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Clinical Cancer Research 13, 2811, May 1, 2007. doi: 10.1158/1078-0432.CCR-06-1844
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

c-Myc–Induced Chemosensitization Is Mediated by Suppression of Cyclin D1 Expression and Nuclear Factor-{kappa}B Activity in Pancreatic Cancer Cells

Hector Biliran, Jr., Sanjeev Banerjee, Archana Thakur, Fazlul H. Sarkar, Aliccia Bollig, Fakhara Ahmed, Jiusheng Wu, Yuan Sun and Joshua D. Liao

Authors' Affiliation: Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan

Requests for reprints: Joshua D. Liao, Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912. Phone: 507-437-9665; Fax: 507-437-9606; E-mail: djliao{at}hi.umn.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Pancreatic cancer is a highly aggressive disease that remains refractory to various chemotherapeutic agents. Because the proto-oncogene c-myc can modulate apoptosis in response to cytotoxic insults and is commonly overexpressed in pancreatic cancer, we investigated the value of c-myc as a potential modulator of cellular response to various chemotherapeutic agents.

Experimental Design: Stable overexpression or small interfering RNA (siRNA)–mediated knockdown of c-myc and restoration of cyclin D1 were done in the Ela-myc pancreatic tumor cell 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-diphenyltetrazolium bromide assay and drug-induced apoptosis was measured by DNA fragmentation, sub-G1, and poly(ADP-ribose) polymerase cleavage analyses. Protein expression profile after cisplatin treatment was determined by Western blotting and DNA binding activity of nuclear factor-{kappa}B was examined 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 chemotherapeutic drugs. Increased sensitivity to cisplatin in c-myc–overexpressing cells was due, in part, to the marked increase in cisplatin-induced apoptosis. Conversely, down-regulation of c-myc expression in stable c-myc–overexpressing cells by c-myc siRNA resulted in decreased sensitivity to cisplatin-induced cell death. These results indicate an important role of c-myc in chemosensitivity of pancreatic cancer cells. The c-myc–induced cisplatin sensitivity correlated with inhibition of nuclear factor {kappa}B activity, which was partially restored by ectopic cyclin D1 overexpression.

Conclusions: Our results suggest that the c-myc–dependent sensitization to chemotherapy-induced apoptosis involves suppression of cyclin D1 expression and nuclear factor {kappa}B activity.

As seen in a variety of neoplasias, dysregulation of the c-myc proto-oncogene expression is a common event in human pancreatic cancer. Amplification and overexpression of c-myc gene have been observed in primary pancreatic carcinoma as well as in metastatic lesions (1–6). In vitro, c-myc gene amplification was evident in the majority of previously established pancreatic cancer cell lines (7). These data suggest that this proto-oncogene may be involved in the development and progression of this malignancy. A more direct and convincing evidence for the role of c-myc in the development of pancreatic cancer comes from an in vivo study by Sandgren et al. (8) reporting that mice carrying a c-myc transgene under the control of elastase gene promoter (Ela-myc) develop pancreatic tumor at an early age (2-7 months) with 100% penetrance.

Numerous studies have been published addressing the prognostic values of c-myc in various malignancies, but the data are still largely controversial and confusing. Positive, null, and negative correlations of c-Myc overexpression or amplification with prognosis or patient survival of various types of cancer have all been reported (9–24). The dual functions of c-Myc (i.e., promotion of both cell proliferation and apoptosis) may be one main reason for these conflicts because c-Myc–induced apoptosis may lead to a better prognosis whereas c-Myc–induced proliferation may lead to a poorer outcome (7). A thorough understanding of at what situations c-Myc protein directs a cell to proliferation and apoptosis is crucial for the treatment of cancers by manipulating the c-Myc levels. With regard to the pancreatic cancer, the prognostic value of c-myc remains unexplored.

