c-myc^inducedchemosensitizationismediatedbysuppression ... · myc clones were routinely cultured in...

c-Myc^ Induced Chemosensitization Is Mediated by Suppressionof Cyclin D1Expression and Nuclear Factor-KBActivity inPancreatic Cancer CellsHector Biliran, Jr., Sanjeev Banerjee, ArchanaThakur, Fazlul H. Sarkar, Aliccia Bollig, Fakhara Ahmed,JiushengWu,Yuan Sun, andJoshua D. Liao

Abstract Purpose: Pancreatic cancer is a highly aggressive disease that remains refractory to various che-motherapeutic agents. Because the proto-oncogene c-myc can modulate apoptosis in responseto cytotoxic insults and is commonly overexpressed in pancreatic cancer, we investigated thevalue of c-myc as a potential modulator of cellular response to various chemotherapeutic agents.Experimental Design: Stable overexpression or small interfering RNA (siRNA)^ mediatedknockdown of c-myc and restoration of cyclin D1were done in the Ela-myc pancreatic tumorcell line. Cell viability after cisplatin treatment of c-myc ^ overexpressing, control, and siRNA-transfected cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and drug-induced apoptosis was measured by DNA fragmentation, sub-G1, andpoly(ADP-ribose) polymerase cleavage analyses. Protein expression profile after cisplatin treat-ment was determined byWestern blotting and DNA binding activity of nuclear factor-nB wasexamined by electrophoretic mobility shift assay.Results: Ectopic overexpression of c-myc in murine and human pancreatic cancer cell lines, Ela-myc and L3.6pl, respectively, resulted in increased sensitivity to cisplatin and other chemothera-peutic drugs. Increased sensitivity to cisplatin in c-myc ^ overexpressing cells was due, in part, tothe marked increase in cisplatin-induced apoptosis. Conversely, down-regulation of c-myc ex-pression in stable c-myc ^ overexpressing cells by c-myc siRNA resulted in decreased sensitivityto cisplatin-induced cell death. These results indicate an important role of c-myc in chemosensi-tivity of pancreatic cancer cells.The c-myc ^ induced cisplatin sensitivity correlatedwith inhibitionof nuclear factor nB activity, which was partially restored by ectopic cyclin D1overexpression.Conclusions: Our results suggest that the c-myc ^ dependent sensitization to chemotherapy-induced apoptosis involves suppression of cyclin D1expression and nuclear factor nB activity.

As seen in a variety of neoplasias, dysregulation of the c-mycproto-oncogene expression is a common event in humanpancreatic cancer. Amplification and overexpression of c-mycgene have been observed in primary pancreatic carcinoma aswell as in metastatic lesions (1–6). In vitro, c-myc geneamplification was evident in the majority of previouslyestablished pancreatic cancer cell lines (7). These data suggestthat this proto-oncogene may be involved in the developmentand progression of this malignancy. A more direct and con-vincing evidence for the role of c-myc in the development ofpancreatic cancer comes from an in vivo study by Sandgren et al.

(8) reporting that mice carrying a c-myc transgene under thecontrol of elastase gene promoter (Ela-myc) develop pancreatictumor at an early age (2-7 months) with 100% penetrance.

Numerous studies have been published addressing theprognostic values of c-myc in various malignancies, but the dataare still largely controversial and confusing. Positive, null, andnegative correlations of c-Myc overexpression or amplificationwith prognosis or patient survival of various types of cancer haveall been reported (9–24). The dual functions of c-Myc(i.e., promotion of both cell proliferation and apoptosis) maybe one main reason for these conflicts because c-Myc–inducedapoptosis may lead to a better prognosis whereas c-Myc–induced proliferation may lead to a poorer outcome (7). Athorough understanding of at what situations c-Myc proteindirects a cell to proliferation and apoptosis is crucial for thetreatment of cancers by manipulating the c-Myc levels. Withregard to the pancreatic cancer, the prognostic value of c-mycremains unexplored.

In this report, we examined whether high levels of c-Myccould modulate the response of pancreatic cancer cells tochemotherapeutic agents. We found that ectopic overexpressionof c-myc in murine Ela-myc and human L3.6pl pancreatic cancercell lines enhanced the sensitivity of the cells o cisplatin andother chemotherapeutic drugs. The c-myc –induced sensitization

Cancer Therapy: Preclinical

Authors’Affiliation: Department of Pathology,Wayne State University School ofMedicine, Detroit, MichiganReceived 7/26/06; revised11/8/06; accepted11/22/06.Grant support: NIH grant R01CA100864 (D.J. Liao).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Joshua D. Liao, Hormel Institute, University of Minnesota,80116th Avenue NE, Austin, MN 55912. Phone: 507-437-9665; Fax: 507-437-9606; E-mail: [email protected].

F2007 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-06-1844

www.aacrjournals.org Clin Cancer Res 2007;13(9) May1, 20072811

was associated with marked induction of cisplatin-inducedapoptosis and concomitant inhibition of cyclin D1 level andnuclear factor nB (NF-nB) activity. Restoration of cyclin D1expression attenuated c-myc –induced cisplatin chemosensitiza-tion but only partially restored NF-nB activity. These resultssuggest that overexpression of c-Myc may sensitize pancreaticcancer cells to several chemotherapeutic agents. Mechanistically,these effects may be due, in part, to the c-Myc inhibition ofNF-nB activity via both c-myc inhibition of cyclin D1 expressionand other cyclin D1–independent mechanisms.

