Retinoblastoma Protein-mediated Apoptosis After (cid:1) -Irradiation*

Restoration of expression of the retinoblastoma gene to DU-145 prostate-cancer cells sensitizes them to apoptosis induced by (cid:1) -irradiation. In contrast, RB expres-sion-protected cells from UV-induced cell death. RB, a caspase substrate, remained intact during apoptosis in (cid:1) -irradiated DU-145 cells because serine proteases, but not caspases, were activated. In DU-145 cells, RB-medi-ated apoptosis involved biphasic activation of ABL kinase. ABL kinase was activated within minutes of irradiation, but in the presence of RB expression ABL kinase activation was enhanced 48 h after irradiation, coincident with the onset of cell death. Apoptosis was inhibited by RB mutants with constitutive ABL binding, but ABL overexpression overcame the effect of the RB mutant constructs. Expression of kinase-dead ABL had a dominant-negative effect on RB-mediated cell death. Activation of JUN N-terminal kinase depended on the presence of RB and occurred withi n 8 h ofirradiation. Mutant JUN proteins that lacked the N-terminal transactivation domain and serine substrates for JUN N-ter-minal kinase inhibited cell death in a dominant-nega-tive manner. Irradiation of DU-145 cells caused activation of p38 MAPK independent of the expression of RB. Inhibitors of p38 MAPK blocked apoptosis after irradiation of RB-expressing cells. The data show that after (cid:1) -irradiation,

Restoration of expression of the retinoblastoma gene to DU-145 prostate-cancer cells sensitizes them to apoptosis induced by ␥-irradiation. In contrast, RB expression-protected cells from UV-induced cell death. RB, a caspase substrate, remained intact during apoptosis in ␥-irradiated DU-145 cells because serine proteases, but not caspases, were activated. In DU-145 cells, RB-mediated apoptosis involved biphasic activation of ABL kinase. ABL kinase was activated within minutes of irradiation, but in the presence of RB expression ABL kinase activation was enhanced 48 h after irradiation, coincident with the onset of cell death. Apoptosis was inhibited by RB mutants with constitutive ABL binding, but ABL overexpression overcame the effect of the RB mutant constructs. Expression of kinase-dead ABL had a dominant-negative effect on RB-mediated cell death.

Activation of JUN N-terminal kinase depended on the presence of RB and occurred within 8 h of irradiation. Mutant JUN proteins that lacked the N-terminal transactivation domain and serine substrates for JUN N-terminal kinase inhibited cell death in a dominant-negative manner. Irradiation of DU-145 cells caused activation of p38 MAPK independent of the expression of RB. Inhibitors of p38 MAPK blocked apoptosis after irradiation of RB-expressing cells. The data show that
after ␥-irradiation, intact RB mediates transcriptional activation that leads to activation of JNK and late activation of ABL kinase. In addition, p38 MAPK activation occurred independent of RB. ABL kinase, JUN N-terminal kinase, and p38 MAPK activity were all required for RB-mediated DU-145 cell death after ␥-irradiation.
Retinoblastoma protein (RB) 1 is a multifunctional protein that binds to transcription factors and kinases to regulate both cell growth and apoptosis. Binding of proteins to discrete sites on RB is controlled by phosphorylation at a large number of phosphoacceptor sites on the RB protein. RB is a tumor-suppressor gene and is a target for disruption in human retinoblastoma and other malignancies. RB is also a target for transforming proteins of various DNA tumor viruses that bind to RB and interfere with cell-cycle control.
RB exerts control over G 1 /S cell-cycle transition by binding to members of the E2F family of transcription factors and colo-calizing at E2F binding sites on promoter regions to act as a transcriptional repressor and recruiter of histone acetylase. The region of the RB protein responsible for E2F family binding is termed the large pocket and spans amino acids 379 -870 (1,2). Control of RB binding to E2F family members is exerted during late G 1 phase of the cell cycle by cyclin D in association with cyclin-dependent kinases 4 and 6 (3). Maintenance of RB phosphorylation is carried out by other kinase such as cyclin E-cdk-2 (4). RB phosphorylation is also affected by p16 INK4 , an inhibitor of cdk4-cdk6, an important tumor suppressor in human cancer (5,6).
