1998;58:4510-4514. Cancer Res   Sudha Iyer, Dai J. Chaplin, Dean S. Rosenthal, et al.   A-4by the Tumor-specific Antiangiogenesis Agent Combretastatin Induction of Apoptosis in Proliferating Human Endothelial Cells

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(CANCER RESEARCH 58. 451(1-4514. October 15. 1998]

Advances in Brief

Induction of Apoptosis in Proliferating Human Endothelial Cells by theTumor-specific Antiangiogenesis Agent Combretastatin A-41

Sudha Iyer, Dai J. Chaplin, Dean S. Rosenthal, A. Mainiti Boulares, Lu-Yuan Li, and Mark E. Smulson2

Dejnirlnieni of HioclicniiMn- unti Molecular riioioi>\-, Georgetown University School of Medicine. Washington, DC 20007 IS. 1., D. S. R.. A. H. B.. L-Y.L. M. E. S.¡.ami TninonrMicrocircidfiiion Group. Gru\ Laboratory Cancer Research Trust, Mount Vernon Hospital. Ntn'thwood, Middlese\ HA6 2JR, United Kingdom ¡D.J. C.]

Abstract

The antiangiogenic, tubulin-binding drug combretastatin A-4 exhibits a

selective toxicity for proliferating endothelial cells in vitro and inducesvascular shutdown in tumor models in vivo. The mechanism of combretastatin A-4 cytotoxicity has now been investigated with cultured prolifer

ating human umbilical vein endothelial cells by examining various markers of apoptosis. Incubation of cells with 0.1 HIM combretastatin A-4

induced the conversion (first detected after 6 h) of the CPP32 proenzymeto active caspase-3, a cysteine protease that plays an important role inapoptosis in many cell types; the drug also increased caspase-3 activity.

Another early event observed was the binding of annexin V to 50% of thecells 8 h after drug treatment. Internucleosomal DNA fragmentation,another hallmark of apoptosis, was detected in cells incubated with 0.1HIM combretastatin A-4 for 24 h. Staining with Hoechst 33258 revealed

that about 75% of cells exhibited a nuclear morphology characteristic ofapoptosis after incubation with drug for 24 h. Incubation of cells for up to8 h with combretastatin A-4 did not induce the release of lactate dehy-

drogenase or increase the uptake of propidium iodide, both indicators ofmembrane integrity. These results indicate that the selective cytotoxiceffect of combretastatin A-4 is mediated by the induction of apoptosis

rather than by necrosis and may provide an enhanced clinical strategy incancer chemotherapy with this new agent.

Introduction

Angiogenesis is critical to both the survival and continued growthof solid tumors and therefore represents a prime target for new cancertreatments. We have shown recently that combretastatin A-4 exhibits

selective toxicity toward endothelial cells of tumor vasculature (1).Our in vitro studies revealed a marked toxicity of this compound forproliferating endothelial cells but not for cells that were quiescentbefore and during drug exposure. A 93% reduction in functionalvascular volume was also observed in an experimental cancer modelin vivo 6 h after systemic drug administration. In our previous study,we assessed cell viability with the neutral red technique. This assayrevealed a concentration-dependent toxic action of combretastatin A-4

on normal human proliferating endothelial cells in culture and, to alesser extent, on cultured tumor cells ( 1). However, the neutral redassay cannot distinguish whether cell death occurs by necrosis orapoptosis. The morphological changes associated with apoptosis arehighly similar among diverse cell types and include cytoplasmicvacuolization, cell shrinkage, plasma membrane blebbing, chromatincondensation and fragmentation, and the formation of membrane-

bound apoptotic bodies. A family of cysteine proteases, known as

Received 7/17/98: accepted 8/31/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely (o indicate Ihis fact.