Materials andMethods

Cell culture and transfection. The Ela-mycT1 pancreatic tumor cellline used herein was generated from a pancreatic tumor tissue derivedfrom a mouse carrying a c-myc transgene under the control of theelastase-1 gene promoter (Ela-myc). This cell line is similar to the Ela-myc tumor cell line (designated as Ela-mycT4) previously described(25), but it showed much lower c-Myc and higher cyclin D1 levels thanEla-mycT4 cells and thus is an ideal system for us to manipulate c-myclevel and observe the effect of c-Myc on cyclin D1. In this study, Ela-mycT4 cells were maintained in DMEM supplemented with 10% fetalbovine serum at 37jC in humidified air with 5% CO2. The humanpancreatic cancer cell lines PANC-28, BxPC-3, L3.6pl, and HPAC werealso maintained in DMEM supplemented with 10% fetal bovine serumat 37jC in humidified air with 5% CO2. To generate stable c-myc –overexpressing clones, the pancreatic cancer cell lines Ela-myc andL3.6pl were transfected in a stable manner with the pcDNA3.1c-mycplasmid or the pcDNA3.1Hygro vector control plasmid using Lipofect-amine 2000 per manufacturer’s instructions (Life Technologies). After48 h of incubation, Ela-myc – and L3.6pl-transfected cells were selectedin DMEM containing 100 and 300 Ag/mL hygromycin (Life Technol-ogies), respectively. After 2 to 3 weeks, single independent clones wererandomly isolated and each individual clone was plated separately.After clonal expansion, cells from each independent clone were testedfor c-myc expression by immonoblotting and reverse transcription-PCR(RT-PCR). The c-myc –overexpressing (M) and vector control (H) Ela-myc clones were routinely cultured in DMEM containing 10% fetalbovine serum in the presence of 50 Ag/mL hygromycin at 37jC inhumidified air with 5% CO2. The c-myc –overexpressing (CM) andvector control (CH) L3.6pl clones were routinely cultured in DMEMcontaining 10% fetal bovine serum in the presence of 150 Ag/mLhygromycin at 37jC in humidified air with 5% CO2.

To ectopically restore cyclin D1 expression in c-myc –overexpressingcells, the M8 clone was stably transfected with the pcDNA3.CCND1plasmid or the pcDNA3.1Neo vector control plasmid using Lipofect-amine 2000 as prescribed by the manufacturer (Life Technologies).Forty-eight hours posttransfection, medium was replaced with DMEMcontaining 500 Ag/mL G418 (Life Technologies). G418-resistantcolonies were selected and screened for cyclin D1 expression byimmunoblot analysis. The M8/Neo1 and M8/D1 clones were subse-quently maintained in DMEM with 10% fetal bovine serum containing250 Ag/mL G418 and 50 Ag/mL hygromycin.

Protein extraction and Western blotting. Whole or fractionated(nuclear and cytoplasmic) proteins (25) were resolved by SDS-PAGEand electrophoretically transferred to a nitrocellulose membrane. Afterblocking with 5% nonfat milk in PBS-Tween 20 for 1 h at roomtemperature, the membranes were blotted with primary antibody,followed by incubation with a peroxidase-conjugated secondaryantibody. Bound antibodies were visualized by enhanced chemilumi-nescence (Pierce). Densitometric analysis was done by scanningimmunoblots and quantitating protein bands using the AlphaEaseFCsoftware (Alpha Innotech). The primary antibodies used were rabbitpolyclonal antibody to cyclin D1 (Santa Cruz Biotechnology, Inc.;1:1,000 dilution), rabbit polyclonal antibody to c-myc (Santa Cruz

Biotechnology; 1:1,000), rabbit polyclonal antibody to cyclin E (SantaCruz Biotechnology; 1:500), rabbit polyclonal antibody to cyclin A(Santa Cruz Biotechnology; 1:1,000), mouse monoclonal antibody tocyclin B1 (Santa Cruz Biotechnology; 1:1,000), mouse monoclonalantibody to actin (Santa Cruz Biotechnology; 1:2,500 dilution), rabbitpolyclonal antibody to poly(ADP-ribose) polymerase (Santa CruzBiotechnology; 1:1,000), and rabbit polyclonal antibody to p65 (SantaCruz Biotechnology; 1:1,000 dilution).

RT-PCR analysis. Total RNA was isolated from exponentiallygrowing cells using the RNeasy Isolation Kit (Qiagen). The extractedRNA (1 Ag) was reverse transcribed with the TaqMan reversetranscriptase in the presence of oligo(dT)15 primer as described bythe manufacturer (Roche, Applied Biosystems). The resulting cDNApreparation was subjected to PCR amplification using an exogenousc-myc primer set with the forward primer (5¶-TAGAAGGCACAGTC-GAGG-3¶) identifying a hygro-specific sequence located upstream of thec-myc cDNA sequence and the reverse primer (5¶-CACCGCCTA-CATCCTGTCCATTCAAGC-3¶) specific to a c-myc exon for 25 cycles.Each PCR cycle included a denaturation step at 94jC for 30 s, a primerannealing step at 55jC for 45 s, and an extension step at 72jC for 45 s.Reactions were done in an Eppendorf AG Mastercycler. Additionalprimers used for PCR were cyclin D1 sense (5¶-CCCTCGGTGTCCTA-CTTCAA-3¶), cyclin D1 antisense (5¶-TGGCATTTTGGAGAGGAAGT-3¶),h-actin sense (5¶-ACGGATTTGGTCGTATTGGG-3¶), and h-actin anti-sense (5¶-TGATTTTGGAGGGATCTCGC-3¶). The PCR products wereanalyzed by electrophoresis on 1% agarose gel containing ethidiumbromide and photographed under UV light.

