Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-07-11
2003-05-06
Bansal, Geetha P. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007230, C435S007400, C435S007920, C435S023000, C436S063000, C436S064000
Reexamination Certificate
active
06558900
ABSTRACT:
ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT
Not applicable.
BACKGROUND OF THE INVENTION
The field of this invention is the area of apoptosis (programmed cell death) and methods for the study of the regulation thereof. Specifically, the present invention provides an in vitro system for the analysis of apoptosis and specific regulators of the apoptotic pathway.
Apoptosis is a distinct form of cell death controlled by an internally encoded suicide program [reviewed by Steller, H. (1995)
Science
267, 1445-1449; White, E. (1996)
Gene
&
Dev.
10, 1-15]. Morphologic changes associated with apoptosis include condensation of nucleoplasm and cytoplasm, blebbing of cytoplasmic membranes, and fragmentation of the cell into apoptotic bodies that are rapidly phagocytosed by neighboring cells [Kerr, J. (1971)
J. Pathol.
105, 13-20; Wyllie et al. (1980)
Int. Rev. Cytol.
68, 251-305]. Biochemical markers of apoptosis include DNA fragmentation into nucleosomal fragments [Wyllie, A. (1980)
Nature
284, 555-556], activation of the interleukin lb converting enzyme (ICE)-family of proteases [Duan et al. (1996)
J. Biol. Chem.
271, 1621-1625; Wang et al. (1996)
EMBO J.
15, 1012-1020], and cleavage of substrates of the ICE-family of proteases, including poly(ADP-ribose) polymerase (PARP) [Tewari et al. (1995)
Cell
81, 801-809; Nicholson et al. (1995)
Nature
376, 37-43] sterol regulatory element binding proteins (SREBPs) [Wang et al. (1995)
J. Biol. Chem.
270, 18044-18050; Wang et al. 1996, supra], nuclear lamin [Lazebnik et al. (1995)
Proc. Natl. Acad. Sci. USA
92, 9042-9046], and the U1 associated 70 kDa protein [Casciola-Rosen et al. (1994)
J. Biol. Chem.
269, 30757-30760].
The cell suicide program is illustrated by genetic studies in the nematode
Caenorhabditis elegans
[Hengartner and Horvitz (1994)
Philos. Trans. R. Soc. London Ser. B
345, 243-246]. Two genes involved in the control of programmed cell death in
C. elegans
have been well characterized. One gene (ced-9) encodes a protein that prevents cells from undergoing apoptosis [Hengartner et al. (1992)
Nature
356, 494-499], and the ced-3 gene encodes a protease required for initiation of apoptosis [Yuan and Horvitz (1990)
Dev. Biol.
138, 33-41].
The bcl-2 family of genes are mammalian counterparts of ced-9 [Hengartner and Horvitz (1994)
Cell
76, 665-676]. Over-expression of bcl-2 coding sequences prevents mammalian cells from undergoing apoptosis in response to a variety of stimuli [reviewed by Reed, J. C. (1994)
J. Cell Biol.
124, 1-6]. The BCL-2 protein is located primarily on the outer membranes of mitochondria [Monaghan et al. (1992)
J. Hist. Cytochem.
40, 1819-1825; Krajewski et al. (1993)
Cancer Res.
53, 4701-4714; de Jong et al. (1994)
Cancer Res.
54, 256-260]. The presence of BCL-2 on the mitochondria surface is correlated with a block in the release of cytochrome c in response to triggers of apoptosis in cells which do not express the BCL-2 protein on the mitochondrial surface [Yang et al. (1997)
Science
275, 1129-1132]. Holocytochrome c, but not apocytochrome c, triggers activation of CPP32 and the apoptotic cascade. Without wishing to be bound by theory, it is believed that the Bcl-2 protein inhibits apoptosis by preventing release of holocytochrome c from the mitochondrial membrane and also prevents depolarization of the mitochondrial membrane.