In this report, we examined whether high levels of c-Myc could modulate the response of pancreatic cancer cells to chemotherapeutic agents. We found that ectopic overexpression of c-myc in murine Ela-myc and human L3.6pl pancreatic cancer cell lines enhanced the sensitivity of the cells o cisplatin and other chemotherapeutic drugs. The c-myc–induced sensitization was associated with marked induction of cisplatin-induced apoptosis and concomitant inhibition of cyclin D1 level and nuclear factor {kappa}B (NF-{kappa}B) activity. Restoration of cyclin D1 expression attenuated c-myc–induced cisplatin chemosensitization but only partially restored NF-{kappa}B activity. These results suggest that overexpression of c-Myc may sensitize pancreatic cancer cells to several chemotherapeutic agents. Mechanistically, these effects may be due, in part, to the c-Myc inhibition of NF-{kappa}B activity via both c-myc inhibition of cyclin D1 expression and other cyclin D1–independent mechanisms.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection. The Ela-mycT1 pancreatic tumor cell line used herein was generated from a pancreatic tumor tissue derived from a mouse carrying a c-myc transgene under the control of the elastase-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 than Ela-mycT4 cells and thus is an ideal system for us to manipulate c-myc level and observe the effect of c-Myc on cyclin D1. In this study, Ela-mycT4 cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37°C in humidified air with 5% CO2. The human pancreatic cancer cell lines PANC-28, BxPC-3, L3.6pl, and HPAC were also maintained in DMEM supplemented with 10% fetal bovine serum at 37°C in humidified air with 5% CO2. To generate stable c-myc–overexpressing clones, the pancreatic cancer cell lines Ela-myc and L3.6pl were transfected in a stable manner with the pcDNA3.1c-myc plasmid or the pcDNA3.1Hygro vector control plasmid using Lipofectamine 2000 per manufacturer's instructions (Life Technologies). After 48 h of incubation, Ela-myc– and L3.6pl-transfected cells were selected in DMEM containing 100 and 300 µg/mL hygromycin (Life Technologies), respectively. After 2 to 3 weeks, single independent clones were randomly isolated and each individual clone was plated separately. After clonal expansion, cells from each independent clone were tested for 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% fetal bovine serum in the presence of 50 µg/mL hygromycin at 37°C in humidified air with 5% CO2. The c-myc–overexpressing (CM) and vector control (CH) L3.6pl clones were routinely cultured in DMEM containing 10% fetal bovine serum in the presence of 150 µg/mL hygromycin at 37°C in humidified air with 5% CO2.

To ectopically restore cyclin D1 expression in c-myc–overexpressing cells, the M8 clone was stably transfected with the pcDNA3.CCND1 plasmid or the pcDNA3.1Neo vector control plasmid using Lipofectamine 2000 as prescribed by the manufacturer (Life Technologies). Forty-eight hours posttransfection, medium was replaced with DMEM containing 500 µg/mL G418 (Life Technologies). G418-resistant colonies were selected and screened for cyclin D1 expression by immunoblot analysis. The M8/Neo1 and M8/D1 clones were subsequently maintained in DMEM with 10% fetal bovine serum containing 250 µg/mL G418 and 50 µg/mL hygromycin.

Protein extraction and Western blotting. Whole or fractionated (nuclear and cytoplasmic) proteins (25) were resolved by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk in PBS-Tween 20 for 1 h at room temperature, the membranes were blotted with primary antibody, followed by incubation with a peroxidase-conjugated secondary antibody. Bound antibodies were visualized by enhanced chemiluminescence (Pierce). Densitometric analysis was done by scanning immunoblots and quantitating protein bands using the AlphaEaseFC software (Alpha Innotech). The primary antibodies used were rabbit polyclonal 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 (Santa Cruz Biotechnology; 1:500), rabbit polyclonal antibody to cyclin A (Santa Cruz Biotechnology; 1:1,000), mouse monoclonal antibody to cyclin B1 (Santa Cruz Biotechnology; 1:1,000), mouse monoclonal antibody to actin (Santa Cruz Biotechnology; 1:2,500 dilution), rabbit polyclonal antibody to poly(ADP-ribose) polymerase (Santa Cruz Biotechnology; 1:1,000), and rabbit polyclonal antibody to p65 (Santa Cruz Biotechnology; 1:1,000 dilution).

RT-PCR analysis. Total RNA was isolated from exponentially growing cells using the RNeasy Isolation Kit (Qiagen). The extracted RNA (1 µg) was reverse transcribed with the TaqMan reverse transcriptase in the presence of oligo(dT)15 primer as described by the manufacturer (Roche, Applied Biosystems). The resulting cDNA preparation was subjected to PCR amplification using an exogenous c-myc primer set with the forward primer (5'-TAGAAGGCACAGTCGAGG-3') identifying a hygro-specific sequence located upstream of the c-myc cDNA sequence and the reverse primer (5'-CACCGCCTACATCCTGTCCATTCAAGC-3') specific to a c-myc exon for 25 cycles. Each PCR cycle included a denaturation step at 94°C for 30 s, a primer annealing step at 55°C for 45 s, and an extension step at 72°C for 45 s. Reactions were done in an Eppendorf AG Mastercycler. Additional primers used for PCR were cyclin D1 sense (5'-CCCTCGGTGTCCTACTTCAA-3'), cyclin D1 antisense (5'-TGGCATTTTGGAGAGGAAGT-3'), ß-actin sense (5'-ACGGATTTGGTCGTATTGGG-3'), and ß-actin antisense (5'-TGATTTTGGAGGGATCTCGC-3'). The PCR products were analyzed by electrophoresis on 1% agarose gel containing ethidium bromide and photographed under UV light.

Cell viability assay. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described above. Briefly, 3.0 x 103 cells per well were plated in 100 µL of maintenance medium. The next day, the cells were treated with cisplatin according to the experimental design. The cells were then incubated with 0.2 µg/mL MTT for 2 h in the dark at 37°C. After removal of the medium, the dye crystals were dissolved in isopropanol and the absorbance was measured at 570 nm with an Ultra Multifunctional Microplate Reader (Tecan). Three independent experiments were done in quadruplicate wells.

