Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-09-11
2001-02-06
Huff, Sheela (Department: 1642)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600
Reexamination Certificate
active
06184202
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the identification, purification, and isolation of a novel protein which interacts with bcl-2 protein to form heteromultimers (heterodimers) in vivo and more particularly to the purification, isolation and use of bcl-2 associated protein, herein called Bax, bcl-2 muteins which comprise a BH1 and/or BH2 domain which has an amino sequence substitution, addition, or deletion and which exhibits a substantially reduced binding to Bax (or other bcl-2 protein family members) and/or a substantially reduced death repressor activity, bcl-2 fragments comprising a BH1 and/or BH2 domain, methods for identifying agents which modulate (e.g., inhibit) binding of Bax to bcl-2 and/or other bcl-2 protein family members.
BACKGROUND OF THE INVENTION
Description of the Related Art
Cell death is an important aspect during the embryonic or post-natal development of major organ systems. Apoptosis, or programmed cell demise, also plays a critical role in maintaining homeostasis in many adult tissues. Within vertebrates, bcl-2 is the best understood gene in a cell death pathway and functions as a cell death repressor.
Apoptosis is a term used to refer to the process(es) of programmed cell death and has been described in several cell types (Waring et al. (1991)
Med. Res. Rev
. 11: 219; Williams G T (1991)
Cell
65: 1097; Williams G T (1992)
Trends Cell Biol
. 2: 263; Yonisch-Rouach et al. (1991)
Nature
352: 345). Apoptosis is likely involved in controlling the amount and distribution of certain differentiated cell types, such as lymphocytes and other cells of the hematopoietic lineage. The mechanism(s) by which apoptosis is produced in cells is incompletely understood, as are the regulatory pathways by which the induction of apoptosis occurs.
Apoptosis Mechanism(s)
Apoptosis was first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation (Kerr et al., 1972). One hallmark pattern early in the process of cell death is internucleosomal DNA cleavage (Wyllie, 1980). The death-sparing effecte; of interrupting RNA and protein synthesis and the stereotyped patterns of cell death during development were consistent with a cell autonomous genetic program for cell death (Wyllie et al. (1980)
Int. Rev. Cytol
. 68: 251; Sulston, J. and Horvitz, H. (1977)
Develop. Biol
. 56: 110; Abrams et al. (1993)
Development
117: 29). The isolation of mutants defective for developmental cell death in the nematode Caenorhabditis elegans supported this view (Ellis, H. and Horvitz, H. (1986)
Cell
44: 817; Hengartner et al. (1992)
Nature
356: 494).
The consistency of the morphologic and biochemical patterns defined as apoptosis within different cell types and species, during normal development and as a response to external stimuli are consistent with a common cause of cellular mortality. This thesis is supported by the concept of an endogenous program responsible for cell death and the presence of gene products which are positive and negative regulators of apoptosis. The best studied negative regulator of apoptosis is the bcl-2 proto-oncogene product. It provides the strongest evidence for a shared mammalian pathway of death by its ability to block a wide variety of cell death models.
bcl-2
The protein encoded by the bcl-2 proto-oncogene has been reported to be capable of inhibiting apoptosis in many hematopoietic cell systems;. The proto-oncogene bcl-2 was isolated and characterized as a result of its frequent translocation adjacent to the immunoglobulin heavy chain enhancer in the t(14;18) chromosome translocation present in more than 80% of human follicular lymphomas (Tsugimoto et al. (1985)
Science
; Chen-Levy et al. (1989)
Mol. Cell. Biol
. 9: 701; Cleary et al. (1986)
Cell
47: 19). These neoplasias are characterized by an accumulation of mature resting B cells presumed to result from a block of apoptosis which would normally cause turnover of these cells. Transgenic mice expressing bcl-2 under the control of the E&mgr; enhancer similarly develop follicular lymphomas which have a high incidence of developing into malignant lymphomas (Hockenbery et al. (1990)
Nature
348: 334; McDonnell T J and Korsmeyer S J (1991)
Nature
349: 254; Strasser et al. (1991)
Cell
67: 889).