The CED-3 protein is a cysteine protease related to the ICE-family of proteases in mammalian cells [Yuan et al. (1993)
Cell
75, 641-652]. The closest mammalian homolog of CED-3 is CPP32 [Fernandes-Alnemri et al. (1994)
J. Biol. Chem.
269, 30761-30764], which cleaves PARP and SREBPs in cells undergoing apoptosis [Tewari et al. (1995) supra; Nicholson et al. (1995) supra; Wang et al. (1996) supra]. CPP32, which is also called caspase-3, is closely related to CED-3 in terms of amino acid sequence identity and substrate specificity [Xue and Horvitz (1995)
Nature
377, 248-251]. Like CED-3 in
C. elegans,
CPP32 normally exists in the cytosolic fraction as an inactive precursor; that precursor is activated proteolytically in cells undergoing apoptosis [Schlegel et al. (1996)
J. Biol. Chem.
271, 1841-1844, 1996; Wang et al. (1996) supra]. Further evidence for the requirement for active CPP32 in apoptosis is that a tetrapeptide aldehyde inhibitor that specifically inhibits CPP32 activity blocks the ability of cytosol from apoptotic cells to induce apoptosis-like changes in normal nuclei in vitro. [Nicholson et al. (1995) supra].
Triggering of apoptosis by activated CPP32 is part of the highly regulated mechanism for initiation of apoptosis; careful regulation of this pathway is necessary to prevent unwanted cell death. CPP32 is activated by multiple proteolytic cleavages of its 32 kDa precursor form, generating the 17/11 kDa or 20/11 kDa active form [Nicholson et al. (1995) supra; Wang et al. (1995) supra]. CPP32 is activated by cleavage at aspartic acid residues, a hallmark of ICE-like proteases [Thomberry et al. (1992)
Nature
356, 768-774], and a cascade of ICE-like proteolytic cleavages leading to apoptosis has been proposed [Tewari et al. (1995) supra; Wang et al. (1996) supra]. Activated CPP32 from HeLa cell extracts cleaves the CPP32 precursor [Wang et al. (1996) supra], indicating that CPP32 can be activated through autocatalysis. Autocatalytic cleavage is probably responsible for active enzyme when the CPP32 precursor is expressed in large quantity in bacteria [Xue and Horvitz (1995) supra]. Recently, another ICE-family protease has been identified that may be responsible for cleaving the CPP32 precursor into the 20/11 kDa active form. This enzyme has been purified from hamster liver extracts and identified as the hamster homolog of Mch2a [Liu et al. (1996)
J. Biol. Chem.
271, 13371-13376; Fernandes-Alnemri et al. (1995)
Cancer Res.
55, 2737-2742]. Autocatalysis and the protease cascade may provide the signal amplification necessary for rapid and irreversible apoptosis, but the intracellular factors that trigger this amplification have yet to be identified.
There have been several previous reports of cell-free apoptosis systems that induce apoptotic changes in the added nuclei [Lazebnik et al. (1993)
J. Cell Biol.
123, 7-22; Newmeyer et al. (1994)
Cell
79, 353-364; Eeari et al. (1995)
EMBO. J.
14, 5201-5208; Martin et al. (1995)
EMBO J.
14, 5191-5200]. These systems require cytosol from cells that are already undergoing apoptosis in vivo; thus, they cannot be used to detect triggering factors.
There is a need in the art for in vitro methods for the analysis of compounds and biological factors which trigger or accelerate apoptosis or which interfere with the induction of apoptosis, as well as those which can increase the apoptotic effect of chemotherapeutic agents in cancers, especially those expressing oncogenic bcl-2. This need is met by the present invention, which allows the study of apoptosis and regulators thereof in a cell-free system in which the analysis is not complicated by previous induction of the apoptotic pathway in the cells used to prepare the test extracts.
SUMMARY OF THE INVENTION
The present invention provides an in vitro system and methods for the analysis of the regulation of apoptosis and for the identification of activators and inhibitors of the apoptotic pathway; the present system is improved over prior art systems for the study of apoptosis in that the prior art systems depended on cell free extracts prepared from organisms in which the apoptosis pathway had already been induced. Thus, the present system and methods permit freedom from the potential interference of apoptosis-inducing factors or other conditions on which prior art systems have relied.
As exemplified herein, the present invention provides an in vitro system fo
Liu Xuesong
Wang Xiaodong
Bansal Geetha P.
Emory University
Greenlee Winner and Sullivan PC
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