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WO1999006588A1 - Systeme exempt de cellules relatif a l'apoptose a dependance mitochondriale et techniques d'application - Google Patents

Systeme exempt de cellules relatif a l'apoptose a dependance mitochondriale et techniques d'application Download PDF

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Publication number
WO1999006588A1
WO1999006588A1 PCT/US1998/001957 US9801957W WO9906588A1 WO 1999006588 A1 WO1999006588 A1 WO 1999006588A1 US 9801957 W US9801957 W US 9801957W WO 9906588 A1 WO9906588 A1 WO 9906588A1
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mitochondria
apoptosis
compound
cell
dependent
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PCT/US1998/001957
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English (en)
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Dale E. Bredesen
H. Michael Ellerby
Lisa M. Ellerby
Douglas R. Green
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The Burnham Institute
La Jolla Institute For Allergy And Immunology
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Priority to AU60538/98A priority Critical patent/AU6053898A/en
Publication of WO1999006588A1 publication Critical patent/WO1999006588A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)
    • G01N2333/96469—Interleukin 1-beta convertase-like enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • Apoptosis is the term used to describe a type of cellular death that occurs in many tissues as a normal physiological process. Also, referred to as "programmed cell death, " this form of cellular demise involves the activation in cells of a built-in genetic program for cell suicide wherein cells essentially autodigest. The remnants of these dead cells are then cleared almost without a trace by neighboring phagocytic cells, without resulting in inflammation or scarring. Apoptosis thus stands in marked contrast to cell death caused, for example, by oxygen-deprivation in the settings of myocardial infarction or stroke, where cells lose their energy supplies, rupture, and spill their contents into the extracellular milieu.
  • Parkinson's disease Mochizuki et al . , J. Neurol. Sci. 137:120-123 (1996)
  • Alzheimer's disease Cotman and Anderson Mol . Neurobiol . 10:19-45 (1995); Dragunow et al . , euroreport 6:1053-1057 (1995); Smale et al . , Exp . Neurol . 133:225-230 (1995); La Ferla et al . , Nat . Genet . 9:21-30 (1996); Su et al . , Neurpreport 5:2529-2533
  • ALS amyotrophic lateral sclerosis
  • human immunodeficiency virus encephalopathy Gelbard et al . , Neuropathol. Appl . Neurobiol. 21:208-217. (1995); Petito et al . , Am. J. Pathol ..146 : 1121-1130 (1995)
  • cerebral trauma Rink et al . , Amer. J. Path. 147:1-9, (1995)
  • Ferrer et al . Brain Path. 4:115-122 (1994)
  • Huntington's disease Yamayama et al .
  • NAIP neuroal apoptosis-inhibitory protein
  • Alzheimer's disease- associated mutations at amyloid precursor protein residue 717 have been shown to be pro- apoptotic in a subclone of cos cells (Yamatsuji et al . ,
  • presenilin-2 mutants enhance the pro-apoptotic effect of presenilin-2 ( olozin et al . ,
  • cell-free methods for identifying compounds that modulate mitochondria-dependent apoptosis are provided. Also provided are bioassays that can distinguish a particular test-compounds activity between mitochondria-dependent, pre-mitochondria-dependent and post-mitochondria- dependent stages of apoptosis.
  • Figure 1 shows staurosporine and tamoxifen- activated neural apoptosis.
  • A Percent apoptotic cells versus time in hours. CSM-25 cells were incubated at 34°C with 10 ⁇ M staurosporine (white rectangles) , or 100 ⁇ M tamoxifen (black rectangles) for the indicated times. Apoptotic cells were judged morphologically.
  • B Percent apoptotic cells versus time in hours. CSM-25 cells were incubated at 34°C with 10 ⁇ M staurosporine (white rectangles) , or 100 ⁇ M tamoxifen (black rectangles) for the indicated times. Apoptotic cells were judged morphologically.
  • B Percent apoptotic cells versus time in hours. CSM-25 cells were incubated at 34°C with 10 ⁇ M staurosporine (white rectangles) , or 100 ⁇ M tamoxifen (black rectangles) for the indicated times. Apoptotic
  • Proteolytic profile of protein substrates selectively cleaved during staurosporine-initiated neural apoptosis.
  • CSM-25 cells were incubated at 34°C with 10 ⁇ M staurosporine for the indicated times.
  • Cell lysates were made at the indicated time points, and subjected to Western blot analysis.
  • Figure 2 shows that tamoxifen-primed extract activates neural cell-free apoptosis.
  • A Nuclear morphological changes in CSM nuclei incubated between 0 and 2 hours at 34°C in a 16,000 g extract made from tamoxifen-primed NSC-19 cells.
  • B Percent apoptotic nuclei incubated as in (A) in either normal or primed extract.
  • C Agarose gel electrophoresis of internucleosomal DNA fragmentation of rat liver nuclei incubated for 2 hours at 34 °C in a 16,000 g extract made from tamoxifen-primed CSM-25 cells.
  • FIG. 3 shows that atractyloside activates neural cell-free apoptosis.
  • Atractyloside (5 mM) was incubated in a 3,000 g extract made from CSM-25 cells for 1.5 h at 37°C. The activation of apoptosis was measured by fodrin cleavage.
  • Atractyloside also induced cell-free apoptosis in a system composed of rat liver mitochondria and 16,000 g extract from CSM-25 cells. However, atractyloside incubated in a 16,000 g extract alone did not lead to cell-free apoptosis.
  • FIG. 4 shows that mastoparan activates neural apoptosis.
  • Mastoparan induces apoptosis in cultured rat cerebellar neuron precursors (the R2 cell line) as measured by cell death using propidium iodide staining of DNA in cells with a compromised plasma membrane (see
  • FIG. 5 shows that mastoparan activates neural and neuronal cell-free apoptosis.
  • A Fodrin cleavage and CPP32 processing in a neural cell-free system composed of a 3,000 g extract (containing mitochondria) made from CSM-25 cells, in a neural cell-free system composed of mouse liver mitochondria in a 16,000 g extract from NT2 cells, and in a neuronal cell-free system composed of rat neuronal mitochondria in a 16,000 g extract from primary cerebellar neurons. All systems were incubated for 1.5 h at 37°C with 100 ⁇ M mastoparan. Mastoparan did not prime a 16,000 g extract without mitochondria.
  • B Mastoparan induced release of cytochrome c from mitochondria.
  • Figure 6 shows that cytochrome c and dATP activate neural and neuronal cell-free apoptosis.
  • FIG. 7 shows that CPP32 processing in mitochondria-dependent activation of cell-free apoptosis.
  • Tamoxifen Tamoxifen (Tam) induces apoptosis at the pre-mitochondrial level (cells) , but does not induce apoptosis at the mitochondrial level (mitochondria and extract) , or the post-mitochondrial level (extract) . Similar results were obtained for staurosporine.