1This work was supported by Grant 97AI08 (to M. S.) and Grant 97A105-I07 (to

D. J. C.) from Oxigene, Inc.. Lund. Sweden.2 To whom requests for reprints should be addressed, at Department of Biochemistry

and Molecular Biology, Georgetown School of Medicine, Basic Science Building. Room351, 3900 Reservoir Road. NW. Washington. DC 20007. Phone: (202)687-1718; Fax:(202)687-7186; E-mail: smulsonfe'bc.georgetown.edu.

caspases, is known to play an important role in apoptotic cell death(2). We previously contributed to the purification and characterizationof one such human enzyme, now known as caspase-3, that catalyzesthe cleavage of PARP' during apoptosis (3). PARP is a nuclear

enzyme that is activated by DNA strand breaks and catalyzes thepoly(ADP-ribosyl)ation of various nuclear proteins. We have shownrecently that a transient burst of poly(ADP-ribosyI)ation of nuclearproteins early in apoptosis, which is followed by caspase-3-mediated

cleavage and inactivation of PARP. is essential for subsequent progression of the death program in various cell types (4, 5). Given thepotential clinical importance of combretastatin A-4 and related drugs,

we have now investigated the mechanism by which this compoundinduces the death of proliferating endothelial cells. Specifically, wehave examined various biochemical and morphological markers ofapoptosis to determine whether the toxic effect of combretastatin A-4

results from the induction of programmed cell death.

Materials and Methods

Systemic Administration of Combretastatin and Measurement of Percentage of Vascular Shutdown. Six hours after i.p. administration of combretastatin A-4 (25 or 100 mg/kg), the mice were injected with Hoechst 33342

(10 mg/kg, i.v.) and, 1 min later, were euthani/.ed. The tumors were excisedand frozen at —¿�20°C,sections were prepared and examined under UV illumi

nation, and vascular volume was determined with a random point scoringsystem as described previously (1).

Cells. HUVECs were obtained from Clonetics and maintained in EGM-2

BulletKit medium in a 5% CO2 incubator. Cells were grown in tissue culturedishes, and assays were performed when they reached a density of 1.3 X IO4cells/cm".

Drug Treatment. HUVECs were incubated with 0.1 mM combretastatinA-4 (Oxigene, Inc.) for various times and then assayed for various markers of

apoptosis or necrosis. Jurkat cells were exposed to antibodies to Fas (50 ng/ml:Kamiya Biologicals) as a control for cells undergoing apoptosis.

Analysis of DNA Fragmentation. Total DNA was isolated from cells asdescribed previously (4) and analyzed by electrophoresis in 1.5% agarose gelsand ethidium bromide staining.

PARP-Cleavage Assay. Caspase-3 activity in cytosolic extracts of cellswas assayed by measuring the cleavage of |'5S]methionine-labeled recombi

nant PARP as described previously (4). Briefly, 5 /^g of cytosolic protein wereincubated for 1 h at 37°Cin a reaction mixture (25 ^1) containing [15S]PARP(~5 X 10J cpm). 50 mM PIPES-KOH (pH 6.5), 2 mM EDTA. 0.1% 3-[(3-

choiamidopropyl)dimethylamino]-l-propanesulfonate detergent, and 5 mM

DTT. The reaction was terminated by the addition of an equal volume of 2XSDS sample buffer [0.25M Tris-CI (pH 6.8). 2% SDS. 8% BME, Bromophenolblue 0.02%], and PARP-cleavage products were detected by SDS-PAGE on

10% gel followed by fluorography.Hoechst or Propidium Iodide Staining. HUVECs were isolated by expo

sure to trypsin followed by resuspension in serum-containing media. The cellswere centrif'uged (800 X g for 5 min), and fixed in 4% formalin for 10 min.

After washing twice with PBS. the cells were stained either with Hoechst33258 (24 /j.g/ml) in PBS containing 80% (v/v) glycerol or with propidium

1The abbreviations used are: PARP. poly(ADP-ribose) polymerase: HUVEC. human

umbilical vein endothelial cell; LDH, lactate dehydrogenase.

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INDUCTION OF APOPTOSIS BY COMBRETASTATIN A-4

iodide (according to the manufacturer's specifications I in PBS. mounted on a

slide, and observed with a fluorescence microscope.Annexin-V and Propidium Iodide Staining. HUVECs were isolated by

exposure to trypsin and centrifugation. washed with ice-cold PBS. and stained

for 15 min in the dark at room temperature with propidium iodide andFITC-labeled annexin-V (both, according to the manufacturer's specifications;

Trevigen) in a solution containing 10X binding buffer and water. The cellswere then examined with a fluorescence microscope.

