Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-01-25
2001-04-24
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S324000, C530S327000
Reexamination Certificate
active
06221615
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of cell physiology, and more particularly, to programmed cell death, or apoptosis. The novel peptides and compositions of the invention are useful for modulating apoptosis in cells.
BACKGROUND OF THE INVENTION
The phenomenon of programmed cell death, or “apoptosis,” is known to be involved in and important to the normal course of a wide variety of developmental processes, including immune and nervous system maturation. Apoptosis also plays a role in adult tissues having high cell turnover rates (Ellis, R. E., et al.,
Annu. Rev. Cell. Biol.
7: 663-698 (1991); Oppenheim, R. W.,
Annu. Rev. 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)). A number of different physiological signals normally activate programmed cell death in these contexts, but non-physiological insults, such as irradiation and exposure to drugs which damage DNA, also can trigger apoptosis (Eastman, A.,
Cancer Cells
2: 275-280 (1990); Dive, C., et al.,
Br. J. Cancer
64: 192-196 (1991); Lennon, S. V., et al.,
Cell Prolif.
24: 203-214 (1991)).
In addition to its role in development, apoptosis has been implicated as an important cellular safeguard against tumorigenesis (Williams, G. T.,
Cell
65: 1097-1098 (1991); Lane, D. P.,
Nature
362: 786-787 (1993)). Under certain conditions, cells die by apoptosis in response to high-level or deregulated expression of oncogenes (Askew, D., et al.,
Oncoaene
6: 1915-1922 (1991); Evan, G. I., et al.,
Cell
69: 119-128 (1992); Rao, L., et al.,
Proc. Natl. Acad. Sci. U.S.A.
89: 7742-7746 (1992); Smeyne, R. J., et al.,
Nature
363: 166-169 (1993); Tanaka, S., et al.,
Cell
77: 829-839 (1994); Wu, X., et al.,
Proc. Natl. Acad. Sci. U.S.A.
91: 3602-3606 (1994)). Suppression of the apoptotic program, by a variety of genetic lesions, may contribute to the development and progression of malignancies. This is well illustrated by the frequent mutation of the p53 tumor suppressor gene in human tumors (Levine, A. J., et al.,
Nature
351: 453-456 (1991)). Wild-type p53 is required for efficient induction of apoptosis following DNA damage (Clarke, A. R., et al.,
Nature
362: 849-852 (1993); Lowe, S. W., et al.,
Cell
74: 957-967 (1993); Lowe, S. W., et al.,
Nature
362: 847-849 (1993)) and cell death induced by constitutive expression of certain oncogenes (Debbas, M., et al.,
Genes
&
Dev.
7: 546-554 (1993); Hermeking, H., et al.,
Science
265: 2091-2093 (1994); Tanaka, S., et al.,
Cell
77: 829-839 (1994); Wu, X., et al.,
Natl. Acad. Sci. U.S.A.
91: 3602-3606 (1994)). The cytotoxicity of many commonly used chemotherapeutic agents is mediated by wild-type p53 (Lowe, S. W., et al.,
Cell
74: 957-967 (1993); Fisher, D. E.,
Cell
78: 539-542 (1994)). Thus, loss of p53 function may contribute to the clinically significant problem of drug resistant tumor cells emerging following chemotherapy regimens.
The expression product of the bcl-2 oncogene functions as a potent suppressor of apoptotic cell death (McDonnell, T. J., et al.,
Cell
57: 79-88 (1989); Hockenbery, D., et al.,
Nature
348: 334-336 (1990)). Constitutive Bcl-2 expression can suppress apoptosis triggered by diverse stimuli, including growth factor withdrawal, oncogene expression, DNA damage, and oxidative stress (Vaux, D. L., et al.,
Nature
335: 440-442 (1988); Sentman, C. L., et al.,
Cell
67: 879-888 (1991); Strasser, A., et al.,
Cell
67: 889-899 (1991); Fanidi, A., et al.,
Nature
359: 554-556 (1992); Hockenbery, D. M., et al.,
Cell
75: 241-251 (1993)). There is also conservation of Bcl-2 function across species. For example, the ced-9 gene of the nematode
C. elegans
appears to be a structural and functional homolog of bcl-2 (Hengartner, M. O., et al.,
Cell
76: 665-676 (1994)) and bcl-2 can complement ced-9 mutations in transgenic animals (Vaux, D. L., et al.,
Science
258: 1955-1957 (1991)). These observations suggest that Bcl-2 is intimately connected with an evolutionarily conserved cell death program.