The bcl-2 protein is a 25 kD membrane-associated cytoplasmic protein (Tsujimoto et al. (1987)
Oncogene
2: 3; U.S Pat. Nos. 5,202,429 and 5,015,568; Chen-Levy (1989) op.cit; Hockenbery (1990) op.cit). Unlike many other proto-oncogene products, the bcl-2 protein apparently functions, at least in part, by enhancing the survival of hematopoietic cells of T and B origins rather than by directly promoting proliferation of these cell types (Vaux et al. (1988)
Nature
335: 440; Tsujimoto Y (1989)
Proc. Natl. Acad. Sci
. (
U.S.A
.) 86: 1958; Tsujimoto Y (1989)
Oncogene
4: 1331; Reed et al. (1989)
Oncogene
4: 1123; Nunez et al. (1989)
Proc. Natl. Acad. Sci
. (
U.S.A
.) 86: 4589; Nunez et al. (1990)
J. Immunol
. 144: 3602; Reed et al. (1990)
Proc. Natl. Acad. Sci
. (
U.S.A
.) 87: 3660; Alnemri et al. (1992)
Proc. Natl. Acad. Sci
. (
U.S.A
.) 89: 7295). The capacity of bcl-2 to enhance cell survival is related to its ability to inhibit apoptosis initiated by several factors, such as cytokine deprivation, radiation exposure, glucocorticoid treatment, and administration of anti-CD-3 antibody (Nunez et al. (1990) op.cit; Hockenbery et al. (1990) op.cit; Vaux et al. (1988) op.cit; Alnemri et al. (1992)
Cancer Res
. 52: 491; Sentman et al. (1991)
Cell
67: 879; Strasser et al. (1991) op.cit). Upregulation of bcl-2 expression also inhibits apoptosis of EBV-infected B-cell lines (Henderson et al. (1991)
Cell
65: 1107). The expression of bcl-2 has also been shown to block apoptosis resulting from expression of the positive cell growth regulatory proto-oncogene, c-myc, in the absence of serum or growth factors (Wagner et al (1993)
Mol. Cell. Biol
. 13: 2432). However, the precise mechanism(s) by which bcl-2 is able to inhibit apoptosis is not yet fully defined.
The bcl-2 proto-oncogene is rather unique among cellular genes in its ability to block apoptotic deaths in multiple contexts (Korsmeyer, S. (1992)
Blood
80: 879). Overexpression of bcl-2 in transgenic models leads to accumulation of cells due to evasion of normal cell death mechanisms (McDonnell et al. (1989)
Cell
57: 79). Induction of apoptosis by diverse. stimuli, such as radiation, hyperthermia, growth factor withdrawal, glucocorticoids and multiple classes of chemotherapeutic agents is inhibited by bcl-2 in in vitro models (Vaux et al. (1988)
Nature
335: 440; Tsujimoto, Y. (1989)
Oncogene
4: 1331; Nunez et al. (1990)
J.Immunol
. 144: 3602; Hockenbery et al. (1990)
Nature
348: 334; Sentman et al. (1991)
Cell
67: 879; Walton et al. (1993)
Cancer Res
. 53: 1853; Miyashita T and Reed J (1993)
Blood
81: 151). These effects are proportional to the level of bcl-2 expression. Additionally, the endogenous pattern of bcl-2 expression is highly suggestive of a role in the regulation of cell survival in vivo (Hockenbery et al. (1991)
Proc. Natl. Acad. Sci. USA
88: 6961; LeBrun et al. (1993)
Am. J. Pathol
. 142: 743). The bcl-2 protein seems likely to function as an antagonist of a central mechanism operative in cell death.
bcl-2 is unique among protooncogenes by being localized to the mitochondrial membrane as defined by Hockenbery, D. M., Nunez, G., Milliman, C., Schreiber, R. D. and Korsmeyer, S. J. “bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.” Nature 378, 334-336, 1990. bcl-2 has been shown to have the onclogenic function of blocking programmed cell death whereas a deregulated bcl-2 extends the survival of certain hematopoietic cell lines following growth factor deprivation. When pro-B-cell or promyelocyte cell lines are deprived of interleukin 3 they normally succumb to a programmed demise entitled apoptosis. This pattern of morphologic cell death is characterized by a dramatic plasma
Howell & Haferkamp L.C.
Huff Sheela
Washington University
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