Ionizing Radiation-induced, Bax-mediated Cell DeathIs Dependent on Activation of Cysteine andSerine Proteases1

Bendi Gong, Quan Chen, Brian Endlich,Suparna Mazumder, and Alex Almasan2

Department of Cancer Biology, Lerner Research Institute [B. G., Q. C.,S. M., A. A.], and Department of Radiation Oncology, The ClevelandClinic Foundation [B. G., Q. C., B. E., S. M., A. A.], Cleveland, Ohio44195

AbstractBcl-2 family proteins and interleukin-1-b convertingenzyme/Caenorhabditis elegans cell death gene-3 (ICE/CED-3) family proteases (caspases) represent the basicregulators of apoptosis. However, the precisemechanism by which they interact is unclear. In thisstudy, we found that g-radiation-induced apoptosis ofleukemia cells was associated with activation ofmultiple caspases and bax up-regulation. Membranechanges and caspase activities were suppressed byspecific caspase inhibitors. Similarly, the serineprotease inhibitors z-Ala-Ala-Asp-cmk (AAD) and tosyl-lysine chloromethyl ketone (TLCK) also preventedcaspase activation and poly(ADP-ribose) polymerasecleavage in vivo but had no effect on caspase activityin vitro. TLCK also prevented bax up-regulation as aresult of its inhibitory effect on p53 function. Inhibitors ofcaspases and serine proteases partially prevented celldeath, suggesting a caspase involvement in Bax-mediated cell death. We propose an ordering of signalingevents in Bax-mediated cell death, including stepsupstream and downstream of p53 and bax up-regulation.

IntroductionThe response of eukaryotic cells to IR3 includes cell-cyclearrest and cell death. Experimental evidence suggests that

the cytotoxic effects of IR and many forms of chemotherapyare mediated through a final common pathway that involvesthe activation of apoptosis (1, 2). Few insights, however, areavailable regarding the signals that control induction of ap-optosis after IR. Two well-characterized proteins known toregulate IR-mediated apoptosis are Bcl-2 and p53. Bcl-2 isthe prototype of a family of proteins, related to the Caenorh-abditis elegans ced-9 gene, that are involved in the regulationof a distal step in an evolutionarily conserved pathway forphysiological cell death and apoptosis, with some membersfunctioning as suppressors of apoptosis and others as pro-moters of cell death (3). Bcl-2 and its homologues, Bcl-x andMcl-1 encode membrane-associated proteins that protectneoplastic cells from DNA damage-induced apoptosis, in-cluding that caused by IR (4, 5). In contrast, Bax is a pro-moter of cell death. The relative ratios of these various pro-and anti-apoptotic members of the Bcl-2 family have beenshown to determine the ultimate sensitivity or resistance ofcells to diverse apoptotic stimuli, including IR (6). Althoughvarious family members can interfere with each other’s func-tions by heterodimeric interactions (6), they can also functionindependently of each other to regulate cell death (7, 8).

The p53 tumor suppressor protein has an essential role incontrolling cell-cycle progression or apoptosis, and its dys-function has profound consequences because about 50% ofall human tumors produce aberrant p53 protein [see Ref. 9for a review]. Studies with p53-null mice showed that p53 isnecessary for thymocytes to undergo apoptosis in responseto DNA damage (1, 10). Moreover, the levels and activity ofp53 have been shown to increase in response to IR and otherDNA-damaging agents (11–15). Although p53 is required foroptimal apoptosis induced by IR (1, 2) the precise role of thistumor suppressor in regulating cell death is poorly under-stood. It is known that p53 transcriptionally activates bax insome types of cells after treatment with IR, chemotherapeu-tic drugs, and other forms of genotoxic stress (16, 17). Otherp53-regulated genes may also contribute to cell death (18).

Genetic studies in the nematode C. elegans have providedevidence that the ced-3 gene is indispensable for cell deathduring development and has provided the first evidence forthe involvement of aspartate-specific cysteine proteases inthe induction of apoptosis. Related Ced-3 homologues inmammalian cells include members of a new family of ICEfamily proteases (19), now called caspases (20). These in-clude: (a) regulator caspases, such as caspase 8 [FADD-likeinterleukin-1-b converting enzyme (FLICE)/MORT1-associ-ated CED-3 homologue (MACH)/Mch5 (21, 22) and caspase9 [ICE-Lap6, MCH6 (23)]; and (b) effector caspases, such ascaspase 3 [CPP32/YAMA/Apopain (24–26)] and caspase 6[MCH2, (27)]. Several proteins, including PARP (28, 29) aswell as many others (see Ref. 30 for review and additional

Received 12/7/98; revised 3/4/99; accepted 4/21/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 Supported by a research grant from the NIH (CA 81504).2 To whom requests for reprints should be addressed, at Department ofCancer Biology, The Cleveland Clinic Foundation, NB40, Cleveland, OH44195. Phone (216) 444-9970; Fax: (216) 445-6269; E-mail:almasaa@ccf.org.3 The abbreviations used are: IR, ionizing radiation; TBS, Tris-bufferedsaline; TBST, TBS with 0.05% Tween #20; AAD, z-Ala-Ala-Asp; AAPD,succinyl-Ala-Ala-Pro-Asp; DEVD, acetyl-Asp-Glu-Val-Asp; CPI, calpaininhibitor I; cmk, chloromethyl ketone (CH2Cl); EMSA, electromobility shiftassays; fmk, fluorometyl ketone; IETD, N-acetyl-Ile-Glu-Thr-Asp; LEHD,acetyl-Leu-Glu-His-Asp; NAC, N-acetyl-L-cysteine; PS, phosphatidyl-serine; PARP, poly(ADP)-ribose polymerase; pNA, p-nitroanilide; PI, pro-pidium iodide; PDTC, pyrrolidine dithiocarbamate; TLCK, tosyl-lysinecmk; TPCK, N-tosyl-L-phenylalanine cmk; VEID, Ac-Val-Glu-Ile-Asp;YVAD, acetyl-Tyr-Val-Ala-Asp ICE, interleukin-1-b converting enzyme;CED, Caenorhabditis elegans cell death gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

491Vol. 10, 491–502, July 1999 Cell Growth & Differentiation

references), have been shown to be cleaved during apopto-sis after a specific aspartate residue (25–27). Recent reportsimplicate caspase activation in p53-dependent apoptosis(31–33); however, the pathways regulating these events arepoorly understood.

