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Cell Growth & Differentiation Vol. 10, 491-502, July 1999
© 1999 American Association for Cancer Research

Ionizing Radiation-induced, Bax-mediated Cell Death Is Dependent on Activation of Cysteine and Serine 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 Cleveland Clinic Foundation [B. G., Q. C., B. E., S. M., A. A.], Cleveland, Ohio 44195


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Bcl-2 family proteins and interleukin-1-ß converting enzyme/Caenorhabditis elegans cell death gene-3 (ICE/CED-3) family proteases (caspases) represent the basic regulators of apoptosis. However, the precise mechanism by which they interact is unclear. In this study, we found that {gamma}-radiation-induced apoptosis of leukemia cells was associated with activation of multiple caspases and bax up-regulation. Membrane changes and caspase activities were suppressed by specific caspase inhibitors. Similarly, the serine protease inhibitors z-Ala-Ala-Asp-cmk (AAD) and tosyl-lysine chloromethyl ketone (TLCK) also prevented caspase activation and poly(ADP-ribose) polymerase cleavage in vivo but had no effect on caspase activity in vitro. TLCK also prevented bax up-regulation as a result of its inhibitory effect on p53 function. Inhibitors of caspases and serine proteases partially prevented cell death, suggesting a caspase involvement in Bax-mediated cell death. We propose an ordering of signaling events in Bax-mediated cell death, including steps upstream and downstream of p53 and bax up-regulation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The response of eukaryotic cells to IR3 includes cell-cycle arrest and cell death. Experimental evidence suggests that the cytotoxic effects of IR and many forms of chemotherapy are mediated through a final common pathway that involves the activation of apoptosis (1 , 2) . Few insights, however, are available regarding the signals that control induction of apoptosis after IR. Two well-characterized proteins known to regulate IR-mediated apoptosis are Bcl-2 and p53. Bcl-2 is the prototype of a family of proteins, related to the Caenorhabditis elegans ced-9 gene, that are involved in the regulation of a distal step in an evolutionarily conserved pathway for physiological cell death and apoptosis, with some members functioning as suppressors of apoptosis and others as promoters of cell death (3) . Bcl-2 and its homologues, Bcl-x and Mcl-1 encode membrane-associated proteins that protect neoplastic cells from DNA damage-induced apoptosis, including that caused by IR (4 , 5) . In contrast, Bax is a promoter of cell death. The relative ratios of these various pro- and anti-apoptotic members of the Bcl-2 family have been shown to determine the ultimate sensitivity or resistance of cells to diverse apoptotic stimuli, including IR (6) . Although various family members can interfere with each other’s functions by heterodimeric interactions (6) , they can also function independently of each other to regulate cell death (7 , 8) .

The p53 tumor suppressor protein has an essential role in controlling cell-cycle progression or apoptosis, and its dysfunction has profound consequences because about 50% of all human tumors produce aberrant p53 protein [see Ref. 9 for a review]. Studies with p53-null mice showed that p53 is necessary for thymocytes to undergo apoptosis in response to DNA damage (1 , 10) . Moreover, the levels and activity of p53 have been shown to increase in response to IR and other DNA-damaging agents (11, 12, 13, 14, 15) . Although p53 is required for optimal apoptosis induced by IR (1 , 2) the precise role of this tumor suppressor in regulating cell death is poorly understood. It is known that p53 transcriptionally activates bax in some types of cells after treatment with IR, chemotherapeutic drugs, and other forms of genotoxic stress (16 , 17) . Other p53-regulated genes may also contribute to cell death (18) .

Genetic studies in the nematode C. elegans have provided evidence that the ced-3 gene is indispensable for cell death during development and has provided the first evidence for the involvement of aspartate-specific cysteine proteases in the induction of apoptosis. Related Ced-3 homologues in mammalian cells include members of a new family of ICE family proteases (19) , now called caspases (20) . These include: (a) regulator caspases, such as caspase 8 [FADD-like interleukin-1-ß converting enzyme (FLICE)/MORT1-associated CED-3 homologue (MACH)/Mch5 (21 , 22) and caspase 9 [ICE-Lap6, MCH6 (23) ]; and (b) effector caspases, such as caspase 3 [CPP32/YAMA/Apopain (24, 25, 26) ] and caspase 6 [MCH2, (27) ]. Several proteins, including PARP (28 , 29) as well as many others (see Ref. 30 for review and additional references), have been shown to be cleaved during apoptosis after a specific aspartate residue (25, 26, 27) . Recent reports implicate caspase activation in p53-dependent apoptosis (31, 32, 33) ; however, the pathways regulating these events are poorly understood.

Serine proteases have also been suggested to participate in apoptosis. The best characterized system for the involvement of a serine protease in apoptosis is cytotoxic T-cell-mediated cell death, which has been shown to require granzyme B. Moreover, granzyme B can process and directly activate a number of caspases, providing evidence for a proteolytic cascade initiated by a serine protease [see Ref. 34 for review and additional references]. Inhibitors of other serine proteases were shown to prevent apoptosis by a variety of stimuli, including staurosporine, DNA-damaging agents, and dexamethasone in immature thymocytes (35) , tumor necrosis factor in various cell lines, Fas ligand in T lymphocytes, serum withdrawal and T-cell receptor-crosslinking in T cells [Ref. 36 and refs. therein]. TLCK is a serine protease inhibitor with specificity for trypsin-like activities, which can inhibit apoptosis induced by diverse stimuli at an early stage before both DNA fragmentation and cytoplasmic changes in immature rat thymocytes (37) . In contrast, TPCK a chymotrypsin-specific protease inhibitor has no effect on cell death in these cells (37) . The effect of TLCK and TPCK could be, however, cell type-dependent because TLCK does not suppress apoptosis in other myeloid cells and TPCK has the opposite effect (38) . Nevertheless, it is becoming apparent that the activation of these proteases is a crucial event in the cellular execution of apoptosis (for review, see Ref. 30 ). However, with the exception of granzyme B, the identity and role of other serine proteases in apoptosis is not understood.

