Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Oxidoreductases
Reexamination Certificate
1996-09-03
2001-05-15
Minnifield, Mita (Department: 1645)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Oxidoreductases
C424S613000, C424S617000, C424S616000, C424S175100, C424S138100, C424S174100, C424S195110, C514S002600, C514S885000, C435S242000
Reexamination Certificate
active
06231852
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods for controlling cell death when a cell is exposed to one or more potentially lethal cellular insults. More particularly, the present invention is directed to the discovery that the cellular production of reactive oxygen species plays an important role in apoptosis or necrosis of a cell when it is exposed to a potentially lethal insult. By controlling the production of reactive oxygen species, cells may be saved which would otherwise die due to the production of excessive reactive oxygen species. Alternatively, cells which resist intentional lethal insults may be rendered more susceptible to death by limiting their ability to prevent production of reactive oxygen species when they are subjected to such intentional insults.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
The phenomenon of cell death has been, and continues to be, the subject of a great deal of investigation. Many studies have been made to ascertain, identify and understand the mechanism(s) involved in cell death. Researchers have hoped that once the underlying mechanisms of cell death are understood that it will then be possible to construct procedures for controlling cell death. This would allow researchers to prevent cells from dying which otherwise would die when exposed to a potentially lethal cellular insult. Conversely, researchers could utilize their knowledge of the mechanism of cell death to hasten death of cancer cells or other undesirable cells which resist death even when subjected to a variety of cellular insults which normally would be lethal.
Some of the research regarding the mechanisms of cell death have centered around the identification and study of proteins which appear to play a role in inhibiting cell death when cells are exposed to potentially lethal insults. An example of such a protein is bcl-2. The protooncogene bcl-2 was discovered by translocation analysis of B-cell lymphomas (1). Subsequently it was reported that its protein product, bcl-2, is targeted to the inner membrane of mitochondria and that apoptosis of B cells, induced by the withdrawal of serum and growth factors, is inhibited by bcl-2 (2). The normal function of bcl-2 is unknown, as is the mechanism by which bcl-2 inhibits apoptosis. Furthermore, bcl-2 has little homology with other known proteins except the Epstein-Barr virus protein BHRF1 [for which no function is known (3)], although some homology to ras has been suggested (4).
Bcl-2 inhibits apoptosis in only a subset of hematopoietic cells. Bcl-2 has also been reported to inhibit the death of sympathetic neurons cultured in the absence of NGF (8).
There is a present and continuing need to discover and investigate the mechanism(s) which are involved in cell death. Once an understanding of a given cell death mechanism is understood, it would be desirable to be able to use this understanding to develop methods for controlling cell death. The control of cell death would not only allow one to prolong the life of desirable cells, but would also allow one to shorten the life of undesirable cells.
SUMMARY OF THE INVENTION
In accordance with the present invention, methods are provided for controlling cell death when the cell is subjected to one or more potentially lethal cellular insults. The present invention is based upon the discovery that an important aspect of both apoptotic and necrotic cell death is the production of reactive oxygen species. The increase in reactive oxygen species which occurs in normal neural cells when they are exposed to cellular insults results in cell death. It was discovered that cell death can be inhibited or avoided entirely if the increase in reactive oxygen species is prevented or at least limited.
As one feature of the present invention, bcl-2 protein is introduced into neural cells which are naturally lacking in this protein. It was discovered that bcl-2 is effective in preventing the production of lethal levels of reactive oxygen species within the cells when they were subjected to cellular insult. The bcl-2 protein was introduced into the cell by infecting the cell with a genetically engineered vector that was capable of expressing bcl-2.
Bcl-2 and related proteins may be used to inhibit cell death during exposure of cells to a variety of cellular insults. Cellular insult situations where the use of bcl-2 inhibits cell death include: withdrawal of serum or growth factor; withdrawal of glucose; exposure to calcium ionophore; membrane peroxidation; free radical-induced damage; and treatment of the cell to reduce its ability to protect itself from oxidative injury (e.g. exposure of cell to enzyme inhibitors such as buthionine sulfoximine).
As another feature of the present invention, cells which naturally include bcl-2 or other proteins which provide protection against oxidative cellular insults may be treated to remove the protection provided by such proteins. This is accomplished in accordance with the present invention by treating the cell with reductants and/or metal chelators which neutralize the effect of the protective protein. The resulting unprotected cell may then be killed by exposure to a selected insult protocol.
The invention is well-suited for increasing the susceptibility of cancer cells to attack by chemotherapy. Many cancer cells include protective compounds, such as bcl-2, which are necessary to the mechanism which protects the cancer cells against death due to oxidative injury. In accordance with the present invention, cancer cells may be treated to neutralize the protective compounds. This eliminates the cancer cells protective mechanism and renders it susceptible to attack by agents which cause oxidative injury due to the production of reactive oxygen species.
The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.
REFERENCES:
patent: 4481195 (1984-11-01), Rabin
patent: 4939178 (1990-07-01), Muller
patent: 5002755 (1991-03-01), Mitchell
patent: 5677135 (1997-10-01), Bredesen
patent: 5681711 (1997-10-01), Bredesen
patent: 5834457 (1998-11-01), Bredesen et al.
patent: WO94/27426 (1994-12-01), None
patent: WO95/05750 (1995-03-01), None
Hockenbery et al., “Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death,” Nov. 22, 1990, Nature, vol. 348, pp. 334-336.
Zhong et al., “BCL-2 Inhibits Apoptosis in Multiple Neural Cell Types,” Abst. 25.11, Society for Neuroscience Abstracts, vol. 18, 1992.
Nuñez et al., “Deregulated Bcl-2 Gene Expression Selectively Prolongs Survival of Growth Factor-Deprived Hemopoietic Cell Lines,” May 1, 1990, Journal of Immunology, vol. 144, No. 9 pp. 3602-3610.
Zhong et al., “bcl-2 inhibits death of central neural cells induced by multiple agents,” May 1993, Proc. Natl. Acad. Sci., vol. 90, pp. 4533-4537.
Mah et al. “The Protooncogene bcl-2 Inhibits Apoptosis in PC12 Cells,” 1993, J. Neurochem, vol. 60, No. 3, pp. 1183-1185.
Garcia et al., “Prevention of Programmed Cell Death of Sympathetic Neurons by the bcl-2 Proto-Oncogene,” Oct. 9, 1992, Science, vol. 258, pp. 302-304.
Martinou et al., “The bcl-2 Proto-Oncogene Protects Neurons From Naturally Occurring Cell Death In Vivo,” Abst. 279.16, Society for Neuroscience Abstracts, vol. 19, 1993.
Merry et al., “bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS,” Development 120, 301-311 (1994).
Allsopp et al., “The Proto-Oncogene bcl-2 Can Selectively Rescue Neurotrophic Factor-Dependent Neurons From Apoptosis,” Cell, vol. 73, 295-307, 1993.
Kane et al., “Bcl-2 Inhibits Neural Cell Death by Decreasin
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Minnifield Mita
The Regents of the University of California
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