RB control over the cell cycle can be interrupted by binding to the transforming proteins of DNA tumor viruses such as SV40 large T antigen, adenovirus E1A, or HPV E7. The viral proteins share a common LXCXE amino acid motif that binds to the A/B pocket of RB extending from amino acids 379 -772 (7)(8)(9). The binding site of the proteins is located in the B box (amino acids 646 -772) and is stabilized by the A box (9,10). The A/B pocket, which is the site for a large number of activating RB mutations in human tumors, is also a site for binding of cellular proteins. Therefore, the A/B pocket is likely to play an important role in RB tumor-suppressor functions (2,11).
In addition to its role in cell cycle regulation, RB influences the function of at least two proteins that are important in control of cell death, the c-Jun N-terminal kinase (JNK) and ABL kinase. The C pocket region of RB that spans amino acids 772-870 binds to the ABL protein (12). ABL is a tyrosine kinase with both cytoplasmic and nuclear localization (13). In the nucleus, ABL kinase is activated in response to DNA damage by binding to ataxia-telangicctasia protein (14,15). ABL can interact with a variety of downstream substrates to stimulate cell death pathways (16 -18). The C-terminal end of RB, amino acids 768 -928, is the site of interaction with JNK, a kinase mediator of cell death (19). JNK, also called stressactivated protein kinase, is an effector of the MAP kinase phosphorylation cascade that phosphorylates JUN at serines 63 and 73 after exposure of the cell to UV or irradiation (20,21). In what may be a mechanism to amplify an apoptotic signal, JNK is also activated by a complex of ABL with JUN that has been phosphorylated at tyrosine 170 by its interaction with ABL (22).
In response to cell stress and DNA damage RB has a variety of roles. After DNA damage, RB is hypophosphorylated and induces growth arrest by blocking cell-cycle progression (23,24). As a cell becomes committed to apoptosis, RB is a target of caspases and undergoes degradation (25)(26)(27). This is consistent with the notion that RB protects cells from apoptosis, as has been demonstrated in a number of experimental systems (24, 28 -37).
We have reported that DU-145 prostate cancer cells, which have one RB allele deleted and one allele that lacks exon 21 (38), are resistant to apoptosis induced by ␥-irradiation. Expression of exogenous RB did not alter DU-145 cell growth in vitro but conferred sensitivity to radiation-induced cell death (38,39). At 72 h after irradiation, we observed 30 -50% apoptosis of DU-145 cells stably transfected with RB. However, caspases associated with the intrinsic cell death pathway were not activated, and expression of intact RB persisted after irradiation. Irradiation-induced apoptosis in DU-145 cells expressing RB was insensitive to zVAD but was blocked by serine protease inhibitors (39). This paper describes the interaction of RB with signaling pathways during DU-145 cell death induced by ␥-irradiation. The data show that, in the appropriate cellular milieu, RB may play a role in death-signal transduction.

MATERIALS AND METHODS
Cell Culture-Growth of DU-145 cells and derivation of RB-expressing cell lines has been previously described (39). DU-2.16 cells were derived in our lab by similar procedures. DU-2.16 and DU-3.12 were two of several clones selected for sustained growth and retention of RB expression during prolonged passage. Cells were irradiated with a JL Shepherd Mark I Irradiator. UV exposure was done at 60 J/m 2 48 h after transfection with GFP and RB expression plasmids. Cell death was assayed 14 h after UV exposure.
Plasmids and Transient Transfection Experiments-RB expression plasmids have been described previously (40,41). RB-N757F has been described previously (42). These were all generously provided by Jean Wang, University of California, San Diego. ABL and ABL-K290R were a gift from Charles Sawyers, UCLA School of Medicine. ABL-S465E, ABL Ϫ (NES) Ϫ , and ABL Ϫ (NLS) Ϫ have been described previously and were a gift from Jean Wang (13,43). JUN expression vectors have been described previously (44 -46). DU-145 cells and RB-2.16 cells were transfected with appropriate expression vectors and with a green fluorescent protein (GFP) expression vector using Superfect TM transfection reagent (Qiagen). Cells were exposed to ␥-radiation 48 h after transfection. Immediately before assay for apoptosis, cells were sorted into fluorescent and nonfluorescent pools. Before sorting, cells were suspended in phosphate-buffered saline at 2 ϫ 10 6 cells per ml. The cells were passed through a 35-m nylon mesh and sorted with a FACSTAR-PLUS flow cytometer (BD Biosciences). Excitation was set at 488 nm with a DF530/30 emission filter. Parallel cell death assays were performed on GFP Ϫ and GFP ϩ pools. In situ end labeling for apoptosis was done as previously described (39). Statistical analysis was used to compare two groups of triplicate apoptosis results with Student's t-test.