Cell viability assay. Cell viability was evaluated by 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay asdescribed above. Briefly, 3.0 � 103 cells per well were plated in 100AL of maintenance medium. The next day, the cells were treated withcisplatin according to the experimental design. The cells were thenincubated with 0.2 Ag/mL MTT for 2 h in the dark at 37jC. Afterremoval of the medium, the dye crystals were dissolved in isopropanoland the absorbance was measured at 570 nm with an UltraMultifunctional Microplate Reader (Tecan). Three independent experi-ments were done in quadruplicate wells.

Clonogenic survival assay. The effects of c-myc overexpression andcisplatin on long-term growth of Ela-myc cells were assessed by clonogenicassays. Briefly, cells were plated at a density of 2.0 � 105 in a 24-well plateand allowed to adhere overnight. The cells were then treated with escalatingconcentrations of cisplatin (0.5, 1.0, 2.5, and 5.0 Amol/L). Twelve hoursafter cisplatin addition, cells were trypsinized, counted, and reseeded at alow density (10,000 in a 10-cm dish) in triplicate. Medium was replacedevery 3 days and the cells were allowed to grow for 10 days. The colonieswere fixed with methanol-acetic acid (3:1), stained with 1% crystal violet,and counted. The survival fraction was determined by dividing the numberof colonies formed in the presence of the drugs by the number of coloniesformed in the untreated control cells. Each dose was done in triplicate andthe experiments were done at least thrice.

Apoptosis analysis. Cells were subjected to proapoptotic conditions asspecified in the text and figure legends. Both attached and floating cellswere collected and subjected to the following apoptosis assays: (a) Thequantitation of cytoplasmic histone-associated DNA fragments was doneusing the Cell Death Detection ELISA Kit (Roche). Briefly, cells were lysedand cell lysates were overlaid and incubated in microtiter plate modulescoated with anti-histone antibody. Samples were subsequently incubatedwith anti–DNA peroxidase followed by color development with (2,2¶-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid substrate)). The absor-bance of the samples was determined with the Ultra MultifunctionalMicroplate Reader (Tecan) at 405 nm. (b) The percentage of cells withsub-G0-G1 DNA content was determined by a Coulter EPICS 753 flowcytometer following staining with propidium iodide using a ModFit 5.2computer program. (c) The proportion of Annexin V–positive cells wasdetermined by Annexin V-FITC apoptosis detection kit according to themanufacturer’s instructions (BD Biosciences PharMingen). Briefly, cellswere labeled with FITC-conjugated Annexin V and propidium iodidewithout permeabilization and subsequently analyzed by a Coulter EPICS


www.aacrjournals.orgClin Cancer Res 2007;13(9) May1, 2007 2812

753 flow cytometer. Propidium iodide–positive, Annexin V–positive(necrotic) cells were excluded from analysis. (d) The cleavage ofpoly(ADP-ribose) polymerase was examined by immunoblotting asdescribed above.

Small interfering RNA studies. Chemically synthesized murine

c-myc –specific small interfering RNA (siRNA), cyclin D1–specific

siRNA, and the control siRNA (sense strand, 5¶-CGAACUCACUGGU-CUGACCdtdt-3 ¶; antisense strand, 5 ¶-GGUCAGACCAGUGA-

GUUCGdtdt-3¶) were purchased from Santa Cruz Biotechnology. The

second set of mouse c-myc –specific siRNA and cyclin D1–specificsiRNA were purchased from Ambion and Qiagen, respectively. For

siRNA transfection, 5 � 105 cells per well were plated in six-well plates

and transfected with 100 pmol of the siRNA duplex for 48 h usingLipofectamine 2000 as a transfection mediator according to the

manufacturer’s instructions (Life Technologies). To assess the effect of

c-myc down-regulation on cisplatin-induced apoptosis, untransfected,

control siRNA–transfected, and c-myc siRNA–transfected M8 cellswere plated in 24-well plates, allowed to recover for 24 h in complete

medium, and then treated with 5.0 Amol/L cisplatin for 24 h. To

determine the effect of cyclin D1 down-regulation on cisplatin-mediated apoptosis, untransfected, control siRNA–transfected, and

cyclin D1 siRNA–transfected M8/D1 cells were plated in 24-well

plates, allowed to recover for 24 h, and treated with cisplatin. The cellswere subsequently subjected to apoptotic assays as described above.

Electrophoretic mobility shift assay. H1, M8/Neo1, and M8/D1 cellswere incubated in the presence or absence of 2.5 Amol/L cisplatin for24 h. Following treatment, the cells were collected and nuclear proteinswere extracted as previously described (25). Electrophoretic mobilityshift assay was done by incubating 5 Ag of nuclear extract with IRDye-700–labeled NF-nB oligonucleotide. The incubation mixture included2 Ag of poly(deoxyinosinic-deoxycytidylic acid) in a binding buffer.The DNA-protein complex formed was separated from free oligonu-cleotide on 8.0% native polyacralyamide gel using buffer containing50 mmol/L Tris, 200 mmol/L glycine (pH 8.5), and 1 mmol/L EDTA,and then visualized by Odyssey Infrared Imaging System usingOdyssey Software Release 1.1. For loading control, 10 Ag of nuclearprotein from each sample were subjected to Western immunoblottingfor retinoblastoma protein. To identify proteins in the DNA-proteincomplex, a supershift experiment was done with polyclonal p65subunit–specific antibody. The anti–cyclin D1 antibody was used asthe nonspecific, negative control antibody. Briefly, nuclear proteinswere incubated for 30 min with different antibodies and assayed forsupershift by gel shift assay as described above. The anti-p65 and anti–cyclin D1 antibodies were purchased from Santa Cruz Biotechnology.