Clonogenic survival assay. The effects of c-myc overexpression and cisplatin on long-term growth of Ela-myc cells were assessed by clonogenic assays. Briefly, cells were plated at a density of 2.0 x 105 in a 24-well plate and allowed to adhere overnight. The cells were then treated with escalating concentrations of cisplatin (0.5, 1.0, 2.5, and 5.0 µmol/L). Twelve hours after cisplatin addition, cells were trypsinized, counted, and reseeded at a low density (10,000 in a 10-cm dish) in triplicate. Medium was replaced every 3 days and the cells were allowed to grow for 10 days. The colonies were fixed with methanol-acetic acid (3:1), stained with 1% crystal violet, and counted. The survival fraction was determined by dividing the number of colonies formed in the presence of the drugs by the number of colonies formed in the untreated control cells. Each dose was done in triplicate and the experiments were done at least thrice.

Apoptosis analysis. Cells were subjected to proapoptotic conditions as specified in the text and figure legends. Both attached and floating cells were collected and subjected to the following apoptosis assays: (a) The quantitation of cytoplasmic histone-associated DNA fragments was done using the Cell Death Detection ELISA Kit (Roche). Briefly, cells were lysed and cell lysates were overlaid and incubated in microtiter plate modules coated with anti-histone antibody. Samples were subsequently incubated with anti–DNA peroxidase followed by color development with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) substrate. The absorbance of the samples was determined with the Ultra Multifunctional Microplate Reader (Tecan) at 405 nm. (b) The percentage of cells with sub-G0-G1 DNA content was determined by a Coulter EPICS 753 flow cytometer following staining with propidium iodide using a ModFit 5.2 computer program. (c) The proportion of Annexin V–positive cells was determined by Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions (BD Biosciences PharMingen). Briefly, cells were labeled with FITC-conjugated Annexin V and propidium iodide without permeabilization and subsequently analyzed by a Coulter EPICS 753 flow cytometer. Propidium iodide–positive, Annexin V–positive (necrotic) cells were excluded from analysis. (d) The cleavage of poly(ADP-ribose) polymerase was examined by immunoblotting as described 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'-CGAACUCACUGGUCUGACCdtdt-3'; antisense strand, 5'-GGUCAGACCAGUGAGUUCGdtdt-3') were purchased from Santa Cruz Biotechnology. The second set of mouse c-myc–specific siRNA and cyclin D1–specific siRNA were purchased from Ambion and Qiagen, respectively. For siRNA transfection, 5 x 105 cells per well were plated in six-well plates and transfected with 100 pmol of the siRNA duplex for 48 h using Lipofectamine 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 cells were plated in 24-well plates, allowed to recover for 24 h in complete medium, and then treated with 5.0 µmol/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 cells were subsequently subjected to apoptotic assays as described above.

Electrophoretic mobility shift assay. H1, M8/Neo1, and M8/D1 cells were incubated in the presence or absence of 2.5 µmol/L cisplatin for 24 h. Following treatment, the cells were collected and nuclear proteins were extracted as previously described (25). Electrophoretic mobility shift assay was done by incubating 5 µg of nuclear extract with IRDye-700–labeled NF-{kappa}B oligonucleotide. The incubation mixture included 2 µg of poly(deoxyinosinic-deoxycytidylic acid) in a binding buffer. The DNA-protein complex formed was separated from free oligonucleotide on 8.0% native polyacralyamide gel using buffer containing 50 mmol/L Tris, 200 mmol/L glycine (pH 8.5), and 1 mmol/L EDTA, and then visualized by Odyssey Infrared Imaging System using Odyssey Software Release 1.1. For loading control, 10 µg of nuclear protein from each sample were subjected to Western immunoblotting for retinoblastoma protein. To identify proteins in the DNA-protein complex, a supershift experiment was done with polyclonal p65 subunit–specific antibody. The anti–cyclin D1 antibody was used as the nonspecific, negative control antibody. Briefly, nuclear proteins were incubated for 30 min with different antibodies and assayed for supershift 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-{kappa}B assay. In addition to electrophoretic mobility shift assay, an ELISA-based kit was used for quantitative detection of NF-{kappa}B activity (EZ-Detect Transcription Factor Kits for NF-{kappa}B p50; Pierce). For each sample, 5 µg of nuclear extract were used according to the instructions of the manufacturer. For the detection of activated NF-{kappa}B, antibodies against the p50 subunit were used, 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-{kappa}B competitor duplex was used according to the instructions of the manufacturer to ensure signal specificity.