The present experiments were designed to examine the temporal relationship of events caused by IR-triggered, Bax-induced apoptosis and to establish the role of cysteine and serine proteases in this process. We confirmed that four known caspases were present, the activity of which could be prevented by specific inhibitors. Furthermore, we found involvement of serine proteases inhibitable by AAD-cmk and TLCK. By using a variety of cleavage site inhibitors, we were able to further separate p53 and bax induction from caspase activation and cell death. These studies place Bax and p53 downstream of a TLCK-inhibitable step but upstream of an AAD-inhibitable protease, all of these events being present before caspase activation. On the basis of these data, we are proposing an ordering of the molecular events involved in IR-signaling leading to cell death. Most importantly, we describe distinct steps, upstream and downstream of p53 and bax activation. Because Bax-induced events are only partially inhibitable by caspase or serine protease inhibitors, this suggests a protease-independent step in addition to the protease-dependent events.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Inhibitors of Serine and Cysteine Proteases Prevent Radiation-induced Apoptosis of MOLT-4 Cells.
Previous studies (39 , 40) have demonstrated that treatment with IR efficiently killed MOLT-4 cells. Cells treated with 4–10 Gy {gamma}-irradiation showed features of apoptosis characterized by the 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 the cell surface, including the translocation of PS from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V was used to determine PS exposure on the cell membrane in combination with PI to establish the integrity of the cell membrane, as described previously (41) . Using this method, we detected FITC+/PI- cells, characteristic of early apoptosis, in a significant number of cells by 8 h, with the majority of cells becoming FITC+ by 12 h (Fig. 1DCitation , upper and lower right panels).



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

 
It is known that the process of PS export to the outer leaflet of the plasma membrane is a caspase-dependent event during apoptosis of cells from various lineages (41 , 42) . We confirmed this in MOLT-4 cells by showing that the caspase 3-specific cell-permeable inhibitor DEVD-fmk was able to prevent PS expression significantly (Fig. 1E)Citation . Next, we sought to examine whether serine proteases might also be involved in this process. We found that the AAD-cmk serine protease inhibitor substantially reduced the appearance of FITC+/PI- (Fig. 1FCitation , lower right panel) and FITC+/PI+ cells (Fig. 1FCitation , upper right panel). Similarly, we found that TLCK, a trypsin-like serine protease inhibitor, also inhibited the appearance of FITC+/PI- (Fig. 1GCitation , lower right panel). In contrast to TLCK, another closely related serine protease inhibitor TPCK, in fact, substantially promoted the appearance of FITC+/PI+ cells ((Fig. 1HCitation , upper right panel). These data implicate a serine protease in the early events of IR-induced apoptosis that affects the function of the plasma membrane. Therefore, both serine and cysteine protease activities can contribute to events that lead to membrane changes.

To further examine the effect of these inhibitors on cell viability, we analyzed loss of metabolic cellular activity by determining NADH activity, measured by tetrazolium reduction. Irradiation (4 Gy) led to a marked decrease in viable cells by 24 h (Fig. 2)Citation . Cell death was prevented by DEVD-fmk in a dose-dependent manner, with cells treated with 100 µM DEVD-fmk having a 3.5-fold increase in cell viability 24 h after IR. Similarly, the serine-protease inhibitor AAD-cmk (100 µM) also protected from cell death (3.5-fold). Cell death was blocked to a lesser extent by TLCK and the caspase 8 and 6 inhibitors, IETD-fmk and VEID-fmk, respectively. Thus, apparently in MOLT-4 cells, the activity of both cysteine and serine proteases is required for cell death. However, both caspase and serine protease-dependent and independent steps may exist inasmuch as none of the inhibitors could completely prevent cell death.



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Fig. 2. Serine and cysteine protease inhibitors prevent cell death. Cells were either left untreated (-) or irradiated with 4 Gy (+) in the absence or presence of the following inhibitors added 1 h before IR: (a) the caspase inhibitors DEVD-fmk (10–100 µM), VEID-fmk, and IETD-fmk (100 µM); and (b) the serine protease inhibitors AAD-cmk (100 µM) and TLCK (300 µM). The MTS cell proliferation assay (Promega) was used to determine the percentage of biochemically active cells 24 h after IR. Each experiment was done in triplicate; values, means ± SD (n = 3).

 
Radiation Activates Multiple Caspases and a Serine Protease.
To follow caspase activation during apoptosis, cytosols from cell lysates obtained after increasing lengths of time after IR were incubated with the chromogenic substrate DEVD-pNA, which mimics the PARP cleavage site P1-P4 tetrapeptide (25) . As seen in Fig. 3ACitation , there was a time-dependent increase in DEVD-pNA cleavage activity starting at 4 h and reaching higher levels after 10 h. Remarkably, the kinetics of DEVD-pNA cleavage showed a linear, time-dependent increase between 4 and 10 h. In contrast to the marked increase in DEVD-pNA cleavage activity, there was no significant change in the cleavage activity for YVAD-pNA (Fig. 3A)Citation . Because DEVD is a substrate for caspase 3 and YVAD is a substrate for caspase 1, this demonstrates that apoptosis induced by IR in MOLT-4 cells is dependent on caspase 3 and not caspase 1 protease activity. To determine which other caspases might be activated during the IR-mediated apoptosis of MOLT-4 cells, we examined VEID-pNA and IETD-pNA cleavage activities. These represent cleavage sites in lamin (43) and procaspase 3 and are considered to be preferred substrates for caspase 6 and 8, respectively. Both the VEID-pNA and IETD-pNA cleavage activities were induced substantially (Fig. 3B)Citation . Finally, we examined the activation of caspase 9 by determining the cleavage of its specific substrate LEHD-AFC. Caspase 9 is a regulator caspase whose a activity has recently been shown to be an essential mediator of apoptosis induced as a result of cytochrome c release from mitochondria (23) . All of the caspase activities were detectable by 4 h and then continued to increase steadily up to at least 8 h (Fig. 3C)Citation . These results show that multiple caspases were activated after the irradiation of MOLT-4 cells.