ELISA-based nuclear factor-kB assay. In addition to electrophoretic

mobility shift assay, an ELISA-based kit was used for quantitative

detection of NF-nB activity (EZ-Detect Transcription Factor Kits for

NF-nB p50; Pierce). For each sample, 5 Ag of nuclear extract were used

according to the instructions of the manufacturer. For the detection of

activated NF-nB, antibodies against the p50 subunit were used,

Fig. 1. c-myc overexpression increases sensitivity of Ela-myc pancreatic cancercells to cisplatin by enhancing cisplatin-induced apoptosis.A,Western blot analysisof c-myc. Serum-starved vector control (H1and H5) and c-myc ^ overexpressing(M4 andM8) clones were collected, and total cellular protein (50 Ag) wassubjected to immunoblot analysis with a specific anti ^ c-myc antibody.Themembrane was reprobed with an anti ^h-actin antibody to confirm equal loading.Shown below each blot is densitometric quantification as ratio of c-Myc protein toh-actin. B, cell viability of vector control and c-myc ^ overexpressing clonesfollowing cisplatin treatment was determined by the MTTassay as described inMaterials andMethods. Percent cell survival comparedwith correspondinguntreated controls. Columns, mean of triplicate determinations of three separateexperiments; bars SE. *, P < 0.05, compared with H1and H5 control clones.C, clonogenic survival assayof vector control and c-myc stable clones on treatmentwith cisplatin.The percent survival is plotted against the drug concentration asindicated (columns, mean; bars, SE).D, vector control and c-myc ^ overexpressingcells were incubated in the absence or presence of 5 Amol/L cisplatin for 24 h.Following drug treatment, floating and attached cells were collected and subjectedto sub-G1DNA content analysis (top) andWestern blotting of poly(ADP-ribose)polymerase (PARP ; bottom). For poly(ADP-ribose) polymerase cleavageimmunoblotting, 50 Ag of protein were subjected to 7% SDS-PAGE andimmunoblotted with an anti ^ poly(ADP-ribose) polymerase antibody.Themembrane was reprobed with anti ^h-actin antibody to ensure equal proteinloading. C andD, points and columns, mean of at least three independentexperiments; bars, SE. *, P < 0.01, compared with vector control clones.

Inhibition of c-myc ^Dependent Apoptosis by Cyclin D1


followed by a secondary horseradish peroxidase–conjugated antibody.

A chemiluminescent substrate using SuperSignal Technology was

added to the wells and the resulting signal was detected using a

luminometer (Tecan). The wild-type NF-nB competitor duplex was

used according to the instructions of the manufacturer to ensure signal

specificity.Immunofluorescent staining for NF-kB p65 localization. Cells were

cultured on coverslips and treated with or without 2.5 Amol/L cisplatinfor 24 h. The cells were then fixed with 10% formalin for 10 min,washed once with PBS, and stored at 4jC. For staining, coverslips weretreated with 0.5% Triton X-100 in PBS for 10 min, washed with 0.05%Triton X-100 in PBS, and incubated at 37jC for 2.5 h with antibody(Santa Cruz Biotechnology) in PBS with 0.05% Triton X-100. Afterthree washes in the same buffer, cells were incubated with FITC-conjugated anti-rabbit antibody at 1:100 (Molecular Probes), alongwith 0.1 Ag/mL 4¶,6-diamidino-2-phenylindole (Sigma) in the samebuffer for 1 h at 37jC. After three washes and air-drying, coverslipswere mounted with antifade (Molecular Probes). Images were capturedon a Zeiss 510 laser scanning inverted confocal microscope systemusing 488- and 364-nm laser wavelengths to detect the FITC and 4¶,6-diamidino-2-phenylindole stains, respectively.

Statistics. Statistical analysis was done with GraphPad PrismSoftware (El Camino Real). Results are expressed as mean F SE andStudent’s t test was used for statistical analysis. P < 0.05 was consideredto be significant.

Results

Ela-mycT1 mouse pancreatic cancer cells were established tooverexpress c-myc. The Ela-mycT1 mouse pancreatic cancer cellline was stably transfected with an exogenous c-myc cDNA orits empty vector to force high levels of c-myc expression.Following transfection and selection for hygromycin resistance,several vector control and c-myc –overexpressing clones werescreened and selected (Fig. 1). Because the endogenous c-mycexpression is highly dependent on serum and mitogenic factors,the stable clonal lines were first subjected to serum-starvedconditions and subsequently screened for exogenous c-mycexpression. As shown in Fig. 1A, two different c-myc clones, M4and M8, showed elevated levels of c-Myc protein than thevector control clones, H1 and H5. RT-PCR analysis withprimers for exogenous c-myc showed a specific 250-bp productthat was detected only in c-myc stable clonal lines (M1, M4, andM8) and not in vector control cells, thus confirming theexpression of exogenous c-myc gene in c-myc –overexpressingclones (data not shown). Based on their high c-myc expressionlevels, the M4 and M8 clones were used for subsequent studies.Overexpression of c-myc in Ela-mycT1 cells enhanced chemo-

sensitivity by augmenting drug-induced apoptosis. The MTTanalysis showed that the c-myc –overexpressing clones exhibited

