J. Biol. Chem.-2005-Rahmani-35217-27

Apoptosis Induced by the Kinase Inhibitor BAY43-9006in Human Leukemia Cells Involves Down-regulation of Mcl-1 through Inhibition of Translation*Received for publication,June16,2005,and in revised form,August15,2005Published,JBC Papers in Press,August18,2005,DOI10.1074/jbc.M506551200 Mohamed Rahmani‡,Eric Maynard Davis‡,Cheryl Bauer‡,Paul Dent§,and Steven Grant‡§¶1From the Departments of‡Medicine,§Biochemistry,and¶Pharmacology,Virginia Commonwealth University,School of Medicine, Richmond,Virginia23298BAY43-9006is a kinase inhibitor that induces apoptosis in a variety of tumor cells.Here we report that treatment with BAY 43-9006results in marked cytochrome c and AIF release into the cytosol,caspase-9,-8,-7,and-3activation,and apoptosis in human leukemia cells(U937,Jurkat,and K562).Pronounced apoptosis was also observed in blasts from patients with acute myeloid leukemia. These events were accompanied by ERK1/2inactivation and caspase-independent down-regulation of Mcl-1.Inducible expres-sion of a constitutively active MEK1construct did not prevent Mcl-1down-regulation,suggesting that this event is not related to MEK/ERK pathway inactivation.Furthermore,BAY43-9006did not induce major changes in Mcl-1mRNA levels monitored by real-time PCR or Mcl-1promoter activity demonstrated by luciferase reporter assays,but it did enhance Mcl-1down-regulation in acti-nomycin D-treated cells.Inhibition of protein synthesis by cyclo-heximide or proteasome function with MG132and pulse-chase studies with[35S]methionine demonstrated that BAY43-9006did not diminish Mcl-1protein stability,nor did it enhance Mcl-1ubiq-uitination,but instead markedly attenuated Mcl-1translation in association with the rapid and potent dephosphorylation of the eIF4E translation initiation factor.Finally,ectopic expression of Mcl-1in leukemic cells markedly inhibited BAY43-9006-mediated cytochrome c cytosolic release,caspase-9,-7,and-3activation,as well as cell death,indicating that Mcl-1operates upstream of cyto-chrome c release and caspase activation.Together,these findings demonstrate that BAY43-9006mediates cell death in human leu-kemia cells,at least in part,through down-regulation of Mcl-1via inhibition of translation.The Ras/Raf/mitogen-activated protein kinase(MEK)2/extracellular-signal-regulated kinase(ERK)cascade plays a critical role in relaying signals from cell surface receptors to various cytoplasmic and nuclear proteins involved in diverse biological process such as cell growth,transformation,differentiation,and apoptosis(1).Aberrant activation of this pathway has been implicated in the development of many tumor types,and constitutive activation of this pathway has been observed in ϳ30%of all human cancer.The serine/threonine Raf kinase family, which consists of three proteins,C-Raf(also referred to as Raf-1),B-Raf, and A-Raf,is an essential component of this pathway(1,2).Strikingly, B-Raf-activating mutations have been observed inϳ70%of malignant melanomas(3,4)and at lower frequencies in a number of other human cancer types,including colorectal(3,5),ovarian,and papillary thyroid carcinomas(3,6,7).Moreover,overexpression of constitutively active c-Raf is sufficient to induce transformation of NIH3T3cells(8). Increased Raf/MEK/ERK activity has also been observed in a variety of leukemias,including acute myeloid leukemia(AML)and chronic mye-loid leukemia(9,10).In addition,constitutive activation of this pathway diminishes apoptosis in hematopoietic cells(11)and abrogates the cyto-kine dependence of several human and murine cytokine-dependent hematopoietic cells lines(e.g.TF-1,FDC-P1,and FL5.12)(12).Con-versely,inhibition of this pathway by pharmacologic MEK inhibitors such as PD98059or U0126enhances apoptosis induction by a variety of agents,including paclitaxel(13)UCN01(14),STI571(15),proteasome inhibitors(16),and lovastatin(17).For these reasons,disrupting the Ras/Raf/MEK/ERK pathway represents an attractive anticancer strat-egy,particularly in leukemia cells.BAY43-9006,a novel bi-aryl urea,has shown promising preclinical activity against a variety of tumor