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Fig. 3. Multiple caspase activation during IR-induced apoptosis. Cells were lysed at the indicated times after IR (4 Gy) and lysates (20 µg of protein) used for caspase assays as described in "Materials and Methods" with: (A) DEVD-pNA ({blacktriangleup}) and YVAD-pNA ({triangleup}); (B) VEID-pNA ({circ}), IETD-pNA ({square}), and AAPD-pNA (•); 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 Granzyme B, respectively. The substrate concentration was 100 µM, except for AAPD (500 µM). The cleavage activities were determined colorimetrically after 2-h incubation at 37°C (ELISA, 410 nm) for all substrates, except for LEHD, which was determined fluorometrically. D, Caspase mRNA expression. The steady state 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 no detectable hybridization signal detected for Granzyme B. E, PARP proteolysis. Immunoblot analysis of PARP protein cleavage was done using anti-PARP C2-10 and -actin antibodies to examine protein samples from total lysates subjected to SDS-PAGE (7% gel) and transferred to nitrocellulose. The Mr 85,000 fragment is a result of proteolytic cleavage of PARP from its Mr 116,000 native form.

 
We also detected a robust induction of AAPD-pNA cleavage activity (Fig. 3B)Citation . AAPD has been previously shown to be a specific substrate for the serine protease granzyme B in vitro (44) . Because granzyme B has been previously implicated in cytotoxic T lymphocyte (CTL) killing in conjunction with perforin activity, we next examined whether granzyme B was expressed in MOLT-4 cells. Steady-state mRNA levels were determined by the multiprobe RNase protection assay in exponentially growing cells. With this method, we examined simultaneously templates for granzyme B and several caspases using the hApo-1B template set. As seen in Fig. 3DCitation , there was no detectable 361-bp mRNA species for granzyme B, with low levels detected for caspase 1 and 5. This would suggest that since there was no granzyme B expression, the AAPD-pNA cleavage activity was due to another enzyme, most likely a granzyme B-like serine protease. In contrast, there was abundant expression of caspases 3, 6, and 8. Interestingly, there were significant differences between the caspase mRNAs detected in MOLT-4 cells and the multiple myeloma cell line IM-9, most notably caspase 1 and 6, which indicates a differential expression of individual caspases in these cell lines.

Previous studies (reviewed by Ref. 30 ) have also identified a number of cellular polypeptides that are cleaved during apoptosis. The best characterized of these cellular substrates is PARP (28) , a nuclear apoptotic landmark cleaved by caspase 3 (29) . There was no detectable PARP cleavage 2 h after IR, inasmuch as all of the protein detected had the size of the intact Mr 116,000 PARP protein. However, PARP was cleaved to the Mr 85,000 signature fragment starting at 3 h, a process that was complete by 8 h (Fig. 3E)Citation . The kinetics of PARP cleavage in vivo corresponds, therefore, to the cleavage of the in vitro substrate DEVD, both reflecting caspase 3 activity.

Caspase Activation after Irradiation Is Blocked by Cysteine and Serine Protease Inhibitors.
It has been previously shown (25) that caspase activation can be blocked by specific cell-permeable caspase cleavage-site peptide inhibitors. To further examine the specific involvement of caspases and serine proteases in IR-induced apoptosis of MOLT-4 cells, we tested the effect of caspase inhibitors on DEVD-pNA cleavage activity in apoptotic cell extracts obtained 8 h after IR. All of the inhibitors could effectively block DEVD-pNA cleavage (Fig. 4A)Citation . In addition, the serine protease inhibitors AAD-cmk and TLCK also efficiently prevented cleavage of the DEVD-pNA substrate. The effect of TLCK was not limited to MOLT-4 cells because 200 or 300 µM TLCK effectively prevented cleavage of DEVD-pNA in the IM-9 multiple myeloma cells irradiated with 10 Gy (data not shown). Similarly, we have also examined the effect of DEVD-fmk and AAD-cmk on caspase 9 cleavage activity. As shown in Fig. 4BCitation , both inhibitors completely prevent caspase 9 activation.



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Fig. 4. Caspase and serine protease inhibitors prevent caspase activation in vivo.A, DEVD-pNA cleavage. The following inhibitors were added 1 h before 4-Gy irradiation at 100 µM: DEVD-fmk; IETD-fmk; VEID-fmk; and AAD-cmk; and at 300 µM: TLCK. Caspase activation was measured as DEVD-pNA cleavage activity. B, LEHD-AFC cleavage. Caspase-9 activation was measured as LEHD cleavage activity, using the fluorometric AFC substrate. Cleavage activities in A and B were determined as described in "Materials and Methods." C, PARP proteolysis. Immunoblot analyses were done on the above cell extracts (A) from cells treated with various inhibitors harvested at 8 h after IR using anti-PARP C2-10 and -actin antibodies.

 
To determine the effect of caspase and serine protease inhibitors on in vivo target substrates, we choose to analyze their effect on PARP cleavage. Cleavage of PARP was prevented by DEVD-fmk, which blocks the enzymatic activity of the caspase 3-like proteases, which demonstrates that caspase 3 cleaved relevant substrates in vivo (Fig. 4C)Citation . PARP cleavage was also blocked by IETD-fmk, VEID-fmk, and TLCK, which indicates that caspase 8 and 6 and a serine protease were also important for PARP cleavage. Taken together, these findings indicate that IR-induced apoptosis is associated with the activation of multiple cysteine and serine proteases and can be prevented by specific peptide inhibitors.