  • Mastoparan induces apoptosis at the pre- mitochondrial level (cells) and at the mitochondrial level (mitochondria and extract) , but does not induce apoptosis at the post-mitochondrial level (extract) . Similar results were obtained for atractyloside. Cytochrome c/dATP (Cytc) induces apoptosis at the mitochondrial level (mitochondria and extract) and at the post-mitochondrial level (extract) , but does not induce apoptosis at the pre-mitochondrial level (cells) . DETAILED DESCRIPTION OF THE INVENTION
  • cell-free methods for identifying compounds that inhibit mitochondria-dependent apoptosis comprising: a) providing a non-activated cell extract, wherein said cell extract further comprises mitochondria, and a test-compound; b) contacting said cell extract with a mitochondria-dependent apoptosis-inducing agent; and c) identifying a compound that inhibits apoptosis.
  • the cell extracts employed are neuronal cell extracts.
  • cell-free refers to a system that does not include whole, intact cells.
  • mitochondria mitochondria present in the system, such as in cell-free extracts having mitochondria added therein, whereas cell- free apoptosis does not occur in the absence of mitochondria.
  • non-activated cell extract refers to a normal cytosolic cell extract obtained from a cell that has not been primed or otherwise induced to undergo apoptosis. It has been found that a non-activated cell extract in combination with mitochondria added therein is particularly advantagous in bioassays for identifying compounds that modulate mitochondria-dependent apoptosis.
  • the cell extracts are preferably obtained from neuronal cells.
  • the non-activated cell extracts employed herein are preferably prepared so that the pelleted mitochondria is not disrupted such that- cytochrome c, or any other contaminating priming agent, is released into the cytosol causing the extract to self-prime.
  • the non- activated cell extract is prepared by centrifuging cell lysates at about 16,000 g for about 10-30 minutes, as described in Example l.C.
  • the cell extracts are substantially free of whole cells, organelles, and membrane fractions that pellet at about 16,000g for 10-30 minutes, such as nuclei, mitochondria, lysosomes, and the like.
  • the invention cell-free assay system is substantially free of non-mitochondria membrane organelles that are greater than or equal to the weight of mitochondria, which are typically present in the cytosol.
  • the phrase "substantially free of non-mitochondria membrane organelles” refers to cell extracts that do not include, for example, nuclei, mitochondria, lysosomes, and any contaminating inducing agents (such as cytochrome c, AIF, and the like) that might cause the cell extract to self- prime.
  • Cell extracts that are substantially free of non- mitochondria membrane organelles can be prepared as described, e.g., in Example l.C.
  • Neuronal refers to primary mammalian neuronal cells and mammalian neural cell-lines, and the like.
  • Neuronal cells suitable for use in the invention methods include, for example, primary cultures of mammalian cerebellar neurons, the human teratocarcinoma- derived neuronal precursor cell line NT2/D1 (Pleasure and Lee, J. Neurosci. Res. 35, 585-602 (1993)), the mouse motor neuron-like cell lines NSC-34 and NSC-19 (Cashman, Devel. Dvn.194.209-221. (1992)), and the rat central nervous system (CNS) neuron-like cell line CSM-25 (Durand et al . ,
  • mammalian refers to the variety of species from which neuronal cells for use in the invention methods can be obtained, e.g., human, rat, mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and the like.
  • Mitochondria employed in the invention methods can be obtained from either the same or different species as the cell extracts.
  • the mitochondria can be obtained from either the same or differenct cell-types as the cell extracts. Methods for isolating mitochondria are described, for example, in Example I.F., and are otherwise well-known in the art (see, e.g., Hovius et al . , Biochem. Biophys . Acta . 1021:217-226 (1990);
  • test-compound refers to a compound to be tested in the invention bioassays.
  • the test-compounds can be obtained from diverse variety of compound libraries that are generally available to those of skill in the art. Single compounds can thus be identified and selected from the test-compounds subjected to the invention bioassays as "compounds" having the desired biological activity.
  • mitochondria-dependent apoptosis-inducing agent refers to agents that induce apoptosis when, preferably only when, there is mitochondria present in the system, such as in whole cells or in a cell-free extracts having mitochondria therein.
  • Suitable agents include, for example, those compounds that can disrupt- the inner and/or outer membrane of the mitochondria, such as by forming pores in, or by lysing, the membrane.
  • Such agents can include proteins or peptides having "mitochondrial presequences" that are rich in basic amino acids, adopt an ⁇ -helical conformation, and result in an amphipathic structure (see, e.g., Nicolay et al . , J. Bioen ⁇ .
  • An exemplary mitochondrial presequence is the 25-residue presequence of cytochrome c-oxidase subunit IV (Maduke et al . , Science. 260:364-367 (1993)).
  • mitochondria-dependent apoptosis-inducing agents include mastoparan, atractyloside, cardiolipin (see, e.g., Hovius et al . , FEBS Lett .. 330:71-76 (1993); Hovius et al . , Biochim. Biophys. Acta. 1021:217-226 (1990)), adrenodoxin precursor (see, e.g., Ou et al . , J. Biochem. 103:589-595 (1988)), synthetic mitochondrial presequences (see, e.g., Roise, PNAS . USA. 89:608-612 (1992); and Furuya et al . ,
  • a preferred mitochondria-dependent apoptosis-inducing agent, for use herein, is mastoparan.
  • the invention methods described herein can be used to identify additional mitochondria- dependent apoptosis-inducing agents.
  • compounds can be identified that induce apoptosis in non- activated cell extracts only in the presence of mitochondria.
  • Such compounds can be identified using an invention cell-free method for identifying compounds that activate mitochondria-dependent neuronal apoptosis described hereinafter.
  • apoptosis refers to the well-known process of programmed cell death. There is a variety of well-known, generally accepted in vi tro indicia of apoptosis, including nuclear morphological changes, internucleosomal fragmentation of DNA, the selective proteolysis of substrates, and the activation of CPP32-like caspases.
  • the substrates of the caspase family of cysteine proteases have received considerable attention because cleavage of these substrates offers molecular mechanisms for many of the hallmark morphological and functional changes exhibited by apoptotic cells (Casiano et al . , J . Ex .
  • the cleavage of fodrin leads to morphological alterations such as process retraction, cellular rounding, and bleb formation.
  • the cleavage of nuclear substrates such as the lamins, NuMA, and topoisomerase I, is believed to be associated with the dissolution of the nuclear membrane, chromatin condensation, and nuclear fragmentation.
  • the cleavage of substrates during apoptosis can lead to activation, not just inactivation.