Transcription Inhibition Assay-Rb2.16 cells were treated by transcriptional inhibitors rifamycin SV (Sigma) or D-5,6-dichlorobenzimidazole riboside (Sigma). After one hour of incubation of the cells with the transcriptional inhibitors, cells were irradiated with 20 Gy of radiation. As a control, a parallel group of cells were treated with corresponding transcriptional inhibitors in the absence of radiation. Cells were then harvested and the percentage of apoptotic cells was characterized by in situ end labeling assay 72 h after radiation.
E2F Transcription Assay-E2F luciferase reporter vector was derived from E2F-CAT vectors that were a gift from Srikumar Chellappan (Department of Biological Sciences, Columbia University, NY) (47). An E2F-luciferase reporter vector was constructed by insert E2F promoter and enhancer region into pGL3 luciferase reporter vector at SacI and HindIII sites. Luciferase assay was performed with the Promega Luciferase Assay System according to the manufacturer's protocol. Luciferase activity was corrected for Renilla luciferase activity to normalize the transfection efficiency.
Western Blotting-Cells were lysed in 20 mM Tris pH 8.0, 1% Triton X-100, 100% glycerol, 137 mM NaCl, 1.5 mM MgCl 2 , 1 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , and 1 mM phenylmethylsulfonyl fluoride at 4°C for 30 min and clarified by centrifugation. Protein content was assayed using Dc protein assay (Bio-Rad Laboratories, Hercules, CA). 20 -40 g of protein were electrophoresed on 10 -20% Tris-glycine gradient gels. Proteins were electrotransferred to nitrocellulose membranes that were incubated sequentially with 5% dry milk in phosphate-buffered saline for 30 min, primary antibody for 1-2 h, and 1:3000 horseradish peroxidase-conjugated secondary antibody (Pierce) for 30 min. Bands were revealed by Enhanced Chemiluminescence using SuperSignal substrate or SuperSignal West Dura Extended Duration Substrate (Pierce). Anti-RB monoclonal antibody G3-245 (BD Biosciences) was used at a dilution of 1:1000. Antisera to caspase-7 and -9 were purchased from Cell Signal Technology (Beverly, MA) and were used at dilution of 1:1000 and 1:2000, respectively. Antiserum to caspase 3 was purchased from PerkinElmer Life Sciences and was used at a dilution of 1:1000. ABL antibody was diluted at 1:500 (Santa Cruz Biotechnol-ogy, Inc., Santa Cruz, CA). P38 MAPK assay was purchased from NEB. IGF-I antibody H-70 and IGFBP3 antibody C-19 were from Santa Cruz. For the IGF-I slot blot, 450 g of total protein was loaded in each well. Antibodies to total and phosphorylated protein kinase B (AKT) were from PerkinElmer Life Sciences. Antibody 1288 for P73 were derived in Jean Wang's laboratory and used for Western blotting. Anti-human APAF1 monoclonal antibody from R&D System Inc. Minneapolis, MN. Antibody SC-44 to JUN was from Santa Cruz Biotechnology. Antibody A-5441 to ␤-actin was from Sigma.
Kinase Assays-Stress-activated protein kinase/JNK kinase activity was processed with SAPK/JNK kinase assay (PerkinElmer Life Sciences). Treated cells were lysed with 1ϫ cell lysis buffer plus 1 mM phenylmethylsulfonyl fluoride and sonicated, and supernatant was removed for kinase assay. 250 l of cell lysate (500 g of total protein) were added to 2 g of GST-JUN fusion protein beads and incubated with gentle rocking overnight at 4°C. After rinsing with lysis buffer twice and kinase buffer twice, the pellet was suspended in 50 l of 1ϫ kinase buffer supplemented with 100 M ATP and incubated for 30 min at 30°C. The reaction was terminated with 25 l of 3ϫ SDS sample buffer. Western blotting was used to detect kinase activity. Phospho-c-Jun (serine 63) was diluted at 1:1000 in Western blotting. MAPK/p38 kinase activity was analyzed with p38 MAP kinase assay (PerkinElmer Life Science). 400 g of total protein was incubated with 20 l of immobilized phospho-p38 MAP kinase monoclonal antibody/protein A/G beads at 4°C overnight. After rinsing with lysis buffer and kinase buffer sequentially, the pellet was suspended with 50 l of 1ϫ kinase buffer with 200 M ATP and 2 g of activating transcription factor-2 (ATF)-2 fusion protein and incubated overnight at 4°C. P38 MAP kinase activity was assayed by Western blot with anti-phospho-ATF-2 antibody at dilution of 1:1000.