The in vivo inhibitor studies above, however, addressed only whether cleavage site inhibitors could prevent caspase activation. To get an insight into whether these inhibitors affected the activity rather than the activation of the proteases involved in apoptosis, we next sought to determine the effect of the same inhibitors on the DEVD-pNA cleavage activity in vitro. A concentration of 10 nM DEVD-fmk effectively blocked DEVD-pNA cleavage activity (Fig. 5A)Citation . We also tested CrmA, a cowpox viral serpin product that is a preferred inhibitor of caspase 1 and is known to block Fas-mediated but not IR-induced apoptosis (45) . As predicted, concentrations up to 1 µM CrmA had no significant effect on DEVD-pNA cleavage activity (Fig. 5A)Citation . Similarly, VEID-fmk and IETD-fmk did not significantly interfere with DEVD-pNA cleavage activity at concentrations up to 0.1 µM (Fig. 5, A and B)Citation . Higher concentrations of inhibitors were also tested: up to 100 µM for AAD-cmk and 300 µM for TLCK. There was a 1000-fold lower inhibitory effect of AAD-cmk as compared with DEVD-fmk on caspase inhibition (Fig. 5C)Citation . This finding, taken together with the ability of 100 µM AAD-cmk to substantially increase cell viability (3.5-fold; Fig. 2Citation ), indicates that AAD is unlikely to directly affect caspases. There was only a limited inhibitory effect of TLCK, for which only about 10% inhibition was detected at 300 µM (Fig. 5D)Citation . These data strongly suggest that AAD-cmk and TLCK do not directly act on caspases.



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Fig. 5. In vitro effects of caspase and serine protease inhibitors on caspase activity. The effect on DEVD-pNA cleavage was evaluated for: (A) DEVD-fmk ({blacktriangleup}), VEID-fmk ({circ}), or CrmA ({blacksquare}); (B) IETD-fmk ({square}) and TLCK ({triangledown}); (C) AAD-cmk; and (D) TLCK. The concentrations of inhibitors used were of 0.01–1 µM (A, B), 1–100 µM (C), and 100–300 µM (D). Cell lysates (20 µg) were incubated for 30 min with various concentrations of inhibitors before the addition of the caspase substrates followed by a 2-h incubation and colorimetric determination of pNA release. Values, means ± SD (n = 3).

 
Bax Levels Are Up-Regulated after Irradiation.
We sought to explore signals upstream of caspase activation responsible for triggering the cell death of irradiated MOLT-4 cells. We chose to analyze the kinetics of induction of the pro-apoptotic gene bax and the anti-apoptotic genes bcl-2, bcl-x, and mcl-1. The time course chosen for this analysis was 1–8 h because there was no significant loss of cell viability during this time. Steady-state levels of mRNA were determined in exponentially growing cells using the multiprobe RNase protection assay to generate simultaneously templates for bax, bcl-2, bcl-x, and mcl-1 as well as for six other mRNAs. Most significantly, IR induced a time- and dose-dependent increase of bax mRNA expression. There was 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 doses tested (Fig. 6)Citation . In contrast to the robust increase in bax expression, there was only a modest increase (less than 2-fold) in expression levels of other bcl-2 family members, such as bcl-2, bcl-x, and mcl-1. Moreover, bax was expressed continuously at later time points when in fact cells were undergoing apoptosis.4 The kinetics of bax induction, as well as its prolonged expression during the time of cell death, is consistent with its potential role in triggering apoptosis.



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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 multiprobe RNase 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 (•), bax ({circ}), bcl-2 ({square}), and mcl-1 ({square}). All of the data are from a single gel, with only representative portions shown. The ordinate shows fold induction, representing values 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.

 
Protease Inhibitors Can Prevent bax Up-Regulation and p53 Function.
To address whether the effect of various inhibitors shown above was direct or rather was influenced by upstream events, we first examined their effect on bax gene expression. Levels of bax mRNA were determined after IR alone or preceded by treatment with cysteine or serine protease inhibitors. We found that the DEVD-fmk and AAD-fmk did not suppress bax expression, which indicated that their effect on caspase 3 was an event downstream of bax expression (Fig. 7Citation , Lanes 9–11). In contrast, however, 300 µM TLCK was able to completely prevent the up-regulation of bax (Fig. 7Citation , Lane 7); 100 µM TLCK only partially prevented bax induction (Fig. 7Citation , Lanes 5 and 6).



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Fig. 7. Effect of inhibitors on bax induction. The multiprobe RNase protection assay was used as described in Fig. 6Citation to determine mRNA levels after IR alone (4 or 8 h; Lanes 2 and 3), or preceded by treatment with TLCK [100 (+), or 300 µM (+++); Lanes 4–7], DEVD-fmk [100 µM (+); Lanes 8 and 9], or AAD-cmk [100 µM (+); Lanes 10 and 11]. On the right, the molecular weight of the protected fragments.

