Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA. The stress-activated ..... in MM patients with advanced disease with high serum levels of IL-6 .... chemiluminescence (ECL; Amersham, Arlington Heights,. IL). Preparation ...
Oncogene (1997) 15, 837 ± 843 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Dexamethasone induces apoptosis of multiple myeloma cells in a JNK/SAP kinase independent mechanism Dharminder Chauhan1, Pramod Pandey2, Atsushi Ogata1, Gerrard Teoh1, Steven Treon1, Mitsuyoshi Urashima1, Surender Kharbanda2 and Kenneth C Anderson1 1
Divisions of Hematologic Malignancies and 2Cancer Pharmacology, Dana-Farber Cancer Institute, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA
The stress-activated protein kinases (SAPKs), also known as c-Jun amino-terminal kinases (JNKs), are activated in response to diverse stimuli including DNA damage, heat shock, interleukin-1, tumor necrosis factora and Fas. Although all these inducers cause apoptosis, whether SAPK/JNK activation is required for apoptosis is controversial. In this study, we demonstrate that ionizing radiation (IR) and dexamethasone (Dex) induce apoptosis in multiple myeloma (MM) derived cell lines, as well as in patient cells. IR-induced apoptosis is associated with activation of SAPK/JNK and p38 kinase, in contrast to Dex-induced apoptosis, which is not associated with activation of stress kinases. Moreover, Dex-induced apoptosis is associated with a signi®cant decrease in the activities of mitogen activated protein kinase (MAPK) and p70S6K, whereas IRtreatment does not alter the activity of these kinases. Both IR and Dex induce poly (ADP ribose) polymerase (PARP) cleavage, a signature event of apoptosis. Finally, interleukin-6 (IL-6) inhibits Dex-induced apoptosis, downregulation of MAP and p70S6K growth kinases and PARP cleavage; in contrast, IL-6 does not inhibit IRinduced apoptosis, activation of SAPK/JNK, and PARP cleavage. Taken together, our ®ndings suggest that SAPK/JNK activation is not required for apoptosis in MM cells, and that there are at least two distinct apoptotic signaling pathways: (i) SAPK/JNK-associated, which is induced by IR and unaected by IL-6; and (ii) SAPK/JNK-independent, which is induced by Dex, associated with downregulation of MAPK and p70S6K and inhibited by IL-6. Keywords: multiple myeloma; irradiation; dexamethasone; apoptosis
Introduction Treatment of eukaryotic cells with ionizing radiation (IR) induces cell cycle arrest, activation of DNA repair and apoptosis. IR aects cells either by a direct interaction with DNA or through the formation of hydroxyl radicals and superoxides (Limoli et al., 1993; Hall, 1988). The ®nding that treatment with IR induces transcription of early response genes, such as c-jun and Egr-1 (Sherman et al., 1990; Hallahan et al., 1991; Datta et al., 1992), suggests the involvement of nuclear signals. To date, multiple protein kinases have been Correspondence: KC Anderson Received 22 January 1997; revised 2 May 1997; accepted 6 May 1997
identi®ed which transduce IR-triggered signals to the nucleus. For example, we and others have previously demonstrated that IR induces activation of SAPK/ JNK in diverse cell types (Kharbanda et al., 1995a,b; Chen et al., 1996). The stress activated protein kinase (SAPK/JNK) and p38 kinase belong to a family of serine/threonine protein kinases and share a signi®cant homology with mitogen activated protein (MAP) kinases (Anderson et al., 1990; Boulton et al., 1991; Derijard et al., 1994; Kyriakas et al., 1994; Lee et al., 1994; Sluss et al., 1994; Han et al., 1994; Gupta et al., 1995). However, the biological responses elicited by the activation of each of these kinases are distinct. For example, activation of SAPK/JNK and p38 kinases is associated with environmental stress, in¯ammatory cytokine and withdrawal of growth factor-induced apoptosis (Derijard et al., 1994; Kyriakis et al., 1994; Raingeaud et al., 1995; Verheij et al., 1996; Xia et al., 1995), whereas activation of MAPK is associated with proliferation and dierentiation triggered by growth factors and phorbol