Measurement of Levels of Toxicity. Levels of cell toxicity were assessedby measuring the release of the cytosolic enzyme láclatedehydrogenase intothe medium with the use of a Cytotox 96 kit (Promega).

Immunoblot Analysis. Cytosolic extracts prepared from HUVECs for thePARP-cleavage assay were also subjected to immunoblot analysis for measurement of CPP32 processing to caspase-3. Samples containing 30 ;j.g ofprotein were fractionated by SDS-PAGE on 4-209c gradient gels, and the

separated proteins were transferred to a nitrocellulose membranes. Membraneswere stained with Ponceau S to confirm equal loading and transfer of proteinsamong lanes. After blocking of nonspecific sites using 5% nonfat milk inPBS + 0.05% Tween 20. the membrane was incubated for l h at roomtemperature with rabbit polyclonal antibodies that recogni/e both CPP32 andthe pi7 subunit of caspase-3 (1:10.000 dilution: kindly provided by Dr. D.Nicholson. Merck Labs. Pointe Claire-Dorval. Quebec, Canada). Immunecomplexes were then detected with horseradish peroxidase-conjugated goat

antibodies to rabbit immunoglobulin G (1:2000 dilution; Amersham LifeSciences) and enhanced chemiluminescence (ECL: Pierce).

Results

Effect of Combretastatin A-4 on Perfused Tumor VascularVolume in Mice. We determinedpreviously the time course for theeffect of systemic administration of combretastatin A-4 at a dose of100 mg/kg on the perfused vascular volume of the murine carcinomaNT growing s.c. in CBA/Gy (TO mice (1). To confirm this action forthe present study, we assessed the effects of two doses of the drug (25and 100 mg/kg) 6 h after i.p. administration (Fig. 1). At the lowerdose, combretastatin A-4 reduced functional vascular volume in thetumor mass by ~30%. At the higher dose, the drug induced essen

tially complete vascular shutdown, consistent with the results of ourprevious study ( 1).

Effects of Combretastatin A-4 on CPP32 Processing andCaspase-3 Activity in HUVECs. Similar to other members of thecaspase family, caspase-3 is synthesized in cells as an inactive Mr32,000 proenzyme (CPP32). Early during apoptosis, CPP32 is cleavedat specific aspartate residues to yield the active enzyme (caspase-3),

Time(h)

Combretastatin

Anti-Fas

24 24

CPP32

BTime (h) 24 1 4 6 24

Combretastatin - + + + +

116kDa

89kDa

—¿�24 kDa

C HUVEC JurkatCombretastatin I. ++ +1 I . I

AntiFas .... +

Time (h) 48 6 24 48 6

I PPW

100-,

80-

I2 60.eU)

I 40J

gcore> 20-

Õ

control 25mg/kg 100mg/kg

Fig. 1. Effect of comhretastatin A-4 on Ihe perfused vascular volume of the murinecarcinoma NT growing s.c. in CBA/Gy (TO mice. Six h after i.p. administration ofcombretastatin A-4 (25 or 100 mg/kg). the mice were injected with Hoechsi 33342 (10mg/kg. i.v.) and. I min later, killed. The tumors were excised and frozen at -20°C.

sections were prepared and examined under UV illumination, and vascular volume wasdetermined with a random point scoring system as described previously (1). Dala areexpressed as percentage vascular shutdown relative to the mean vascular volume ofuntreated tumors and are means for 5 to 13 tumors; hors, SE.