Serine proteases have also been suggested to participatein apoptosis. The best characterized system for the involve-ment of a serine protease in apoptosis is cytotoxic T-cell-mediated cell death, which has been shown to require gran-zyme B. Moreover, granzyme B can process and directlyactivate a number of caspases, providing evidence for aproteolytic cascade initiated by a serine protease [see Ref.34 for review and additional references]. Inhibitors of otherserine proteases were shown to prevent apoptosis by avariety of stimuli, including staurosporine, DNA-damagingagents, and dexamethasone in immature thymocytes (35),tumor necrosis factor in various cell lines, Fas ligand in Tlymphocytes, serum withdrawal and T-cell receptor-crosslinking in T cells [Ref. 36 and refs. therein]. TLCK is aserine protease inhibitor with specificity for trypsin-like ac-tivities, which can inhibit apoptosis induced by diverse stim-uli at an early stage before both DNA fragmentation andcytoplasmic changes in immature rat thymocytes (37). Incontrast, TPCK a chymotrypsin-specific protease inhibitorhas no effect on cell death in these cells (37). The effect ofTLCK and TPCK could be, however, cell type-dependentbecause TLCK does not suppress apoptosis in other myeloidcells and TPCK has the opposite effect (38). Nevertheless, itis becoming apparent that the activation of these proteasesis a crucial event in the cellular execution of apoptosis (forreview, see Ref. 30). However, with the exception of gran-zyme B, the identity and role of other serine proteases inapoptosis is not understood.

The present experiments were designed to examine thetemporal relationship of events caused by IR-triggered, Bax-induced apoptosis and to establish the role of cysteine andserine proteases in this process. We confirmed that fourknown caspases were present, the activity of which could beprevented by specific inhibitors. Furthermore, we found in-volvement of serine proteases inhibitable by AAD-cmk andTLCK. By using a variety of cleavage site inhibitors, we wereable to further separate p53 and bax induction from caspaseactivation and cell death. These studies place Bax and p53downstream of a TLCK-inhibitable step but upstream of anAAD-inhibitable protease, all of these events being presentbefore caspase activation. On the basis of these data, we areproposing an ordering of the molecular events involved inIR-signaling leading to cell death. Most importantly, we de-scribe distinct steps, upstream and downstream of p53 andbax activation. Because Bax-induced events are only par-tially inhibitable by caspase or serine protease inhibitors, thissuggests a protease-independent step in addition to theprotease-dependent events.

ResultsInhibitors of Serine and Cysteine Proteases Prevent Ra-diation-induced Apoptosis of MOLT-4 Cells. Previousstudies (39, 40) have demonstrated that treatment with IRefficiently killed MOLT-4 cells. Cells treated with 4–10 Gy

g-irradiation showed features of apoptosis characterized bythe appearance of acridine orange- or Hoechst 33342-stained cells, chromatin margination, and apoptotic bodies(data not shown) similar to previous reports (39, 40).Changes during the early stages of apoptosis occur at thecell surface, including the translocation of PS from the innerside of the plasma membrane to the outer layer, by which PSbecomes exposed at the external surface of the cell. AnnexinV was used to determine PS exposure on the cell membranein combination with PI to establish the integrity of the cellmembrane, as described previously (41). Using this method,we detected FITC1/PI2 cells, characteristic of early apop-tosis, in a significant number of cells by 8 h, with the majorityof cells becoming FITC1 by 12 h (Fig. 1D, upper and lowerright panels).

It is known that the process of PS export to the outer leafletof the plasma membrane is a caspase-dependent eventduring apoptosis of cells from various lineages (41, 42). Weconfirmed this in MOLT-4 cells by showing that the caspase3-specific cell-permeable inhibitor DEVD-fmk was able toprevent PS expression significantly (Fig. 1E). Next, we soughtto examine whether serine proteases might also be involvedin this process. We found that the AAD-cmk serine proteaseinhibitor substantially reduced the appearance of FITC1/PI2(Fig. 1F, lower right panel) and FITC1/PI1 cells (Fig. 1F,upper right panel). Similarly, we found that TLCK, a trypsin-like serine protease inhibitor, also inhibited the appearanceof FITC1/PI2 (Fig. 1G, lower right panel). In contrast toTLCK, another closely related serine protease inhibitorTPCK, in fact, substantially promoted the appearance ofFITC1/PI1 cells (Fig. 1H, upper right panel). These dataimplicate a serine protease in the early events of IR-inducedapoptosis that affects the function of the plasma membrane.Therefore, both serine and cysteine protease activities cancontribute to events that lead to membrane changes.

To further examine the effect of these inhibitors on cellviability, we analyzed loss of metabolic cellular activity bydetermining NADH activity, measured by tetrazolium reduc-tion. Irradiation (4 Gy) led to a marked decrease in viable cellsby 24 h (Fig. 2). Cell death was prevented by DEVD-fmk in adose-dependent manner, with cells treated with 100 mM

DEVD-fmk having a 3.5-fold increase in cell viability 24 h afterIR. Similarly, the serine-protease inhibitor AAD-cmk (100 mM)also protected from cell death (3.5-fold). Cell death wasblocked to a lesser extent by TLCK and the caspase 8 and 6inhibitors, IETD-fmk and VEID-fmk, respectively. Thus, ap-parently in MOLT-4 cells, the activity of both cysteine andserine proteases is required for cell death. However, bothcaspase and serine protease-dependent and independentsteps may exist inasmuch as none of the inhibitors couldcompletely prevent cell death.

Radiation Activates Multiple Caspases and a SerineProtease. To follow caspase activation during apoptosis,cytosols from cell lysates obtained after increasing lengths oftime after IR were incubated with the chromogenic substrateDEVD-pNA, which mimics the PARP cleavage site P1–P4tetrapeptide (25). As seen in Fig. 3A, there was a time-dependent increase in DEVD-pNA cleavage activity startingat 4 h and reaching higher levels after 10 h. Remarkably, the

492 Proteases Mediate Bax-induced Apoptosis

kinetics of DEVD-pNA cleavage showed a linear, time-de-pendent increase between 4 and 10 h. In contrast to themarked increase in DEVD-pNA cleavage activity, there wasno significant change in the cleavage activity for YVAD-pNA(Fig. 3A). Because DEVD is a substrate for caspase 3 andYVAD is a substrate for caspase 1, this demonstrates that

apoptosis induced by IR in MOLT-4 cells is dependent oncaspase 3 and not caspase 1 protease activity. To determinewhich other caspases might be activated during the IR-mediated apoptosis of MOLT-4 cells, we examined VEID-pNA and IETD-pNA cleavage activities. These representcleavage sites in lamin (43) and procaspase 3 and are con-sidered to be preferred substrates for caspase 6 and 8,respectively. Both the VEID-pNA and IETD-pNA cleavageactivities were induced substantially (Fig. 3B). Finally, weexamined the activation of caspase 9 by determining thecleavage of its specific substrate LEHD-AFC. Caspase 9 is aregulator caspase whose a activity has recently been shownto be an essential mediator of apoptosis induced as a resultof cytochrome c release from mitochondria (23). All of thecaspase activities were detectable by 4 h and then continuedto increase steadily up to at least 8 h (Fig. 3C). These resultsshow that multiple caspases were activated after the irradi-ation of MOLT-4 cells.