esters (Kharbanda et al., 1994; Kolch et al., 1991). Moreover, recent studies have demonstrated that these kinases have dierent upstream activators and substrate speci®cities. For example, transcription factor c-Jun is a substrate of SAPK/JNK (Kyriakis et al., 1994; Lee et al., 1994), AFT2 is phosphorylated by p38 and SAPK/JNK (Raingeaud et al., 1995, 1996; Derijard et al., 1995), and Elk-1 is a substrate of all three members of this family (Marais et al., 1993; Whitmarsh et al., 1995). These kinases may also be coordinately regulated. For example, a recent report demonstrates that apoptosis in pheochromocytoma cells was associated not only with sustained activation of SAPK/JNK and p38 kinase, but also with downregulation of MAPK, suggesting that activation of SAPK/JNK/p38 kinases and concomitant inhibition of MAPK is a determining factor for cells to undergo apoptosis (Xia et al., 1995). Furthermore, we have shown previously that Fasinduced apoptosis in multiple myeloma (MM) cells is associated with activation of both SAPK/JNK and p38 kinase (Chauhan et al., 1997). To date, however, it remains controversial as to whether SAPK/JNK activation is required during apoptosis. Speci®cally, the role of SAPK/JNK activation during apoptosis in MM cells induced by IR or other agents is not yet de®ned. Recent emphasis has focused on the family of aspartate-speci®c cysteine proteases (ASAPs) that mediates all apoptotic cell death (Henkart, 1996). Once activated, ASAPs eciently cleave poly (ADPribose) polymerase (PARP), thereby preventing DNA
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repair mechanisms during apoptosis. Activation of ASAPs and related PARP cleavage in MM cells undergoing apoptosis induced by IR or other agents has also not yet been characterized. In the present study, we characterized the signaling mechanisms involved during apoptosis induced by IR and by Dexamethasone (Dex) in MM derived cell lines and patient cells. Although both IR and Dex treatment of MM cells induce apoptosis and PARP cleavage, only IR-induced apoptosis involves activation of SAPK/JNK and p38 kinase. In contrast, Dex-induced apoptosis is associated with decreased MAPK and p70S6K activity, whereas IR treatment does not alter the activity of these kinases. Finally, interleukin-6 (IL-6) blocks Dex-induced apoptosis, downregulation of MAPK and p70S6K activities, and PARP cleavage in MM cells but does not inhibit IR-induced apoptosis, SAPK/JNK activation, and PARP cleavage. Our data therefore demonstrate that SAPK/JNK activation is not required for apoptosis in MM cells and, in particular, that Dex induces apoptosis in a SAPK/ JNK independent mechanism.
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Results and discussion Previous studies have demonstrated that treatment of diverse cell types with IR induces apoptosis. We and others have shown that Fas or glucocorticoids triggers apoptosis in MM cells (Chauhan et al., 1997; Westerndorf et al., 1995; Shima et al., 1995; Hata et al., 1995; Lichtenstein et al., 1995). In the present study, OCI-MY5 MM cells were exposed to IR for 48 h and DNA fragmentation assays were performed at various intervals (Figure 1a). Increased cleavage of DNA by 24 h was noted in IR-treated, but not in untreated control MM cells. To quantify cell death, ¯ow cytometric analyses using propidium iodide (Ormerod et al., 1992) were next performed. After exposure of OCI-MY5 MM cells to IR for 48 h, the majority (67+3%, n=3) of cells underwent apoptosis. Acridine orange staining (Duke et al., 1992) con®rmed changes characteristic of apoptosis. Since IR and U.V. have been shown to triggers activation of SAPK/JNK and p38 kinase in other systems (Kharbanda et al., 1995; Derijard et al., 1994), we next assayed for IR-induced activation of SAPK/ JNK and p38 kinase in MM cells. OCI-MY5 MM cells were treated with IR, and in vitro immune complex kinase assays were performed (Figure 1b, upper panel). Analyses of anti-SAPK/JNK immunoprecipitates demonstrated a transient 8 ± 10-fold activation of SAPK/ JNK at 1 h in MM cells. Similar results were