It is known that bcl-2 is a member of a family of related genes, at least some of which also modulate apoptosis. Of these, bcl-x bears the highest degree of homology to bcl-2, and is differentially spliced to produce a long form, termed bcl-x
L
, and a shorter form, bcl-x
S
, harboring an internal deletion (Boise, L. H., et al.,
Cell
74: 597-608 (1993)). Bcl-x
L
functions to suppress apoptosis, whereas the deleted form, Bcl-x
S
, inhibits the protection against cell death provided by Bcl-2 expression. A second Bcl-2 homolog, Bax, forms heterodimers with Bcl-2 (Oltvai, Z. N., et al.,
Cell
74: 609-619 (1993)) and has been shown to counteract Bcl-2 and accelerate apoptosis. Mutational analysis of Bcl-2 has suggested that the interaction with Bax is required for Bcl-2 to function as an inhibitor of cell death (Yin, X. -M., et al.,
Nature
369: 321-323 (1994)).
The isolation and characterization of a bcl-2 related gene, termed bak, is described in co-pending U.S. application Ser. No. 08/321,071, filed Oct. 11, 1994, now U.S. Pat. No. 5,672,686, which is a continuation-in-part of U.S. application Ser. No. 08/287,427, filed Aug. 9, 1994 (bak is referred to therein as bcl-y), now abandoned, the disclosures of which are incorporated herein by reference. Ectopic Bak expression accelerates the death of an IL-3 dependent cell line upon cytokine withdrawal, and opposes the protection against apoptosis afforded by Bcl-2. In addition, enforced expression of Bak is sufficient to induce apoptosis of serum deprived fibroblasts, raising the possibility that Bak directly activates, or is itself a component of, the cell death machinery.
The known cellular Bcl-2 related genes, where analyzed, have distinct patterns of expression and thus may function in different tissues. While Bcl-2 expression appears to be required for maintenance of the mature immune system, it is desirable to identify other genes which may govern apoptotic cell death in other lineages. In addition, the identification of particular regions or domains of the proteins encoded by such genes may provide a basis for understanding their structural and functional characteristics and allow the development of valuable diagnostics and therapeutics. For example, the identification of agents capable of restoring or inducing apoptosis in tumor cells (in which loss of p53 tumor suppressor gene function may be implicated in tumorigenesis and in clinically significant drug resistance) would be of significant therapeutic value, particularly where such restoration or induction was independent of p53 function. Similarly, the development of agents capable of counteracting the anti-apoptotic function of oncogenes such as bcl-2, the activation of which is implicated in tumorigenesis (e.g., lymphoma) and in chemotherapeutic drug resistance, would be of great potential value.
SUMMARY OF THE INVENTION
The present invention is directed to a novel protein domain of general significance to the actions of multiple cell death regulatory molecules, which has been identified and mapped to a short subsequence in the central portion of the Bak molecule. This heretofore unrecognized protein domain, which the inventor has designated the “GD domain,” is essential both to Bak's interaction with Bcl-x
L
, and to Bak's cell killing function. Truncated Bak species encompassing the GD domain are themselves sufficient to bind to Bcl-x
L
and to kill cells in transfection assays.
The GD domain has been identified in two other Bcl-2 binding proteins that function to induce apoptosis: Bax and Bip1a. As with Bak, mutation of the homologous GD domain elements in Bax and Bip1a diminishes cell killing and protein binding function. Thus, the GD domain is responsible for mediating key protein/protein interactions of significance to the actions of multiple cell death regulatory molecules.
In one aspect, then, the invention is directed to purified and isolated peptides comprising the GD domain and to molecules th
Chittenden Thomas D.
Lutz Robert J.
Apoptosis Technology, Inc.
Hale and Dorr LLP
Lacourciere Karen A.
Schwartzman Robert A.
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