ABL tyrosine kinase activity was analyzed by peptide phosphorylation assay. Cells were co-transfected with GFP and appropriate expression vectors. GFP-positive cells were lysed with 1ϫ cell lysis buffer as described above. Anti-cABL immunoprecipitation was performed by adding 400 g of total protein and 2.5 g of anti-ABL antibody (24 -11) (Santa Cruz Biotechnology) for 2 h at 4°C and adding 30 l (50%) of protein A/G for another 1 h at 4°C. Beads with immune complexes were rinsed with lysis buffer and then kinase buffer. Immune complexes were incubated in kinase buffer with 100 M peptide (EAIYAAP-FAKKK) (PerkinElmer Life Science), 100 M ATP, and 1 Ci [␥-32 P]ATP for 5 min at 30°C. After incubation, 15-l aliquots were spotted onto phosphocellulose discs followed by washing with 1% (v/v) phosphoric acid and then distilled water. The incorporated 32 P was determined by scintillation counting.
Detection of Cytochrome c-Cytochrome c was detected by Western blot analysis. In brief, cells were disrupted by a Dounce homogenizer with 20 strokes using 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, 2 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml pepstatin. The homogenates were separated into cytosol and membrane fractions by ultracentrifugation. Equal amount of protein were subjected to Western blot analysis using anti-cytochrome c antibody (Santa Cruz Biotechnology) and anti-␤-actin antibody (Sigma).
DNA Laddering-After drug exposure, pelleted cells (2 ϫ 10 7 cells) were lysed in 1% Nonidet P-40, 20 mM EDTA, 50 mM Tris-HCl, pH 7.4), kept on ice for 10 min, and centrifuged for 10 min at 1600 ϫ g. SDS was added to the supernatant (10%) to a final concentration of 1%, and RNase A was added to a final concentration of 200 g/ml. The mixture was then incubated for 2h at 37°C. Reaction was added with proteinase K to a concentration of 200 g/ml and incubated for 2 h at 56°C. DNA was ethanol-precipitated, dissolved in 20 g of TE buffer (10 mM Tris-HCl, 1 mM EDTA), and run on a 1% agarose gel in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0).

RESULTS
Cell Death Mediated by RB-We extended our observations of the effect of RB on DU-145 cell death by deriving additional DU-145 clones expressing RB. Newly derived DU-2.16 cells had a very similar death response to ␥-irradiation compared with the previously described DU-1.1 cells and B5 cells. Although a low level of DNA fragmentation could be seen after irradiation, no low molecular weight DNA fragments ("laddering") were seen (Fig. 1A). We were not able to demonstrate activation of caspases in the intrinsic cell-death pathway (Fig. 1B). We previously showed that DU-145 cells are able to undergo robust caspase activation during apoptosis induced by okadaic acid and therefore had an intact caspase cascade (39). We were able to detect a small increase in cytoplasmic cytochrome c in DU-2.16 cells after irradiation, suggesting that the intrinsic mitochondrial-death pathway had been activated (Fig. 1C). We previously showed that RB-mediated cell death could be blocked by serine protease inhibitors such as N ␣ -p-tosyl-L-lysylchloromethyl ketone (TLCK), but not by caspase inhibitors like z-Val-Ala-Asp (OMe)-CH 2 F (zVAD) (39).