obtained when anti-p38 immunoprecipitates from control and IR-treated cells were analysed for phosphorylation of GST-ATF2 (Figure 1c, upper panel). To determine whether IR induces SAPK/JNK and p38 kinase activity in a dose dependent fashion, we treated OCIMY5 MM cells for 1 h with IR doses ranging from 5 ± 33 Gy. Although IR induces SAPK/JNK and p38 kinase activity at 5 Gy, maximal activity of these kinases was observed when cells were treated with 20 Gy (data not shown). In order to con®rm whether IR aects JNK/SAPK and p38 kinase protein levels, total cell lysates were also subjected to immunoblotting with respective antibodies. The results demonstrated
Figure 1 IR induces DNA fragmentation and activation of SAPK and p38 kinase in OCI-MY5 MM cells. Cells were cultured in media alone for the indicated times with an IR dose of 20 Gy. Genomic DNA was isolated, end-labeled with 0.5 mCi of [g32P]dCTP, and analysed by 1.8% agarose gel electrophoresis (a). OCI-MY5 MM cells were treated with IR (20 Gy) for 1 h, 3 h and 6 h. Lysates from control and IR-treated OCI-MY5 MM cells were immunoprecipitated with anti-SAPK antibody (b) and anti-p38 kinase antibody (c). Immune complex kinase assays were performed by addition of GST-Jun (b) or GST-ATF2 (c) and [g32P]ATP and incubation for 15 min at 308C. The phosphorylated proteins were resolved by 10% SDS ± PAGE and analysed by Coomassie blue staining and autoradiography (Exposure time for autoradiography was 30 mins for panel b). Total cell lysates from treated and untreated cells were also subjected to immunoblotting with either SAPK/JNK (b, lower panel) or p38 kinase antibody (c, lower panel)
that IR treatment of MM cells does not alter the protein level of both JNK/SAPK and p38 kinase (Figure 1b, lower panel and Figure 1c, lower panel). Our ®ndings that IR-induced apoptosis involves activation of SAPK/JNK suggest a role of SAPK/ JNK in at least IR-induced apoptosis in MM cells. These ®ndings are in concert with those of Verheij et al. (1996) which demonstrated a role of SAPK/JNK in ceramide- or TNF-induced apoptosis. Furthermore, it has been demonstrated that coordinate regulation of SAPK/JNKs, p38 and ERK kinases facilitates apoptosis induced by withdrawal of growth factor, suggesting that activation of SAPK/JNK may be essential for apoptosis (Xia et al., 1995). In contrast, a recent report demonstrated that TNF-induced activation of SAPK/JNK is not linked to TNFinduced apoptosis (Liu et al., 1996). It therefore remains controversial as to whether activation of SAPK/JNK is an obligatory event during apoptosis induced by diverse apoptotic stimuli in all cell types. To determine whether distinct inducers of apoptosis utilize the SAPK/JNK pathway, we next studied Dexinduced apoptosis in our MM cell line and patient model system. OCI-MY5 MM cells were treated with
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Dex (10 mM) for 72 h and DNA cleavage was analysed by agarose gel electrophoresis of the 32P-labeled genomic DNA at various intervals (Figure 2a). In contrast to control untreated cells, signi®cant DNA fragmentation was detected in OCI-MY5 MM cells treated with Dex for 72 h. Both FACS analysis and acridine orange staining con®rmed apoptosis in Dex treated MM cells (data not shown). We next examined whether Dex-induced apoptosis also involves activation of SAPK/JNK or p38 kinase. OCI-MY5 MM cells were treated with Dex for the indicated periods and assayed for the activity of these kinases. In contrast to IR, Dex did not induce activation of either SAPK/JNK (Figure 2b, upper panel) or p38 kinase (Figure 2c, upper panel) in OCI-MY5 MM cells. Furthermore, Dex treatment did not alter the protein levels of either SAPK/JNK or p38 kinase (Figure 2b, lower panel, and Figure 2c, lower panel, respectively). However, downregulation of SAPK/JNK activity was observed at 12 h, 24 h and 48 h after Dex treatment (Figure 2b, upper panel). The lack of activation of SAPK/JNK or p38 kinase in MM cells treated with Dex suggests that Dex-induced apoptosis in MM cells is independent of SAPK/JNK and p38 kinase pathways.