  • PARP cleavage has been reported to lead to inactivation, Lazebnik et al . , Nature , 371:346-347, (1994)
  • the cleavage of PKC- ⁇ results in the activation of the enzyme (Emoto et al . , The EMBO J .. 24:6148-6156, (1995).
  • apoptosis in cell-free systems can be assessed by detecting the relative levels of: caspase processing (i.e., the cleavage of the pro-caspase to active forms; see, e.g., Casciola-Rosen et al . , 1996, J. Ex . Med .. ., 183: 1957-1964; Tewari et al, 1995, J. Biol . Chem.. 32:18738-18741; Tewari et al, 1995, Cell.
  • caspase processing i.e., the cleavage of the pro-caspase to active forms; see, e.g., Casciola-Rosen et al . , 1996, J. Ex . Med .. ., 183: 1957-1964; Tewari et al, 1995, J. Biol . Chem.. 32:18738-18741; Tewari et al, 1995, Cell.
  • cytosolic substrate cleavage the release of cytochrome c from mitochondria, and the like.
  • cytosolic substrates that are cleaved as a result of apoptosis are set forth in Table 1, and include: fodrin, CPP32, PKC- ⁇ , and the like.
  • nuclei When nuclei is optionally included in the cell- free system, the occurrence of apoptosis can be assessed by, in addition to the methods described above, detecting: chromatin condensation, shrinkage and fragmentation of the nuclei, and the like (see, for example, Zanzami et al . , J. Ex . Med.. 183:1533-1544 (1995); Newmeyer et al . , Cell. 79:353-364 (1994)).
  • nuclear substrates that are cleaved as a result of apoptosis are also set forth in Table 1, and include: DNA topoisomerase (Liu, Ann. Rev. Biohem. 58, 351-375
  • modulates refer to either the inhibition (such as with antagonists) or activation (such as with agonist) of apoptosis.
  • the modulation of apoptosis at the mitochondria-dependent stage can be determined, for example, by methods described herein, such as in Example 3, or the like.
  • cell-free methods for identifying a compound that activates mitochondria-dependent apoptosis comprising: a) providing a non-activated cell extract in the presence and absence of mitochondria; b) contacting said cell extract with a test-compound; and c) identifying a compound that activates mitochondria-dependent apoptosis.
  • a compound is identified as a mitochondria-dependent activator of apoptosis if, for example, it activates apoptosis in the presence of mitochondria, and does not activate apoptosis in the absence of mitochondria.
  • the cell extract is obtained from neuronal cells.
  • cell-free methods for identifying a compound that modulates neuronal apoptosis comprising: a) providing a neuronal cell extract containing mitochondria, b) contacting said cell extract with a test- compound, and c) identifying a compound that modulates apoptosis.
  • the modulating compound identified inhibits apoptosis and the extract further comprises a mitochondria-dependent apoptosis inducing agent.
  • the modulating compounds identified are mitochondria-dependent, as such compounds activate apoptosis only in the presence of mitochondria and do not activate apoptosis in the absence of mitochondria.
  • cell -free methods for identifying compounds that inhibit mitochondria- dependent neuronal apoptosis comprising: a) providing a- non-activated neuronal cell extract in the presence and absence of a test-compound, wherein said cell extract further comprises mitochondria, and a mitochondria-dependent apoptosis-inducing agent ; and b) identifying a compound that inhibits apoptosis.
  • a compound is identified as an inhibitor of apoptosis if, for example, it decreases the levels of caspase processing or substrate cleavage when the test- compound is present compared to such levels in the absence of test-compound.
  • an invention method for identifying a compound that specifically modulates mitochondria-dependent apoptosis comprises:
  • test-compound modulates pre-mitochondria-dependent apoptosis
  • test-compound modulates post-mitochondria-dependent apoptosis
  • identifying a compound that modulates mitochondria-dependent apoptosis refers to the stage of apoptotic induction that requires components other than mitochondria, such as contained in whole cells.
  • pre-mitochondria- dependent apoptosis refers to the stage of apoptotic induction that requires components other than mitochondria, such as contained in whole cells.
  • staurosporine and tamoxifen induce apoptosis in whole cells from which active extracts may then be prepared, but do not induce apoptosis directly in cell extracts. This indicates that induction of apoptosis by these agents requires an intact signaling mechanism that is absent in the invention cell-free systems described herein.
  • neuronal cell-free apoptotic systems that include additional purified fractions such as plasma membranes (Meier et al . , J. Cell Biol . 998:991-1000 (1984) and lysosomes (Ohshita and Kido, Anal. Biochem. 230:41-47 (1995) ) . Since this type of cell-free system could represent a more upstream system, agents like tamoxifen might then induce apoptosis without the need for intact cells .
  • mitochondrial inner membrane permeability transition As used herein, the phrase “mitochondria-dependent apoptosis” refers to apoptotic induction that requires the presence of mitochondria. As set forth in Example 3, unlike staurosporine and tamoxifen, atractyloside and mastoparan, both of which induce the mitochondrial inner membrane permeability transition (Zamzami et al . , J. Exp. Med. 183:1533-1544 (1996);
  • Example 4 it has been demonstrated that cytochrome c and dATP, added together, activate neural cell extracts in a manner that is independent of mitochondria. These results indicate that cytochrome c and dATP induce apoptosis at a point distal to those of staurosporine, tamoxifen, atractyloside, and mastoparan. With respect to cytochrome c/dATP activation, it should be noted that not all forms of cytochrome c activate the system. Yeast (ISO-1) and partially acetylated horse cytochrome c are incapable of activating the system. In yeast, lysine 72 is trimethylated (Brayer and Murphy, Scott, R.A.
  • lysine 72 is a good candidate as a residue required for cytochrome c to activate cell-free apoptosis.
  • Bel-2 produces an anti-apoptotic effect at the pre-mitochondria-dependent and mitochondria-dependent stages, but cannot protect against the post-mitochondria-dependent activation of apoptosis by cytochrome c.
  • the phrase "specifically modulates mitochondria-dependent apoptosis” refers to a compound that modulates only mitochondria-dependent apoptosis and does not modulate pre- or post- mitochondria-dependent apoptosis.
  • Methods for determining whether a test-compound activates mitochondria-dependent apoptosis are described, for example, in Example 3.
  • mastoparan a peptide toxin from wasp ( Vespula lewisii ) venom (Hirai et al . , Chem. Pharm. Bull.
  • Example 2 Methods for determining whether a test-compound activates pre-mitochondria-dependent apoptosis are described in, e.g., Example 2. As set forth in Example 2, it has been found that tamoxifen, an anti-oestrogenic and anti-neoplastic agent (Fenwick et al . , Br . J. Pharmacol . 59:191-199 (1977); Pollak, Digestion 57
  • test- compounds that produce the same results as tamoxifen or staurosporine can readily be identified by the methods of, e.g., Example 2 as pre-mitochondria-dependent apoptosis activating compounds.