 
The effect of TLCK on bax expression could be direct or rather a reflection of the p53 activity because p53 function is required for transcriptional activation of bax in multiple cell systems (17 , 46) . To address this issue, we first examined one well-studied characteristic of p53, to increase its protein levels after genotoxic damage, such as from IR. As expected, the irradiation of MOLT-4 cells led to increased p53 protein levels (Fig. 8A)Citation . The specificity of this change was tested using ß-actin, for which no change was observed. Next, we examined the effect of serine and cysteine protease inhibitors on IR-mediated p53 induction. We found that TLCK and TPCK were able to effectively prevent nuclear p53 protein stabilization (Fig. 8B)Citation . In contrast, CPI (a calcium-activated cysteine protease unrelated to caspases), AAD-cmk, and DEVD-fmk had no effect. Finally, we investigated the ability of serine and cysteine protease inhibitors to affect a second function of p53 as a DNA-binding dependent transcriptional activator (47) . We, thus, examined whether a change in p53 protein levels was mirrored by changes in DNA binding activity. The DNA binding activity in untreated or irradiated (4 Gy) MOLT-4 cells was determined by a gel retardation assay. Consistent with the Western analyses, we could detect a shift in the position of oligonucleotides containing the p53 protein-binding site, in lysates isolated from cells 1, 2, or 6 h after 4 Gy IR (Fig. 9Citation , Lanes 2–4). Two additional DNA-damaging agents, H2O2 and etoposide (VP16), also caused increased DNA binding of p53. In order to visualize the p53-specific complexes, the anti-p53 antibody pAb421 was used to stabilize and supershift the p53 protein-oligonucleotide complexes. Similar results were obtained with the antibody DO1, which recognizes a different epitope of p53 (data not shown). No change in the p53-specific bands could be detected in extracts from irradiated MOLT-4 cells that were treated with the protease inhibitors DEVD-fmk and AAD-cmk (Fig. 9Citation , Lanes 7 and 8). In contrast, TLCK, TPCK, or CPI all prevented DNA binding activity after IR. As a positive control, we used two antioxidants, PDTC and NAC, known to abrogate p53 DNA binding (48) ; as expected, they abrogated DNA binding.



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Fig. 8. Effect of inhibitors on p53 activation by IR. A, the effect of IR on p53 protein levels. Immunoblot analyses were done on cells lysed at the indicated times after 4 Gy IR using anti-p53 (DO1, top) and anti-ß-actin (bottom). B, the effect of inhibitors on nuclear p53 levels. Nuclear extracts were prepared as described previously (67) 6 h after 4 Gy IR (+). The following inhibitors were added 1 h before IR: DEVD-fmk; AAD-cmk; TPCK; and CPI (100 µM); and TLCK (300 µM); cells were harvested; and cell lysates were immunoblotted with the anti-p53 DO1 and -actin antibodies.

 


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Fig. 9. Effect of inhibitors on p53 DNA binding. The nuclear extracts used were those prepared for Fig. 8Citation after IR (4 Gy), except Lane 1 (no treatment), Lanes 2–4 (1–6 h after IR), Lane 5 (H2O2, 100 µM), and Lane 6 (VP16, 10 µM). The following inhibitors were added 1 h before IR: DEVD-fmk (Lane 7, 100 µM); AAD-cmk (Lane 8, 100 µM); TLCK (Lane 9, 300 µM); TPCK (Lane 10, 100 µM); CPI (Lane 11, 100 µM); PDTC (Lane 12, 100 µM); and NAC (Lane 13, 30 mM). EMSA was done as described in "Materials and Methods" using p53-binding 32P-oligonucleotides and the anti-p53 pAb421 antibodies to supershift the p53-specific oligonucleotide-binding complexes (arrow).

 
Taken together, the above data support distinct steps in the IR-triggered Bax-mediated apoptosis pathway of MOLT-4 cells: (a) one inhibitable by TLCK, upstream of p53 and bax induction; and (b) a second, inhibitable by AAD-cmk, downstream of bax up-regulation but before caspase activation. These findings are summarized in a model (Fig. 10)Citation .



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Fig. 10. Model of protease activation in IR-triggered, bax-mediated cell death. IR leads to p53-dependent bax expression and the activation of multiple cysteine proteases. Inhibitors of serine proteases can prevent caspase activation either upstream of p53 and bax (TLCK) or downstream of them (AAD-cmk). Activated caspase 3 cleaves proteolytically multiple cellular substrates, including DFF45, which results in the release of the active DFF40 nuclease and DNA fragmentation (67) . The requirement of cleavage of procaspase 3 at an IETD site for caspase 3 activation places caspase 8 upstream of caspase 3. A parallel pathway of cell death can be also activated when the plasma membrane cell-death receptors come in contact with their respective activating ligands.7 .

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we evaluated the temporal relationship between IR-triggered, bax-induced apoptotic events and the ability of cysteine 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) after 20 Gy or 4 Gy, respectively. We clearly demonstrate here that multiple caspases are activated after irradiation of MOLT-4 cells by using in vitro caspase cleavage substrates and inhibitors and monitoring the cleavage of PARP protein in vivo. The DEVD tetrapeptide used for these assays has been shown to serve in vitro as a cleavage substrate for caspases other than caspase 3, such as caspase 2 and 7 (30) . However, the major active caspases identified in a variety of hematopoietic cell types analyzed including MOLT-4 are caspase 3 and 6 (50) . Because caspase 6 does not recognize the DEVD motif, it suggests that the DEVD cleavage activity in vitro and PARP cleavage in vitro both reflect caspase 3 activity. Moreover, we found that IR also activated caspase 6, as measured by increased VEID cleavage activities. The caspase 3 and 6 activities are likely to be the end product of the caspase activity, carrying out the main function of the whole protease system and, therefore, representing the effector caspases directly involved in cell destruction (50) .

What is less clear is the identity of the critical upstream factor(s) leading to caspase-3 and -6 activation. One indication for the order of activation is the increased LEHD and IETD cleavage activities that we detected after IR. LEHD is a preferred substrate for caspase 9, which is activated by cytochrome c and Apaf-1, a likely initiating event in genotoxic stress-induced apoptosis. Cleavage at the IETD site is also essential to activate caspase 3. Such a site is located at residue 172 of procaspase 3, and it is proteolytically cleaved by a caspase to generate the large and small subunits that constitute the active caspase 3. Because the IETD cleavage activity corresponds to caspase 8, this suggests that this regulatory caspase is also acting upstream of caspase 3. Caspase 8 has been shown previously to have an essential role in Fas and tumor necrosis factor-mediated caspase cascade leading to apoptosis (21 , 22) .