significantly decreased cell viability as compared with vectorcontrol clones on treatment with cisplatin (Fig. 1B). At 5 and10 Amol/L of cisplatin, the cell viability of c-myc clones wasdecreased by 20% to 50% (P < 0.05) compared with controlclones. Similarly, treatment of vector control and c-myc –overexpressing clones with two other drugs, gemcitabine andhistone deactylase inhibitor FK228, showed the same response.Microscopically cisplatin-treated c-myc –overexpressing clonesdisplayed cell shrinkage and detachment with significantnumber of floating cells. The clonogenic survival assay showedthat the c-myc –overexpressing clones were highly sensitive tocolony inhibition after treatment with various doses of cisplatin.At the concentration range of 1.0 to 5.0 Amol/L, the c-myc –overexpressing clones displayed significantly decreased survivalof f40% to 50% (P < 0.01) compared with vector controlcells (Fig. 1C). Consistent with the chemosensitive phenotypeof c-myc clones, the cisplatin treatment of c-myc clones resultedin a marked increase in cisplatin-induced apoptosis, as judgedby increased percentage of sub-G1 cells and increased proteo-lytic cleavage of poly(ADP-ribose) polymerase (Fig. 1D) ascompared with vector control cells. Furthermore, the cisplatin-treated c-myc clones exhibited increased cytoplasmic histone-associated DNA fragmentation and Annexin V positive staining(data not shown) as compared with control cells, thus con-firming the enhanced apoptotic sensitivity of c-myc clones tocisplatin.

We then down-regulated c-myc expression in the M8 clone ofc-myc stable cells using two different mouse c-myc –specificsiRNAs (Fig. 2A). c-myc siRNA–treated M8 cells exhibiteddecreased percentage of sub-G1 DNA population (Fig. 2A) andAnnexin V–positive cells (Fig. 2A) compared with controlsiRNA–treated or untransfected cells on cisplatin treatment.These results indicate that elevated c-myc expression mayaccount for enhanced susceptibility to cisplatin-induced apo-ptosis in c-myc –overexpressing Ela-myc pancreatic cancer cells.Elevated c-myc expression in human pancreatic cancer cells

enhances chemosensitivity and drug-induced apoptosis. Thelevel of endogenous c-myc expression in a panel of humanpancreatic cancer cell lines was examined by Western blotanalysis (Fig. 2B). We observed that the human PANC-28 cellsthat exhibited the highest basal level of c-myc were highlysensitive to either 2-methoxyestradiol (Fig. 2B) or FK228(Fig. 2B), two new chemotherapeutic agents that are currentlyused for clinical trials for several types of cancer (26, 27). ThePANC-28 cells showed the highest percentage of growthinhibition even under low drug concentrations (1 Amol/L 2-methoxyestradiol and 10 nmol/L FK228) compared with otherhuman pancreatic cancer cell lines.

Fig. 2. Down-regulation of c-myc in c-myc ^ overexpressing Ela-myc cells enhances resistance to cisplatin-induced apoptosis. A, the c-myc ^ overexpressing M8 cellswere transfectedwith either c-myc siRNA or control siRNA. After 48 h, the cells were collected and total cellular protein (50 Ag) was subjected to immunoblot analysis witha specific anti ^ c-myc antibody.The membrane was reprobed with an anti ^h-actin antibody to confirm equal loading (top). Untransfected, control siRNA ^ transfected,and c-myc siRNA ^ transfected cells were subsequently treated with cisplatin for 24 h and subjected to sub-G0-G1DNA content analysis (middle) and AnnexinV staining(bottom). Columns, mean of at least three independent experiments; bars, SE. *, P < 0.05, comparedwith control siRNA ^ transfected M8 cells.B, the relationship betweenc-myc levels and chemosensitivity in human pancreatic cancer cells.Western blot of c-myc in a panel of human pancreatic cancer cell lines (top).The cell viability of severalhuman pancreatic cancer cell lines following treatment with 2-methoxyestradiol (2-ME ;middle) or FK228 (bottom) was determined by the MTTassay. Percent growthinhibition compared with corresponding untreated controls. Columns, mean of triplicate determinations of three separate experiments; bars, SE. *, P < 0.01, compared withBxPC-3 cells. C, top,Western blot analysis of c-myc in vector control (CH5 and CH6) and c-myc ^ overexpressing (CH18 and CH19) L3.6pl stable clones. Bottom, cellviability of vector control and c-myc ^ overexpressing clones following treatment with cisplatin was determined by theMTTassay. Percent cell survival compared withcorresponding untreated controls. Columns, mean of triplicate determinations of three separate experiments; bars, SE. *, P < 0.05, compared with CH5 and CH6 controlclones.D, vector control and c-myc ^ overexpressing cells were incubated in the absence or presence of 20 Amol/L cisplatin for 24 h. Following drug treatment, floating andattached cells were collected and subjected to sub-G1DNA content analysis (top) and AnnexinV staining (bottom). *, P < 0.01, compared with vector control clones.