Immunofluorescent staining for NF-{kappa}B p65 localization. Cells were cultured on coverslips and treated with or without 2.5 µmol/L cisplatin for 24 h. The cells were then fixed with 10% formalin for 10 min, washed once with PBS, and stored at 4°C. For staining, coverslips were treated with 0.5% Triton X-100 in PBS for 10 min, washed with 0.05% Triton X-100 in PBS, and incubated at 37°C for 2.5 h with antibody (Santa Cruz Biotechnology) in PBS with 0.05% Triton X-100. After three washes in the same buffer, cells were incubated with FITC-conjugated anti-rabbit antibody at 1:100 (Molecular Probes), along with 0.1 µg/mL 4',6-diamidino-2-phenylindole (Sigma) in the same buffer for 1 h at 37°C. After three washes and air-drying, coverslips were mounted with antifade (Molecular Probes). Images were captured on a Zeiss 510 laser scanning inverted confocal microscope system using 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 Prism Software (El Camino Real). Results are expressed as mean ± SE and Student's t test was used for statistical analysis. P < 0.05 was considered to be significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Ela-mycT1 mouse pancreatic cancer cells were established to overexpress c-myc. The Ela-mycT1 mouse pancreatic cancer cell line was stably transfected with an exogenous c-myc cDNA or its 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 were screened and selected (Fig. 1 ). Because the endogenous c-myc expression is highly dependent on serum and mitogenic factors, the stable clonal lines were first subjected to serum-starved conditions and subsequently screened for exogenous c-myc expression. As shown in Fig. 1A, two different c-myc clones, M4 and M8, showed elevated levels of c-Myc protein than the vector control clones, H1 and H5. RT-PCR analysis with primers for exogenous c-myc showed a specific 250-bp product that was detected only in c-myc stable clonal lines (M1, M4, and M8) and not in vector control cells, thus confirming the expression of exogenous c-myc gene in c-myc–overexpressing clones (data not shown). Based on their high c-myc expression levels, the M4 and M8 clones were used for subsequent studies.


Figure 1
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  		Fig. 1. c-myc overexpression increases sensitivity of Ela-myc pancreatic cancer cells to cisplatin by enhancing cisplatin-induced apoptosis. A, Western blot analysis of c-myc. Serum-starved vector control (H1 and H5) and c-myc–overexpressing (M4 and M8) clones were collected, and total cellular protein (50 µg) was subjected to immunoblot analysis with a specific anti–c-myc antibody. The membrane was reprobed with an anti–ß-actin antibody to confirm equal loading. Shown below each blot is densitometric quantification as ratio of c-Myc protein to ß-actin. B, cell viability of vector control and c-myc–overexpressing clones following cisplatin treatment was determined by the MTT assay as described in Materials and Methods. Percent cell survival compared with corresponding untreated controls. Columns, mean of triplicate determinations of three separate experiments; bars SE. *, P < 0.05, compared with H1 and H5 control clones. C, clonogenic survival assay of vector control and c-myc stable clones on treatment with cisplatin. The percent survival is plotted against the drug concentration as indicated (columns, mean; bars, SE). D, vector control and c-myc–overexpressing cells were incubated in the absence or presence of 5 µmol/L cisplatin for 24 h. Following drug treatment, floating and attached cells were collected and subjected to sub-G1 DNA content analysis (top) and Western blotting of poly(ADP-ribose) polymerase (PARP; bottom). For poly(ADP-ribose) polymerase cleavage immunoblotting, 50 µg of protein were subjected to 7% SDS-PAGE and immunoblotted with an anti–poly(ADP-ribose) polymerase antibody. The membrane was reprobed with anti–ß-actin antibody to ensure equal protein loading. C and D, points and columns, mean of at least three independent experiments; bars, SE. *, P < 0.01, compared with vector control clones.

 
Overexpression of c-myc in Ela-mycT1 cells enhanced chemosensitivity by augmenting drug-induced apoptosis. The MTT analysis showed that the c-myc–overexpressing clones exhibited significantly decreased cell viability as compared with vector control clones on treatment with cisplatin (Fig. 1B). At 5 and 10 µmol/L of cisplatin, the cell viability of c-myc clones was decreased by 20% to 50% (P < 0.05) compared with control clones. Similarly, treatment of vector control and c-myc–overexpressing clones with two other drugs, gemcitabine and histone deactylase inhibitor FK228, showed the same response. Microscopically cisplatin-treated c-myc–overexpressing clones displayed cell shrinkage and detachment with significant number of floating cells. The clonogenic survival assay showed that the c-myc–overexpressing clones were highly sensitive to colony inhibition after treatment with various doses of cisplatin. At the concentration range of 1.0 to 5.0 µmol/L, the c-myc–overexpressing clones displayed significantly decreased survival of ~40% to 50% (P < 0.01) compared with vector control cells (Fig. 1C). Consistent with the chemosensitive phenotype of c-myc clones, the cisplatin treatment of c-myc clones resulted in a marked increase in cisplatin-induced apoptosis, as judged by increased percentage of sub-G1 cells and increased proteolytic cleavage of poly(ADP-ribose) polymerase (Fig. 1D) as compared 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 confirming the enhanced apoptotic sensitivity of c-myc clones to cisplatin.