We also detected a robust induction of AAPD-pNA cleav-age activity (Fig. 3B). AAPD has been previously shown to bea specific substrate for the serine protease granzyme B invitro (44). Because granzyme B has been previously impli-cated in cytotoxic T lymphocyte (CTL) killing in conjunctionwith perforin activity, we next examined whether granzyme Bwas expressed in MOLT-4 cells. Steady-state mRNA levelswere determined by the multiprobe RNase protection assayin exponentially growing cells. With this method, we exam-ined simultaneously templates for granzyme B and severalcaspases using the hApo-1B template set. As seen in Fig.3D, there was no detectable 361-bp mRNA species for gran-zyme B, with low levels detected for caspase 1 and 5. Thiswould suggest that since there was no granzyme B expres-

Fig. 1. Membrane changes represent an early event in IR-induced apoptosis. Expression of PS on the plasma membrane was measured by staining thecells with FITC-labeled Annexin V, in conjunction with PI to assess cell membrane permeability. At the indicated times after IR (4 Gy), cells were preparedfor flow cytometry (A–D). To determine the effect of inhibitors on PS exposure, cells were treated 1 h before irradiation (4 Gy) with 100 mM DEVD-fmk (E),AAD-cmk (F), or TPCK (H) or with 300 mM TLCK (G). The data were obtained from the cell population from which debris were gated out against forwardand side scatter. Numbers represent the proportion of early apoptotic cells (FITC1/PI2, lower right panel), and late apoptotic or necrotic cells (FITC1/PI1,upper right panel). Flow cytometric measurements were performed by bivariate flow cytometry using a FACScan and analyzed with CellQuest software(Becton Dickinson).

Fig. 2. Serine and cysteine protease inhibitors prevent cell death. Cellswere either left untreated (2) or irradiated with 4 Gy (1) in the absence orpresence of the following inhibitors added 1 h before IR: (a) the caspaseinhibitors DEVD-fmk (10–100 mM), VEID-fmk, and IETD-fmk (100 mM); and(b) the serine protease inhibitors AAD-cmk (100 mM) and TLCK (300 mM).The MTS cell proliferation assay (Promega) was used to determine thepercentage of biochemically active cells 24 h after IR. Each experimentwas done in triplicate; values, means 6 SD (n 5 3).

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sion, the AAPD-pNA cleavage activity was due to anotherenzyme, most likely a granzyme B-like serine protease. Incontrast, there was abundant expression of caspases 3, 6,and 8. Interestingly, there were significant differences be-tween the caspase mRNAs detected in MOLT-4 cells and themultiple myeloma cell line IM-9, most notably caspase 1 and6, which indicates a differential expression of individualcaspases in these cell lines.

Previous studies (reviewed by Ref. 30) have also identifieda number of cellular polypeptides that are cleaved duringapoptosis. The best characterized of these cellular sub-strates is PARP (28), a nuclear apoptotic landmark cleavedby caspase 3 (29). There was no detectable PARP cleavage2 h after IR, inasmuch as all of the protein detected had thesize of the intact Mr 116,000 PARP protein. However, PARPwas cleaved to the Mr 85,000 signature fragment starting at3 h, a process that was complete by 8 h (Fig. 3E). The kinetics

of PARP cleavage in vivo corresponds, therefore, to thecleavage of the in vitro substrate DEVD, both reflectingcaspase 3 activity.

Caspase Activation after Irradiation Is Blocked by Cys-teine and Serine Protease Inhibitors. It has been previ-ously shown (25) that caspase activation can be blocked byspecific cell-permeable caspase cleavage-site peptide inhib-itors. To further examine the specific involvement ofcaspases and serine proteases in IR-induced apoptosis ofMOLT-4 cells, we tested the effect of caspase inhibitors onDEVD-pNA cleavage activity in apoptotic cell extracts ob-tained 8 h after IR. All of the inhibitors could effectively blockDEVD-pNA cleavage (Fig. 4A). In addition, the serine prote-ase inhibitors AAD-cmk and TLCK also efficiently preventedcleavage of the DEVD-pNA substrate. The effect of TLCKwas not limited to MOLT-4 cells because 200 or 300 mM

TLCK effectively prevented cleavage of DEVD-pNA in the

Fig. 3. Multiple caspase activation during IR-induced apoptosis. Cells were lysed at the indicated times after IR (4 Gy) and lysates (20 mg of protein) usedfor caspase assays as described in “Materials and Methods” with: (A) DEVD-pNA (Œ) and YVAD-pNA (‚); (B) VEID-pNA (E), IETD-pNA (h), and AAPD-pNA(F); and (C) LEHD-AFC substrates. DEVD, YVAD, VEID, IETD, LEHD, and AAPD are preferred peptide substrates for caspase 3, 1, 6, 8, and 9 and GranzymeB, respectively. The substrate concentration was 100 mM, except for AAPD (500 mM). The cleavage activities were determined colorimetrically after 2-hincubation at 37°C (ELISA, 410 nm) for all substrates, except for LEHD, which was determined fluorometrically. D, Caspase mRNA expression. The steadystate mRNA expression was analyzed in exponentially growing MOLT-4 and IM-9 cells by RNase protection, using the hApo-1B multiprobe template set.Numbers on the right, the size of the protected fragments for caspase 8, 3, 6, 2S, 5, 7, 1, and 2L (S and L represent two different isoforms). There was nodetectable hybridization signal detected for Granzyme B. E, PARP proteolysis. Immunoblot analysis of PARP protein cleavage was done using anti-PARPC2-10 and -actin antibodies to examine protein samples from total lysates subjected to SDS-PAGE (7% gel) and transferred to nitrocellulose. The Mr 85,000fragment is a result of proteolytic cleavage of PARP from its Mr 116,000 native form.

494 Proteases Mediate Bax-induced Apoptosis

IM-9 multiple myeloma cells irradiated with 10 Gy (data notshown). Similarly, we have also examined the effect ofDEVD-fmk and AAD-cmk on caspase 9 cleavage activity. Asshown in Fig. 4B, both inhibitors completely prevent caspase9 activation.