cell types and is currently undergoing phase II/III clinical evaluation(18–20).Although it was initially devel-oped as a specific inhibitor of C-Raf and B-Raf,subsequent studies revealed that this compound also inhibits several other important tyro-sine kinases involved in tumor progression,including vascular epider-mal growth factor receptor-2,vascular epidermal growth factor recep-tor-3,platelet-derived growth factor receptor-␤,Flt3,and c-Kit(21). Interestingly,BAY43-9006has been shown to inhibit C-Raf and wild type as well as mutant V600E B-Raf kinase activities in vitro and to diminish MEK/ERK activation in various tumor cell lines,including those harboring mutant Ras or B-Raf(21–23).Several studies have shown that myeloid cell leukemia-1(Mcl-1),a Bcl-2family member,plays a pivotal role in cell survival,particularly in hematopoietic cells.For example,depletion of Mcl-1using antisense oligonucleotides rapidly triggers apoptosis in U937cells(24).Moreover, inducible deletion of Mcl-1in mice resulted in loss of early bone marrow progenitor populations,including hematopoietic stem cells(25).Dele-tion of Mcl-1during early lymphocyte differentiation also increased apoptosis and arrested development at the pro-B-cell and double-neg-ative T-cell stages.In addition,specific ablation of Mcl-1in peripheral B-and T-cell populations resulted also in their rapid loss(26).On the other hand,selective overexpression of Mcl-1in hematopoietic tissues of transgenic mice promotes the survival of hematopoietic cells and*This work was supported by Public Health Service Grants CA-63753,CA-93738, CA-100866,and CA-88906from NCI,National Institutes of Health(NIH),Grant DK52825from NIH,Award6045-03from the Leukemia and Lymphoma Society of America,Award DAMD17-03-1-0209from the Department of Defense,and a Trans-lational Research award from the V-foundation.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 with18U.S.C.Section1734solely to indicate this fact.1To whom correspondence should be addressed:Division of Hematology/Oncology, MCV Station Box230,VA Commonwealth University,Richmond,VA23298.Tel.:804-828-5211;Fax:804-828-8079;E-mail:stgrant@.2The abbreviations used are:MEK,mitogen-activated protein kinase/extracellular sig-nal-regulated kinase kinase;ERK,extracellular signal-regulated kinase;AML,acute myeloid leukemia;Mcl-1,myeloid cell leukemia-1;PARP,poly(ADP-ribose)polymer-ase;z-VAD-FMK,benzyloxycarbonyl-VAD-fluoromethyl ketone;HA,hemagglutinin;JNK,c-Jun N-terminal kinase;mTOR,mammalian target of rapamycin;CA,constitu-tively active;AIF,apoptosis inducing factor;FAB,French-American-British.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.280,NO.42,pp.35217–35227,October21,2005©2005by The American Society for Biochemistry and Molecular Biology,Inc.Printed in the U.S.A.by guest on September 11, 2016/Downloaded fromenhances the outgrowth of myeloid cell lines (27).Finally,overexpres-sion of Mcl-1protects cells from apoptosis induced by a variety of agents,including UV,tumor necrosis factor-related apoptosis-inducing ligand,etoposide,staurosporine,actinomycin D,among others (28–31).Such evidence suggests that Mcl-1may play a critical role in the survival of leukemia and possibly other malignant hematopoietic cells.Interestingly,expression of Mcl-1has been shown to be dependent upon an intact MEK/ERK pathway in both hematopoietic (32)and non-hematopoietic cells (33).Currently,the one or more mechanisms by which BAY 43-9006induces cell death in human leukemia cells remain to be fully elucidated.Here we report that BAY 43-9006potently induces mitochondrial injury and apoptosis in these cells in association with a pronounced and MEK/ERK-independent reduction in Mcl-1expression.Moreover,pre-vention of BAY 43-9006-mediated Mcl-1down-regulation by ectopic expression of an Mcl-1construct substantially diminishes BAY 43-9006-induced mitochondrial injury and apoptosis.Finally,the pres-ent results indicate that BAY 43-9006down-regulates Mcl-1expression through inhibition of translation,rather than through a transcriptional,post-translational,or caspase-dependent mechanism.MATERIALS AND