Inhibitor studies reinforced the critical role we found for caspase activation in the commitment to apoptosis. Most importantly, these cleavage-site inhibitors prevented not only caspase activity but also cell death. Thus, DEVD-fmk completely blocked caspase-3 activity and partially blocked cell death in a dose-dependent manner. Consistent with our findings, other studies have also shown that bax-mediated cell death can be prevented by caspase inhibitors (51) . These findings contrast, however, with two recent reports that suggest a bax-dependent, protease-independent mechanism of cell death in cell lines with exogenously expressed bax (52 , 53) . It has been suggested that, because caspase inhibitors are apparently unable to prevent bax-mediated cytochrome c release (53) but can prevent caspase activation, an alternative caspase-independent pathway of cell death must function. The discrepancy between these studies may be explained by the relative dominance of caspase-dependent and -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 role played by serine protease(s) in apoptosis by analyses of AAPD cleavage activity. AAPD was previously shown to be an effective substrate for granzyme B in vitro (44) . Because we could not detect any granzyme B mRNA and because granzyme B is not expected to be expressed in cultured cells, we believe that the target of these inhibitors is another serine protease, most likely a granzyme B-like protease. Moreover, the in vivo inhibitory effect of AAD-cmk, a preferred granzyme-B inhibitor, provides further support for the role of a serine protease in this process.

An essential issue to address is whether AAD and TLCK are only inhibiting serine proteases or are they targeting a caspase as well. Several lines of evidence make a direct effect on a caspase unlikely, favoring the notion that these inhibitors are, in fact, targeting a serine protease. First, we found that TLCK is effective only when added relatively early after IR. Moreover, TLCK (up to 300 µM) was unable to prevent caspase cleavage activity in vitro. Furthermore, we also found that TLCK efficiently prevented PARP cleavage in vivo, similar to a previous report (54) . In addition, TLCK was able to also prevent the appearance of FITC+/PI- cells, further supporting the inhibitory role of TLCK in early apoptosis. In contrast, another serine protease inhibitor, TPCK, did not prevent any of the hallmarks of protease activation or cell death, even though it could prevent bax mRNA induction. Importantly, TPCK was quite cytotoxic to these cells and is likely to cause necrosis. This could explain why, although both inhibitors prevented p53 activation, only TLCK prevented cell death. However, the fact that TLCK was less potent in preventing cell death than DEVD-fmk suggests that parallel pathways of cell death may converge in a common downstream caspase activation. In addition, TLCK itself—at 300 µM—had some cytotoxic effect after longer exposures (more than 12 h), which is likely to be manifested in the form of necrosis.

In this investigation, we found that bax was induced significantly following 2–10 Gy IR. Bax is a death agonist, previously shown to be essential for apoptosis in some cell systems (6) . Moreover, expression of Bax, in the absence of any other death stimulus is sufficient to induce apoptosis (52) . In addition, there is recent evidence that bax suppresses tumorigenesis and induces apoptosis in vivo (55) . Our findings are consistent with previous studies that found that IR induced an increase in bax mRNA in several leukemia and lymphoma cell lines that contain wild-type p53 but failed to induce bax expression in most solid tumor lines or in leukemia and lymphoma cell lines that lacked p53, or in those that contained mutant p53 (16) . Moreover, in some p53-deficient solid tumor lines, IR-induced apoptosis did not enhance expression of p53 or Bax (56) . To further illustrate the cell-type specificity of IR-triggered events, bax induction was observed in lymphoid and other radiosensitive organs in mice but not in other radioresistant tissues (57) . However, the up-regulation of bax, as well as p21WAF1 and gadd45,5 all known to be transcriptionally activated by wild-type p53 after IR, was somewhat surprising in view of a report of a mutant p53 allele in MOLT-4 cells (58) . Because it has been reported that MOLT-4 cells are heterozygous, harboring both a wild-type and a mutant p53 allele (248 codon change, Arg -> Gln), we have sequenced the cDNA of our MOLT-4 cells. The cDNA sequence analysis has revealed only a wild-type DNA sequence, similar to what was recently reported (59) . The discrepancy with earlier studies could have resulted from a substantially lower, or lack of, expression of the mutant allele, or from the loss of the mutated allele.

In our study, the effect of IR was observed predominantly for bax, with only a small increase in the expression levels of anti-apoptotic genes bcl-2, bcl-x, and mcl-1. We show results of a sensitive and quantitative approach for simultaneous analysis of multiple bcl-2 family transcripts. This approach should be useful for examining the levels of both pro- and anti-apoptotic genes in other experimental systems. In the hematopoietic cell line used in this study, the outcome of increased bax expression after IR was cell death. However, we found that irradiation (1–4 Gy) of a highly radioresistant fibroblast cell line in which Bax expression was unchanged led to increased levels of Bcl-2 and related anti-apoptotic proteins that provided significant protection from Fas-mediated cell death (60) . Moreover, in those cells, the Fas-induced proteolytic caspase-3 activity was diminished, providing a significant protection from Fas cytotoxicity as measured by clonogenic assays.

It is still unclear how the apoptotic signal is transmitted from bax to caspases. Recent studies have shown that mitochondria may play an important role in the induction of apoptosis by regulating the release of cytochrome c into the cytosol (41 , 61, 62, 63) . Cytochrome c release is also essential for apoptosis induced by IR (Ref. 64 and Chen et al.6 ). It has been suggested that the way by which bax works is to actively facilitate the release of cytochrome c and other factors essential for activation of the caspase cascade (53) . An alternative, caspase-independent pathway of cell death could be functional because caspase inhibitors were unable to completely prevent cell death.