The endogenous RB protein has antiapoptotic effects and is a target for caspase cleavage in most cells (27, 48 -54). For example, irradiation of TSU-Pr1 cells is known to activate caspases that cleave RB (Fig. 1D). In contrast, RB is not cleaved after irradiation of B5 cells (39) or of DU-2.16 cells, perhaps due to the minimal level of caspase activation. Fig. 1D shows that RB was present in both phosphorylated and unphosphorylated states in DU-145 transfected cells and that irradiation caused some dephosphorylation of RB within 8 h of exposure. The truncated RB in DU-145 cells is expressed at low levels and is difficult to detect with Western blotting (39). As an index of RB activity, we assayed a reporter construct under the control of an E2F1-responsive promoter. Baseline activity of the promoter in DU-lux cells exceeded activity in DU-2.16 cells. Irradiation had no effect on the E2F1-responsive promoter, consistent with the finding that phosphorylation of RB was not appreciably affected by irradiation of DU-145 cells (Fig. 1E). We previously showed that ␥-irradiation did not substantially affect cell cycle distribution of DU-145 cells that did or did not express wild-type RB (39). Finally, the proapoptotic effect of RB in DU-145 cells may be restricted to damage caused by ␥-irradiation since RB expression inhibited DU-145 cell death after UV exposure (Fig. 1F).
We assayed the expression of candidate pro-and antiapoptotic proteins whose expression has been reported to be affected by RB and, therefore, could contribute to the effect of RB on apoptosis of irradiated DU-145 cells. The growth factor IGF-I has been shown to act as a survival factor and can be regulated by E2F1 (55)(56)(57). We also assayed the expression of the inhibitory binding protein IGFBP3, which can have proapoptotic effects (58). The overall level of IGFBP3 was lower in B5 cells than in DU-lux cells and, interestingly, increased after irradiation, but not until 48 h (Fig. 2A). We also examined the levels of AKT and phosphorylated AKT in DU-lux and B5 cells. This was done by immunoprecipitating AKT and then Western blotting the immunoprecipitated with either AKT antibody or antibody specific for phosphorylated AKT. We saw no differences in levels of AKT or AKT phosphorylation at the time of irradiation or at 48 h after irradiation in either cell line (Fig.   2B). APAF1, the proapoptotic mediator of death signaling by cytochrome c egress from the mitochondria, can have altered expression due to RB (59). However, we found no differences in baseline expression of APAF1 in our control or RB-transfected cell lines (Fig. 2C). Finally, we analyzed expression of P73, a proapoptotic protein whose expression is controlled by E2F1 and ABL (60 -64). Although we found higher baseline p73 expression in DU-145 than the transfected cells, there was essentially no detectable p73 in DU-lux, B5, and DU-3.12 (Fig.  2D). The increased baseline P73 expression in DU-145 cells did not correlate with cellular response to ␥-irradiation because neither DU-145 nor DU-lux cells underwent apoptosis, but B5 and DU-3.12 cells did.
Mutations That Affect Interactions of RB with Other Proteins Attenuate Its Proapoptotic Effects-RB is a multifunctional regulator of transcription factors and of the transcriptional complex. RB can bind to E2F-1 and inhibit E2F-1-mediated transcription (65)(66)(67). RB can also form a transcriptional repression complex by binding and recruiting molecules such as histone deacetylase I to E2F-1-binding promoter regions (68 -70). We used mutants of RB to analyze RB functions important for its proapoptotic effects (Fig. 3A). These experiments were conducted by transient cotransfection of the RB-expression vector and a GFP selection marker that was used to isolate transfected cells by sorting (see "Materials and Methods"). To demonstrate that the various mutant RB constructs used in these experiments were expressed in DU-145 cells, we performed transient transfection along with green fluorescent protein-selectable marker and analyzed RB expression in the transfected cells. As shown in Fig. 3B, six RB plasmid expression vectors engendered protein expression in DU-145 cells.
RB mutant 13S lost the ability to bind to ABL but retained interaction with E2F and inhibition of cell growth and colony formation (40). The 13S construct conferred sensitivity to DU-145 cell death (Fig. 3C). This result suggested that retention of E2F-1 binding and regulation of cell growth were consistent with the proapoptotic affect of RB. In contrast, PSM.9I and PSM.2S both had minimal effects on cell death (Fig. 3C) (41). The PSM.2S mutation eliminates serines 807 and 811, thus conferring constitutive ABL binding and loss of Rat-1 growth suppression, but retaining regulation of E2F-1 binding by phosphorylation (41,71). The PSM.2S construct, however, retains the ability to modulate the phenotype of SAOS-2 cells and is more resistant to the effects of cyclin A overexpression than wild-type RB (71). The PSM.9I mutant has constitutive binding to E2F-1 and ABL and retains growth suppression of Rat-1 cells (41). The RB mutant RB(N757F) has been shown to elim-

FIG. 2. Expression of pro-and antiapoptotic proteins that were potentially affected by RB analyzed by immunoblotting.
