Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis.

The response of eukaryotic cells to ionizing radiation (IR) includes induction of apoptosis. However, the signals that regulate this response are unknown. The present studies demonstrate that IR treatment of U-937 cells is associated with: (i) internucleosomal DNA fragmentation; (ii) cleavage of poly(ADP-ribose) polymerase; (iii) cleavage of protein kinase C δ; and (iv) induction of an Ac-DEVD-p-nitroanilide cleaving activity. Overexpression of the cowpox protein CrmA blocked tumor necrosis factor (TNF)-induced apoptosis but had no effect on IR-induced DNA fragmentation or cleavage of poly(ADP-ribose) polymerase and protein kinase C δ. By contrast, overexpression of the baculovirus p35 protein blocked both IR- and TNF-induced apoptosis. The results further demonstrate that the IR-induced proteolytic activity is directly inhibited by the addition of purified recombinant p35, but not by CrmA. We show that the CPP32 protease is sensitive to p35 and not CrmA. We also show that IR induces activation of CPP32 and that this event, like induction of apoptosis, is sensitive to overexpression of p35 and not CrmA. These findings indicate that IR-induced apoptosis involves activation of CPP32 and that this CrmA-insensitive apoptotic pathway is distinct from those induced by TNF and certain other stimuli.

The response of eukaryotic cells to ionizing radiation (IR) 1 includes cell cycle arrest and activation of DNA repair. The available evidence suggests that IR induces these effects by direct interaction with DNA or through the formation of reactive oxygen intermediates that damage DNA and cell membranes (1). In the event of irreparable damage, IR-treated cells also undergo programmed cell death or apoptosis. Few insights, however, are available regarding the signals that control induction of apoptosis in the IR response. While p53 is required for optimal apoptosis induced by IR (2,3), the precise role of this tumor suppressor in regulating cell death is poorly understood. Other studies have shown that Bcl-2 and Bcl-x L inhibit IR-induced apoptosis (4 -7). Several proteins, including poly(ADP-ribose) polymerase (PARP) (8), lamin B1 (9), DNA-dependent protein kinase (10), the 70-kDa protein component of the U1 small nucleoprotein (11), and topoisomerase I (12), have been shown to be cleaved during apoptosis. Recent studies have also shown that protein kinase C (PKC) ␦ is proteolytically activated during IR-induced apoptosis (13). Cleavage of PARP and PKC␦ is blocked by overexpression of Bcl-2 and Bcl-x L (13,14). The mechanistic basis for the anti-apoptotic effects of Bcl-2 and Bcl-x L is unclear.
The present studies demonstrate that, in contrast to TNF, IR induces apoptosis by a CrmA-insensitive, p35-sensitive mechanism. The results support a distinct signaling cascade responsible for IR-induced cell death.