Overall, our data indicate that the increase in bax gene expression in leukemic cells—in which IR induces rapid apoptosis—occurs downstream of events sensitive to TLCK and upstream of an AAD-sensitive serine protease or of caspases. Of the inhibitors tested, AAD-cmk, DEVD-fmk, and TLCK could block IR-mediated cell death effectively, but only TLCK could block p53 function and bax expression. Additionally, because blocking bax expression may not be sufficient to completely block cell death, there may be other pathways of cell death in these cells. These studies also demonstrate that p53 and bax function downstream of a TLCK-inhibitable serine protease. However, they act upstream of an AAD-inhibitable serine protease and of multiple caspases and cause apoptosis through the regulation of caspase activity. It would be important to identify and characterize such TLCK and AAD-sensitive endogenous serine protease(s) in order to understand the death-signaling pathway in IR-triggered, bax-mediated apoptosis. Additionally, further studies may lead to the development of immunological or molecular methods of activating caspases, which may be effective at inducing apoptosis in certain cancers that are resistant to conventional radio- or chemotherapy.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture and Treatments.
MOLT-4 cells, derived from an individual with acute lymphoblastic leukemia, and the IM-9 multiple myeloma cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 with 10% (vol/vol) heat-inactivated fetal bovine serum, 50 units/ml penicillin and 50 mg/ml streptomycin (Life Technologies, Inc.). Exponentially growing cells were adjusted to a density of 1–2 x 105 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-rate of 2.8 Gy/min, as described previously (65) . All of the chemicals, unless specified otherwise, were obtained from Sigma Chemical Co. (St. Louis, MO).

RNase Protection Assay.
Total RNA was isolated from cells at various intervals after irradiation using the Trizol reagent (Life Technologies, Inc.). To determine the steady-state levels of RNA, we used the RiboQuant system (Pharmingen) for RNase protection assay with a Multi-Probe Template set, which allows simultaneous quantitation and characterization of multiple RNA molecules. The hStress-1 and hAPO-1B template sets (Pharmingen) were used for the T7 polymerase-directed synthesis of high specific activity [32P]-antisense RNA probes. Each probe set contained 10 probes, including two housekeeping gene products, L32 and GAPDH. The multiprobe RNase protection approach assures that the proper quantitative analyses can be obtained from a single gel after PhosphorImage analysis of the mRNA levels for all of the probes, normalized to L32 or GAPDH levels. Probes (4 x 105 cpm) were synthesized using T7 polymerase labeled with [32P]UTP and hybridized to 10 µg of total RNA overnight at 56°C. RNA hybrids were digested with RNAse A and T1, purified, and resolved on 6% denaturing polyacrylamide gels. The level of each mRNA species was determined by PhosphorImage analysis based on signal intensities given by the appropriately sized, protected probe fragments, which were also normalized to expression levels of the housekeeping genes.

Immunoblotting.
Cell lysates were resolved by one-dimensional SDS-PAGE under reducing conditions, followed by transfer onto 0.45-µm nitrocellulose membranes (Schleicher and Schull) in transfer buffer at 0.2 A for 2 h. For PARP analysis, the cells were resuspended in a sample buffer [62.5 mM Tris-HCl (pH 6.8), 6 M urea, 10% glycerol, 2% SDS, 0.00125% bromphenol blue, and 5% ß-mercaptoethanol] and were sonicated to effectively dissociate PARP protein/DNA interactions. After transfer, residual binding sites were blocked by incubating the membrane in TBS containing 10% nonfat dry milk for 1 h at room temperature. The blots were then incubated with the appropriate primary antibody in TBST containing 5% nonfat dry milk for 16 h at 4°C. The blots were then washed three times for 10 min in TBST, followed by incubation with the secondary antibody conjugated to horseradish peroxidase in TBST containing 5% nonfat dry milk for 1 h at 25°C. After three 10-min washes in TBST, the blots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham) according to the manufacturer’s protocol and exposed 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 ß-actin (1:5000 dilution, Sigma), with a sheep antimouse IgG-conjugated to horseradish peroxidase (1:5000 dilution, Amersham) used as secondary antibody.

Apoptosis Assays.
To detect PS exposure on cell membranes, cells were stained with FITC-Annexin V (25 ng/ml, green fluorescence, R&D Systems, Minneapolis, MN), simultaneously with dye exclusion of PI (negative for red fluorescence). The test described discriminates intact cells (FITC-/PI-), early apoptotic cells (FITC+/PI-), and late apoptotic or necrotic cells (FITC+/PI+). Flow cytometric measurements were performed by bivariate flow cytometry as described previously (41) , using a FACScan, and analyzed with CellQuest software (Becton Dickinson) on mean values obtained from the cell population from which debris were gated out. The comparative experiments were performed at the same time, and the results were normalized against data from the 0-h time point.

Caspase activity was measured using the following pNA-derived chromogenic substrates for caspases: YVAD; DEVD; VEID; and IETD as preferred substrates for caspase 1, 3, 6, or 8, respectively (Calbiochem). For caspase-9 activity, the more sensitive fluorogenic substrate LEHD- AFC.TFA was used. Additionally, AAPD-pNA was used as a preferred substrate for the serine protease granzyme B (Sigma). In brief, cells were lysed on ice in 0.2% NP40, 2.5 mM digitonin, 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 phenylmethane-sulfonylfluoride, and 1 µg/ml each leupeptin, aprotinin, and pepstatin for 10 min and centrifuged at 14,000 rpm for 3 min. Protease assays included 20 µg of protein (in 20 µl of lysis buffer), 80 µl of reaction buffer [100 mM HEPES (pH 7.6), 20% glycerol, 5 mM DTT, and 0.5 mM EDTA], and 1 µl (100 µM final concentration) of 10 mM pNA peptide substrates. Control experiments (not shown) established that the release of substrate was linear with time and with protein concentration under the conditions specified.