To further examine whether elevated c-myc can modulatechemosensitivity of human pancreatic cancer cells, the humanL3.6pl cells, which express low basal level of c-myc (Fig. 2B),were stably transfected with an exogenous c-myc cDNA.Following selection for hygromycin resistance, two differentL3.6pl c-myc clones, CM18 and CM19, showed elevated levelsof c-Myc protein f2- to 3-fold higher than the vector controlclones, CH6 and CH5 (Fig. 2C). The MTT analysis showed thatthe c-myc –overexpressing L3.6pl clones exhibited significantlydecreased cell viability as compared with vector control cloneson treatment with cisplatin (Fig. 2C). At 20 Amol/L cisplatin,the cell viability of c-myc clones was decreased by 20% to 30%(P < 0.05) compared with control clones. Treatment of vectorcontrol and c-myc –overexpressing clones with FK228 similarlyshowed the same response (data not shown). Consistent withtheir chemosensitive phenotype, the c-myc –overexpressingL3.6pl clones exhibited a marked increase in cisplatin-inducedapoptosis, as judged by the increased percentage of sub-G1 (Fig.2D) and Annexin V–positive (Fig. 2D) cells, as compared withcontrol clones. These results indicate that elevated c-mycexpression in the L3.6pl cells increases cisplatin sensitivity, atleast in part, by sensitizing cells to cisplatin-induced apoptosis.c-Myc–dependent sensitization to cisplatin-induced cell death is

partly mediated by suppression of cyclin D1. Because we recentlyshowed that cyclin D1 imposed resistance of pancreatic cells tochemotherapeutic agents (25), we wondered whether cyclin D1was involved in c-Myc–rendered drug sensitivity. We foundthat the mRNA and protein levels of cyclin D1 weredramatically reduced in c-myc –overexpressing Ela-myc stableclones M4 and M8, as compared with the corresponding vector

clones (H1 and H5; Fig. 3A and B). Cyclin E level was alsoslightly decreased, cyclin A level was slightly increased, andcyclin B1 level was unchanged (Fig. 3A). We ectopicallyrestored cyclin D1 expression in the M8 clone of c-myc –overexpressing Ela-myc cells by stable transfection withpcDNA3.1cyclin D1 or empty vector construct. The resultantM8/D1 clones expressed cyclin D1 f3- to 5-fold higher thanthe corresponding vector control clones (M8/Neo1) whereasthe expression levels of c-myc remained unchanged (Fig. 4A).When treated with either 2.5 or 5 Amol/L cisplatin for 24 h andsubjected to sub-G1 analysis (Fig. 4B) and Annexin V staining(Fig. 4C), the M8/D1 cells (M8/D1.4 clone) displayed lowerapoptotic frequency as compared with M8/Neo1 cells. Analysesof other M8/D1 clones showed similar data [Fig. 4B (bottom)and C (bottom)], confirming that cyclin D1 overexpressionconferred enhanced resistance to cisplatin-induced cell death inthese cells. Further, we depleted cyclin D1 in M8/D1 cells viasiRNA strategy (Fig. 4D). Treatment with cisplatin for24 h significantly enhanced the drug-induced cell death incyclin D1 siRNA–transfected M8/D1.4 cells, as evidenced byincreased amount of cytoplasmic histone-associated DNAfragments (Fig. 4D, middle) and increased percentage of sub-G1 DNA population (Fig. 4D, bottom). These results suggest thatcyclin D1 overexpression may account for enhanced cisplatinresistance in M8/D1 cells. Interestingly, restoration of cyclin D1in M8/D1 cells also inhibited the proliferative advantageconferred by c-myc (data not shown).NF-kB activity was decreased in c-myc–expressing cells and

was only partially restored by restoration of cyclin D1. As shownby both electrophoretic mobility shift assay (Fig. 5A) and anELISA-based NF-nB DNA binding activity assay (Fig. 5A,bottom), the c-myc –overexpressing M8 cells exhibited decreasedNF-nB DNA binding activity in the presence or absence ofcisplatin as compared with the vector control clone (H1). Incontrast, the stable M8/D1 cells exhibited a higher basal andcisplatin-induced NF-nB activity compared with the M8 cells.Consistent with the electrophoretic mobility shift assay find-ings, immunoblot analysis of cytoplasmic and nuclear extracts(Fig. 5B), as well as confocal microscopy assay (Fig. 5C),showed that the c-myc stable clones exhibited reduced p65nuclear translocation (punctated nuclear staining) comparedwith vector control cells in the presence or absence of cisplatin,in association with increased cytosolic levels of InBa proteinand a concomitant decrease in phospho-InBa (Fig. 5D). Incontrast, M8/D1 cells exhibited a slight increase of NF-nB p65nuclear translocation in the presence or absence of cisplatintreatment compared with M8 cells as evidenced by Western blotand immunocytochemical analysis (Fig. 5D). Interestingly, nosignificant differences in the steady-state levels of cytosolic InBaand phospho-InBa proteins were observed between M8 andM8/D1 cells.

The protein levels of Bcl-2 and Bcl-xl, two NF-nB down-stream antiapoptotic effectors that are known apoptotic targetsof c-myc (11, 12, 19), were significantly reduced in theuntreated as well as cisplatin-treated c-myc stable M8 cellscompared with that of control clonal line, whereas the stableM8/D1 cells showed enhanced levels of these proteins in thepresence or absence of cisplatin. On the other hand, thesteady-state levels of Bax protein, a proapoptotic Bcl-2 familymember, were markedly induced in both untreated andcisplatin-treated c-myc –overexpressing (M8/Neo1) cells and

Fig. 3. Effect ofc-myc overexpressionon the expressionof cyclinsD1, A, B1, and E.A, total cell lysates were prepared from exponentially growing vector control(H1and H5) and c-myc ^ overexpressing Ela-myc clones (M1, M4, and M8) andsubjected toWestern blot analysis of different cyclins with h-actin shown as aloading control. B, expression of cyclin D1by RT-PCR analysis in vector control andc-myc ^ overexpressing clones.Total RNA (1 Ag) from control and c-myc cloneswas analyzed by RT-PCR with primers for cyclin D1and h-actin. RT-PCR produceda 300-bp cyclin D1fragment and a 200-bp actin fragment.