METHODSCells —The human leukemia U937,Jurkat,and K562cells were cul-tured as previously reported (34).U937cells stably overexpressing Mcl-1were kindly provided by Dr.Ruth Craig (Dartmouth Medical School,Hanover).These cells were obtained by transfecting U937cells with a pCEP4-Mcl-1construct that encodes for the 40-kDa Mcl-1pro-tein.Stable single cell clones were selected in the presence of 400␮g/ml hygromycin.Thereafter,cells from each clone were analyzed for Mcl-1expression by Western blot.A Tet-On Jurkat cell line inducibly express-ing constitutively active MEK1under doxycycline control was previ-ously described (34).Isolation of Patient-derived Leukemic Blasts —Leukemic blasts were obtained with informed consent from the peripheral blood of several patients with acute myeloblastic leukemia (AML),FAB subtype M2.These studies have been sanctioned by the Investigational Review Board of Virginia Commonwealth University/Medical College of Virginia,and all patients provided informed consent.In each case,the percentage of blasts in the peripheral blood was Ͼ70%.Blood was collected into hep-arinized syringes,diluted 1:3with RMPI 1640medium,and transferred as an overlayer to centrifuge tubes containing 10ml of Ficoll-Hypaque (specific gravity,1.077–1.081).After centrifugation at room tempera-ture for 30min,the interface layer,containing predominantly leukemic blasts,was extracted with a sterile Pasteur pipette,suspended in RPMI medium,and washed three times.Leukemic blasts,which displayed Ͼ90%viability by trypan blue exclusion,were then diluted into RPMI medium containing 10%fetal calf serum at a concentration of 106cells/ml,and exposed to drugs as described in the case of continuously cul-tured cell lines.Reagents —BAY 43-9006(Bayer,West Haven,CT)was provided by Dr.John Wright,Cancer Treatment and Evaluation Program,NCI,National Institutes of Health (Bethesda,MD).It was dissolved in Me 2SO,and aliquots were maintained at Ϫ80°C.MG132was pur-chased from Calbiochem;cycloheximide and actinomycin D were pur-chased from Sigma.SB202190was purchased from Alexis Corp.(San Diego,CA).Rapamycin and U0126were purchased from Cell Signaling Technology (Beverly,MA).The broad spectrum cell-permeable caspase inhibitor,z-VAD-FMK was purchased from Enzyme Systems Products (Livermore,CA).All reagents were prepared and used as recommended by their suppliers.Assessment of Apoptosis —Apoptotic cells were routinely identified by Annexin V-fluorescein isothiocyanate staining as previously described (35).Briefly,105cells were collected,washed in cold phosphate-buffered saline,and then resuspended in binding buffer (10m M Hepes/NaOH,pH 7.4,140m M NaCl,2.5m M CaCl 2)containing fluorescein-labeled annexin V (BD Pharmingen)and propidium iodide.Samples were incu-bated for 15min and then analyzed by flow cytometer (BD Biosciences FACScan).Quantitative Real-time PCR —U937cells were left untreated or treated with 10␮M BAY 43-9006for the indicated period after which they were lysed and total RNA was extracted using the RNeasy mini kit (Qiagen).Quantitative real-time PCR analysis was carried out on the ABI Prism ௡7900Sequence Detection System (Applied Biosystems,Fos-ter City,CA)using the TaqMan ௡One Step PCR Master Mix Reagents Kit (polynucleotide:4309169)as recommended by the manufacturer.The cycling conditions were:48°C/30min;95°C/10min;and 40cycles of 95°C/15s and 60°C/1min.The cycle threshold was determined to provide the optimal standard curve values (0.98–1.0).The probes (5Ј-TCAAGTGTTTAGCCACAAAGGCACCAAAAG-3Ј)and Mcl-1-specific primers (forward,GGGCAGGATTGTGACTCTCATT;reverse,5Ј-GATGCAGCTTTCTTGGTTTATGG-3Ј)were designed using the Primer Express ௡2.0version.The probes were labeled at the 5Ј-end with 6-carboxyfluoresceine and at the 3Ј-end with 6-carboxytet-ramethylrhodamine.Ribosomal RNA (18S rRNA)was used as endog-enous control.Each sample was tested in triplicate,and the Mcl-1mRNA level was normalized to that of 18S rRNA.Transient Transfection and Reporter Gene Assay —K562cells were transiently transfected using Amaxa nucleofector TM (Koeln,Germany)as previously described (36).Constitutively active