Fig. 2. Effects of combretastatin A-4 on the proteolytic processing of CPP32 (Ai, oncaspase-3 activity (fl). and on internucleosomal DNA fragmentation IO in HUVECs. A,HUVECs (I X IO6) were incubated in the presence of O.I mM combretaslatin A-4 for 6

or 24 h or in Ihe absence of drug for 24 h. as indicated. The cells were then washed withserum-free medium and homogenized in I ml of a solution containing 10 mM HEPES-KOH (pH 7.4). 2 mM EDTA, 0.1% 3-[(3-choIamidopropyl)dimethylamino)-l-propanesul-

fonate detergent. 5 mM DTT. 1 imi phenylmethylsulfonyl fluoride. 10 /¿g/mlpepstalin A,20 ¿¿g/mlleupeptin. and 10 ^tg/ml aprotinin. The homogenate was centrifuged at100,000 X K. and the resulting supernatant was subjected to immunoblot analysis withantibodies to CPP32. The positions of CPP32 (M, 32.000) and pl7 (M, 17,000) areindicated. B. HUVECs (I x IO6) were incubated with 0.1 mM comhretastatin A-4 for the

indicated times or in its absence for 24 h. Cytosolic extracts were prepared as describedin A and assayed for caspase-3 activity as described in "Materials and Methods." The

positions of the full-length PARP substrate (M, 116.000) and its M, 89.000 and M, 24.000cleavage products arc indicated. C HUVECs (I X 10") were incubated with 0.1 mM

combretastatin A-4 for the indicated times or in its absence for 48 h. As a positive control.Jurkat cells were treated with antibodies to Fas for 6 h. DNA was extracted from the cells,subjected to electrophoresis on a 1.5% agarose gel, and stained with ethidium bromide.

which is a heterodimer of a A/r 17.000 subunit (pi7) and a M, 12,000subunit (pl2). To determine whether CPP32 is proteolytically processed to pi 7 during exposure of HUVECs to combretastatin A-4, weincubated the cells with the drug for various times and then subjectedcytosolic extracts to immunoblot analysis with antibodies to CPP32.

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INDUCTION OF APOPTOSIS BY COMBRETASTATIN A-4

(-) Combretastatin (+) Combretastatin (6 h)

B

(+) Combretastatin (16 h)

Propidium iodide

(+) Combretastatin (24 h)

Phase contrast

24 h

48h

Fig. 3. Effects of comhretastatin A-4 on staining of HUVECs by Hoechst 33258 (A) or propidium iodide (SI. A, cells were incubated in the absence of drug for 24 h or in its presence(0.1 mM) for 6, 16. or 24 h. as indicated. They were then isolated by exposure to trypsin and centrifugation. The cells were fixed with formalin, washed with PBS. and stained withHoechst 33258. Arrou's. cells exhibiting chromalin condensation. B, cells were incubated with 0.1 mM Combretastatin A-4 for 24 h (upper /WMC/.V)or 48 h (lower panels), after whichthey were exposed to trypsin. collected by centrifugation. fixed with formalin, washed with ice-cold PBS. and incubated with propidium iodide for IO min in the dark. Left panels.propidium iodide staining: ri^ht panel*, phase-contrast micrographs. X40.

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INDUCTION OF APOPTOSIS BY COMBRETASTAT1N A-4

Time Phase contrast Annexin + propidiumiodide

Oh

Fig. 4. Effect of combretastatin A-4 on stainingof HUVECs by annexin-V and propidium iodide.

Cells were incubated with 0.1 HIMcombretastatinA-4 for 0, 8, or 24 h as indicated, after which theywere exposed to trypsin, collected by centrifuga-lion, washed with ice-cold PBS, and incubatedwith annexin-V and propidium iodide for 15 minin the dark. Left panels, phase-contrast micrographs; right panels, annexin-V (green) and pro

pidium iodide (reti} staining. Filled arrows, lateapoptosis; unfilled arrows, early apoptosis. X40.

8h

24h

As a positive control, we induced apoptosis in human Jurkat T cells byincubation with antibodies to Fas; such treatment resulted in thegeneration of pl7 from CPP32. Whereas incubation of HUVECs inthe absence of drug for 24 h did not result in the conversion of CPP32to p 17, incubation of the cells with 0.1 mM combretastatin A-4induced CPP32 processing in a time-dependent manner (Fig. 2A).

Processing of CPP32 was substantial after incubation of HUVECswith the drug for 6 h and appeared maximal at 24 h.