Fig. 4. Ectopic cyclin D1inhibits cisplatin-mediated apoptosis in c-myc ^ overexpressing cells. A, exponentially growing cultures of M8, M8/Neo1, and M8/D1Ela-mycclones were lysed and total cellular protein (50 Ag)was subjected to immunoblotting analysis with anti ^ cyclin D1or anti ^ c-myc antibody. h-Actin is shown as a control forprotein loading.B andC, M8,M8/Neo1, andM8/D1cells were cultured inmaintenancemediumwith or without cisplatin for 24 h and subjected to sub-G1DNA and AnnexinVapoptotic assays. B, top, representative fluorescence-activated cell sorting profile of M8/Neo1and M8/D1.4 cells illustrating the percentage of sub-G1cells. Bottom,columns, mean of at least three independent experiments; bars, SE. C, top, representative density plots of propidium iodide labeling versus AnnexinV-FITC staining inM8/Neo1and M8/D1.4 cells showing the proportion of propidium iodide ^ negative, AnnexinV ^ positive cells. Bottom, columns, mean of at least three independentexperiments; bars, SE. *, P < 0.05; **, P < 0.01. D, siRNA-directed suppression of cyclin D1sensitizes M8/D1cells to cisplatin-induced apoptosis.Western blot analysis ofcyclin D1after M8/D1.4 cells were transfected with either cyclin D1siRNAs or control siRNA. After 48 h, the cells were collected and total cellular protein (50 Ag) wassubjected to immunoblotting analysis with a specific anti ^ cyclin D1antibody.The membrane was reprobed with an anti ^h-actin antibody to confirm equal loading (top).Untransfected, control siRNA ^ transfected, and cyclin D1^ specific siRNA ^ transfected M8/D1.4 cells were treated with 5.0 Amol/L cisplatin for 24 h, and subjected toCell Death Detection ELISA (middle) and sub-G1DNA (bottom) apoptotic assays. Columns, meanof at least three independent experiments; bars, SE. *, P < 0.01, comparedwith control siRNA ^ transfected M8/D1.4 cells.



were barely detectable in vector control cells before and aftercisplatin treatment. This up-regulation of Bax protein was notsignificantly altered in M8/D1 cells.

Discussion

c-Myc exhibits a dual function in stimulation of cellproliferation and apoptosis. In this study, we showed thatectopic overexpression of c-myc in pancreatic cancer cellsresults in a marked increase in chemotherapy-induced celldeath. This observation is similar to what was reported incolonic cancer that c-myc amplification sensitized cells to5-fluorouracil–based adjuvant therapy (9, 28), although itconflicts with many other reports that high c-Myc levelsindicates a poor prognosis of several types of cancer. Moreover,we also showed that c-Myc suppresses cyclin D1 expression,which dovetails with the reports in fibroblasts (29–31) andwith our observation of an inverse expression pattern for c-mycand cyclin D1 in mammary tumors derived from MMTV-c-myctransgenic mice (32). In our system, restoration of cyclin D1expression in the c-myc –overexpressing pancreatic cancer cellssignificantly attenuated cisplatin-induced cell death, whereasdown-regulation of cyclin D1 in the c-myc/cyclin D1 stableclones by siRNA mitigated the cyclin D1–induced resistance tocisplatin. These findings, together with the study showing thatcyclin D1 rescued fibroblasts from c-myc –dependent apoptoticcell death (33), suggest the possibility that suppression of

cyclin D1 is a downstream event underlying c-myc –inducedsensitivity to chemotherapeutic agents, and thus may play amechanistic role in c-myc –dependent cell death.

Another novel finding of our study is that c-Myc inhibitsNF-nB activity and the expression of its downstream antiapop-totic effectors, Bcl-2 and Bcl-xl, in pancreatic cancer cells.Mechanistically, the inhibition of NF-nB is related to itsinterference with the nuclear translocation of the p65 NF-nBsubunit. Our observation of increased levels of cytoplasmic InBaprotein in c-myc –overexpressing cells presumably resulted inenhanced cytoplasmic sequestration and reduced nucleartranslocation of p65. Furthermore, the reduced phosphorylationof InBa in c-myc –overexpressing cells, as evidenced by lowlevels of phospho-InBa, suggests that this endogenous NF-nBinhibitor is subjected to less proteolytic degradation. BecauseNF-nB is a well-known survival factor for cancer cells, partly viaits stimulation of Bcl-2 and Bcl-xl and suppression of Bax, c-Mycinhibition of NF-nB may be a mechanism underlying theobserved sensitization of pancreatic cancer cells to chemother-apeutic agents by a c-myc –dependent process (Fig. 6A).However, the question remains about the mechanistic role ofcyclin D1 in relation to c-Myc and NF-nB during cell deathobserved in our studies (Fig. 6A). Whereas it is well known thatNF-nB can induce cyclin D1 in other systems (34, 35), we haverecently found that cyclin D1 can also induce NF-nB activity inpancreatic cancer cells (25). Consistent with cyclin D1 as adownstream target of NF-nB, the suppression of cyclin D1 in

Fig. 6. Model illustrating how c-myc and itsdownstream effectors (NF-nB and cyclin D1)may affect pancreatic cancer cell survival andchemosensitivity. A, overexpression of c-myc mayinhibit cyclin D1expression andNF-nBactivity, leadingto enhanced apoptotic potential and decreased cellsurvival. Concomitant overexpression of cyclin D1,presumably through activation of growth factorsignaling, may override the c-myc ^ mediatedinhibition of cyclin D1expression and NF-nB activityand render cells weakly apoptotic and highly resistantto chemotherapeutic agents. B, pancreatic tumor cellsmay exhibit three different phenotypes in the contextof c-Myc and cyclin D1expression.1, tumor cellsexhibiting overexpression of cyclin D1are highlyresistant to chemotherapy. 2, in certain tumor cells,c-myc overexpression may render these cells highlysensitive to drug-induced cell death due, at least inpart, to its suppression of cyclin D1. 3, some tumorcells with dysregulated c-myc may gain cyclin D1expression, presumably through activation ofgrowth factor signaling, which can antagonizec-Myc ^ imposed drug sensitivity.