We then down-regulated c-myc expression in the M8 clone of c-myc stable cells using two different mouse c-myc–specific siRNAs (Fig. 2A ). c-myc siRNA–treated M8 cells exhibited decreased percentage of sub-G1 DNA population (Fig. 2A) and Annexin V–positive cells (Fig. 2A) compared with control siRNA–treated or untransfected cells on cisplatin treatment. These results indicate that elevated c-myc expression may account for enhanced susceptibility to cisplatin-induced apoptosis in c-myc–overexpressing Ela-myc pancreatic cancer cells.


Figure 2
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  		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 cells were transfected with either c-myc siRNA or control siRNA. After 48 h, the cells were collected and total cellular protein (50 µg) was subjected to immunoblot analysis with a specific anti–c-myc antibody. The membrane was reprobed with an anti–ß-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-G1 DNA content analysis (middle) and Annexin V staining (bottom). Columns, mean of at least three independent experiments; bars, SE. *, P < 0.05, compared with control siRNA–transfected M8 cells. B, the relationship between c-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 several human pancreatic cancer cell lines following treatment with 2-methoxyestradiol (2-ME; middle) or FK228 (bottom) was determined by the MTT assay. Percent growth inhibition compared with corresponding untreated controls. Columns, mean of triplicate determinations of three separate experiments; bars, SE. *, P < 0.01, compared with BxPC-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, cell viability of vector control and c-myc–overexpressing clones following treatment with cisplatin was determined by the MTT assay. Percent cell survival compared with corresponding untreated controls. Columns, mean of triplicate determinations of three separate experiments; bars, SE. *, P < 0.05, compared with CH5 and CH6 control clones. D, vector control and c-myc–overexpressing cells were incubated in the absence or presence of 20 µmol/L cisplatin for 24 h. Following drug treatment, floating and attached cells were collected and subjected to sub-G1 DNA content analysis (top) and Annexin V staining (bottom). *, P < 0.01, compared with vector control clones.

 
Elevated c-myc expression in human pancreatic cancer cells enhances chemosensitivity and drug-induced apoptosis. The level of endogenous c-myc expression in a panel of human pancreatic cancer cell lines was examined by Western blot analysis (Fig. 2B). We observed that the human PANC-28 cells that exhibited the highest basal level of c-myc were highly sensitive to either 2-methoxyestradiol (Fig. 2B) or FK228 (Fig. 2B), two new chemotherapeutic agents that are currently used for clinical trials for several types of cancer (26, 27). The PANC-28 cells showed the highest percentage of growth inhibition even under low drug concentrations (1 µmol/L 2-methoxyestradiol and 10 nmol/L FK228) compared with other human pancreatic cancer cell lines.

To further examine whether elevated c-myc can modulate chemosensitivity of human pancreatic cancer cells, the human L3.6pl 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 different L3.6pl c-myc clones, CM18 and CM19, showed elevated levels of c-Myc protein ~2- to 3-fold higher than the vector control clones, CH6 and CH5 (Fig. 2C). The MTT analysis showed that the c-myc–overexpressing L3.6pl clones exhibited significantly decreased cell viability as compared with vector control clones on treatment with cisplatin (Fig. 2C). At 20 µmol/L cisplatin, the cell viability of c-myc clones was decreased by 20% to 30% (P < 0.05) compared with control clones. Treatment of vector control and c-myc–overexpressing clones with FK228 similarly showed the same response (data not shown). Consistent with their chemosensitive phenotype, the c-myc–overexpressing L3.6pl clones exhibited a marked increase in cisplatin-induced apoptosis, as judged by the increased percentage of sub-G1 (Fig. 2D) and Annexin V–positive (Fig. 2D) cells, as compared with control clones. These results indicate that elevated c-myc expression in the L3.6pl cells increases cisplatin sensitivity, at least 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 recently showed that cyclin D1 imposed resistance of pancreatic cells to chemotherapeutic agents (25), we wondered whether cyclin D1 was involved in c-Myc–rendered drug sensitivity. We found that the mRNA and protein levels of cyclin D1 were dramatically reduced in c-myc–overexpressing Ela-myc stable clones M4 and M8, as compared with the corresponding vector clones (H1 and H5; Fig. 3A and B ). Cyclin E level was also slightly decreased, cyclin A level was slightly increased, and cyclin B1 level was unchanged (Fig. 3A). We ectopically restored cyclin D1 expression in the M8 clone of c-myc–overexpressing Ela-myc cells by stable transfection with pcDNA3.1cyclin D1 or empty vector construct. The resultant M8/D1 clones expressed cyclin D1 ~3- to 5-fold higher than the corresponding vector control clones (M8/Neo1) whereas the expression levels of c-myc remained unchanged (Fig. 4A ). When treated with either 2.5 or 5 µmol/L cisplatin for 24 h and subjected to sub-G1 analysis (Fig. 4B) and Annexin V staining (Fig. 4C), the M8/D1 cells (M8/D1.4 clone) displayed lower apoptotic frequency as compared with M8/Neo1 cells. Analyses of other M8/D1 clones showed similar data [Fig. 4B (bottom) and C (bottom)], confirming that cyclin D1 overexpression conferred enhanced resistance to cisplatin-induced cell death in these cells. Further, we depleted cyclin D1 in M8/D1 cells via siRNA strategy (Fig. 4D). Treatment with cisplatin for 24 h significantly enhanced the drug-induced cell death in cyclin D1 siRNA–transfected M8/D1.4 cells, as evidenced by increased amount of cytoplasmic histone-associated DNA fragments (Fig. 4D, middle) and increased percentage of sub-G1 DNA population (Fig. 4D, bottom). These results suggest that cyclin D1 overexpression may account for enhanced cisplatin resistance in M8/D1 cells. Interestingly, restoration of cyclin D1 in M8/D1 cells also inhibited the proliferative advantage conferred by c-myc (data not shown).