MNK1T332D and eIF4E (wild type)were kindly provided by Dr.J.A.Cooper (Fred Hutchinson Cancer Research Center,Seattle,WA)(37).PcDNA3.1-Mcl-1was a generous gift from Dr.R.W.Craig.Empty vector pcDNA3.1was purchased from Invitrogen.Reporter gene assays were carried out as previously described (38).Briefly,cells were cotransfected with a Ϫ203/ϩ10-Mcl-1-pGL2plasmid (39)in which firefly luciferase is driven by the Ϫ203to ϩ10element of the Mcl-1gene promoter,or the pGL2-basic empty vector (Promega,Madison,WI)and pRL-TK-luc plasmid encoding for Renilla luciferase.Cells were incubated for 6h and then treated with BAY 43-9006for an additional 20h,after which the activity of firefly and Renilla luciferases was measured using the Dual-Luciferase reporter assay system (Promega).Values for firefly luciferase activity were normalized to those obtained for Renilla luciferase activity.Immunoprecipitation and Immunoblotting —For immunoprecipita-tion,cells were lysed in buffer containing 20m M Tris (pH 7.5),150m M NaCl,1m M EDTA,1m M EGTA,antiproteases (10␮g/ml of leupeptin and aprotinin,1m M phenylmethylsulfonyl fluoride),and 1%Triton X-100after which 500␮g of protein lysate was subjected to immuno-precipitation using the designated antibodies.Immunoblotting was per-formed using the immunoprecipitates or the whole cells lysates as pre-viously described in detail (35).The primary antibodies used in this study were as follows:caspase-3,Bax,caspase-7,Bcl-2,and Mcl-1(BD Pharmingen);Caspase-8(Alexis Corp.);poly(ADP-ribose)polymerase (PARP,Biomol Research Laboratories,Plymouth Meeting,PA);Bcl-x L XIAP,total and Phospho-ERK1/2(Thr-202/Tyr-204),ubiquitin,cleaved caspase-9,cleaved caspase-3,phospho-4EBP1(Ser-65),phos-pho-eIF4E (Ser-209),phospho-eIF4G (Ser-1108),phospho-p90RSK (Ser-380),phospho-p70S6K (Thr-389),phospho-p38(Thr-180/Tyr-182),and phospho-JNK1(Thr-183/Tyr-185)(Cell Signaling Technol-ogy);eIF4G (BD Transduction Laboratories);Bim,HA,cytochrome c ,AIF,total and phosphorylated Bcr-abl,eIF4E,p70S6K,myc,JNK1,andBAY 43-9006-mediated Apoptosis Involves Mcl-1by guest on September 11, 2016/Downloaded fromp38(Santa Cruz Biotechnology,Santa Cruz,CA);and Bak and␣-tubulin (Calbiochem).Mcl-1Protein Stability—U937cells were washed in phosphate-buff-ered saline and cultured at a density of5ϫ106cells,in methionine-free RPMI for15min,then labeled with100␮Ci/ml[35S]methionine(ICN, Biomedicals,Inc.,Irvine,CA)for60min.Cells were then washed in phosphate-buffered saline and cultured in complete RPMI containing fetal bovine serum and excess of cold methionine(10m M)and cysteine (5m M)for the indicated periods in the presence or absence of BAY 43-9006with or without the proteasome inhibitor MG132.At the end of the indicated intervals,107cells were collected and subsequently sub-jected to immunoprecipitation using Mcl-1antibodies as described above.The immunoprecipitates were subjected to SDS-PAGE followed by autoradiography.m7-GTP-Sepharose Chromatography—Following stimulation,cells were lysed in lysis buffer as indicated above.500␮g of protein lysates was incubated with50␮l of m7-GTP-Sepharose beads(Amersham Bio-sciences)for2h at4°C after which the beads were washed three times and boiled in Laemmli buffer for5min,and following centrifugation, the supernatants containing the proteins were subjected to Western blot analysis.Subcellular Fractionation—Leukemic cells(4ϫ106)were lysed using digitonin buffer(35),after which cytosolic and membrane fractions were separated by centrifugation,solubilized in Laemmli buffer,and boiled for5min.Proteins were analyzed by Western blot to evaluate cytochrome c release into the cytosol.Statistical Analysis—The significance of differences between exper-imental conditions was determined using the Student’s t test for unpaired observations.RESULTSTreatment with BAY43-9006Results in a Marked Induction of Mito-chondrial Injury and Apoptosis in Human Leukemia Cells—To charac-terize the effects of BAY43-9006in U937cells,dose-response and time-course studies were performed(Fig.1).As shown in Fig.1A,exposure of U937cells to increasing concentrations of BAY43-9006for24h revealed a