to its role in developmental processes, apoptosis is an important cellular safeguard against tumorigenesis (Williams, G. T.,
Cell
65:1097-1098 (1991); Lane, D. P.
Nature
362:786-787 (1993)). Defects in the apoptotic pathway may contribute to the onset or progression of malignancies. Suppression of the apoptotic pathway(s), by a variety of genetic lesions, occurs frequently in a broad range of human tumors. In particular, loss of the p53 tumor suppressor gene function, either through deletion or mutation, occurs in more than 50% of human cancers. p53 gene function is also indicated in normal cell cycle events. Reviews of p53 function include Levine, A. J., et al.,
Nature
351:453-456 (1991); Hollstein M., et al.,
Science
253:49-53 (1991); Donehower, et al.,
Biochem. BioPhys. Acta
1155:181-205 (1993); Lane, D. P.
Nature
362:786-787 (1993); Zambetti, et al.,
FASEB J.
7:855-865 (1993); and Greenblatt M. S., et al.,
Cancer Res.,
54: 4855-4878 (1994).
p53 may exert its tumor suppressor function, at least in part, by directing cells that have sustained genomic damage to undergo apoptosis (Lowe S. W., Jacks T., Housman D. E. and Ruley H. E. (1994)
Proc. Natl. Acad. Sci. USA,
91, 2026-2030). p53 is a sequence-specific DNA binding protein that functions both as a transcriptional activator and repressor (Donehower L. A. and Bradley A. (1993)
Biochim. Biophys. Acta.,
1155, 181-205; Prives C. and Manfredi J. (1993)
Genes Dev.,
7, 529-534; Fields S. and Jang S. K. (1990)
Science,
249, 1046-1048; Raycroft L., Wu H. and Lozano G. (1990)
Science,
249, 1049-1051). Although there is some evidence that transcription may not be required in p53-mediated apoptosis (Caelles C., Helmberg A. and Karin M. (1994)
Nature,
370, 220-223), several p53-regulated genes have been identified to date (Kastan M. B., Zhan Q., El-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B. and A. J. Fornace Jr. (1992)
Cell,
71, 587-597, 1992; El-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W. and Vogelstein B. (1993)
Cell,
75, 817-825; Barak Y., Juven T., Haffner R. and Oren M. (1993)
EMBO J.,
12, 461-468; Wu X., Bayle J. H., Olson D. and Levine A. J. (1993)
Genes
&
Dev.,
7, 1126-1132; Zambetti G. P., Bargonetti J., Walker K., Prives C. and Levine A. J. (1992)
Genes
&
Dev.,
6, 1143-1152; Okamoto K. and Beach D. (1994)
EMBO J,
13, 4816-4822; Buckbinder L., Talbott R., Seizinger B. R. and Kley N. (1994)
Proc. Natl. Acad. Sci. USA,
91, 10640-10644, and two of these genes, bcl-2 and bax (Miyashita T. and teed J. (1995)
Cell,
80, 293-299; Miyashita T., Krajewski S., Krajewska M., Wang H., Lin H., Hoffman B., Lieberman K. and Reed J. (1994)
Oncogene,
9, 1799-1805; Zhan Q., Fan S., Bae I., Guillouf C., Liebermann D. A., O'Connor P. M. and A. J. Fornace Jr. (1994) Oncogene, 9, 3743-3751), have been clearly implicated in apoptosis (Oltvai Z. and Korsmeyer S. (1994)
Cell,
79, 189-192).
In addition to cancer, deregulation of apoptosis may contribute to a number of other human diseases. A variety of degenerative disorders may involve aberrant apoptosis, resulting in premature or inappropriate cell death (Barr, P. J., et al.,
Biotechnology
12:487-493 (1994)) Productive infection by certain viruses may depend on suppression of host cell death by anti-apoptotic viral gene products (Rao, L., et al.,
Proc. Natl. Acad. Sci. USA
89:7742-7746 (1992); Ray, C. A., et al.,
Cell
69:597-604 (1992); White, E., et al.,
Mol. Cell. Biol.
12:2570-2580 (1992); Vaux, D. L., et al.,
Cell
76:777-779 (1994), and inhibition of apoptosis can alter the course (i.e. lytic vs. latent) of viral infection; Levine, B., et al.,
Nature
361:739-742 (1993)). Widespread apoptosis of T lymphocytes triggered by HIV infection may, at least in part, be responsible for the immune system failure associated with AIDS (Gougeon M., et al.,
Science
260:1269-1270 (1993)).
The ability of p53 to suppress tumorigenesis appears linked to its activity as a transcriptional activator, since tumor-derived mutant p53 molecules almost invariably have lost transactivation potential (Kern, S. E., et al.,
Science
256:827-830 (1992)). Thus, the function of the p53 tumor suppressor appears to depend, at least in part, on the ability to activate the expression of one or more target genes. Genes activated by p53 may in turn mediate one or more aspects of p53's tumor suppressor function, which including cell cycle arrest and apoptosis, depending on the cellular context. Consistent with the notion, certain p53-activated Menses identified to date have been implicated in cell cycle control (gadd45, cyclin G, p21/WAF) and at least one p53-activated gene (bax) is linked to the regulation of apoptosis.
Tumor cells frequently have lost wild-type p53 function. Consequently, activation of p53 target genes and associated tumor suppressor functions, such as cell cycle arrest and apoptosis, is defective in cancer cells. Therefore, from the perspective of pharmaceutical development, identification of genes which are regulated (e.g., induced or repressed) by p53 may permit development of agents that activate, restore or suppress p53-dependent tumor suppression functions such as apoptosis or cell cycle regulation, depending on the clinical setting.
SUMMARY OF THE INVENTION
In a broad aspect, the present invention is directed to a gene, termed EI24, whose RNA was induced in NIH3T3 cells following treatment with etoposide. Experiments performed with cells derived from p53-deficient mice demonstrate that induction of the EI24 mRNA is dependent on expression of functional p53 in vertebrate cells including etoposide-treated fibroblasts, and gamma-irradiated thymocytes. Expression of a conditional p53-fusion protein demonstrates that activation of wild-type p53 function in E1A and T24 H-ras-transformed p53-deficient fibroblasts is sufficient for induction of the EI24 mRNA.
The novel EI24 gene and the corresponding EI24 protein of mammals have thus been isolated and characterized, and are described in a number of embodiments herein. The present invention thus relates to an apoptosis associated protein EI24, products and processes involved in the cloning, preparation and expression of genes for EI24; antibodies with specificity to EI24; and nucleotide probes corresponding to the EI24 nucleotide sequence or portions thereof. The EI24 polypeptide is useful for producing antibodies thereto. The antibodies and probes are useful for detecting and isolating EI24 in biological specimens including for example, cells from all human tissues including heart tissue, lung tissue, tumor cells, brain tissue, placenta, liver, skeletal muscle, kidney, and pancreas.
The present invention further relates to species homologs and viral homologs of EI24.
In a particular embodiment, th

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