A, slot blot analysis of IGF-1 expression in cell lysates and supernatants. Also shown is a Western blot of IGFBP3 as affected by 20 Gy of irradiation over 48 h. B, analysis of AKT and phospho-AKT expression by immunoprecipitation of AKT followed by blotting with either a phospho-AKT-specific antibody or an AKT antibody. C, Western blot for APAF1 in DU-145 derivative cell lines. D, Western blotting for p73 using two different p73 antibodies. HRP-1 cells provide the positive control.
inate binding of the A/B pocket to proteins with LXCXE motifs such as histone deacetylase I and the ability to induce cellgrowth arrest, but retain E2F-binding (42). The RB(N757F) mutant was unable to mediate cell death and was as devoid of proapoptotic activity as PSM.9I. The compound mutant PSM.9I(RB)N757F also had no effect on cell death. It thus appeared that disruption of binding to histone deacetylase I or constitutive binding to ABL resulted in loss of the proapoptotic functions of RB. Both PSM.2S and PSM.9I were able to compete with wild-type RB and block the mediation of DU-145 cell death (Fig. 3D). PSM.2S and PSM.9I both have constitutive ABL binding. PSM.9I, which cannot be phosphorylated, was a more effective competitor of apoptosis.
The Role of ABL in RB-mediated Apoptosis-Because RB mutants with constitutive ABL binding were competitive inhibitors of DU-145 cell death, we studied the role of ABL in DU-145 cell apoptosis induced by ␥-irradiation. The ABL tyrosine kinase, a target for RB binding, is activated by ␥-irradiation and is proapoptotic when localized to the nucleus (13,18,72). Overexpression of ABL itself in DU-145 cells predisposed to radiation-induced cell death but to a lesser degree than wild-type RB. A kinase-dead ABL-K290R The constructs used are indicated below the histogram. D, apoptosis of DU-2.16 cells that were transiently transfected with the indicated expression plasmids. Transfection was carried out so that the cells were exposure to a constant amount of DNA. Cells were sorted for the cotransfected selectable marker GFP immediately before assay for apoptosis 72 h after 20 Gy of irradiation. had no effect on cell death (Fig. 4A). Moreover, increasing expression of ABL could overcome the antiapoptotic effects of PSM.2S, consistent with the notion that PSM.2S was inhibiting apoptosis by complexing with ABL (Fig. 4B).
Activation of ABL kinase was induced rapidly by ␥-irradiation of DU-145 cells even though the parental cells did not undergo apoptosis after irradiation. In the presence of RB expression, ABL kinase underwent a second increase 24 -48 h after irradiation (Fig. 4C). The inset in Fig. 4C shows that levels of endogenous ABL were not altered by irradiation or by the presence of RB. The interaction of ABL and RB was further analyzed by coexpression of the two in transient transfection experiments. Whereas overexpression of ABL by itself was permissive for a low to moderate degree of cell death, RB alone facilitated a higher level of apoptosis, and coexpression of ABL enhanced the effect of RB (left side of Fig. 4D). RB constructs PSM.2S and PSM.9I with constitutive ABL binding had a dominant-negative effect on ABL and abrogated the modest degree of cell death enhancement mediated by ABL alone, consistent with the data in Fig. 4B. Furthermore, kinase-dead ABL had a dominant-negative effect that blocked the propapoptotic effect of RB.
ABL kinase has both nuclear localization and nuclear export sequences that mediate shuttling of the ABL protein between nucleus and cytoplasm (13). Nuclear ABL kinase is activated by radiation via ATM to induce p73 in the process of mediating radiation-induced cell death (60,73,74). Removal of the ABL nuclear export sequence restricts the BCR-ABL oncoprotein to the nucleus and is proapoptotic (75). In DU-2.16, ABL with a disrupted nuclear export sequence is as propapoptotic as wildtype ABL, but ABL restricted to the cytoplasm due to loss of a nuclear localization sequence has reduced apoptotic activity (Fig. 4E). To confirm that the effects seen were caused by expression of the wild-type and mutant ABL vectors, we showed that each vector engendered similar levels of ABL expression in DU-145 cells (Fig. 4F).