moderate induction of apoptosis at concentration as low as5␮M as indicated by annexin V analysis.Higher concentration of BAY 43-9006resulted in more pronounced cell death(e.g.80%at15␮M). Virtually identical results were obtained in Jurkat lymphoid leukemia cells(Fig.1B).Exposure of U937cells to10␮M BAY43-9006at varying intervals resulted in induction of apoptosis that was detected as early as 4h after drug treatment.Longer exposure intervals resulted in a marked increase in cell death(e.g.55and70%at24and48h,respectively). Essentially equivalent findings were observed when Jurkat cells were examined(data not shown).Furthermore,exposure to BAY43-9006 resulted in a release of cytochrome c and AIF into the cytosol(Fig.1D) accompanied by cleavage of caspases-7,-8,-9,and-3as well as PARP (Fig.1E).These events were readily apparent after8h of treatment andbecame more pronounced after20h.Together,these findings indicate that BAY43-9006results in a striking induction of caspase activation, mitochondrial injury,and apoptosis in human myeloid and lymphoid leukemic cells.Exposure of U937Cells to BAY43-9006Is Associated with a Decrease in ERK Phosphorylation—Dose-response studies(Fig.2)revealed that exposure of U937cells to BAY43-9006at concentration as low as5␮M resulted in a discernable decrease in ERK phosphorylation as early as half an hour after beginning of exposure,and this decrease persisted at4 and8h of drug administration.Exposure to7.5and10␮M BAY43-9006 produced even more pronounced reductions in ERK phosphorylation.In contrast,total levels of ERK were unchanged.These findings confirmthat,as previously described in other cell types(21,22)BAY43-9006 inactivates ERK in human leukemia cells.Treatment of U937Cells with BAY43-9006Results in Rapid Decreasein Mcl-1Protein Level and Late Bcl-2,Bax,and XIAP Cleavage—In viewof the critical role that Bcl-2family proteins play in apoptosis regulation (40,41),expression of these proteins was monitored in U937cells fol-lowing treatment of cells with BAY43-9006(10␮M)for varying inter-vals(Fig.3A).After20h of treatment,a decline in protein levels ofBcl-x L,Bak,Bim,as well as cleavage of Bcl-2,Bax,and XIAP were detected,however,no major changes were observed at earlierintervals. FIGURE1.Treatment with BAY43-9006results in a striking mitochondrial damage, caspase activation,and apoptosis in human leukemia cells.U937(A)and Jurkat(B)cells were exposed to the designated concentration of BAY43-9006for24h after whichthe percentage of apoptotic cells was determined by annexin V analysis as describedunder“Materials and Methods.”C,U937cells were exposed to10␮M BAY43-9006for the designated period after which apoptosis was determined as above.D,U937cells weretreated with BAY43-9006for the designated period,after which mitochondria-free cyto-solic fractions were obtained as described under“Materials and Methods”and subjectedto Western blot analysis to monitor release of cytochrome c and AIF.Alternatively,wholecell lysates were obtained,and subjected to Western blot analysis to monitor expressionof procaspase-3,caspase-8,caspase-7,cleaved caspase-9(c-casp-9),and PARP.The blotswere subsequently re-probed with anti-tubulin(Tub)antibodies to document equiva-lent loading and transfer.The results of a representative study are shown;two additional experiments yielded equivalentresults.FIGURE2.Treatment with BAY43-9006resulted in a dose-dependent dephospho-rylation of ERK1/2.U937cells were exposed to BAY43-9006for0.5,4,and8h,afterwhich protein lysates were prepared and subjected to Western blot analysis to monitorERK1/2phosphorylation.Each lane was loaded with20␮g of protein;the blots were subsequently reprobed with antibody directed against tubulin to control for equal load-ing and transfer of proteins.Two additional experiments yielded similar results.BAY43-9006-mediated Apoptosis Involves Mcl-1by guest on September 11, 2016/Downloaded fromThe finding that these changes were detected after caspase activation argues against the possibility of a primary role for these phenomena in BAY 43-9006-mediated cell death.In addition,no change in Bid protein