MATERIALS AND METHODS
Cell Culture and Transfections-Human U-937 myeloid leukemia cells (American Type Culture Collection, Rockville, MD) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. CrmA cDNA was cloned into the pEF1 vector (provided by Dr. Ping Li, BASF, Worcester, MA), which was constructed by ligation of human elongation factor 1␣ promoter from pEF321-CAT (41) into a vector modified from pBluescript. p35 was cloned into the pEF2 vector, which was made by substituting the cytomegalovirus promoter of pcDNA3 with elongation factor 1␣ promoter (33,41). To generate a CrmA-or p35-overexpressing line, U-937 cells were cotransfected by electroporation (Gene Pulsar, Bio-Rad, 0.25 V, 960 microfarads) with pcDNA3 and pEF1-CrmA or pEF2-p35 (33,41). Transfectants were selected in the presence of 400 g/ml geneticin sulfate. Irradiation was performed with a ␥-ray source (cesium 137, Gamma Cell 1000, Atomic Energy of Canada, Ltd., Ontario) at a fixed dose rate of 13 gray/min. Cells were also treated with TNF (42).
Isolation and Analysis of RNA-Total cellular RNA was isolated as described (43). RNA (20 g/lane) was separated in agarose/formaldehyde gels, transferred to nitrocellulose filters, and hybridized to the 32 P-labeled DNA fragments corresponding to the entire CrmA or p35 open reading frames. Hybridizations were performed as described (7). Analysis of DNA Fragmentation-DNA was prepared as described (13) and separated in 2% agarose gels. The DNA was visualized by UV illumination after ethidium bromide staining.
Immunoblot Analysis-Cell lysates were prepared as described (13). Proteins were subjected to electrophoresis in an SDS-10% polyacrylamide gel and then transferred to nitrocellulose membranes. The membranes were blocked with 5% dried milk, 0.1% Tween 20, and phosphate-buffered saline and were incubated with anti-CrmA polyclonal antibody (raised against full-length CrmA protein), anti-PKC␦ (Santa Cruz Biotechnology, Santa Cruz, CA), anti-CPP32 polyclonal antibody (raised against the large subunit of CPP32; amino acids 1-175), or anti-Ich-1 L (Transduction Laboratories, Lexington, KY). Preparation of lysates and immunoblotting for PARP were carried out as described using the C-2-10 anti-PARP monoclonal antibody (44). After washing with phosphate-buffered saline/Tween, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (Amersham) for anti-PARP and anti-Ich-1 L or anti-rabbit IgG (Amersham) for anti-CrmA, anti-PKC␦, and anti-CPP32.
Protein Expression and Purification-CrmA contained an N-terminal polyhistidine linker and was expressed in Escherichia coli MM294 cells under the control of a pL promoter as described (25). Cell lysate supernatant was diluted 1:1 with buffer A (50 mM HEPES, pH 7.5, 10% glycerol, 0.2 M NaCl) and applied to a 5-ml NiSO 4 -charged Hi-Trap column (Pharmacia Biotech Inc.). The column was washed with 2% buffer B (buffer A plus 200 mM imidazole), and the protein was eluted with buffer B. Fractions containing CrmA as judged by SDS-PAGE were pooled and dialyzed at 4°C against 20 mM Tris, pH 7.5. The sample was applied to an 8-ml Mono Q column (Pharmacia), equilibrated with buffer C (20 mM Tris, pH 7.5, 5 mM dithiothreitol), and eluted with a gradient to buffer D (buffer C plus 300 mM NaCl). p35 was expressed in E. coli MM294 cells and purified as described (33). ICE, Ich-1, Ich-2, and CPP32 contained N-terminal polyhistidine linkers and were expressed and purified as described (25).
Protease Assays-Cell lysates were centrifuged at 900 ϫ g for 10 min at 4°C. Protease assays included 178 l of reaction buffer (100 mM HEPES, pH 7.5, 20% v/v glycerol, 5 mM dithiothreitol, and 0.5 mM EDTA), 2 l of 10 mM acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) in Me 2 SO (100 M final concentration; California Peptide Research, Inc., Napa, CA), and 20 l of cell lysate. Samples were incubated at 30°C, and enzyme-catalyzed release of p-nitroanilide was monitored at 405 nm for 30 min in a microtiter plate reader (Molecular Devices Inc., Sunnyvale, CA). In certain experiments, the cell lysate diluted into assay buffer was first incubated with varying amounts of inhibitors at room temperature for 30 min. Inhibition of purified ICE homologs by CrmA was performed as described (33) using Ac-DEVD-pNA (500 M) as a substrate for Ich-1, Ich-2, and CPP32 and Ac-YVAD-pNA (125 M) as a substrate for ICE. Values for nanomoles of pNA released were calculated from those observed in A 405 values using a standard curve.