  • Example 4 Methods for determining whether a test-compound activates post-mitochondria-dependent apoptosis are described, e.g., in Example 4. As set forth in Example 4, it has been found that cytochrome c and dATP, added together, do not activate apoptosis in whole cells. However, they do activate a normal cell extract, whether or not mitochondria are added to the extract .
  • the data provided herein teach a general temporal ordering of neuronal apoptotic events as pre-mitochondria-dependent, mitochondria-dependent, and post-mitochondria-dependent .
  • methods are provided herein for identifying compounds that modulate mitochondria-dependent apoptosis. Such methods are useful, for example, for early high-throughput screening of random test-compounds to reduce the number of test- compounds selected for further research, when it is desired to obtain compounds that exhibit apoptotic activity or lack such activity at a particular mitochondria-dependent-stage of apoptosis.
  • compounds known to modulate apoptosis can be subjected to the invention methods to identify the specific apoptotic stage of such activity.
  • identifying a compound that specifically modulates mitochondria-dependent apoptosis one can select a compound that specifically modulates either pre-mitochondria-dependent apoptosis, or post- mitochondria-dependent apoptosis, or any combination thereof.
  • methods for identifying a compound that modulates a specific mitochondria-dependent-stage of apoptosis comprising:
  • determining whether a test-compound modulates mitochondria-dependent apoptosis (2) determining whether said test-compound modulates pre-mitochondria-dependent apoptosis; (3) determining whether said test-compound modulates post-mitochondria-dependent apoptosis; and (4) identifying a compound that modulates a specific mitochondria-dependent-stage of apoptosis .
  • step (4) above can be identifying a compound that modulates mitochondria-dependent apoptosis, wherein the compound does not modulate pre-mitochondria-dependent or post- mitochondria-dependent apoptosis.
  • step (4) above can be identifying a compound that modulates pre-mitochondria-dependent apoptosis, wherein the compound does not modulate mitochondria-dependent or post-mitochondria-dependent apoptosis.
  • step (4) above can be identifying a compound that modulates post-mitochondria-dependent apoptosis, wherein the compound does not modulate pre-mitochondria-dependent or mitochondria-dependent apoptosis .
  • Example 1 The general methods described in Example 1 were employed to carry out Examples 1-5 described herein.
  • the cerebellar tissue was inserted into the top of the bag, and gently teased through the mesh with very light strokes of a glass rod into the same medium as used for the dissection, only now containing 10% heat-inactivated (56°C for 30 min) fetal bovine serum (FBS) (Sigma Chemical Company) and 1% Penicillin-Streptomycin (10,000 units/ml penicillin G sodium and 10.0 ⁇ g/ml streptomycin sulfate; GibcoBRL) .
  • FBS fetal bovine serum
  • Penicillin-Streptomycin 10,000 units/ml penicillin G sodium and 10.0 ⁇ g/ml streptomycin sulfate; GibcoBRL
  • the cell/media suspension was then poured through a sieve with 25 openings per linear inch, and then poured through a sieve with 45 openings per linear inch.
  • the cells were plated at a density of about 10 6 / ⁇ r ⁇ l on "2000 mm 2 tissue culture plates (Falcon Integrid)
  • cytosine ?-D-arabinoside Sigma
  • 40 mM final concentration
  • CSM 14.1 cells (Durand et al . , Soc Neurosci Aba, 16:40 (1990); Zhong et al . , Mol Brian Res.. 19:353- 355 (1993a); Proc. Natl. Acad. Sci. USA. 90:4533-4537 (1993b) were made using E14 rat mesencephalon primary cultures, and immortalized by using the pSVtsA58 retrovirus. This line is neural by neurofilament staining (NF-H) at the restrictive temperature 39°C, and by expression of tyrosine hydroxylase mRNA, suggesting that it is a dopaminergic precursor.
  • NF-H neurofilament staining
  • CSM-25 a subclone of CSM 14.1 was selected for its high propensity to undergo apoptosis following serum withdrawal.
  • CSM-25 cells were grown at 34°C, with a 5% C0 2 /95% air mixture, in the same medium as that used for the cerebellar neurons .
  • the NSC-34 and NSC-19 cell lines mimic selected aspects of motor neuron development in an immortalized clonal system. These cell lines are mouse-mouse neural hybrids, developed by fusing the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12 - 14 spinal cord cells (Cashman et al . , Devel .
  • the NSC-34 and NSC-19 hybrids display a mutipolar neuron-like phenotype, express choline acetyltransferase, and induce twitching in cocultured mouse myotubes . Beyond this, they express the additional properties expected of motor neurons, including generation of action potentials, expression of neurofilament triplet proteins, and acetylcholine synthesis, storage, and release. Furthermore, NSC-34 cells induce acetylcholine receptor clusters on cocultured myotubes, and undergo a vimentin-neuro- filament switch with maturation in culture, similar to that occurring in neuronal development.
  • NSC-34 and NSC- 19 cells were grown at 37°C, with a 5% C0 2 / 95% air mixture, in Dulbecco's modified Eagle's medium (DMEM) (GibcoBRL) , supplemented with 4.5 g/1 glucose and L-glutamine (Mediatech cellgro) and containing 10% heat- inactivated (56 ⁇ for 30 min) fetal bovine serum (FBS) (Summit Biotechnology, Ft.- Collins Col.) and the penicillin/streptomycin formulation given above.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • the human teratocarcinoma cell line NT2/D1 can be manipulated following treatment of retinoic acid to yield > 99% pure cultures of terminally differentiated NT2-N neurons (Pleasure and Lee, J. Neurosci. Res. 35:585-602 (1993)).
  • the NT2/D1 neuronal precursor line is a stem cell line that cannot be induced to yield derivatives other than neurons. This is in sharp contrast to other teratocarcinoma cell lines which yield multiple cell types from multiple germ cell layers.
  • NT2/D1 cells were grown at 37°C, with a 5% C0 2 / 95% air mixture, in Opti-MEM supplemented with 10% heat- inactivated (56 ⁇ C for 30 min) fetal bovine serum (FBS) (Sigma Chemical Company) and containing the penicillin/streptomycin formulation given above.
  • FBS heat- inactivated fetal bovine serum
  • the R2 cell line is a conditionally immortalized cerebellar neural line (Rabizadeh et al . , J.
  • the R2 cells were grown at 34 °C in the same medium as that used for the cerebellar neurons.
  • Jurkat and Hela cells were grown at 37°C, with a 5% C0 2 / 95% air mixture in RPMI 1640 medium supplemented with L-glutamine (Mediatech cellgro) and containing 10% heat-inactivated (56°C for 30 min) fetal bovine serum (FBS) (Sigma Chemical Company) and the penicillin/streptomycin formulation given above.