To determine the effect of caspase and serine proteaseinhibitors on in vivo target substrates, we choose to analyzetheir effect on PARP cleavage. Cleavage of PARP was pre-vented by DEVD-fmk, which blocks the enzymatic activity ofthe caspase 3-like proteases, which demonstrates thatcaspase 3 cleaved relevant substrates in vivo (Fig. 4C). PARP

cleavage was also blocked by IETD-fmk, VEID-fmk, andTLCK, which indicates that caspase 8 and 6 and a serineprotease were also important for PARP cleavage. Takentogether, these findings indicate that IR-induced apoptosis isassociated with the activation of multiple cysteine and serineproteases and can be prevented by specific peptide inhibi-tors.

The in vivo inhibitor studies above, however, addressedonly whether cleavage site inhibitors could prevent caspaseactivation. To get an insight into whether these inhibitorsaffected the activity rather than the activation of the pro-teases involved in apoptosis, we next sought to determinethe effect of the same inhibitors on the DEVD-pNA cleavageactivity in vitro. A concentration of 10 nM DEVD-fmk effec-tively blocked DEVD-pNA cleavage activity (Fig. 5A). We alsotested CrmA, a cowpox viral serpin product that is a pre-ferred inhibitor of caspase 1 and is known to block Fas-mediated but not IR-induced apoptosis (45). As predicted,concentrations up to 1 mM CrmA had no significant effect onDEVD-pNA cleavage activity (Fig. 5A). Similarly, VEID-fmkand IETD-fmk did not significantly interfere with DEVD-pNAcleavage activity at concentrations up to 0.1 mM (Fig. 5, A andB). Higher concentrations of inhibitors were also tested: up to100 mM for AAD-cmk and 300 mM for TLCK. There was a1000-fold lower inhibitory effect of AAD-cmk as comparedwith DEVD-fmk on caspase inhibition (Fig. 5C). This finding,taken together with the ability of 100 mM AAD-cmk to sub-stantially increase cell viability (3.5-fold; Fig. 2), indicates thatAAD is unlikely to directly affect caspases. There was only alimited inhibitory effect of TLCK, for which only about 10%inhibition was detected at 300 mM (Fig. 5D). These datastrongly suggest that AAD-cmk and TLCK do not directly acton caspases.

Bax Levels Are Up-Regulated after Irradiation. Wesought to explore signals upstream of caspase activationresponsible for triggering the cell death of irradiated MOLT-4cells. We chose to analyze the kinetics of induction of thepro-apoptotic gene bax and the anti-apoptotic genes bcl-2,bcl-x, and mcl-1. The time course chosen for this analysiswas 1–8 h because there was no significant loss of cellviability during this time. Steady-state levels of mRNA weredetermined in exponentially growing cells using the multi-probe RNase protection assay to generate simultaneouslytemplates for bax, bcl-2, bcl-x, and mcl-1 as well as for sixother mRNAs. Most significantly, IR induced a time- anddose-dependent increase of bax mRNA expression. Therewas no significant increase in bax mRNA up to 2 h, with a 5-to 6.5-fold increase in bax levels reached by 8 h at all dosestested (Fig. 6). In contrast to the robust increase in baxexpression, there was only a modest increase (less than2-fold) in expression levels of other bcl-2 family members,such as bcl-2, bcl-x, and mcl-1. Moreover, bax was ex-pressed continuously at later time points when in fact cellswere undergoing apoptosis.4 The kinetics of bax induction,as well as its prolonged expression during the time of cell

4 B. Endlich, B. Gong, S. Mazumder, Q. Chen, S. Abraham, and A. Al-masan, unpublished observations.

Fig. 4. Caspase and serine protease inhibitors prevent caspase activa-tion in vivo. A, DEVD-pNA cleavage. The following inhibitors were added1 h before 4-Gy irradiation at 100 mM: DEVD-fmk; IETD-fmk; VEID-fmk;and AAD-cmk; and at 300 mM: TLCK. Caspase activation was measuredas DEVD-pNA cleavage activity. B, LEHD-AFC cleavage. Caspase-9 ac-tivation was measured as LEHD cleavage activity, using the fluorometricAFC substrate. Cleavage activities in A and B were determined as de-scribed in “Materials and Methods.” C, PARP proteolysis. Immunoblotanalyses were done on the above cell extracts (A) from cells treated withvarious inhibitors harvested at 8 h after IR using anti-PARP C2-10 and-actin antibodies.

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death, is consistent with its potential role in triggering apo-ptosis.

Protease Inhibitors Can Prevent bax Up-Regulationand p53 Function. To address whether the effect of variousinhibitors shown above was direct or rather was influencedby upstream events, we first examined their effect on baxgene expression. Levels of bax mRNA were determined afterIR alone or preceded by treatment with cysteine or serineprotease inhibitors. We found that the DEVD-fmk and AAD-fmk did not suppress bax expression, which indicated thattheir effect on caspase 3 was an event downstream of baxexpression (Fig. 7, Lanes 9–11). In contrast, however, 300 mM

TLCK was able to completely prevent the up-regulation ofbax (Fig. 7, Lane 7); 100 mM TLCK only partially prevented baxinduction (Fig. 7, Lanes 5 and 6).

The effect of TLCK on bax expression could be direct orrather a reflection of the p53 activity because p53 function isrequired for transcriptional activation of bax in multiple cellsystems (17, 46). To address this issue, we first examinedone well-studied characteristic of p53, to increase its proteinlevels after genotoxic damage, such as from IR. As expected,the irradiation of MOLT-4 cells led to increased p53 proteinlevels (Fig. 8A). The specificity of this change was testedusing b-actin, for which no change was observed. Next, weexamined the effect of serine and cysteine protease inhibi-tors on IR-mediated p53 induction. We found that TLCK andTPCK were able to effectively prevent nuclear p53 proteinstabilization (Fig. 8B). In contrast, CPI (a calcium-activatedcysteine protease unrelated to caspases), AAD-cmk, andDEVD-fmk had no effect. Finally, we investigated the abilityof serine and cysteine protease inhibitors to affect a secondfunction of p53 as a DNA-binding dependent transcriptionalactivator (47). We, thus, examined whether a change in p53protein levels was mirrored by changes in DNA binding ac-tivity. The DNA binding activity in untreated or irradiated(4 Gy) MOLT-4 cells was determined by a gel retardationassay. Consistent with the Western analyses, we could de-

tect a shift in the position of oligonucleotides containing thep53 protein-binding site, in lysates isolated from cells 1, 2, or6 h after 4 Gy IR (Fig. 9, Lanes 2–4). Two additional DNA-damaging agents, H2O2 and etoposide (VP16), also causedincreased DNA binding of p53. In order to visualize the p53-specific complexes, the anti-p53 antibody pAb421 was usedto stabilize and supershift the p53 protein-oligonucleotidecomplexes. Similar results were obtained with the antibodyDO1, which recognizes a different epitope of p53 (data notshown). No change in the p53-specific bands could be de-tected in extracts from irradiated MOLT-4 cells that weretreated with the protease inhibitors DEVD-fmk and AAD-cmk(Fig. 9, Lanes 7 and 8). In contrast, TLCK, TPCK, or CPI allprevented DNA binding activity after IR. As a positive control,we used two antioxidants, PDTC and NAC, known to abro-gate p53 DNA binding (48); as expected, they abrogatedDNA binding.