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Figure 2 Dex treatment induces DNA fragmentation without aecting SAPK and p38 kinase activity in OCI-MY5 MM cells. Cells were cultured for the indicated times in media alone and with Dex (10 mM). Genomic DNA was isolated, end-labeled with 0.5 mCi of [g32P]dCTP, and analysed by 1.8% agarose gel electrophoresis (a). MW, molecular weight markers (100 bp ladders and HindIII digest of 1-DNA). OCI-MY5 MM cells were treated with DEX (10 mM) for 1 h, 3 h, 6 h, 12 h, 24 h and 48 h. Lysates from control and Dex-treated OCI-MY5 MM cells were immunoprecipitated with anti-SAPK antibody (b) and antip38 kinase antibody (c). Immune complex kinase assays were performed by addition of GST-Jun (b) or GST-ATF2 (c) and [g32P]ATP and incubation for 15 min at 308C. The phosphorylated proteins were resolved by 10% SDS ± PAGE and analysed by Coomassie blue staining and autoradiography (Exposure time for autoradiography was 24 h for panel b). The results are representative of three independent experiments. Total cell lysates from treated and untreated cells were also subjected to immunoblotting with either SAPK/JNK (b, lower panel) or p38 kinase antibody (c, lower panel)
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Figure 3 Eects of IR or DEX on DNA fragmentation as well as on SAPK and p38 kinase activity in PCL patient cells. Cells were cultured for the indicated times in media alone and with either IR (20 Gy) or Dex (10 mM). Genomic DNA was isolated, end-labeled with 0.5 mCi of [g32P]dCTP and analysed by 1.8% agarose gel electrophoresis (a). PCL patient cells were treated with IR (20 Gy) or Dex (10 mM) for the indicated time period. Lysates from control and IR or Dex-treated PCL patient cells were immunoprecipitated with anti-SAPK antibody (b and d), or anti-p38 kinase antibody (c and e). Immune complex kinase assays were performed by addition of GST-Jun (b and d) or GSTATF2 (c and e) and [g32P]ATP and incubated for 15 min at 308C. The phosphorylated proteins were resolved by 10% SDS ± PAGE and analysed by Coomassie blue staining and autoradiography. The results are representative of three independent experiments
We next performed similar experiments using MM patient (PCL) cells. As noted in OCI-MY5 MM cells, treatment of PCL cells with IR or Dex is associated with increased DNA fragmentation (Figure 3a). Treatment of PCL cells with IR activates both SAPK/JNK and p38 kinase (Figure 3b and c), whereas Dex treatment does not activate either of these kinases (Figure 3d and e). In addition, protein levels of SAPK/JNK and p38 kinases were not altered by treatment of PCL cells with either IR or Dex (data not shown). Taken together, our results therefore suggest that there are at least two dierent apoptotic pathways in MM cell lines as well as patient cells: one which involves activation of SAPK/JNK and p38 kinase, as induced by IR; and another which is independent of SAPK/JNK and p38 kinase, as triggered by Dex. Since Dex-induced apoptosis in MM cells does not involve activation of SAP/JNK or p38 kinase, we next
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Figure 4 Dex downregulates MAPK and p70S6K activity in OCIMY5 cells. Cells were cultured for the indicated times in media alone or with Dex (10 mM). Lysates from control and Dex-treated OCI-MY5 cells were immunoprecipitated with anti-MAPK antibody. Immune complex kinase assays were performed by addition of MBP as a substrate (a, upper panel) and incubated for 15 min at 308C. The phosphorylated proteins were resolved by 10% SDS ± PAGE and analysed by Coomassie blue staining and autoradiography. Anti-MAPK immunoprecipitates were also analysed by immunoblotting with anti-MAPK antibody (a, lower panel). Lysates from control and Dex-treated OCI-MY5 cells were immunoprecipitated with anti-p70S6K antibody and in vitro immune complex kinase assays were performed utilizing S6 peptide as an exogenous substrate. The results are expressed as percent inhibition in p70S6K activity (mean+s.d. of three independent experiments; indicated as ±&± Dex; ±*± IR)