Figure 3
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  		Fig. 3. Effect of c-myc overexpression on the expression of cyclins D1, A, B1, and E. A, total cell lysates were prepared from exponentially growing vector control (H1 and H5) and c-myc–overexpressing Ela-myc clones (M1, M4, and M8) and subjected to Western blot analysis of different cyclins with ß-actin shown as a loading control. B, expression of cyclin D1 by RT-PCR analysis in vector control and c-myc–overexpressing clones. Total RNA (1 µg) from control and c-myc clones was analyzed by RT-PCR with primers for cyclin D1 and ß-actin. RT-PCR produced a 300-bp cyclin D1 fragment and a 200-bp actin fragment.

 

Figure 4
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  		Fig. 4. Ectopic cyclin D1 inhibits cisplatin-mediated apoptosis in c-myc–overexpressing cells. A, exponentially growing cultures of M8, M8/Neo1, and M8/D1 Ela-myc clones were lysed and total cellular protein (50 µg) was subjected to immunoblotting analysis with anti–cyclin D1 or anti–c-myc antibody. ß-Actin is shown as a control for protein loading. B and C, M8, M8/Neo1, and M8/D1 cells were cultured in maintenance medium with or without cisplatin for 24 h and subjected to sub-G1 DNA and Annexin V apoptotic assays. B, top, representative fluorescence-activated cell sorting profile of M8/Neo1 and M8/D1.4 cells illustrating the percentage of sub-G1 cells. Bottom, columns, mean of at least three independent experiments; bars, SE. C, top, representative density plots of propidium iodide labeling versus Annexin V-FITC staining in M8/Neo1 and M8/D1.4 cells showing the proportion of propidium iodide–negative, Annexin V–positive cells. Bottom, columns, mean of at least three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01. D, siRNA-directed suppression of cyclin D1 sensitizes M8/D1 cells to cisplatin-induced apoptosis. Western blot analysis of cyclin D1 after M8/D1.4 cells were transfected with either cyclin D1 siRNAs or control siRNA. After 48 h, the cells were collected and total cellular protein (50 µg) was subjected to immunoblotting analysis with a specific anti–cyclin D1 antibody. The membrane was reprobed with an anti–ß-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 µmol/L cisplatin for 24 h, and subjected to Cell Death Detection ELISA (middle) and sub-G1 DNA (bottom) apoptotic assays. Columns, mean of at least three independent experiments; bars, SE. *, P < 0.01, compared with control siRNA–transfected M8/D1.4 cells.

 
NF-{kappa}B activity was decreased in c-myc–expressing cells and was only partially restored by restoration of cyclin D1. As shown by both electrophoretic mobility shift assay (Fig. 5A ) and an ELISA-based NF-{kappa}B DNA binding activity assay (Fig. 5A, bottom), the c-myc–overexpressing M8 cells exhibited decreased NF-{kappa}B DNA binding activity in the presence or absence of cisplatin as compared with the vector control clone (H1). In contrast, the stable M8/D1 cells exhibited a higher basal and cisplatin-induced NF-{kappa}B activity compared with the M8 cells. Consistent with the electrophoretic mobility shift assay findings, 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 p65 nuclear translocation (punctated nuclear staining) compared with vector control cells in the presence or absence of cisplatin, in association with increased cytosolic levels of I{kappa}B{alpha} protein and a concomitant decrease in phospho-I{kappa}B{alpha} (Fig. 5D). In contrast, M8/D1 cells exhibited a slight increase of NF-{kappa}B p65 nuclear translocation in the presence or absence of cisplatin treatment compared with M8 cells as evidenced by Western blot and immunocytochemical analysis (Fig. 5D). Interestingly, no significant differences in the steady-state levels of cytosolic I{kappa}B{alpha} and phospho-I{kappa}B{alpha} proteins were observed between M8 and M8/D1 cells.