RESULTS
Previous studies have demonstrated that treatment of U-937 cells with IR is associated with the induction of apoptosis (6,7,45). To determine whether this event is sensitive to inhibitors of ICE-like proteases, we prepared U-937 cell clones that overexpress CrmA and p35 (Fig. 1A). IR treatment of both U-937 and U-937/CrmA cells resulted in a pattern of internucleosomal DNA fragmentation characteristic of apoptosis (Fig. 1B). By contrast, there was little if any DNA fragmentation in irradiated U-937/p35 cells (Fig. 1B). Immunoblot analysis of CrmA in U-937/CrmA cell lysates and comparison of signals that were obtained with recombinant CrmA indicated a concentration of approximately 1 ng/20 g of lysates (data not shown). Since CrmA blocks TNF-induced apoptosis in other cell types (34), we asked whether the U-937/CrmA cells were sensitive to this agent. The results demonstrate that both CrmA and p35 block apoptosis induced by TNF treatment (Fig. 1C). These findings indicated that IR induces apoptosis by a distinct CrmA-insensitive mechanism.
To determine the involvement of CrmA-insensitive proteases in IR-induced apoptosis, we assessed cleavage of the 116-kDa PARP protein to an 85-kDa fragment (8). As expected, IR treatment of U-937 cells resulted in PARP cleavage (Fig. 2). Similar findings were obtained in irradiated U-937/CrmA cells, but there was no detectable cleavage of PARP in IR-treated U-937/p35 cells (Fig. 2). IR also induces cleavage of PKC␦ (13). PKC␦ cleavage to a 40-kDa fragment was unaffected in IRtreated U-937/CrmA cells, and this event was sensitive to overexpression of p35 (Fig. 2). Taken together, these results demonstrate that IR induces cleavage of PARP and PKC␦ by one or more p35-sensitive, CrmA-insensitive protease(s).
To assay for protease activity directly, we incubated cell lysates with Ac-DEVD-pNA and monitored release of p-nitroanilide. Lysates from U-937 cells exhibited an increase in peptide cleavage activity that was detectable at 4 h and maximal at 6 h after IR exposure (Fig. 3A). The kinetics of Ac-DEVD-pNA cleavage corresponded temporally with IR-induced internucleosomal DNA fragmentation in U-937 cells (13). Lysates from irradiated U-937/CrmA cells exhibited a kinetically similar but less pronounced induction of Ac-DEVD-pNA cleavage activity (Fig. 3A). By contrast, there was little if any induction of such activity in lysates from irradiated U-937/p35 cells (Fig. 3A). To further define the effects of CrmA and p35 on Ac-DEVD-pNA cleavage, we incubated lysates from irradiated U-937 cells with the purified recombinant anti-apoptotic proteins. While CrmA had little apparent effect, addition of p35 was associated with complete inhibition (Fig. 3B). CPP32 has been shown to be responsible for proteolytic cleavage of Ac-DEVD-pNA (23). To address the potential role of a CPP32-like activity in IR-induced apoptosis, we compared the effects of the peptidic CPP32 inhibitor Ac-DEVD-cho (23) on IR-induced Ac-DEVD-pNA cleaving activity with those observed after adding purified CPP32 to lysates of unirradiated cells (Fig. 3C). The finding that the IR-induced protease activity in cell lysates is approximately 3-fold more sensitive to Ac-DEVD-cho inhibition than CPP32 suggests that the induced activity is due to CPP32-like enzymes, perhaps including CPP32 itself.
ICE, Ich-1, Ich-2, and CPP32 are all potently inhibited by p35 (33). CrmA, in contrast, displays about 10 4 -fold selectivity for ICE compared with CPP32 (23), although inhibition of CPP32 by CrmA has also been reported (22). To resolve this discrepancy and to address the differential effects of CrmA and p35 observed on IR-induced apoptosis, we measured CrmA inhibition of purified ICE, Ich-1, Ich-2, and CPP32 (Fig. 4A). CrmA inhibited ICE and Ich-2 approximately 50% at a 1:1 molar ratio (approximately 1 M each) but was without effect on Ich-1 and CPP32 at up to a 10:1 molar ratio (Fig. 4A). Since the IR-induced Ac-DEVD-pNA cleaving activity is p35-sensitive and CrmA-insensitive, these results further support involvement of a CPP32-like protease in IR-induced apoptosis. To directly assess activation of CPP32, we assayed lysates from irradiated cells for cleavage of the proenzyme to its active subunits (21,23,31). IR-treated U-937 and U-937/CrmA cells exhibited CPP32 activation, while there was little if any cleavage of the proenzyme in irradiated U-937/p35 cells (Fig. 4B). By contrast, there was no detectable effect of IR treatment on Ich-1 L levels (Fig. 4B). Taken together, the findings indicate that IR-induced apoptosis is associated with activation of CPP32.

DISCUSSION
Eukaryotic cells respond to lethal doses of IR with induction of apoptosis (8,45,46). Bcl-2 blocks IR-induced apoptosis (4 -6). Similar findings have been obtained in cells that overexpress Bcl-x L (7). Other studies have demonstrated that Bcl-2 and Bcl-x L inhibit IR-induced proteolytic cleavage of PKC␦ (13). These results have suggested that Bcl-2 and Bcl-x L function upstream to IR-induced activation of certain members of the ICE/Ced-3 family of cysteine proteases. More recent work has shown that Bcl-2 and Bcl-x L block staurosporine-induced, but not Fas-induced PARP cleavage and CPP32 activation (14). By contrast, the finding that Bcl-2 and Bcl-x L block Fas-induced cleavage of PKC␦ (13) suggests that these anti-apoptotic proteins may function upstream only to certain cysteine proteases. The present studies were performed to further define the ap-

FIG. 3. Induction of a CPP32-like activity in IR-treated U-937 cells.
A, U-937 cells (q) or cells expressing CrmA (E) or p35 (f) were exposed to 20 gray IR and harvested at the indicated times. The cells were lysed by a 1:1 dilution in enzyme reaction buffer containing 1% (v/v) Nonidet P-40. Samples were assayed for protease activity toward the peptide substrate Ac-DEVD-pNA. B, lysates from IR-treated U-937 cells containing the indicated amounts of CrmA (E) or p35 (q) were incubated at room temperature for 30 min and then assayed for protease activity by the addition of substrate. Under standard assay conditions, lysate from irradiated cells released approximately 40 nmol of pNA/min from Ac-DEVD-pNA. The percentage of inhibition was determined by taking the absolute activity as 100%. C, lysates from IRtreated U-937 cells (q) or unirradiated cells (E) to which approximately 30 ng of purified CPP32 was added (giving about 40 nmol of pNA release/min from Ac-DEVD-pNA under standard assay conditions) were tested for inhibition by Ac-DEVD-cho (23) using Ac-DEVD-pNA as a substrate.