  • the Jurkat cells were grown in 225 cm 2 flasks (Costar) at 10 6 cells/ml, containing 200 ml of medium.
  • E_ Preparation of cell lysates.
  • Cells incubated with either 100 ⁇ M tamoxifen or 10 ⁇ M staurosporine were collected at various time points, one 70 - 80% confluent (about 10 7 cells) plate for each time point.
  • the plate was placed on ice, and all subsequent steps were performed either on ice or at 4°C.
  • the 20 ml of media in each plate containing any detached cells was saved in a 50 ml conical centrifuge tube.
  • the adherent cells received 20 ml of PBS and were then gently lifted off the plate with a cell scraper and pooled with the detached cells. A final 10 ml was used to completely wash off the plate.
  • the combined 50 ml was placed on ice and treated with the protease inhibitor cocktail Complete
  • the 16,000 g cytoplasmic extract used in this work is made free of nuclei, mitochondria, and any contaminating apoptotic inducing agent (such as cytochrome c or AIF which can be released from damaged mitochondria), that would lead the extract to self-prime.
  • apoptotic inducing agent such as cytochrome c or AIF which can be released from damaged mitochondria
  • adherent cells between 2 and 40 plates were harvested. The required plates were removed from the incubator and immediately placed on ice, and all subsequent steps were performed either on ice or at 4°C. The 20 ml of media in a plate was removed and discarded, and another 10 ml of ice cold PBS (pH 7.2) was added to the plate. Note that extract of apoptotic cells was made before appreciable cell detachment.
  • the cells were then gently, but quickly, lifted off the plate with a cell scraper, and placed on ice in a 50 ml centrifuge tube until all cells could be harvested for centrifugation.
  • the cells were centrifuged (4°C) at 200 g and the supernatant removed by aspiration. The resulting cell pellet was washed twice in 50 ml of ice cold PBS.
  • the cells were then re-suspended in a 15 ml conical centrifuge tube with 10 ml of hypotonic extraction Buffer [(HEB); 50 mM PIPES, pH 7.4 , 50 mM KCl, 5 mM EGTA, 2 mM MgCl 2 , 1 mM dithiothreitol (DTT) , 10 ⁇ M cytochalasin B, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) ] , formulated to swell the cells in preparation for homogenization.
  • the cells were centrifuged at 1000 g (4°C) to form a tight pellet. The supernatant was aspirated, and the volume of the cell pellet was approximated.
  • HEB HEB was added to a volume between 0.6 - 1 times the pellet volume.
  • the cells were transferred to a 2 ml Dounce homogenizer (Kontes Glass Company) and allowed to swell for 20 - 30 min on ice. Cells were lysed with 20 - 100 gentle strokes of a B-type pestle. The desired extent of lysis (> 90%) was monitored under the microscope by Trypan blue staining. The volume of HEB added, the time of swelling, and the number of pestle strokes all varied according to the cell type. The cell lysate was then transferred to an Eppendorf tube and centrifuged for 30 min at 16,000 g (4°C) in an Eppendorf 5415 C microcentrifuge.
  • the clarified supernatant was carefully removed, leaving approximately 50 ⁇ l behind, so as not to contaminate the extract .
  • the extract was used immediately or stored in aliquots at -84°C. Extracts made this way from primed or apoptotic cells lost little, if any, of their apoptosis inducing activity.
  • the 3,000 g cytoplasmic extract used in this work contains mitochondria, along with pieces of plasma membrane etc., but not whole cells, or nuclei. The method here is the same as that for 16,000 g extracts, with the following distinctions.
  • the lysis buffer is the cell free system buffer (CFS) used by Zamzami et al., J.
  • This buffer is designed not to be hypotonic, so mitochondria do not swell. Yet, at the same time, the cells do not swell either.
  • CSM cells were the only cell line big enough to lyse in the homogenizer without swelling. After lysis of the cells, the lysate was centrifuged for 5 min at 1,000 g (4°C) to remove whole cells and most of the nuclei.
  • the Pierce Coomassie Plus protein assay with BSA standard was used to assay protein concentration in cell extracts using a Shimadzu UV-2101 PC UV-Vis Scanning Spectrophotometer .
  • 16,000 g extracts had a protein concentration between 15 - 20 mg/ml, and 3,000 g extracts between 25 - 30 mg/ml.
  • Rat liver nuclei- were prepared as described (Newmeyer et al . , Cell, (1994)).
  • CSM and HeLa nuclei were prepared as described (Martin et al . , EMBO J.
  • Rat and mouse liver mitochondria were prepared as described by Hovius et al . , Biochem. Biophys. Acta.
  • the livers were quickly removed and submerged in ice cold mitochondria isolation buffer (MIB) [ MIB; 250 mM mannitol (or sometimes 210 mM mannitol and 70 mM sucrose), 0.5 mM EGTA, 5 mM Hepes, 0.1 - 0.05% (w/v) bovine serum albumin (BSA) (pH 7.2 )], supplemented with the protease inhibitors of leupeptin (1 ⁇ g/ml) , pepstatin A (1 ⁇ g/ml) , antipain (50 ⁇ g/ml) , and chymostatin (10 ⁇ g/ml) .
  • the entire mitochondria isolation was performed on ice or at 4°C.
  • the livers were washed in MIB to remove as much blood as possible, and then chopped into 1 - 2 mm 2 cubes with a razor blade .
  • the small cubes were then rinsed off in a sieve (Science Ware Mini-Sieve Microsieve set; Fisher), and transferred to a 15 ml Potter Elvehjem Homogenizer (Kontes Glass Company) which was surrounded by ice.
  • the suspension was loaded on a continuous Percoll gradient .
  • the gradient was made from the diffusion of 4 discontinuous layers (25%, 35%, 45% and 60%) of Percoll in MIB for 3 - 4 h at 25°C, which was then placed at 4oC until needed.
  • the suspension/gradient was then centrifuged at 40,000 g for 40 min using a Beckman JA-12 or JA25.50 rotor.
  • the gradient also contained of 250 mM mannitol, 1 mM EGTA, 25 mM Hepes, 0.1% BSA (pH 7.4), as an osmotic balancer.
  • the mitochondria were removed from the brown band at approximately 1.10 g/ml with a Pasteur pipette. Sometimes the discontinuous gradient method of Boutry and Briquet, Eur. J. Biochem. 127:129-136 (1982) was used in place of the above continuous gradient technique. The mitochondrial pellets were washed with MIB by centrifuging for 10 min at 6300 g in a Beckman JA-12 or JA25.50 rotor.
  • mitochondria storage buffer [MSB; 400 mM mannitol, 10 mM KH 2 P0 4 , 5 mg/ ml BSA, 50 mM Tris-HCl, pH 7.2), and stored on ice for up to 4 h, until needed for the experimentation.