determined whether Dex aects growth-related kinases, such as MAPK and p70S6K. Treatment of OCI-MY5 cells with Dex downregulates MAPK activity, assayed by MBP phosphorylation (Figure 4a, upper panel). Reprobing of these blots with anti-MAPK antibody demonstrated equal amounts of protein in each lane (Figure 4a, lower panel). We also studied p70S6K since previous studies have demonstrated that activation of p70S6K is associated with G1 to S phase transition (Chung et al., 1992). Moreover, the signaling cascade regulating p70S6K is distinct from Ras44Raf44 MEK44MAPK and RSK (Blenis, 1993) and is mediated directly by phosphatidylinositol 3-kinase (PI3K) (Cheatham et al., 1994; Chung et al., 1994). In the present study, Dex treatment of OCI-MY5 MM cells is associated with signi®cant inhibition of p70S6K
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Figure 5 IR and Dex induce proteolytic cleavage of PARP. OCIMY5 MM cells were treated with IR (20 Gy) and Dex (10 mM) for the indicated time periods. Immunoblot analyses of the lysates was performed with anti-PARP antibody
activity at 24 h, as measured directly by S6 peptide phosphorylation (Figure 4b). Furthermore, whether inhibition of p70S6K activity by Dex treatment is PI3K dependent or independent requires further studies. A recent study which demonstrates that Dex blocks the ability of NF-kB to activate transcription of genes that regulate cell proliferation (Scheiman et al., 1995) is consistent with these ®ndings. IR treatment of OCIMY5 MM cells did not downregulate either MAPK or p70S6K (data not shown and Figure 4b). In the present study we next examined whether IR and Dex induce poly (ADP-ribose) polymerase (PARP) cleavage, the hallmark of apoptosis which indicates activation of the protease cascade (Henkart, 1996; Desnoyers et al., 1994). Both IR and Dex induced PARP cleavage, consistent with apoptosis and activation of proteases (Figure 5a and b). Taken together, these ®ndings suggest that Dex-induced apoptosis is independent of SAPK/JNK activation, and involves inhibition of growth-related kinases as well as activation of proteases. In contrast, IR-induced apoptosis is associated with activation of SAP/JNK and proteases, without aecting growth kinases. Finally we attempted to con®rm the existence of distinct pathways using inhibitors of apoptosis. Since IL-6 is an anti-apoptotic factor for MM cells (Lichtenstein et al., 1995), we studied the eect of IL6 on the IR and Dex induced apoptotic signaling events. Treatment of OCI-MY5 MM cells with IL-6 (100 ng/ml) prior to Dex resulted in complete blocking of apoptosis and PARP cleavage (Figure 6a), but did not prevent IR-induced apoptosis and PARP cleavage (Figure 6b). Moreover, IL-6 prevented Dex-induced downregulation of p70S6K and MAPK activities in the absence of any changes in the protein levels of either kinase (Figure 6c, d, and data not shown). In contrast, IR-induced apoptosis and SAP/JNK activation is not inhibited by IL-6 (data not shown). The present study therefore provides evidence for at least two cascades involved in apoptosis in MM cells:
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Figure 6 Eects of IL-6 on IR and Dex-induced proteolytic activity of PARP and on the Dex-induced decrease in p70S6K and MAPK activity. OCI-MY5 MM cells were treated with IR (20 Gy) or with Dex (10 mM) in the presence or absence of IL-6. Immunoblot analyses of the lysates were performed with anti-PARP monoclonal antibody (a and b). Cells were also treated with Dex with or without IL-6, for the indicated times. Anti-p70S6K immunoprecipitates were analysed for S6 peptide phosphorylation as described. (±&± Dex; ±*± Dex+IL-6)
one induced by IR, which is associated with activation of SAPK and p38 and does not involve MAPK and p70S6K; and another triggered by Dex, which downregulates MAPK and p70S6K without activating SAPK or p38 kinase. Additional evidence supporting two signaling pathways associated with apoptosis in MM cells is the ability of IL-6 to inhibit Dex-induced apoptosis and signaling, without altering IR-induced apoptosis or signaling. Furthermore, our ®ndings suggest that Dex downregulates growth kinases in MM cells. The demonstration that IL-6 prevents Dexinduced apoptosis and signaling in MM cells is consistent with the known resistance to Dex-treatment in MM patients with advanced disease with high serum levels of IL-6 (Bataille et al., 1989). Ongoing studies of the mechanisms whereby IR and Dex induce apoptosis in MM cells may not only provide clues to basic growth control, but also suggest new treatment strategies.