FIG. 4. IR induces CPP32 activation by a CrmA-insensitive mechanism.
A, purified ICE (q), Ich-2 (E), CPP32 (å), or Ich-1 (Ç) at 1 M each were incubated with varying amounts of CrmA and assayed as described (33). B, immunoblot analysis of whole cell lysates from the untreated and IR-treated cell lines was performed with anti-CPP32 (upper panel) or anti-Ich-1 L antibodies (lower panel). The two arrows (upper panel) indicate the large subunits of CPP32 (p19/p17). optotic pathway in irradiated cells. The CrmA and p35 proteins were overexpressed in U-937 cells because of their reported functions in inhibiting members of the ICE/Ced-3 family. The results suggest that IR activates a CrmA-insensitive, p35-sensitive pathway.
CrmA is a member of the serpin family that inhibits ICE by forming an active site-directed complex (32,47). CrmA expression blocks apoptosis induced by: (i) nerve growth factor withdrawal of chicken neurons (48); (ii) cytotoxic T cell-mediated cytolysis (49); (iii) detachment of cells from an extracellular matrix (50); (iv) serum withdrawal of Rat1 fibroblasts (20); and (v) activation of Fas or TNF receptors (34). However, the present studies demonstrate that IR-induced apoptosis is not blocked by overexpression of CrmA. This finding is not explainable by insufficient CrmA expression since the U-937/CrmA cells were insensitive to TNF-induced apoptosis. Moreover, a recent report has shown that CrmA has no effect on IR-induced apoptosis of mouse lymphoma cells (51). We also demonstrate that IR-induced PARP and PKC␦ cleavage is not affected by CrmA. Further, IR-induced Ac-DEVD-pNA cleaving activity was detectable in U-937/CrmA cells and was not inhibited by Ͼ10 M CrmA in the assay mixture (Fig. 3C) CPP32 was also not inhibited by CrmA at concentrations of up to 10 M (Fig. 4). Collectively, these data suggest that, in contrast to TNF, IR induces apoptosis via one or more cysteine proteases (such as CPP32) that are CrmA-insensitive.
The baculovirus p35 protein inhibits apoptosis in cells from insects, nematodes, and mammals (33,39,52,53). Other studies have shown that p35 inhibits the proteolytic activity of Ced-3, ICE, CPP32, Ich-1, and Ich-2, but not granzyme B (33,54). The present studies demonstrate that p35 blocks IR-induced Ac-DEVD-pNA cleaving activity. We also found that p35 blocks IR-induced apoptosis and cleavage of PARP and PKC␦. These findings support a role for ICE/Ced-3-like proteases in IR-induced apoptosis. However, the sensitivity of TNF-induced, but not IR-induced, apoptosis to CrmA supports the involvement of distinct proteases in the two processes. Sphingomyelin hydrolysis and ceramide production have been identified in TNF-treated cells (55,56). This pathway is also activated in cells exposed to IR (57). Since ceramide has been shown to mediate apoptosis (58), sphingomyelin hydrolysis may contribute to induction of apoptosis by both IR and TNF. While there may be central signals for inducing apoptosis by diverse agents, the insensitivity of IR-induced apoptosis to CrmA distinguishes the protease(s) activated in irradiated cells from those involved in TNF-treated cells.
Recent work has demonstrated that Fas-mediated apoptosis is associated with sequential activation of ICE and then CPP32-like activity (59). In the present studies, there was little if any effect of IR treatment on YVAD-cleaving activity (data not shown). These findings and the demonstration that IRinduced apoptosis is insensitive to overexpression of CrmA support the lack of ICE involvement. Thus, ICE-like proteases may be involved in Fas-(59) and TNF-induced apoptosis but not in IR-treated cells. Collectively, the present results indicate that IR induces activation of CPP32. While PARP cleavage can be mediated by CPP32 and/or Mch3 (23,28), PKC␦ is cleaved by CPP32 and not ICE, Ich-1, Ich-2, Mch2, Mch3, or ICE rel -III (60). Thus, IR-induced cleavage of PARP and PKC␦ is consistent with activation of CPP32. Studies of IR-induced Ac-DEVD-pNA cleavage and sensitivity of this activity to Ac-DEVD-cho and p35, but not CrmA, also support involvement of CPP32. Moreover, IR-induced cleavage of the CPP32 proenzyme to its subunits is in concert with activation of this protease. Thus, our findings indicate that IR-induced apoptosis is associated with CPP32 activation and that the apoptotic signals induced by irradiation differ from those associated with TNF-or Fasmediated cell death.