  • Cultured cell mitochondria were prepared as described previously (Moreadith and Fiskum, Anal. Biochem. 137:360-367 (1984) incorporated herein by reference in its entirety) .
  • the assay of apoptotic substrate cleavage, DNA fragmentation, and caspase activity in cell-free reactions involved the formulation of the following cell- free systems :
  • reactions of primed (or apoptotic) extract on cytosolic substrates 20 ⁇ l of normal cytoplasmic extract (15 - 25 mg/ml protein) , 5 ⁇ l of primed or apoptotic cytoplasmic extract (15 - 25 mg/ml protein) , and 4 ⁇ l of HEB buffer, or synthetic peptides diluted in this buffer.
  • cytochrome c and dATP For reactions activated by cytochrome c and dATP, a system was reconstituted according to the following formula: 10 ⁇ l of 16,000 g normal extract, 0.1 ⁇ l cytochrome c (1 - 10 ⁇ M final), and 0.1 ⁇ l of dATP (10 mM final) , 1 - 2 ⁇ l of peptide (or other) inhibitors or HEB buffer, and 0.5 - 1 ⁇ l of nuclei (2 x 10 5 ) or HEB buffer.
  • the ionic strength/osmolarity of the extract was then altered to account for the 50% of the extract composed of hypotonic buffer by adding to every 10 ⁇ l of extract, 0.5 ⁇ l of a lOx stock of CFS buffer.
  • the mitochondria were then added to extract with mastoparan or atractyloside according to the following formula: 20 ⁇ l normal extract, 2 ⁇ l of mitochondria (final concentration of 500 ng/ml) , and 2 ⁇ l of atractyloside (5 mM final -concentration) or mastoparan (10 - 50 ⁇ M final concentration) or CFS buffer.
  • reaction tubes were then carried out in 500 ⁇ l o-ring sealed, screw-top microcentrifuge tubes (Continental Biological Supply) , in a heat bath at 30°C or 37°C for various time periods. At the end of the incubation period, reaction tubes were flash frozen on dry ice, or in liquid nitrogen, and stored at -84°C.
  • N-benzyloxycaronyl-Val-Ala- Asp . fluoromethylketone (zVAD.fmk) was purchased from Enzyme Systems, Dublin, CA.. Ac-YVAD aldehyde and Ac- DEVD-adehyde were purchased from BACHEM Bioscience Inc.
  • Bovine heart cytochrome c, horse heart cytochrome c, yeast (ISO-1) cytochrome c, and partially acetylated cytochrome c were purchased from Sigma Chemical Company.
  • bovine heart cytochrome c and horse heart cytochrome c were purchased from Fluka.
  • dATP was purchased from Promega and Gibco. Sodium and potassium atractyloside, mastoparan were purchased by Sigma Chemical Company.
  • Control peptide (DLSLARLATAR- LAI) was purchased from Coast Scientific, San Diego, California. H. Quantification of apoptosis.
  • Electrophoresis of proteins was carried out using either 8% or 12% SDS-polyacrylamide gels. Prior to loading samples on the gel, bromophenol blue dye was added to each sample (0.002% final concentration). Equal amounts of total protein were loaded per lane, and proteins were separated at 4°C under reducing conditions at 50 V through the stacking gel, and 90 V through the separating gel.
  • Anti-Cr--fodrin mouse monoclonal antibody was purchased from Chemicon International.
  • Anti- ⁇ 5PKC-d rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc.
  • Anti-poly (ADP-ribose) polymerase (PARP) rabbit polyclonal antibody was purchased from BIOMOL Research Laboratories, Inc.
  • Anti-CPP32 mouse monoclonal antibody was purchased from Transduction Laboratories, Inc.
  • Anti-CPP32 rabbit polyclonal antibody was purchased from Upstate Biotechnology, Inc.
  • Anti-CPP32 goat polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc.
  • Anti-lamin B mouse monoclonal mouse antibody was purchased from Oncogene Research Products (CALBIOCHEM) .
  • Anti-cytochrome c mouse monoclonal antibody was provided by Dr. Ronald Jemmerson (University of Minnesota Medical School, Minneapolis, Minnesota 55455) .
  • Human sera containing highly specific, high titer auto-antibodies to poly (ADP-ribose) polymerase (PARP), DNA topoisomerase I, Ul-70 kDa, NuMa, Lamin B, Jo-1, rRNP, and PCNA were from the collection of W.M. Keck Autoimmune Disease Center Laboratory (The Scripps Research Institute, La Jolla, CA) serum bank (Casiano et al . , J . Exp . Med . 184:765-770 (1996) incorporated herein by reference in its entirety) .
  • the blots were then washed for 1 h with frequent changes of TBST, followed by incubation in a peroxidase-coupled secondary antibody for 1 h in TBST containing 5% non-fat dried milk, in a Seal-A-Meal bag.
  • the mouse, human, and rabbit peroxidase-coupled secondary antibodies were from Amersham.
  • the blots were washed for 1 h with frequent changes of TBST.
  • Enhanced chemiluminescence detection of the proteins was carried out using Hyperfilm ECL (Amersham) , and with Pierce SuperSignal Substrate Western Blotting reagents, or Amersham ECL reagents .
  • nuclei were lysed in TE buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA) containing 0.5% sodium lauryl sarkosyl and 0.5 mg/ml proteinase K. Digestion was continued for 1-3 h at 50oc, followed by the addition of Rnase A to- 1.0 mg/ml and further incubation for lh.
  • TE buffer 50 mM Tris-HCl, pH 8.0, 10 mM EDTA
  • Running dye (10 mM EDTA, 0.25% bromophenol blue, 50% glycerol) was then added in 1 : 6 ratio of dye: sample, and the DNA preparations were electrophoresed in 1.5 - 2 % agarose gels in TAE buffer (40 mM Tris-acetate, ImM EDTA) , or TBE buffer (40 mM Tris-borate, 1 mM EDTA) , at 4V per cm of gel (30 - 35 V) , in a Biorad Mini-Sub Cell electrophoresis apparatus . DNA was visualized by ethidium bromide staining.
  • the following cell-free system was reconstituted. First, a 100 ⁇ l aliquot (at least) of 16,000 g extract was clarified a second time for 20-30 min at 4°C, to ensure reproducable spectrophotoretric results of fully clarified extract.
  • cell-free system was reconstituted according to the following formula: 10 ⁇ l of 16,000 g normal extract, 0.1 ⁇ l cytochrome c (1 - 10 ⁇ M final), and 0.1 ⁇ l of dATP (1 mM final) , 1 - 2 ⁇ l of peptide (or other) inhibitors or HEB buffer, and 0.5 - 1 ⁇ l of nuclei (2 x 105) or HEB buffer.