Taken together, the above data support distinct steps inthe IR-triggered Bax-mediated apoptosis pathway ofMOLT-4 cells: (a) one inhibitable by TLCK, upstream of p53and bax induction; and (b) a second, inhibitable by AAD-cmk,downstream of bax up-regulation but before caspase acti-vation. These findings are summarized in a model (Fig. 10).

DiscussionIn this study, we evaluated the temporal relationship betweenIR-triggered, bax-induced apoptotic events and the ability ofcysteine and serine proteases to mediate this process.Caspase 3 was reported previously to be activated in U937(45) and in TK6 cell variants with different p53 status (49) after20 Gy or 4 Gy, respectively. We clearly demonstrate here thatmultiple caspases are activated after irradiation of MOLT-4cells by using in vitro caspase cleavage substrates and in-hibitors and monitoring the cleavage of PARP protein in vivo.The DEVD tetrapeptide used for these assays has beenshown to serve in vitro as a cleavage substrate for caspases

Fig. 5. In vitro effects of caspaseand serine protease inhibitorson caspase activity. The effecton DEVD-pNA cleavage wasevaluated for: (A) DEVD-fmk (Œ),VEID-fmk (E), or CrmA (■); (B)IETD-fmk (h) and TLCK (ƒ); (C)AAD-cmk; and (D) TLCK. Theconcentrations of inhibitors usedwere of 0.01–1 mM (A, B), 1–100mM (C), and 100–300 mM (D). Celllysates (20 mg) were incubated for30 min with various concentra-tions of inhibitors before the ad-dition of the caspase substratesfollowed by a 2-h incubation andcolorimetric determination of pNArelease. Values, means 6 SD(n 5 3).

496 Proteases Mediate Bax-induced Apoptosis

other than caspase 3, such as caspase 2 and 7 (30). How-ever, the major active caspases identified in a variety ofhematopoietic cell types analyzed including MOLT-4 arecaspase 3 and 6 (50). Because caspase 6 does not recognizethe DEVD motif, it suggests that the DEVD cleavage activityin vitro and PARP cleavage in vitro both reflect caspase 3activity. Moreover, we found that IR also activated caspase6, as measured by increased VEID cleavage activities. Thecaspase 3 and 6 activities are likely to be the end product ofthe caspase activity, carrying out the main function of thewhole protease system and, therefore, representing the ef-fector caspases directly involved in cell destruction (50).

What is less clear is the identity of the critical upstreamfactor(s) leading to caspase-3 and -6 activation. One indica-tion for the order of activation is the increased LEHD andIETD cleavage activities that we detected after IR. LEHD is apreferred substrate for caspase 9, which is activated bycytochrome c and Apaf-1, a likely initiating event in geno-toxic stress-induced apoptosis. Cleavage at the IETD site isalso essential to activate caspase 3. Such a site is located atresidue 172 of procaspase 3, and it is proteolytically cleaved

by a caspase to generate the large and small subunits thatconstitute the active caspase 3. Because the IETD cleavageactivity corresponds to caspase 8, this suggests that thisregulatory caspase is also acting upstream of caspase 3.Caspase 8 has been shown previously to have an essentialrole in Fas and tumor necrosis factor-mediated caspasecascade leading to apoptosis (21, 22).

Inhibitor studies reinforced the critical role we found forcaspase activation in the commitment to apoptosis. Mostimportantly, these cleavage-site inhibitors prevented notonly caspase activity but also cell death. Thus, DEVD-fmkcompletely blocked caspase-3 activity and partially blockedcell death in a dose-dependent manner. Consistent with ourfindings, other studies have also shown that bax-mediatedcell death can be prevented by caspase inhibitors (51). Thesefindings contrast, however, with two recent reports that sug-gest a bax-dependent, protease-independent mechanism ofcell death in cell lines with exogenously expressed bax (52,53). It has been suggested that, because caspase inhibitorsare apparently unable to prevent bax-mediated cytochromec release (53) but can prevent caspase activation, an alter-

Fig. 6. Induction of Bcl-2-related RNAs by IR. A, time course and dose response of gene expression. Exponentially growing MOLT-4 cells were irradiated(2–10 Gy), and RNA was extracted from cells at the indicated times after treatment. The steady-state mRNA expression was analyzed by the multiprobeRNase protection assay after IR with 2 (A, D), 4 (B, E), and 10 Gy (C, F). The hStress-1 template set was used to determine mRNA levels for bcl-x (F), bax(E), bcl-2 (h), and mcl-1 (h). All of the data are from a single gel, with only representative portions shown. The ordinate shows fold induction, representingvalues obtained by normalizing the levels of mRNA to those of untreated cells and to that of the mRNA levels of a housekeeping gene (L32). On the right,the molecular weight of the protected fragments.

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native caspase-independent pathway of cell death mustfunction. The discrepancy between these studies may beexplained by the relative dominance of caspase-dependentand -independent cell death pathways in various cell lines.Similar to DEVD-fmk, the caspase 8 and 6 inhibitors IETD-fmk and VEID-fmk prevented multiple caspase activities,PARP cleavage, and, to some extent, cell death.

This study also provides evidence for an essential roleplayed by serine protease(s) in apoptosis by analyses ofAAPD cleavage activity. AAPD was previously shown to bean effective substrate for granzyme B in vitro (44). Because

we could not detect any granzyme B mRNA and becausegranzyme B is not expected to be expressed in culturedcells, we believe that the target of these inhibitors is anotherserine protease, most likely a granzyme B-like protease.Moreover, the in vivo inhibitory effect of AAD-cmk, a pre-ferred granzyme-B inhibitor, provides further support for therole of a serine protease in this process.