Materials and methods Cell culture The human MM cell line OCI-MY5 (provided by Dr HA Messner, Ontario Cancer Institute, Toronto, Canada) was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 mM/ml streptomycin and 2 mM L-glutamine. Mononuclear cells were isolated from a patient with MM (patient PCL) by Ficoll-Hypaque density gradient centrifugation, and incubated with HB-7 (anti-CD38) MoAb-biotin-streptavidin and 2H4 (anti-CD45RA) MoAb-¯uorescein isothiocyanate on ice. Tumor cells
(96+2% CD38+45RA7) were isolated using an Epics C cell sorter (Coulter Electronics, Hialeah, FL), washed, and resuspended in RPMI-1640 media containing 10% FBS and antibiotics. Gamma irradiation was performed at room temperature using a Gamma-cell 1000 (Atomic Energy of Canada, Ottawa, Canada) under aerobic conditions, with a 137 Cs source emitting at a ®xed dose rate of 0.76 Gy min71 as determined by dosimetry. DNA fragmentation assays Genomic DNA was isolated from OCI-MY5 MM cell line and from PCL patient cells, and DNA end-labeling was performed as previously described (Tian et al., 1991; Frank, 1992). Labeled DNA probes were electrophoresed for 2 ± 3 h at 90 Volts on 1.8% agarose gels, which were then dried and exposed for autoradiography. In vitro immune complex c-Jun, ATF-2, or myelin basic protein (MBP) phosphorylation assays In vitro immune complex c-Jun kinase assays were performed as previously described (Angel et al., 1988). Lysates were precleared by incubating with 5 mg/ml rabbitanti-mouse IgG for 1 h at 48C and then for an additional 30 min incubation with protein A-Sepharose. The supernatants were incubated with preimmune rabbit serum or speci®c antibodies for SAPK/JNK or MAPK (Santa Cruz Biotechnology, San Diego, CA) or p38 kinase (provided by Dr Roger Davis, University of Mass, Worcester, MA) for 2 h at 48C prior to the addition of protein A-Sepharose. The immune complexes were washed three times with lysis buer, once with kinase buer and resuspended in kinase buer containing [g-32P]ATP (3000 ci/mmol; New England Nuclear, Boston, MA) and GST-Jun (2-100), GST-ATF2 (provided by Dr Davis), or MBP substrates. The reaction was incubated for 15 min at 308C and terminated by the
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addition of SDS sample buer. Proteins were analysed by SDS ± PAGE, Coomassie blue staining and autoradiography. Immunoprecipitation and immunoblotting Immunoprecipitations were performed as previously described (Chauhan et al., 1995). Brie¯y, cell lysates were prepared by resuspending OCI-MY5 MM cells for 30 min on ice in lysis buer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM PMSF, 1 mM sodium vanadate, 1 mM DTT, 10 mM sodium ¯uoride, 1% NP-40 and 10 mg/ml each of leupeptin and aprotinin. Equal amounts of proteins (250 ± 300 mg) were subjected to immunoprecipitation with the indicated antibodies and immune complexes were precipitated with protein A-Sepharose. The resulting protein precipitates were washed three times with lysis buer and resolved by SDS ± PAGE. Proteins were then transferred to nitrocellulose ®lters, blocked by incubation in 5% dry milk in PBST (0.05% Tween-20 in PBS), and probed with the indicated antibodies. p70S6K antibody was provided by Dr John Blenis (Harvard Medical School, Boston, MA). Blots were then developed by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL). Preparation of cell lysates for PARP immunoblot analyses was performed as described using C-2-10 antiPARP monoclonal antibody (Desnoyers et al., 1994). Immune complex protein kinase assays for p70S6K Total cell lysates were prepared with anti-p70 S6K antiserum; immune complexes were separated and subjected to in vitro kinase assays using synthetic S6 (RRRLSSLRA) peptide as substrate (Chung et al., 1992). In brief, the immune complexes were washed twice with hypotonic lysis buer, twice with LiCl buer (0.5M LiCl, 100 mM Tris, pH 7.6) and twice with assay buer (20 mM Tris, pH 7.2, 10 mM MgCl2). After washing, they were suspended in kinase buer (20 mM Tris, pH 7.2, 10 mM MgCl2) and the reaction (50 ml) was started by the addition of 20 m M ATP (1 mCi of [g32-P]ATP) and 100 mM S6 peptide. After incubation for 15 min at 308C, 25 ml was spotted onto phosphocellulose paper (P81, Whatman), followed by washing with 1% phosphoric acid and then distilled water. Incorporation of [32P]phosphate was assayed by scintillation counting.
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