Interaction of RB and JUN-Execution of cell death requires the activity of the c-Jun N-terminal kinase (JNK) (20). We previously showed that apoptosis mediated by RB involved the activation of JNK (39), whereas under other circumstances, RB binds to JNK to block its activity (19). RB can also interact directly with the transcription factor JUN via the JUN leucine zipper (76). We analyzed mutants of JUN in RB-mediated cell death (Fig. 5A). Overexpression of JUN by itself had no effect on cell death (left side of Fig. 5B). However, JUN overexpression slightly enhanced RB-mediated death, and a JUN transactivation mutant TAM-67, lacking amino acids 1-67 of the N-terminal transactivation domain, exerted an inhibitory effect on cell death (Fig. 5B) 5D). Fig. 5E shows that the different JUN expression plasmids were expressed in transfected cells with protein species seen at estimated sizes predicted by the mutant plasmid constructs. Degradation fragments were also detected.
JNK is activated during cell stress and activates JUN by phosphorylating serines 63 and 73 (21). Activation of JNK in DU-145 cells occurred within 8 h of irradiation as shown in Fig.  6A. A low level of JNK activation occurred in DU-lux cells, but this activation was insufficient to induce cell death. In contrast, activation of JNK after irradiation of DU-2.16 cells was easily detected. Activation of JNK affects JUN and its targets for transcriptional control. To explore whether the permissive effect of RB expression on cell death was due to effects on transcription, we added low doses of either of two transcriptional inhibitors, rifamycin SV or D-5,6-dichlorobenzimidazole riboside, to DU-145 cells after irradiation. Either agent was able to block cell death 72 h after irradiation. Rifamycin SV was well tolerated by the cells. At higher doses, D-5,6-dichlorobenzimidazole riboside exposure was toxic by itself (Fig. 6B).
We showed above that irradiation of DU-145 cells induced a low level of ABL kinase activation nearly immediately that was followed at 48 h by further activation of ABL kinase only in the presence of RB (Fig. 4C). Early ABL kinase activation occurred independent of JNK activation. Consistent with this finding, ABL kinase activity at 24 h was not affected by the expression of the JUN63/73 even though JUN63/73 inhibited apoptosis (Figs. 5D and 6C). Taken together, the data show that ABL kinase activity was necessary but not sufficient for cell death and was independent of JNK activity and JUN phosphorylation.
Finally, we studied the activation of the cytoplasmic p38 mitogen-activated protein kinase (p38 MAPK) in the response of DU-145 cells to ␥-irradiation (77)(78)(79)(80)(81). In particular, p38 MAPK has been implicated in both MYC-dependent cell death and caspase-independent death of HeLa cells exposed to photodynamic therapy (82,83). As an example of a kinase whose activation was not dependent on RB, we demonstrated, by substrate phosphorylation, that P38 MAPK was activated in DU-145 cells after ␥-irradiation irrespective of RB expression or cell death (Fig. 7A). Inhibition of p38 MAPK by either of two inhibitors, SB202190 and SB203580, had no effect on JNK activity in irradiated DU-2.16 cells (Fig. 7B), but diminished cell death (Fig. 7C). Therefore, p38 MAPK contributed to celldeath signaling, but did not initiate death pathway activation and was not involved with JNK activation. DISCUSSION Our findings are summarized in the schema shown in Fig. 8. In the absence of RB expression, ␥-irradiation of DU-145 cells activates several signaling pathways that result in activation of JNK, ABL kinase, and p38 MAPK. However, the intensity of the signals is insufficient to execute the cell death program. In the presence of RB expression, JNK activation is markedly enhanced and ABL kinase activation undergoes later activation. p38 MAPK is not affected, but its activation contributes to and is required for apoptosis. In conclusion, RB may play a role in an alternative pathway to cell death when cells fail to activate caspases in response to death stimuli such as ␥-irradiation. In light of the antiapoptotic effect that RB had on DU-145 cells exposed to UV irradiation, death pathways mediated by RB may be specific for signaling downstream from doublestrand DNA breaks induced by ␥-irradiation.