Figure 5
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  		Fig. 5. c-myc–induced cisplatin sensitization was associated with suppression of NF-{kappa}B activity and down-regulation of NF-{kappa}B-regulated antiapoptotic Bcl-2 and Bcl-xl expression. A, H1, M8/Neo1, and M8/D1.4 stable cells were incubated in the presence or absence of 2.5 µmol/L cisplatin for 24 h. Following treatment, floating and attached cells were collected, and nuclear extract was collected and subjected to electrophoretic mobility shift assay; supershift assay was also done to indicate specificity of NF-{kappa}B band (top) and ELISA-based NF-{kappa}B assay (bottom). B, cytoplasmic and nuclear extracts (50 µg) prepared from untreated and cisplatin-treated cells were subjected to Western blotting with a specific antibody against the p65 subunit of NF-{kappa}B. C, untreated and cisplatin-treated cells were subjected to immunocytochemistry and subsequently analyzed by confocal microscopy as described in Materials and Methods. D, cytoplasmic extracts (50 µg) prepared from untreated and cisplatin-treated cells were subjected to Western blotting for detection of I{kappa}B{alpha} and phospho-I{kappa}B{alpha}. Immunoblot analysis of ß-actin was also done to confirm equal loading. The total cell lysate (50 µg) from untreated and cisplatin-treated cells was also subjected to immunoblotting with an antibody against Bcl-2, Bcl-xl, or Bax. The detection of ß-actin was used as control for equal protein loading.

 
The protein levels of Bcl-2 and Bcl-xl, two NF-{kappa}B downstream antiapoptotic effectors that are known apoptotic targets of c-myc (11, 12, 19), were significantly reduced in the untreated as well as cisplatin-treated c-myc stable M8 cells compared with that of control clonal line, whereas the stable M8/D1 cells showed enhanced levels of these proteins in the presence or absence of cisplatin. On the other hand, the steady-state levels of Bax protein, a proapoptotic Bcl-2 family member, were markedly induced in both untreated and cisplatin-treated c-myc–overexpressing (M8/Neo1) cells and were barely detectable in vector control cells before and after cisplatin treatment. This up-regulation of Bax protein was not significantly altered in M8/D1 cells.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
c-Myc exhibits a dual function in stimulation of cell proliferation and apoptosis. In this study, we showed that ectopic overexpression of c-myc in pancreatic cancer cells results in a marked increase in chemotherapy-induced cell death. This observation is similar to what was reported in colonic cancer that c-myc amplification sensitized cells to 5-fluorouracil–based adjuvant therapy (9, 28), although it conflicts with many other reports that high c-Myc levels indicates 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) and with our observation of an inverse expression pattern for c-myc and cyclin D1 in mammary tumors derived from MMTV-c-myc transgenic mice (32). In our system, restoration of cyclin D1 expression in the c-myc–overexpressing pancreatic cancer cells significantly attenuated cisplatin-induced cell death, whereas down-regulation of cyclin D1 in the c-myc/cyclin D1 stable clones by siRNA mitigated the cyclin D1–induced resistance to cisplatin. These findings, together with the study showing that cyclin D1 rescued fibroblasts from c-myc–dependent apoptotic cell death (33), suggest the possibility that suppression of cyclin D1 is a downstream event underlying c-myc–induced sensitivity to chemotherapeutic agents, and thus may play a mechanistic role in c-myc–dependent cell death.

Another novel finding of our study is that c-Myc inhibits NF-{kappa}B activity and the expression of its downstream antiapoptotic effectors, Bcl-2 and Bcl-xl, in pancreatic cancer cells. Mechanistically, the inhibition of NF-{kappa}B is related to its interference with the nuclear translocation of the p65 NF-{kappa}B subunit. Our observation of increased levels of cytoplasmic I{kappa}B{alpha} protein in c-myc–overexpressing cells presumably resulted in enhanced cytoplasmic sequestration and reduced nuclear translocation of p65. Furthermore, the reduced phosphorylation of I{kappa}B{alpha} in c-myc–overexpressing cells, as evidenced by low levels of phospho-I{kappa}B{alpha}, suggests that this endogenous NF-{kappa}B inhibitor is subjected to less proteolytic degradation. Because NF-{kappa}B is a well-known survival factor for cancer cells, partly via its stimulation of Bcl-2 and Bcl-xl and suppression of Bax, c-Myc inhibition of NF-{kappa}B may be a mechanism underlying the observed sensitization of pancreatic cancer cells to chemotherapeutic agents by a c-myc–dependent process (Fig. 6A ). However, the question remains about the mechanistic role of cyclin D1 in relation to c-Myc and NF-{kappa}B during cell death observed in our studies (Fig. 6A). Whereas it is well known that NF-{kappa}B can induce cyclin D1 in other systems (34, 35), we have recently found that cyclin D1 can also induce NF-{kappa}B activity in pancreatic cancer cells (25). Consistent with cyclin D1 as a downstream target of NF-{kappa}B, the suppression of cyclin D1 in c-myc–overexpressing cells may be a consequence of c-Myc–dependent inhibition of NF-{kappa}B activity. Alternatively, this down-regulation of cyclin D1 may result from the direct inhibition of cyclin D1 by c-myc (29–31). Thus, both pathways could be used by c-Myc in conferring drug sensitivity to pancreatic cancer cells.