We next examined the effect of combretastatin A-4 on caspase-3

activity (Fig. 2B). HUVECs were incubated with 0.1 m\i drug forvarious times, after which cytosolic extracts were prepared and assayed for their ability to catalyze cleavage of the caspase-3 substrate[35S]PARP into M, 89,000 and Mr 24,000 products. Extracts prepared

from cells incubated in the absence of drug for 24 h or those derivedfrom cells incubated in the presence of drug for l h containednegligible caspase-3 activity. However, cells treated with combretastatin A-4 for 4 h showed a marked increase in caspase-3 activity,

which increased further in cells exposed to the drug for 6 or 24 h.

These data are consistent with the time course of CPP32 proteolysis incombretastatin A-4-treated HUVECs (Fig. 2A).

Effects of Combretastatin A-4 on Internucleosomal DNA Frag

mentation and Nuclear Morphology in HUVECs. The effect ofcombretastatin A-4 on internucleosomal DNA fragmentation, a biochemical hallmark of upoptosis, in HUVECs was examined by agar-

ose gel electrophoresis of extracted DNA (Fig. 2C). As a positivecontrol, Jurkat cells treated with antibodies to Fas for 6 h exhibitedmarked internucleosomal DNA degradation, as revealed by the characteristic ladder pattern of DNA fragments on electrophoresis. HUVECs incubated in the absence of drug for 48 h or those exposed tocombretastatin A-4 for 6 h showed no evidence of nuclear DNA

fragmentation. In contrast, HUVECs treated with drug for 24 or 48 hexhibited substantial DNA fragmentation. The time course of DNAfragmentation in drug-treated cells is consistent with previous data

suggesting that the processing of CPP32 and cleavage of PARP bycaspase-3 are required before substantial DNA fragmentation can

occur (4).4513

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INDUCTION Ol; APOPTOSIS BY COMBRETASTATIN A-4

Comhrctastatin A-4 also induced changes in nuclear morphology

characteristic of apoptosis. Hoechst 33258 staining and fluorescencemicroscopy revealed that 15% of HUVECs treated with combretasta-tin A-4 for 24 h exhibited chromatin condensation (Fig. 3A), compared with only ~\% of cells incubated in the absence of drug.

Nuclear fragmentation and chromatin condensation were also detectedby propidium iodide staining of cells treated with combretastatin A-4

for 24 or 48 h (Fig. 3ß);no such changes were observed in cellsincubated without drug.

Effects of Combretastatin A-4 on Cell Membrane Asymmetry,

Integrity, and LDH Release in HUVECs. The loss of plasma membrane asymmetry is an early event in apoptosis in many different celltypes, resulting in the exposure of phosphatidyl serine on the outersurface of the membrane. Because annexin-V reacts with high affinity

with phosphatidyl serine in the presence of calcium, it is a useful assay forapoplosis. We therefore stained unfixed HUVECs with annexin-V and

propidium iodide. Untreated HUVEC cells did not stain with eitherannexin-V or propidium iodide (Fig. 4). However, after incubation withthe drug lor 8 h, SO'/f of the cell membranes were bound to annexin-V,while 99</f of the cells excluded propidium iodide, supporting the idea

that apoptosis is the primary mechanism of cell death in this system. Onlyafter a 24-h exposure of cells to combretastatin A-4 do annexin-V-positive cells begin to take up propidium iodide, indicating that loss of

membrane integrity occurs late in apoptosis. after caspase activation.We also investigated the effect of combretastatin A-4 on the release

of LDH from HUVECs into the culture medium. The medium of cellsincubated in the absence or presence of combretastatin A-4 for up to24 h consistently contained —¿�10%of total cellular LDH (data not

shown), again indicating that the drug does not impair membraneintegrity at these time points and that it induces cell death by apoptosisrather than by necrosis.