level was noted.In striking contrast,levels of the anti-apoptotic protein Mcl-1protein declined rapidly (e.g.over 2h)following treatment with BAY 43-9006,and by 8h expression was essentially absent (Fig.3A ).Further analysis revealed that treatment with BAY 43-9006for 4h resulted in the dose-dependent down-regulation of Mcl-1,which roughly paralleled the extent of lethality (Fig.3B ).Furthermore,the pan-caspase inhibitor z-VAD-FMK was ineffective in preventing down-regulation of Mcl-1protein following 4or 8h of treatment with BAY 43-9006.In contrast,late changes in expression of other Bcl-2family members in cells exposed to BAY 43-9006were clearly diminished by z-VAD-FMK (data not shown).Similar results were obtained in Jurkat cells (data not shown).Together,these findings demonstrate that BAY 43-9006induces the rapid and dose-dependent down-regulation of Mcl-1protein through a mechanism independent of caspase activation.Treatment with BAY 43-9006Enhances Cell Death in Bcr-abl ϩCells as Well as in Human Leukemia Blasts in Association with ERK Dephos-phorylation and Mcl-1Down-regulation —To determine whether BAY 43-9006-mediated lethality could be extended to include human Bcr-Abl ϩleukemia cells,parallel studies were performed in K562cells.As shown in Fig.4A ,24-h exposure to BAY 43-9006resulted in a dose-de-pendent cell death as monitored by annexin V staining assay.Apoptosis was initially detected at BAY 43-9006concentrations Ն2.5␮M ,and at 10␮M ,the large majority of cells was apoptotic (ϳ75%).Time-course studies revealed a time-dependent cleavage of PARP that was first detected at 4h of treatment and became more apparent at later expo-sure intervals (8,16,and 24h)(Fig.4B ).Notably,a rapid decline in ERK phosphorylation and Mcl-1protein levels were also observed,analo-gous to results in U937cells.In contrast,no major changes were noted in Bcr-abl expression or phosphorylation until considerably later inter-vals (e.g.24h)when the large majority of cells were apoptotic (Fig.4B ).Lastly,attempts were made to determine whether BAY 43-9006also triggered cell death in primary human leukemia blasts,or whether this phenomenon was restricted to continuously cultured cell lines.Signifi-cantly,treatment with BAY 43-9006resulted in a marked dose-depend-ent increase in cell death in human leukemia blasts isolated from 2patients with AML (FAB classification M2;Fig.4C ).An increase in apoptosis at 24h was detected at a BAY 43-9006concentration as low as 2.5␮M (35–45%)and further increases in cell death were observed at higher concentrations.For example,at 10␮M BAY 43-9006induced 60and 80%apoptosis in blasts from patient #1and patient #2,respectively.Studies involving blasts from two additional patients yielded essentially identical results (data not shown).These events were accompanied by pronounced PARP cleavage (Fig.4D ).Notably,a marked decrease in Mcl-1protein levels and in ERK phosphorylation was also observed in both samples,whereas the total ERK1/2level remained unaffected.Thus,exposure to BAY 43-9006leads to a marked increase in lethality in primary human AML blasts in association with diminished ERK phos-phorylation and Mcl-1down-regulation,analogous to findings in con-tinuously cultured leukemia cell lines.Mcl-1Down-regulation by BAY 43-9006Is Independent of MEK/ERK,p90RSK,mTOR,and p70S6K Inactivation —Previous studies have indi-cated that interruption of the Raf/MEK/ERK pathway by the MEK inhibitor PD98059decreases basal levels of Mcl-1(42)and attenuates Mcl-1protein accumulation in response to various cytokines,including epidermal growth factor,interleukin-5,and stem cell factor (33).Con-sequently,the possibility that BAY 43-9006down-regulates Mcl-1through inhibition of the Raf/MEK/ERK pathway appeared plausible.To test this possibility,Jurkat cells (MT6)inducibly expressing a con-stitutively active MEK1under the control of a doxycycline-responsive promoter was employed.Western blot analysis revealed that addition of doxycycline resulted in a substantial increase in expression of constitu-tively active MEK1and phospho-ERK1/2in both control and BAY 43-9006-treated