For in vivo studies, cell-permeable cysteine or serine protease inhibitors (50–100 µM) were added to cells (2–3 x 105/ml) 1 h before irradiation (unless otherwise stated) and remained in the medium until the time of cell lysis 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 and TPCK, preferably inhibiting trypsin and chymotrypsin-like activities, respectively, and the granzyme-B inhibitor AAD-cmk were used at a concentration of 100 or 300 µM. For in vitro inhibition studies, the caspase and serine protease inhibitors, as well as the cowpox viral serpin product known as cytokine-response modifier (CrmA), were added at the indicated concentrations to apoptotic cell lysates for 30 min at 25°C before incubation with the DEVD-pNA substrate. Samples were then incubated for an additional 2 h at 37°C, and DEVD cleavage was monitored by enzyme-catalyzed release of pNA, by determining absorbance at 410 nm in a microtiter plate reader (Cambridge Tech, Inc.). In the case of caspase 9, the production of AFC was monitored in a Turner Fluorometer (Model 112, Sequoia-Turner Corp.). Values were normalized against the calibration curve obtained with free substrate only.

Cytotoxicity Assays.
Untreated or irradiated cells were seeded (2–3 x 103 to 104 cells) in 96-well plates in the presence or absence of a variety of inhibitors, followed by incubation for the indicated times. Cell viability was determined 24 h after irradiation with the CellTiter 96 AQueous One Solution Reagent (MTS, Promega), an improved variation of the MTT assay used to determine tetrazolium reduction. Measurements consisted 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 extracted as above, and cDNA was synthesized by reverse transcription with M-MLV Reverse Transcriptase (Life Technologies, Inc.) using random hexamers as primer. The PCR primers for p53 amplification by RT-PCR were 5'-GGCCATCTACAAGCAGTC-3' (sense, corresponding to residues 480–497) and 5'-GGAGGCTGTCAGTGGGGAAC-3' (antisense, corresponding to residues 1217–1198). The PCR mixture was denatured at 94°C for 4 min, and amplification was carried out for 35 cycles at 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min, followed by a final elongation step at 72°C for 5 min. The PCR products were purified and sequenced using the primer 5'-CTCGCTTAGTGCTCCCTGG-3' (corresponding to residues 919–909). DNA sequencing was done using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA Polymerase, 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, cells were 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 phenylmethane-sulfonylfluoride, and 1 µg/ml each leupeptin, aprotinin, and pepstatin and incubated on ice for 15 min. After adding NP40 to 0.2%, the cells were incubated on ice for another 15 min and resuspended by vortexing. The nuclei were pelleted in a microfuge at full-speed for 20 s, and the nuclear pellet was resuspended in 30 µl of the above buffer supplemented with 0.2% NP40 and 0.4 M NaCl. Nuclei were then incubated on ice for 15 min and centrifuged again for 15 min. The oligonucleotide 5'-AGCTTAGACATGCCTAGACATGCCTA-3', representing a consensus binding site for p53 (47) was annealed to its complement and end-labeled with [{gamma}-32P]ATP. For binding, 10 µg of extract protein, 1 µg of poly dI-dC (Pharmacia), and 0.5 ng of [{gamma}-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 incubation at 25°C, the reaction products were separated on a 6% PAGE gel in 0.5x Tris-borate EDTA buffer. For supershift experiments, 0.1 µg of pAb421 or DO1 anti-p53 antibodies (Ab-1 or Ab-6, Oncogene Science) were used in the binding reactions; these antibodies recognize the COOH (residue 371–380) or NH2 (residue 11–25) termini of p53. Gels were dried and exposed to X-ray film (Eastman Kodak).


    Acknowledgments
 
We thank Drs. Graham Casey (Cleveland Clinic Foundation) for providing primers and for advice on p53 DNA sequencing and Michael Chernov for advice on the EMSA assays. We also thank Dr. Satya Yadav (Cleveland Clinic DNA Sequencing Core) and Amy Raber (Cleveland Clinic Flow Cytometry Core) for expert assistance. The Becton-Dickinson FACS Vantage Cell Sorter was purchased through a generous gift from the Keck Foundation.


    Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a research grant from the NIH (CA 81504). Back

2 To whom requests for reprints should be addressed, at Department of Cancer Biology, The Cleveland Clinic Foundation, NB40, Cleveland, OH 44195. Phone (216) 444-9970; Fax: (216) 445-6269; E-mail: almasaa{at}ccf.org Back

3 The abbreviations used are: IR, ionizing radiation; TBS, Tris-buffered saline; 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, calpain inhibitor I; cmk, chloromethyl ketone (CH2Cl); EMSA, electromobility shift assays; fmk, fluorometyl ketone; IETD, N-acetyl-Ile-Glu-Thr-Asp; LEHD, acetyl-Leu-Glu-His-Asp; NAC, N-acetyl-L-cysteine; PS, phosphatidylserine; PARP, poly(ADP)-ribose polymerase; pNA, p-nitroanilide; PI, propidium iodide; PDTC, pyrrolidine dithiocarbamate; TLCK, tosyl-lysine cmk; TPCK, N-tosyl-L-phenylalanine cmk; VEID, Ac-Val-Glu-Ile-Asp; YVAD, acetyl-Tyr-Val-Ala-Asp ICE, interleukin-1-ß converting enzyme; CED, Caenorhabditis elegans cell death gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

4 B. Endlich, B. Gong, S. Mazumder, Q. Chen, S. Abraham, and A. Almasan, unpublished observations. Back

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

6 Q. Chen, B. Gong, and A. Almasan. Distinct stages of cytochrome c release from mitochondria: a feedback amplification loop linking caspase activation to mitochondria in genotoxic stress-induced apoptosis, submitted for publication. Back

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

Received for publication 12/ 7/98. Revision received 3/ 4/99. Accepted for publication 4/21/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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