Discussion

We have demonstrated previously that combretastatin A-4. a tubulin-

binding drug, selectively reduces the viability of proliferating HUVECsin culture as well as induces vascular shutdown in tumor models in vivo( 1). However, the neutral red assay used in these experiments to assessthe viability of cultured cells could not determine whether the cytotoxiceffect of the drug resulted in cell death by necrosis or by apoptosis. Wehave now shown that combretastatin A-4 induces the death of prolifer

ating HUVECs, in large part if not completely, by apoptosis. An important difference between apoptosis and necrosis is that the contents of thedying cell are efficiently disposed of in the former and are released intothe surrounding environment in the latter; necrosis, unlike apoptosis, isthus associated with a risk of inflammation and inappropriate exposure ofself-antigens.

We have shown that combretastatin A-4 induced the proteolyticconversion of CPP32 into caspase-3 in proliferating HUVECs.Furthermore, with the use of an in vitro PARP-cleavage assay, weshowed that combretastatin A-4 induced a marked increase incaspase-3-like activity after incubation with the cells for only 4 h.Various lines of evidence indicate that caspase-3 is both necessaryand sufficient to trigger apoptosis: (a) disruption of the caspase-3

gene in knockout mice results in excessive accumulation of neuronal cells, due to lack of apoptosis in the brain (6): (b) variousstrategics for inducing apoptosis. including Fas activation, serumwithdrawal, and exposure to ionizing radiation or pharmacologicalagents, also trigger activation of caspase-3 as a result of proteolyticcleavage of CPP32 (7-11 ); (c) a tetrapeptide aldehyde inhibitor ofcaspase-3 (Ac-DEVD-CHO) blocks initiation of the apoptotic pro

gram in response to various signals (3, 12); and (d) the addition ofactive caspase-3 to a cytosolic fraction derived from normal cells

triggers biochemical events characteristic of apoptosis (13). In addition toPARP, substrates for the protease activity of caspase-3 include U1-70K,DNA-dependent protein kinase, the retinoblastoma protein (Rb), fodrin,actin. lamin, gelsolin, and an inhibitor of a caspase-activated DNase, all

of which play roles in DNA repair, mRNA splicing, regulation of the cellcycle, or the morphological changes or DNA fragmentation associatedwith apoptosis (3, 12-17).

In addition to triggering the cytosolic and membrane events associated with apoptosis. combretastatin A-4 induced internucleosomal

DNA cleavage, chromatin condensation, and nuclear fragmentation inproliferating HUVECs, all of which are nuclear hallmarks of cells inthe final stages of apoptosis. Although we have identified the mechanism of cell death induced by combretastatin A-4 in endothelial cells,

it remains to be determined why proliferating cells, such as thosecontributing to tumor vascularization, are sensitive to the cytotoxicaction of this drug, whereas quiescent cells, which constitute themajority of endothelial cells in the body, are not. The signalingpathway by which combretastatin A-4 triggers caspase-3 activation

and the other apoptotic phenomena described here also remains to beidentified. Nevertheless, our data should contribute to the development of combretastatin A-4 or related drugs as potential cancer

chemotherapeutic agents.

References

1. Dark. G. G.. Hill. S. A.. Prise. V. E., Tozer, G. M, Petlil. G. R.. and Chaplin. D. J.Comhrctastatin A-4. an agent that displays potent and seleetive toxicity toward tumorvasculature. Cancer Res.. 57: 1829-1834, 1997.

2. Alnemri. E.. Livingston. D.. Nicholson. D.. Salvesen. G.. Thornherry. N.. Wong. W.,and Yuan. J. Human ICE/CED-3 protease nomenclature. Cell. K7: 171, 1996.

3. Nicholson. D. W.. Ali. A.. Thornherry. N. A.. Vaillancoun. J. P.. Ding. C. K.. Gallant.M.. Gareau. Y.. Griffin. P. R.. Labelle. M.. Lazebnik. Y. A.. Munday. N. A.. Raju.S. M.. Smulson. M. E.. Yamin. T. T.. Yu. V. L.. and Miller. D. K. Identification andinhihilion of the ICE/CED-3 prolease necessary for mammalian apoptosis. Nature(Land.). 376: 37-43, 1995.

4. Simbulan-Rosenthal, C. M.. Rosenthal. D. S.. Iyer. S.. Boulares. A. H.. and Smulson.M. E. Transient po!y(ADP-rihosyl)ation of nuclear proteins and role of polyiADP-ribose) in the early stages of apoptosis. J. Biol. Chem.. 273: 13703-13712. 1997.