An essential issue to address is whether AAD and TLCKare only inhibiting serine proteases or are they targeting acaspase as well. Several lines of evidence make a directeffect on a caspase unlikely, favoring the notion that theseinhibitors are, in fact, targeting a serine protease. First, wefound that TLCK is effective only when added relatively earlyafter IR. Moreover, TLCK (up to 300 mM) was unable toprevent caspase cleavage activity in vitro. Furthermore, wealso found that TLCK efficiently prevented PARP cleavage invivo, similar to a previous report (54). In addition, TLCK wasable to also prevent the appearance of FITC1/PI2 cells,further supporting the inhibitory role of TLCK in early apo-ptosis. In contrast, another serine protease inhibitor, TPCK,did not prevent any of the hallmarks of protease activation orcell death, even though it could prevent bax mRNA induction.Importantly, TPCK was quite cytotoxic to these cells and islikely to cause necrosis. This could explain why, althoughboth inhibitors prevented p53 activation, only TLCK pre-vented cell death. However, the fact that TLCK was lesspotent in preventing cell death than DEVD-fmk suggests thatparallel pathways of cell death may converge in a commondownstream caspase activation. In addition, TLCK itself—at300 mM—had some cytotoxic effect after longer exposures(more than 12 h), which is likely to be manifested in the formof necrosis.

In this investigation, we found that bax was induced sig-nificantly following 2–10 Gy IR. Bax is a death agonist, pre-viously shown to be essential for apoptosis in some cellsystems (6). Moreover, expression of Bax, in the absence ofany other death stimulus is sufficient to induce apoptosis(52). In addition, there is recent evidence that bax suppressestumorigenesis and induces apoptosis in vivo (55). Our find-ings are consistent with previous studies that found that IRinduced an increase in bax mRNA in several leukemia and

Fig. 7. Effect of inhibitors onbax induction. The multiprobeRNase protection assay wasused as described in Fig. 6 todetermine mRNA levels after IRalone (4 or 8 h; Lanes 2 and 3), orpreceded by treatment withTLCK [100 (1), or 300 mM (111);Lanes 4–7], DEVD-fmk [100 mM

(1); Lanes 8 and 9], or AAD-cmk[100 mM (1); Lanes 10 and 11].On the right, the molecular weightof the protected fragments.

Fig. 8. Effect of inhibitors on p53 activation by IR. A, the effect of IR onp53 protein levels. Immunoblot analyses were done on cells lysed at theindicated times after 4 Gy IR using anti-p53 (DO1, top) and anti-b-actin(bottom). B, the effect of inhibitors on nuclear p53 levels. Nuclear extractswere prepared as described previously (67) 6 h after 4 Gy IR (1). Thefollowing inhibitors were added 1 h before IR: DEVD-fmk; AAD-cmk;TPCK; and CPI (100 mM); and TLCK (300 mM); cells were harvested; andcell lysates were immunoblotted with the anti-p53 DO1 and -actin anti-bodies.

498 Proteases Mediate Bax-induced Apoptosis

lymphoma cell lines that contain wild-type p53 but failed toinduce bax expression in most solid tumor lines or in leuke-mia and lymphoma cell lines that lacked p53, or in those thatcontained mutant p53 (16). Moreover, in some p53-deficientsolid tumor lines, IR-induced apoptosis did not enhanceexpression of p53 or Bax (56). To further illustrate the cell-type specificity of IR-triggered events, bax induction wasobserved in lymphoid and other radiosensitive organs inmice but not in other radioresistant tissues (57). However, theup-regulation of bax, as well as p21WAF1 and gadd45,5 allknown to be transcriptionally activated by wild-type p53 afterIR, was somewhat surprising in view of a report of a mutantp53 allele in MOLT-4 cells (58). Because it has been reportedthat MOLT-4 cells are heterozygous, harboring both a wild-type and a mutant p53 allele (248 codon change, Arg3 Gln),we have sequenced the cDNA of our MOLT-4 cells. ThecDNA sequence analysis has revealed only a wild-type DNAsequence, similar to what was recently reported (59). Thediscrepancy with earlier studies could have resulted from asubstantially lower, or lack of, expression of the mutantallele, or from the loss of the mutated allele.

In our study, the effect of IR was observed predominantlyfor bax, with only a small increase in the expression levels ofanti-apoptotic genes bcl-2, bcl-x, and mcl-1. We show re-sults of a sensitive and quantitative approach for simultane-ous analysis of multiple bcl-2 family transcripts. This ap-proach should be useful for examining the levels of both pro-and anti-apoptotic genes in other experimental systems. Inthe hematopoietic cell line used in this study, the outcome ofincreased bax expression after IR was cell death. However,we found that irradiation (1–4 Gy) of a highly radioresistantfibroblast cell line in which Bax expression was unchangedled to increased levels of Bcl-2 and related anti-apoptoticproteins that provided significant protection from Fas-medi-ated cell death (60). Moreover, in those cells, the Fas-in-duced proteolytic caspase-3 activity was diminished, pro-

viding a significant protection from Fas cytotoxicity asmeasured by clonogenic assays.

It is still unclear how the apoptotic signal is transmittedfrom bax to caspases. Recent studies have shown that mi-tochondria may play an important role in the induction ofapoptosis by regulating the release of cytochrome c into thecytosol (41, 61–63). Cytochrome c release is also essentialfor apoptosis induced by IR (Ref. 64 and Chen et al.6). It hasbeen suggested that the way by which bax works is toactively facilitate the release of cytochrome c and otherfactors essential for activation of the caspase cascade (53).An alternative, caspase-independent pathway of cell deathcould be functional because caspase inhibitors were unableto completely prevent cell death.

Overall, our data indicate that the increase in bax geneexpression in leukemic cells—in which IR induces rapid ap-optosis—occurs downstream of events sensitive to TLCKand upstream of an AAD-sensitive serine protease or ofcaspases. Of the inhibitors tested, AAD-cmk, DEVD-fmk,and TLCK could block IR-mediated cell death effectively, butonly TLCK could block p53 function and bax expression.Additionally, because blocking bax expression may not besufficient to completely block cell death, there may be otherpathways of cell death in these cells. These studies alsodemonstrate that p53 and bax function downstream of aTLCK-inhibitable serine protease. However, they act up-stream of an AAD-inhibitable serine protease and of multiplecaspases and cause apoptosis through the regulation ofcaspase activity. It would be important to identify and char-acterize such TLCK and AAD-sensitive endogenous serineprotease(s) in order to understand the death-signaling path-way in IR-triggered, bax-mediated apoptosis. Additionally,further studies may lead to the development of immunolog-ical or molecular methods of activating caspases, which may

5 B. Gong, and A. Almasan. Differential up-regulation of TP53-responsivegenes by genotoxic stress in hematopoietic cells containing wild-type andmutant p53, submitted for publication.