cells (Fig.5A ).However,exposure to BAY 43-9006resulted in equivalent decreases in Mcl-1expression in the absence or the presence of doxycycline.Similar results were obtained in two addi-tional MEK1-inducible clones and in U937cells stably expressingcon-FIGURE 3.BAY 43-9006caused a time-and dose-dependant but caspase-independ-ent down-regulation of Mcl-1.A ,U937cells were exposed to 10␮M BAY 43-9006for the indicated periods after which protein lysates were prepared and subjected to Western blot analysis as described under “Materials and Methods”using the indicated antibodies.B ,U937cells were treated with BAY 43-9006at the designated concentration for 4h,after which cells lysates were prepared and subjected to Western blot analysis using anti-Mcl-1antibodies.C ,U937cells were treated with 10␮M BAY 43-9006in the presence or absence of 25␮M z-VAD-FMK for 4and 8h,after which Mcl-1protein level was monitored by Western blot analysis.For all studies,each lane was loaded with 20␮g of protein;the blots were subsequently reprobed with antibody directed against tubulin to control for equal loading and transfer of proteins.Arrows indicate cleavage fragments (CF ).Two additional experiments yielded similarresults.FIGURE 4.Exposure to BAY 43-9006results in a marked increase in apoptosis in association with down-regulation of Mcl-1and inactivation of ERK1/2in K562cells as well as in primary human leukemia blasts.A ,K562cells were exposed to the indi-cated concentrations of BAY 43-9006for 24h after which the extent of apoptosis was determined using annexin V staining.B ,K562cells were treated with 6␮M BAY 43-9006for the designated intervals after which cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies.C ,leukemia blasts were isolated as described under “Materials and Methods”from the peripheral blood of two patients with AML (FAB classification M2),exposed to the designated concentration of BAY 43-9006for 24h,after which the extent of apoptosis was determined using annexin V analysis.Each sample was performed in duplicate,and the means ϮS.D.were calculated.D ,alternatively,blasts were lysed after 6h of treatment and subjected to Western blot analysis to monitor the level of phosphorylated ERK1/2,total ERK1/2,and Mcl-1proteins.The protein lysates were also prepared after 24h of exposure to monitor PARP cleavage by Western blot analysis.Total ERK in these experiments also serves as control for loading and transfer of proteins.BAY 43-9006-mediated Apoptosis Involves Mcl-1by guest on September 11, 2016/Downloaded fromstitutively active MEK1(43)(data not shown).Effects of BAY 43-9006on p90RSK and p70S6K were also examined.As shown in Fig.5B ,a time-course study revealed that BAY 43-9006did not have a major effect on p90RSK phosphorylation.Expression and phosphorylation of p70S6K were also unperturbed by treatment with BAY 43-9006.Con-sistent with these findings,induction of constitutively active MEK1resulted in increased phosphorylation of p90RSK (Fig.5C ),whereas BAY 43-9006continued to down-regulate Mcl-1(Fig.5A ).In addition,pretreatment of cells with the MEK inhibitor U0126(10␮M ),which clearly decreased ERK and p90RSK phosphorylation,resulted in only a very modest decline in Mcl-1protein levels (Fig.5D ).Notably,treat-ment of cells with the mTOR inhibitor rapamycin (20n M )also failed to decrease Mcl-1protein levels despite markedly diminishing phospho-rylation of p70S6kinase (Fig.5E ).Together,these findings argue against the possibility that BAY 43-9006down-regulates Mcl-1through inhibi-tion of Raf/MEK/ERK1/2,p90RSK,mTOR,or p70S6K.Finally,activation of the stress-activated protein kinases p38and JNK were examined.Consistent with previous studies (19),BAY 43-9006induced a marked decrease in p38phosphorylation without affecting total protein levels.This was associated with an increase in JNK phos-phorylation (Fig.5F ).However,inhibition of p38or JNK activation with SB202190or SP600125,respectively,did not result in major change in Mcl-1protein levels (data not shown),arguing against the possibility that BAY 43-9006down-regulates Mcl-1through a p38-or JNK-de-pendent