RB is a key regulator of the cell cycle that binds to a number of proteins involved in cell-cycle signaling, apoptosis, differentiation, and transcriptional activation (84). RB plays an important role in G 1 (85) and S phase (24) cell-cycle arrest after normal cells are exposed to ionizing radiation. Moreover, RB can influence the choice between cell-cycle arrest, allowing for DNA after exposure to ionizing radiation, and apoptosis (86). RB is a tumor-suppressor gene whose expression is lost in a wide range of tumors. Restoration of RB expression to tumor cells that have lost RB expression most commonly provides a protective effect against cell death induction by DNA-damaging agents (32,33,87,88). Loss of RB has been shown to predispose to abnormalities of cell-cycle control and cell death (28). The RB protein itself is a target for caspase cleavage during apoptosis (49 -54). Mutation of the caspase cleavage sites in RB protein diminishes the death response and results in hyperproliferation of neuronal cells in genetically altered mice (26,27,86). It was therefore quite unexpected to find that DU-145 prostate cancer cells, which have a very poor apoptosis response to ionizing radiation, were sensitized to apoptosis by restoration of wild-type RB gene expression (39). Clearly in this aggressive cancer cell line with mutant P53 (89), the expression of RB had proapoptotic effect after ␥-irradiation and had minimal effect on cell-cycle arrest (39).
Apoptosis in DU-145 cells expressing RB may have been potentiated by the absence of caspase activation in these cells. Caspase activation may have cleaved RB protein and thereby attenuated DU-145 cell death signaling. Although the caspase cleavage pathways are intact and can be activated by agents such as okadaic acid, ␥-irradiation resulted in no detectable caspase activation in DU-145 cells (39). We did observe cytochrome c egress in irradiated B5 and DU-2.16. Cytochrome c egress from mitochondria accompanies activation of the proapoptotic Omi/HtrA2 serine protease (90,91). We previously showed that the serine protease inhibitor TLCK, but not the caspase inhibitor zVAD, blocked RB-mediated DU-145 cell death, consistent with dependence of DU-145 cell death on Omi/HrtA2 (39).
The experiments in this paper examined downstream targets of RB cell death signaling. One likely mediator of apoptosis is ABL because it is known to be inhibited by RB binding and known to mediate apoptosis when it is localized to the nucleus (13,74). Our data are consistent with an important role for ABL in RB-mediated cell death. However, it appeared that whereas ABL was downstream from RB, ABL was not activated directly by interaction with RB since RB variant proteins that had no ABL binding-mediated cell death, but those with constitutive ABL binding blocked the cell death response. Second, we observed a biphasic activation of ABL kinase activity in irradiation DU-145 cells expressing RB, whereas ABL kinase activation in native DU-145 cells occurred only early after irradiation. Thus it appeared that RB mediated a late and secondary activation of ABL kinase, as diagrammed in Fig. 8.
In the presence of RB expression, 20 Gy of irradiation induced activation of JNK within 8 h. Using JUN phosphorylation site mutants, we also showed that JUN phosphorylation was important for RB-mediated apoptosis as was transactivation, perhaps mediated by the leucine zipper protein interaction domain. A JUN DNA binding mutant did not block RBmediated cell death. JNK activation preceded the peak of ABL kinase activation. It is possible that JNK activation was responsible for ABL kinase activation by inducing interaction of JUN and ABL. JUN and ABL have been shown to interact directly in a circuit of phosphorylation that also involves JNK (22). Although it is possible that JNK mediates activation of ABL kinase by RB, we do not know how RB causes activation of JNK. In cells in which RB is antiapoptotic, JNK is downregulated, perhaps by direct interaction with the N-terminal domain of RB (19,92). Whether under other circumstances RB interaction with JNK can activate the kinase activity remains to be shown. p38 MAPK activation has been shown to inhibit RB repression of E2F1 activation (47). The robust activation of p38 MAPK activity may be the reason we did not observe any activation of E2F1 transcriptional activity after irradiation of DU-lux or RB-2.16 cells (Fig. 1E). It is noteworthy that expression of exogenous RB in DU-145 cells caused only minor alteration in cell growth and did not appreciably alter the cell cycle, consistent with a negligible effect of RB expression on E2F1 activity in these cells (39).