  • the reactions were run as in Example I.G. (Activation of cell-free apoptosis) .
  • N-acetyl-Tyr-Val-Ala-Asp-pNA (Reiter, Inter. J. Peptide and Prot. Res. 43:87-96 (1994)), were made in DMSO, and were purchased from BIOMOL Research Laboratories, Inc.
  • Synchronous cell extracts are required to study effectively the temporal ordering of events in apoptosis (e.g., a protease cascade), or to make a so-called primed extract, which is made from cells committed to, but not yet engaged in, apoptosis, thus representing a more upstream stage of apoptosis than extracts taken from apoptotic cells.
  • apoptosis e.g., a protease cascade
  • primed extract which is made from cells committed to, but not yet engaged in, apoptosis, thus representing a more upstream stage of apoptosis than extracts taken from apoptotic cells.
  • an extract was considered primed if it met the following criteria: 1) the cells showed little or no morphological change at the time of harvest, and 2) there was little or no cleavage of the cytosolic substrate fodrin.
  • DNA topoisomerase I Modification of DNA topology 100 kDa->70 kDa (Topo 1) Liu, L.F. (1989), Ann. Rev. Biohem. 58, 3Sl-3 5.
  • Lamin B Nuclear envelope formation; 68 kDa->45 kDa anchoring chromatin to nuclear matriix, Lazebnik et al . (1995), Proc. Natl. Acad. Sci. USA 92, 9042-9046.
  • NuMA Involved in nuclear structure 210 kDa->160,180 kDa and nuclear re-formation Co pton, D.A., and Cleveland, D.W. (1994), Curr. Opin. Cell Biol.6, 343-6.
  • PARP DNA repair interaction with 110 kDa->85 kDa chromatin in the nuclear matrix Lazebnik, Y.A. et al . (1994), Nature 371,346-347.
  • Figure IB shows the proteolytic profile of 11 substrates in neural cell lines.
  • the nuclear substrate PARP was fully cleaved within the first 3 h of staurosporine-induced neural cell death, and was the first substrate fully cleaved in our kinetic profile. This is consistent with other kinetic studies of substrate cleavage events indicating that PARP is cleaved early during apoptosis (Casiano et al., J. Exp. Med. 184:765-770 (1996); Tewari et al . , Cell 81:801- 809 (1995) ) . After 3 h exposure to staurosporine, small amounts of cleaved PKC-5, lamin B, Ul-70, fodrin, and NuMA appeared.
  • cleavage was complete by 6 h, while Ul-70, NuMA, PKC-5, Topo I, and lamin B were substantially cleaved 12 h into staurosporine-stimulated apoptosis .
  • tamoxifen is an extremely potent inducer of apoptosis in non-glial neural cells.
  • Tamoxifen is effective in treatment of estrogen receptor (ER) -positive, as well as some ER-negative, breast cancers.
  • ER estrogen receptor
  • ER-negative, breast cancers Although the precise mechanism of action of tamoxifen, especially in estrogen-independent cells, remains unclear, like staurosporine, it is a protein kinase C (PKC) inhibitor (Couldwell et al . , FEBS Lett . , 345:43-46 (1994) and Neurosurgery 35:1184-1186. (1996), and such inhibition is known to induce apoptosis (Stanwell et al . , C. Carcinogenesis . 17:1259-1265 (1996)).
  • PKC protein kinase C
  • WITG3 (Iwasaki et al . , Cancer Immun. Immunothe .. 40:228- 234 (1995)), and in some non-neural cell lines: rat osteoclasts T-289 melanoma cell line and the estrogen receptor positive MCF-7 and estrogen receptor negative MDA-231 human mammary carcinoma cell lines.
  • Tamoxifen citrate 4-hydroxytamoxifen, and tamoxifen, were purchased from Sigma Chemical Company. Cells were induced to undergo apoptosis by exposure to 100 ⁇ M tamoxifen citrate (or 4 -hydroxytamoxifen, or tamoxifen) for times ranging from 1 - 24 h at the incubation temperature of the cell line. The treatment of non-glial, neural CSM-25 (and NSC) cells with 100 ⁇ M tamoxifen resulted in the rapid activation of apoptosis ("100% in 3 h) ( Figure 1A) . More importantly, tamoxifen produced a homogeneous detachment of neural cells in about 2 h.
  • Apoptosis is usually accompanied by the cleavage of DNA at internucleosomal sites (Wyllie et al . , Int. Rev. Cytol . 68:251-306 (1980)). This effect has been reproduced in several non-neural cell-free systems (Newmeyer et al . , Cell, 79:353-364 (1994); Lazebnik et al . , J. Cell. Biol. 123:7-22 (1993)).
  • rat liver nuclei incubated in tamoxifen- primed 16,000 g CSM-25 extracts also underwent this type of chromatin destruction involving the fragmentation of DNA into integer multiples of " 200 bp, while nuclei incubated with normal non-primed 16,000 g neural extracts remained unfragmented for several hours .
  • CPP32 is also known as Yama or apopain (Quan et al . , B. Proc. Natl. Acad. Sci. USA 93:1972-1976, (1996) Tewari et al.,Call 81:801-809 (1995b) .
  • caspases The activation of caspases is known to be essential for apoptotic execution.
  • the kinetics of caspase activation was measured spectrophotometrically by assaying the hydrolysis of a substrate that can only be cleaved by either a CPP32-like caspase family member (DEVD-pNA substrate) (Nicholson et al . , Supra) . or an ICE-like caspase family member (YVAD-pNA substrate) (Thornberry et al . , Supra) .
  • DEVD-pNA substrate CPP32-like caspase family member
  • YVAD-pNA substrate an ICE-like caspase family member
  • This Example demonstrates that mastoparan and atractyloside induce a mitochondria-dependent neuronal cell-free apoptosis, as measured by fodrin cleavage, CPP32 processing to active forms, and the CPP32-like caspase hydrolysis of the DEVD-pNA substrate.
  • Atractyloside is an inhibitor of the mitochondrial adenine nucleotide translocator (ANT) , and an inducer of the mitochondrial inner membrane permeability transition (MPT) (Zamzami et al . , Supra : de Macedo et al . , Eur. J. Biochem. 215:595-600 (1993).
  • MPT mitochondrial inner membrane permeability transition
  • incubation of a 3,000 g CSM-25 non- primed extract for 1.5 h at 37°C in the presence of 5 mM atractyloside resulted in the cleavage of fodrin, an event that has been shown previously to be tightly coupled to apoptosis (Martin et al . , Supra) .
  • Atractyloside also induced the cleavage of fodrin in a system composed of rat liver mitochondria and a 16,000 g CSM-25 non-primed extract.
  • atractyloside incubated in a 16,000 g non-primed extract alone did not lead to such cleavage, demonstrating that the cleavage of fodrin was mitochondria-dependent.