5. Rosenthal. D. S.. Ding, R., Simhulan-Rosenlhal, C. M. G.. Vaillancourt. J. P..Nicholson. D. W.. and Smulson. M. E. Intact cell evidence of Ihe early synthesis, andsubsequent late apopain-medialed suppression, of poly(ADP-ribose) during apoptosis. Exp. Cell Res., 232: 313-321. 1997.

6. Kuida. K.. Zheng. T.. Na. S.. Kuan, C.. Yang. D.. Karasuyama. H.. Rakic. P.. andFlavell. R. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature (Lond.). 384: 368-372. 1996.

7. Darmon. A.. Ley. T.. Nicholson. D.. and Bleackley. R. Cleavage of CPP32 bygranzyme B represents a critical role for granzyme B in the induction of target cellDNA fragmentation. J. Biol Chem.. 271: 2I709-2I7I2, 1996.

8. Chinnaiyan. A.. Orth. K.. O'Rourke. K.. Duan. H.. Poirier. G., and Dixit. V. Molec

ular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of theCED-3-like apoptotic proteases. J. Biol Chem.. 271: 4573-4576, 1996.

9. Martin. S.. Amarante-Mendes. G.. Shi, L., Chuang. T., Casiano, C.. O'Brien. G..

Fitzgerald. P.. Tan. E.. Bokoch. G.. Greenberg. A., and Green. D. The cylotoxic cellprotease granzyme B initiates apoptosis in a cell-free system by proteolytic processingand activation of the ICE/CED-3 family protease. CPP32. via a novel two-stepmechanism. EMBO J., 15: 2407-2416. 1996.

10. Schlegel, J.. Peters, I., Orrenius. S., Miller. D.. Thornberry, N.. Yamin, T., andNicholson. D. CPP32/apopain is a key interleukin 1/3 converting en/yme-like protease involved in Fas-mediated apoplosis. J. Biol. Chem., 271: 1841-1844. 1996.

11. Erhardt. P.. and Cooper. G. Activation of CPP32 apoptotic prolease by distinctsignaling pathways with differential sensitivity to Bcl-xL. J. Biol. Chem.. 277:17601-17604. 1996.

12. Tewari. M.. Quan. L. T., O'Rourke. K.. Desnoyers, S., Zeng, Z.. Beidler, D. R.,

Poirier, G. G., Salvesen. G. S., and Dixit. V. M. Yama/CPP32b, a mammalianhomolog of CED-3, is a crmA-inhibitable protease that cleaves the death substratepoly(ADP-ribose) polymerase. Cell. Kl: 801-809, 1995.

13. Janicke. R.. Walker, P., Lin, X.. and Porter. A. Specific cleavage of the retinoblastoma protein by an ICE-like protease in apoptosis. EMBO J.. 15: 6969-6978, 1996.

14. Casciola-Rosen, L.. Nicholson. D.. Chong. T.. Rowan. K.. Thornberry, N.. Miller, D..and Rosen. A. Apopain/CPP32 cleaves proteins that are essential for cellular repair:a fundamental principle of apoptolic death. J. Exp Med.. 183: 1957-1964. 1996.

15. Song, W. C.. Baenschi, S. W.. Boeglin. W. E.. Harris. T. M.. and Brash. A. R.Formation of epoxyalcohols by a purified aliene oxide synthase. Implications for themechanism of aliene oxide synthesis. J. Biol. Chem., 2t>8: 6293-6298. 1993.

16. Kothakota, S., Azuma. T., Reinhard, C.. Klippe!, A., Tang, J.. Chu. K.. McGarry, T.,Kirschner. M.. Koths. K., Kwiatkowski, D., and Williams. L. Caspase-3 generatedfragment of gelsolin: effector of morphological change in apoplosis. Science(Washington DC). 27X: 294-298, 1997.

17. Mariin. S., and Green. D. Protease activation during apoptosis: death by a thousandcuts? Cell. 82: 349-352. 1995.

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