mechanism.Mcl-1Down-regulation by BAY 43-9006Is Largely Independent of Transcription —Mcl-1is known to be regulated at the transcriptional level by a variety of transcription factors,including E2F1,CREB,andETS (44–46).Consequently,Mcl-1promoter activity was monitored using a reporter gene assay.As shown in Fig.6A ,treatment with BAY 43-9006had no significant effect on luciferase driven by an Mcl-1pro-moter (p Ͼ0.05).Essentially equivalent results were obtained in K562cells (data not shown).These findings argue against the possibility that BAY 43-9006inhibits Mcl-1transcription.Next,Mcl-1mRNA was quantified using real-time PCR.Notably,treatment of U937cells with BAY 43-9006resulted in a very modest and transient decline in Mcl-1mRNA levels after 2h of treatment (Fig.6B ).After 4h of treatment,Mcl-1mRNA returned to near basal levels.In addition,inhibition of transcription using actinomycin D (5␮g/ml)resulted in a decrease in Mcl-1protein levels,and co-treatment with BAY 43-9006resulted in a further decline (Fig.6C ),suggesting an alternative,transcription-inde-pendent mechanism of Mcl-1down-regulation by this agent.Collec-tively,these and the preceding findings are most consistent with the notion that BAY 43-9006down-regulates Mcl-1protein at either the translational or at the post-translational level.BAY 43-9006Down-regulates Mcl-1through Inhibition of Translation —Because Mcl-1protein has a short half-life and is known to be degraded by the proteasome system (47,48),we first investigated whether Mcl-1ubiquitination was modified by treatment with BAY 43-9006.Immunoprecipitation followed by Western blot analysis (Fig.7A )revealed no major changes in Mcl-1ubiquitination in BAY43-9006-FIGURE 5.Mcl-1down-regulation by BAY 43-9006is independent of MEK/ERK,p90RSK,mTOR,and p70S6K inactivation.A and C ,Jurkat cells (MT6)inducibly express-ing constitutively active HA-tagged MEK1were left untreated or treated for 24h with 2␮g/ml doxycycline and then exposed to 10␮M BAY 43-9006for an additional 4h.Cells were then analyzed for HA-MEK1,phospho-ERK1/2,phospho-p90RSK,and Mcl-1expres-sion by Western blot.B ,D ,E ,and F ,U937cells were treated with 10␮M BAY 43-9006(B and F ),U0126(D ),or 20n M rapamycin (E )for the designated intervals after which protein lysates were prepared and subjected to Western blot using indicated antibodies.For each experiment,at least two additional studies yielded equivalentresults.FIGURE 6.BAY 43-9006does not substantially diminish Mcl-1mRNA levels or Mcl-1promoter activity.A ,Jurkat cells were cotransfected with (Ϫ203/ϩ10-Mcl-1-pGL2)or pGL2-Basic and pRL-TK-luc plasmids.Cells were incubated for 6h and then treated with BAY 43-9006for an additional 20h after which activity of firefly and Renilla luciferase was monitored as described under “Materials and Methods.”Values for firefly luciferase activ-ity were normalized to those obtained for Renilla luciferase activity,after which values obtained for (Ϫ203/ϩ10-Mcl-1-pGL2)transfected cells were divided by the correspond-ing values obtained for pGL2-Basic transfected cells.The graph shown represents the average of four experiments performed in triplicate ϮS.D.*,not significantly different from values for the control (p Ͼ0.05).B ,U937cells were treated with BAY 43-9006at the designated intervals,after which total RNA were isolated and Mcl-1mRNA were quanti-fied using real-time PCR as described under “Materials and Methods.”Values represent the means ϮS.D.for three separate experiments performed in triplicate and are expressed as a -fold increase relative to the non-treated cells.C ,U937cells were treated with 5␮g/ml actinomycin D (Act D )in the presence or absence of BAY 43-9006(10␮M )for 2and 4h,after which cells lysates were prepared and subjected to Western blot analysis to monitor Mcl-1protein level.Each lane was loaded with 20␮g of protein;blots were subsequently reprobed with antibodies directed against tubulin to control for equal loading and transfer of proteins;two additional experiments yielded similar results.BAY 43-9006-mediated Apoptosis Involves Mcl-1by guest on September 11, 2016/Downloaded from。