  • incubation of the 3,000 g CSM-25 extract alone (or the 16,000 g CSM-25 extract) did not lead to the cleavage of fodrin.
  • the wasp venom peptide toxin mastoparan kills cultured cerebellar granular neurons by apoptosis (Yan et al . , J. Neurochem. 65:2425-2431 (1995).
  • the results in Figure 4 demonstrate that mastoparan also induces cell death in cultured R2 rat cerebellar neuron precursors. This death was determined to be apoptotic within 6 h at a mastoparan concentration of 50 ⁇ M, and necrotic at a concentration of 100 ⁇ M or greater, as measured by morphology.
  • mastoparan induces the MPT (Pfeiffer et al . , J. Biol. Chem.
  • mastoparan interacts with the mitochondria outer membrane to release mitochondrial proteins even before the MPT (Nicolay et al . , J. Bioenergetics Biomembranes 26:327-334 (1994).
  • Mastoparan was assayed for a mitochondria-dependent activation of protease activity unique to apoptosis.
  • a 3,000 g CSM-25 non-primed extract was incubated with 50 ⁇ M mastoparan for 1.5 h at 37°C.
  • the results showed that in addition to the cleavage of the cytoskeletal protein fodrin, the caspase family member CPP32 was processed to the active forms found in apoptosis ( Figure 5A) .
  • Incubation of the 3,000 g CSM-25 non-primed extract alone did not lead to the cleavage of fodrin or the processing of CPP32.
  • Mastoparan induces the release of cytochrome c from mitochondria
  • the neuronal cell-free system described in the previous Example 4A activates apoptosis with horse and bovine cytochrome c, it does not activate with yeast cytochrome c (ISO-1) , nor with partially acetylated horse heart cytochrome c ( Figure 6C) .
  • the cytochrome c acetylation process preferentially acetylates surface lysines.
  • yeast cytochrome c differs from mammalian cytochrome c, not only in the number and distribution of lysines ( Figure 7) , but also in that lysine 72 is naturally tri-methylated in yeast cytochrome c (Clements et. al .
  • lysines are important for the function of cytochrome c in activating cell-free apoptosis, and that mutations of these lysines might affect the mechanism by which cytochrome c initiates apoptosis.
  • JcI-2 inhibits both necrotic and apoptotic cell death in several cell types, including neural cells, and in response to a wide variety of inducers, including serum and growth factor withdrawal, calcium ionophores, glucose withdrawal, membrane peroxidation, glucocorticoids and chemotherapeutic agents, baculovirus infection, free radical inducing agents, and protein kinase inhibitors such as staurosporine.
  • inducers including serum and growth factor withdrawal, calcium ionophores, glucose withdrawal, membrane peroxidation, glucocorticoids and chemotherapeutic agents, baculovirus infection, free radical inducing agents, and protein kinase inhibitors such as staurosporine.
  • inducers including serum and growth factor withdrawal, calcium ionophores, glucose withdrawal, membrane peroxidation, glucocorticoids and chemotherapeutic agents, baculovirus infection, free radical inducing agents, and protein kinase inhibitors such as staurosporine.
  • bcl-2 protects neurons during acute in vivo cerebral ischemia (Martinou et al . , Neuron 13:1017-1030 (1994).
  • the gene Jbcl-2 encodes a 26-kDa membrane-associated protein Bcl-2 that has been ultrastructurally located to the mitochondria, the nuclear membrane, and the endoplasmic reticulum.
  • Bcl-2 can prevent neural cells from undergoing apoptosis induced by such agents as tamoxifen and staurosporine (pre-mitochondrial phase) , and can prevent the cell-free activation of apoptosis by such agents as atractyloside (mitochondrial phase) , it cannot prevent the cell-free initiation of neural apoptosis by cytochrome c/dATP (post-mitochondrial phase) .
  • Example 5 CPP32 processing in mitochondria-dependent activation of cell-free apoptosis.
  • a cell-free system composed of 16,000 g neural non-primed extract and added rat liver mitochondria was incubated at 37°C with 100 ⁇ M tamoxifen, or 50 ⁇ M mastoparan, or 10 ⁇ M cytochrome c and 1 mM dATP for 2, 1, and 1 hours, respectively.
  • mastoparan and cytochrome c/dATP activated the cell-free system, tamoxifen did not.
  • a cell-free system composed of 16,000 g neural extract was incubated at 37°C with 100 ⁇ M tamoxifen, or 50 ⁇ M mastoparan, or 10 ⁇ M cytochrome c and 1 mM dATP, for 2, 2, and 1 hours, respectively. Although cytochrome c/dATP activated the cell-free system, tamoxifen and mastoparan did not .
  • tamoxifen induces apoptosis at the pre-mitochondrial level (cells) , but does not induce apoptosis at the mitochondrial level (mitochondria and extract) , or the post-mito-chondrial level (extract) .
  • Mastoparan induces apoptosis at the pre-mitochondrial level (cells) and at the mitochondrial level (mitochondria and extract) , but does not induce apoptosis at the post-mitochondrial level (extract) .
  • cytochrome c/dATP induces apoptosis at the mitochondrial level (mitochondria and extract) and at the post-mitochondrial level (extract) , but does not induce apoptosis at the pre-mitochondrial level (cells) .

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Abstract

L'invention a trait à des méthodes ne faisant pas appel à des cellules, lesquelles méthodes permettent d'identifier des composés modulant une apoptose à dépendance mitochondriale. Elle concerne également des méthodes d'identification de composés modulant un stade spécifique à dépendance mitochondriale d'apoptose.
PCT/US1998/001957 1997-08-04 1998-01-29 Systeme exempt de cellules relatif a l'apoptose a dependance mitochondriale et techniques d'application WO1999006588A1 (fr)

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US7056735B2 (en) 2000-09-11 2006-06-06 Institut Pasteur Mimetics and inhibitors of the interaction between Vpr (HIV viral protein of regulation) and ANT (Mitochondrial adenine nucleotide translocator)
US7642051B2 (en) 2000-09-11 2010-01-05 Institut Pasteur Screening methods for the identification of inhibitors of human immunodeficiency virus (HIV) viral protein R (Vpr) binding to the adenine nucleotide translocator (ANT)
US7357928B2 (en) 2002-04-08 2008-04-15 University Of Louisville Research Foundation, Inc. Method for the diagnosis and prognosis of malignant diseases
US7541150B2 (en) 2002-04-08 2009-06-02 University Of Louisville Research Foundation, Inc Method for the diagnosis and prognosis of malignant diseases
US8029784B2 (en) 2002-04-08 2011-10-04 University Of Louisville Research Foundation, Inc. Method for the diagnosis and prognosis of malignant diseases
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