6 Q. Chen, B. Gong, and A. Almasan. Distinct stages of cytochrome crelease from mitochondria: a feedback amplification loop linking caspaseactivation to mitochondria in genotoxic stress-induced apoptosis, sub-mitted for publication.

Fig. 9. Effect of inhibitors onp53 DNA binding. The nuclearextracts used were those pre-pared for Fig. 8 after IR (4 Gy),except Lane 1 (no treatment),Lanes 2–4 (1–6 h after IR), Lane5 (H2O2, 100 mM), and Lane 6(VP16, 10 mM). The following in-hibitors were added 1 h beforeIR: DEVD-fmk (Lane 7, 100 mM);AAD-cmk (Lane 8, 100 mM);TLCK (Lane 9, 300 mM); TPCK(Lane 10, 100 mM); CPI (Lane 11,100 mM); PDTC (Lane 12, 100mM); and NAC (Lane 13, 30 mM).EMSA was done as described in“Materials and Methods” usingp53-binding 32P-oligonucleotidesand the anti-p53 pAb421 anti-bodies to supershift the p53-specific oligonucleotide-bindingcomplexes (arrow).

499Cell Growth & Differentiation

be effective at inducing apoptosis in certain cancers that areresistant to conventional radio- or chemotherapy.

Materials and MethodsCell Culture and Treatments. MOLT-4 cells, derived from an individualwith acute lymphoblastic leukemia, and the IM-9 multiple myeloma cellswere obtained from the American Type Culture Collection (Rockville, MD)and grown in RPMI 1640 with 10% (vol/vol) heat-inactivated fetal bovineserum, 50 units/ml penicillin and 50 mg/ml streptomycin (Life Technolo-gies, Inc.). Exponentially growing cells were adjusted to a density of 1–2 3105 cells/ml on the day before the experiment was performed. Irradiation(2–10 Gy) was performed at 25°C, using a Mark I Irradiator (J. C. Shepherd& Associates, Irvine, CA) with a 137Cs source emitting at a fixed dose-rateof 2.8 Gy/min, as described previously (65). All of the chemicals, unlessspecified otherwise, were obtained from Sigma Chemical Co. (St. Louis, MO).

RNase Protection Assay. Total RNA was isolated from cells at vari-ous intervals after irradiation using the Trizol reagent (Life Technologies,Inc.). To determine the steady-state levels of RNA, we used the RiboQuantsystem (Pharmingen) for RNase protection assay with a Multi-Probe Tem-plate set, which allows simultaneous quantitation and characterization ofmultiple RNA molecules. The hStress-1 and hAPO-1B template sets(Pharmingen) were used for the T7 polymerase-directed synthesis of highspecific activity [32P]-antisense RNA probes. Each probe set contained 10probes, including two housekeeping gene products, L32 and GAPDH. Themultiprobe RNase protection approach assures that the proper quantita-tive analyses can be obtained from a single gel after PhosphorImageanalysis of the mRNA levels for all of the probes, normalized to L32 orGAPDH levels. Probes (4 3 105 cpm) were synthesized using T7 poly-merase labeled with [32P]UTP and hybridized to 10 mg of total RNAovernight at 56°C. RNA hybrids were digested with RNAse A and T1,

purified, and resolved on 6% denaturing polyacrylamide gels. The level ofeach mRNA species was determined by PhosphorImage analysis basedon signal intensities given by the appropriately sized, protected probefragments, which were also normalized to expression levels of the house-keeping genes.

Immunoblotting. Cell lysates were resolved by one-dimensionalSDS-PAGE under reducing conditions, followed by transfer onto 0.45-mmnitrocellulose membranes (Schleicher and Schull) in transfer buffer at 0.2A for 2 h. For PARP analysis, the cells were resuspended in a samplebuffer [62.5 mM Tris-HCl (pH 6.8), 6 M urea, 10% glycerol, 2% SDS,0.00125% bromphenol blue, and 5% b-mercaptoethanol] and were son-icated to effectively dissociate PARP protein/DNA interactions. Aftertransfer, residual binding sites were blocked by incubating the membranein TBS containing 10% nonfat dry milk for 1 h at room temperature. Theblots were then incubated with the appropriate primary antibody in TBSTcontaining 5% nonfat dry milk for 16 h at 4°C. The blots were then washedthree times for 10 min in TBST, followed by incubation with the secondaryantibody conjugated to horseradish peroxidase in TBST containing 5%nonfat dry milk for 1 h at 25°C. After three 10-min washes in TBST, theblots were developed using the enhanced chemiluminescence (ECL) de-tection system (Amersham) according to the manufacturer’s protocol andexposed to X-ray film (Eastman Kodak).

Primary antibodies used in immunoblot analysis were the murine anti-PARP, (1:5000 dilution, clone C-2-10, Biomol Research Laboratories), anti-p53 [1:300 dilution, DO1 (Ab-6), Oncogene Science], and antihuman b-actin(1:5000 dilution, Sigma), with a sheep antimouse IgG-conjugated to horse-radish peroxidase (1:5000 dilution, Amersham) used as secondary antibody.

Apoptosis Assays. To detect PS exposure on cell membranes, cellswere stained with FITC-Annexin V (25 ng/ml, green fluorescence, R&DSystems, Minneapolis, MN), simultaneously with dye exclusion of PI (neg-ative for red fluorescence). The test described discriminates intact cells(FITC2/PI2), early apoptotic cells (FITC1/PI2), and late apoptotic ornecrotic cells (FITC1/PI1). Flow cytometric measurements were per-formed by bivariate flow cytometry as described previously (41), using aFACScan, and analyzed with CellQuest software (Becton Dickinson) onmean values obtained from the cell population from which debris weregated out. The comparative experiments were performed at the sametime, and the results were normalized against data from the 0-h time point.

Caspase activity was measured using the following pNA-derived chro-mogenic substrates for caspases: YVAD; DEVD; VEID; and IETD as pre-ferred substrates for caspase 1, 3, 6, or 8, respectively (Calbiochem). Forcaspase-9 activity, the more sensitive fluorogenic substrate LEHD-AFC.TFA was used. Additionally, AAPD-pNA was used as a preferredsubstrate for the serine protease granzyme B (Sigma). In brief, cells werelysed on ice in 0.2% NP40, 2.5 mM digitonin, 20 mM HEPES (pH 7.9), 20mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1 mM

DTT, 0.5 mM phenylmethane-sulfonylfluoride, and 1 mg/ml each leupeptin,aprotinin, and pepstatin for 10 min and centrifuged at 14,000 rpm for 3min. Protease assays included 20 mg of protein (in 20 ml of lysis buffer), 80ml of reaction buffer [100 mM HEPES (pH 7.6), 20% glycerol, 5 mM DTT,and 0.5 mM EDTA], and 1 ml (100 mM final concentration) of 10 mM pNApeptide substrates. Control experiments (not shown) established that therelease of substrate was linear with time and with protein concentrationunder the conditions specified.