Fig. 5. c-myc ^ induced cisplatin sensitization was associated with suppression of NF-nB activity and down-regulation of NF-nB-regulated antiapoptotic Bcl-2 and Bcl-xlexpression. A, H1, M8/Neo1, and M8/D1.4 stable cells were incubated in the presence or absence of 2.5 Amol/L cisplatin for 24 h. Following treatment, floating andattached cells were collected, and nuclear extract was collected and subjected to electrophoretic mobility shift assay; supershift assay was also done to indicate specificityof NF-nB band (top) and ELISA-based NF-nB assay (bottom). B, cytoplasmic and nuclear extracts (50 Ag) prepared from untreated and cisplatin-treated cells were sub-jected toWestern blotting with a specific antibody against the p65 subunit of NF-nB. C, untreated and cisplatin-treated cells were subjected to immunocytochemistry andsubsequently analyzed by confocal microscopy as described in Materials and Methods.D, cytoplasmic extracts (50 Ag) prepared from untreated and cisplatin-treated cellswere subjected toWestern blotting for detection of InBa and phospho-InBa. Immunoblot analysis of h-actin was also done to confirm equal loading.The total cell lysate(50 Ag) fromuntreated and cisplatin-treated cells was also subjected to immunoblottingwith an antibody against Bcl-2, Bcl-xl, or Bax.The detection of h-actinwas used ascontrol for equal protein loading.



c-myc –overexpressing cells may be a consequence of c-Myc–dependent inhibition of NF-nB activity. Alternatively, this down-regulation of cyclin D1 may result from the direct inhibition ofcyclin D1 by c-myc (29–31). Thus, both pathways could be usedby c-Myc in conferring drug sensitivity to pancreatic cancer cells.

The prognostic value of c-myc in various human cancersremains controversial; positive, null, and negative associationsof c-myc amplification or overexpression with tumor aggres-siveness or patient outcome have all been reported (9, 28,36–40). Similar conflicting results have also been reported forcyclin D1 (41–45), just like many other studies exploring theprognostic value of a single gene. Interaction of one protein(e.g., c-Myc) with different proteins under different situationsmay be an underlying mechanism for the conflicting results.The present study, together with our recent report (25),suggests that overexpression of c-myc and cyclin D1, alone orconcomitantly, may affect the sensitivity of cancer cellsdifferentially to chemotherapeutic agents. In the context ofc-Myc and cyclin D1 expression, pancreatic tumor cells mayexhibit three different phenotypes (Fig. 6B): (a) somepancreatic tumor cells exhibit overexpression of cyclin D1,which may happen spontaneously or secondarily due toactivation of k-ras, Notch, or transforming growth factor a/epidermal growth factor receptor signaling, which are knownto occur frequently in human pancreatic cancer, and thus highlevels of cyclin D1 expression may render cancer cells resistantto chemotherapeutic agents. (b) In other pancreatic cancercells, c-myc expression may be increased, which may not onlypromote cell proliferation but also render these cells highlysensitive to drug-induced cell death due, at least in part, to itssuppression of cyclin D1. However, it is noteworthy that thisc-myc –induced chemosensitization may be abrogated by theexistence of other genetic mutations in pancreatic tumor. (c) Athird phenotype may arise by concomitant overexpression of

cyclin D1 and c-myc in such situations wherein transforminggrowth factor a is overexpressed, which overrides thesuppression of cyclin D1 by c-Myc (32). Under this microen-vironment, cyclin D1 can antagonize c-Myc–dependent drugsensitivity. In the two latter scenarios, antagonism of c-mycexpression, which has been widely proposed as a strategy forcancer therapy (46, 47), may actually prevent c-Myc–inducedapoptosis and drug sensitivity and thereby result in adverseeffects.

In summary, we found that ectopic overexpression of c-mycin pancreatic cancer cells resulted in enhanced sensitivity tocisplatin-induced cell death, in association with inhibition ofcyclin D1 expression and NF-nB activity. Restoration of cyclinD1 in c-myc –overexpressing cells significantly attenuates thec-Myc–dependent chemosensitization and partially abrogatesthe inhibition of NF-nB. These results suggest that suppres-sion of NF-nB activity and cyclin D1 expression may beimportant, yet unrecognized, mechanisms underlying thec-Myc–dependent sensitization of cancer cells to chemother-apy. Because many highly malignant tumors often exhibitoverexpression and/or amplification of c-myc and cyclin D1,alone and simultaneously, our findings provide insights intothe potential mechanisms responsible for their seeminglyconflicting behavior in response to chemotherapy, as reflectedby the conflicting prognostic values for c-myc and cyclin D1alone. Based on our results, we suggest that precautionarymeasures should be implemented when considering combin-ing strategies that target elevated c-myc with standardchemotherapy in the treatment of pancreatic cancer.

Acknowledgments

We thank Dr. C.J. Sherr (St. Jude Children’s ResearchHospital, Memphis,TN) forproviding us themurine cyclin D1cDNA construct.

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