Figure 6
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  		Fig. 6. Model illustrating how c-myc and its downstream effectors (NF-{kappa}B and cyclin D1) may affect pancreatic cancer cell survival and chemosensitivity. A, overexpression of c-myc may inhibit cyclin D1 expression and NF-{kappa}B activity, leading to enhanced apoptotic potential and decreased cell survival. Concomitant overexpression of cyclin D1, presumably through activation of growth factor signaling, may override the c-myc–mediated inhibition of cyclin D1 expression and NF-{kappa}B activity and render cells weakly apoptotic and highly resistant to chemotherapeutic agents. B, pancreatic tumor cells may exhibit three different phenotypes in the context of c-Myc and cyclin D1 expression. 1, tumor cells exhibiting overexpression of cyclin D1 are highly resistant to chemotherapy. 2, in certain tumor cells, c-myc overexpression may render these cells highly sensitive to drug-induced cell death due, at least in part, to its suppression of cyclin D1. 3, some tumor cells with dysregulated c-myc may gain cyclin D1 expression, presumably through activation of growth factor signaling, which can antagonize c-Myc–imposed drug sensitivity.

 
The prognostic value of c-myc in various human cancers remains controversial; positive, null, and negative associations of c-myc amplification or overexpression with tumor aggressiveness or patient outcome have all been reported (9, 28, 36–40). Similar conflicting results have also been reported for cyclin D1 (41–45), just like many other studies exploring the prognostic value of a single gene. Interaction of one protein (e.g., c-Myc) with different proteins under different situations may 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 or concomitantly, may affect the sensitivity of cancer cells differentially to chemotherapeutic agents. In the context of c-Myc and cyclin D1 expression, pancreatic tumor cells may exhibit three different phenotypes (Fig. 6B): (a) some pancreatic tumor cells exhibit overexpression of cyclin D1, which may happen spontaneously or secondarily due to activation of k-ras, Notch, or transforming growth factor {alpha}/epidermal growth factor receptor signaling, which are known to occur frequently in human pancreatic cancer, and thus high levels of cyclin D1 expression may render cancer cells resistant to chemotherapeutic agents. (b) In other pancreatic cancer cells, c-myc expression may be increased, which may not only promote cell proliferation but also render these cells highly sensitive to drug-induced cell death due, at least in part, to its suppression of cyclin D1. However, it is noteworthy that this c-myc–induced chemosensitization may be abrogated by the existence of other genetic mutations in pancreatic tumor. (c) A third phenotype may arise by concomitant overexpression of cyclin D1 and c-myc in such situations wherein transforming growth factor {alpha} is overexpressed, which overrides the suppression of cyclin D1 by c-Myc (32). Under this microenvironment, cyclin D1 can antagonize c-Myc–dependent drug sensitivity. In the two latter scenarios, antagonism of c-myc expression, which has been widely proposed as a strategy for cancer therapy (46, 47), may actually prevent c-Myc–induced apoptosis and drug sensitivity and thereby result in adverse effects.

In summary, we found that ectopic overexpression of c-myc in pancreatic cancer cells resulted in enhanced sensitivity to cisplatin-induced cell death, in association with inhibition of cyclin D1 expression and NF-{kappa}B activity. Restoration of cyclin D1 in c-myc–overexpressing cells significantly attenuates the c-Myc–dependent chemosensitization and partially abrogates the inhibition of NF-{kappa}B. These results suggest that suppression of NF-{kappa}B activity and cyclin D1 expression may be important, yet unrecognized, mechanisms underlying the c-Myc–dependent sensitization of cancer cells to chemotherapy. Because many highly malignant tumors often exhibit overexpression and/or amplification of c-myc and cyclin D1, alone and simultaneously, our findings provide insights into the potential mechanisms responsible for their seemingly conflicting behavior in response to chemotherapy, as reflected by the conflicting prognostic values for c-myc and cyclin D1 alone. Based on our results, we suggest that precautionary measures should be implemented when considering combining strategies that target elevated c-myc with standard chemotherapy in the treatment of pancreatic cancer.


   Acknowledgments
 
We thank Dr. C.J. Sherr (St. Jude Children's Research Hospital, Memphis, TN) for providing us the murine cyclin D1 cDNA construct.


   Footnotes
 
Grant support: NIH grant R01 CA100864 (D.J. Liao).

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/26/06; revised 11/ 8/06; accepted 11/22/06.


   References
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 Results
 Discussion
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