For in vivo studies, cell-permeable cysteine or serine protease inhibitors(50–100 mM) were added to cells (2–3 3 105/ml) 1 h before irradiation(unless otherwise stated) and remained in the medium until the time of celllysis for RNA isolation or apoptosis assays. The inhibitors used were:DEVD-fmk; VEID-fmk; and IETD-fmk—specific for caspase 3, 6, and 8,respectively (Calbiochem). The serine protease inhibitors TLCK andTPCK, preferably inhibiting trypsin and chymotrypsin-like activities, re-spectively, and the granzyme-B inhibitor AAD-cmk were used at a con-centration of 100 or 300 mM. For in vitro inhibition studies, the caspase andserine protease inhibitors, as well as the cowpox viral serpin productknown as cytokine-response modifier (CrmA), were added at the indicatedconcentrations to apoptotic cell lysates for 30 min at 25°C before incu-bation with the DEVD-pNA substrate. Samples were then incubated for anadditional 2 h at 37°C, and DEVD cleavage was monitored by enzyme-catalyzed release of pNA, by determining absorbance at 410 nm in amicrotiter plate reader (Cambridge Tech, Inc.). In the case of caspase 9,the production of AFC was monitored in a Turner Fluorometer (Model 112,

7 B. Gong and A. Almasan. Ionizing radiation activates the TRAIL ligandand Fas/APO-1 receptor-mediated cell death pathways in human tumorcell, submitted for publication.

Fig. 10. Model of protease activation in IR-triggered, bax-mediated celldeath. IR leads to p53-dependent bax expression and the activation ofmultiple cysteine proteases. Inhibitors of serine proteases can preventcaspase activation either upstream of p53 and bax (TLCK) or downstreamof them (AAD-cmk). Activated caspase 3 cleaves proteolytically multiplecellular substrates, including DFF45, which results in the release of theactive DFF40 nuclease and DNA fragmentation (67). The requirement ofcleavage of procaspase 3 at an IETD site for caspase 3 activation placescaspase 8 upstream of caspase 3. A parallel pathway of cell death can bealso activated when the plasma membrane cell-death receptors come incontact with their respective activating ligands.7

500 Proteases Mediate Bax-induced Apoptosis

Sequoia-Turner Corp.). Values were normalized against the calibrationcurve obtained with free substrate only.

Cytotoxicity Assays. Untreated or irradiated cells were seeded(2–3 3 103 to 104 cells) in 96-well plates in the presence or absence of avariety of inhibitors, followed by incubation for the indicated times. Cellviability was determined 24 h after irradiation with the CellTiter 96 AQue-ous One Solution Reagent (MTS, Promega), an improved variation of theMTT assay used to determine tetrazolium reduction. Measurements con-sisted of determining absorbance at 490 nm using an ELISA reader(Cambridge Tech, Inc.); all of the determinations were done in triplicate.

p53 Functional Assays. Total RNA from MOLT-4 cells was extractedas above, and cDNA was synthesized by reverse transcription with M-MLV Reverse Transcriptase (Life Technologies, Inc.) using random hex-amers as primer. The PCR primers for p53 amplification by RT-PCR were59-GGCCATCTACAAGCAGTC-39 (sense, corresponding to residues 480–497) and 59-GGAGGCTGTCAGTGGGGAAC-39 (antisense, correspondingto residues 1217–1198). The PCR mixture was denatured at 94°C for 4min, and amplification was carried out for 35 cycles at 94°C for 30 s, 58°Cfor 1 min, and 72°C for 1 min, followed by a final elongation step at 72°Cfor 5 min. The PCR products were purified and sequenced using theprimer 59-CTCGCTTAGTGCTCCCTGG-39 (corresponding to residues919–909). DNA sequencing was done using ABI PRISM BigDye Termi-nator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA Poly-merase, FS by the Molecular Biotechnology Core of Cleveland Clinic,using a 377 Xl upgrade DNA sequencer (Perkin Elmer, Applied Biosystems).

EMSAs were done essentially as described previously (66). Briefly, cellswere resuspended in 20 mM HEPES (pH 7.9), 20 mM NaF, 1 mM Na3VO4,1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM phenylmeth-ane-sulfonylfluoride, and 1 mg/ml each leupeptin, aprotinin, and pepstatinand incubated on ice for 15 min. After adding NP40 to 0.2%, the cells wereincubated on ice for another 15 min and resuspended by vortexing. Thenuclei were pelleted in a microfuge at full-speed for 20 s, and the nuclearpellet was resuspended in 30 ml of the above buffer supplemented with0.2% NP40 and 0.4 M NaCl. Nuclei were then incubated on ice for 15 minand centrifuged again for 15 min. The oligonucleotide 59-AGCTTAGACAT-GCCTAGACATGCCTA-39, representing a consensus binding site for p53(47) was annealed to its complement and end-labeled with [g-32P]ATP. Forbinding, 10 mg of extract protein, 1 mg of poly dI-dC (Pharmacia), and 0.5ng of [g-32P]ATP-labeled p53 oligonucleotide probe was used in 6 mM

HEPES (pH 7.9), 1 mM DTT, and 0.5 mM EDTA. After 30 min of incubationat 25°C, the reaction products were separated on a 6% PAGE gel in 0.53Tris-borate EDTA buffer. For supershift experiments, 0.1 mg of pAb421 orDO1 anti-p53 antibodies (Ab-1 or Ab-6, Oncogene Science) were used inthe binding reactions; these antibodies recognize the COOH (residue371–380) or NH2 (residue 11–25) termini of p53. Gels were dried andexposed to X-ray film (Eastman Kodak).

AcknowledgmentsWe thank Drs. Graham Casey (Cleveland Clinic Foundation) for providingprimers and for advice on p53 DNA sequencing and Michael Chernov foradvice on the EMSA assays. We also thank Dr. Satya Yadav (ClevelandClinic DNA Sequencing Core) and Amy Raber (Cleveland Clinic FlowCytometry Core) for expert assistance. The Becton-Dickinson FACS Van-tage Cell Sorter was purchased through a generous gift from the KeckFoundation.

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502 Proteases Mediate Bax-induced Apoptosis