The Association of CCND1 Overexpression and Cisplatin Resistance in Testicular Germ Cell Tumors and Other Cancers
2010; Elsevier BV; Volume: 176; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.090780
ISSN1525-2191
AutoresElodie Noël, Marc Yeste‐Velasco, Xueying Mao, Jackie Perry, Sakunthala C. Kudahetti, Ningfeng Fiona Li, Swee Y. Sharp, Tracy Chaplin, Liyan Xue, Alan McIntyre, Ling Shan, Thomas Powles, R.T.D. Oliver, Bryan D. Young, Janet Shipley, Daniel M. Berney, Simon P. Joel, Yong‐Jie Lu,
Tópico(s)Prostate Cancer Treatment and Research
ResumoDevelopment of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors. Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors. Cisplatin has dramatically improved the clinical outcome for testicular germ cell tumors (TGCTs) and remains the first line treatment of several other solid tumors such as, ovarian, breast, head and neck, and small cell lung cancers.1Kelland L The resurgence of platinum-based cancer chemotherapy.Nat Rev Cancer. 2007; 7: 573-584Crossref PubMed Scopus (3644) Google Scholar, 2Vermorken JB Mesia R Rivera F Remenar E Kawecki A Rottey S Erfan J Zabolotnyy D Kienzer HR Cupissol D Peyrade F Benasso M Vynnychenko I De Raucourt D Bokemeyer C Schueler A Amellal N Hitt R Platinum-based chemotherapy plus cetuximab in head and neck cancer.N Engl J Med. 2008; 359: 1116-1127Crossref PubMed Scopus (2534) Google Scholar However, in many cases, cancer cells develop a resistant phenotype, and the outcome for these patients is very poor.3Kollmannsberger C Nichols C Bokemeyer C Recent advances in management of patients with platinum-refractory testicular germ cell tumors.Cancer. 2006; 106: 1217-1226Crossref PubMed Scopus (67) Google Scholar, 4Mayer F Honecker F Looijenga LH Bokemeyer C Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors.Ann Oncol. 2003; 14: 825-832Crossref PubMed Scopus (67) Google Scholar Many mechanisms associated with resistance to platinum drugs have been identified, such as alteration of drug transporters, drug uptake, and efflux in cells, abnormalities in DNA damage repair and apoptosis induction.1Kelland L The resurgence of platinum-based cancer chemotherapy.Nat Rev Cancer. 2007; 7: 573-584Crossref PubMed Scopus (3644) Google Scholar, 4Mayer F Honecker F Looijenga LH Bokemeyer C Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors.Ann Oncol. 2003; 14: 825-832Crossref PubMed Scopus (67) Google Scholar, 5Borst P Rottenberg S Jonkers J How do real tumors become resistant to cisplatin?.Cell Cycle. 2008; 7: 1353-1359Crossref PubMed Scopus (179) Google Scholar, 6Houldsworth J Korkola JE Bosl GJ Chaganti RS Biology and genetics of adult male germ cell tumors.J Clin Oncol. 2006; 24: 5512-5518Crossref PubMed Scopus (67) Google Scholar, 7Mayer F Stoop H Scheffer GL Scheper R Oosterhuis JW Looijenga LH Bokemeyer C Molecular determinants of treatment response in human germ cell tumors.Clin Cancer Res. 2003; 9: 767-773PubMed Google Scholar, 8Masters JR Koberle B Curing metastatic cancer: lessons from testicular germ-cell tumours.Nat Rev Cancer. 2003; 3: 517-525Crossref PubMed Scopus (181) Google Scholar However, the evidence for these mechanisms in a clinical setting has not been clearly established.4Mayer F Honecker F Looijenga LH Bokemeyer C Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors.Ann Oncol. 2003; 14: 825-832Crossref PubMed Scopus (67) Google Scholar, 5Borst P Rottenberg S Jonkers J How do real tumors become resistant to cisplatin?.Cell Cycle. 2008; 7: 1353-1359Crossref PubMed Scopus (179) Google Scholar TGCTs, which include seminomas and non-seminomas, provide an ideal model to study the molecular mechanisms of cisplatin resistance, due to their extreme sensitivity to cisplatin-based chemotherapy.4Mayer F Honecker F Looijenga LH Bokemeyer C Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors.Ann Oncol. 2003; 14: 825-832Crossref PubMed Scopus (67) Google Scholar, 8Masters JR Koberle B Curing metastatic cancer: lessons from testicular germ-cell tumours.Nat Rev Cancer. 2003; 3: 517-525Crossref PubMed Scopus (181) Google Scholar However, although TGCT development and pathogenesis have been extensively studied, and some genetic aberrations have been described as characterization markers of adult TGCTs, such as the extra copies of the short arm of chromosome 12,9Looijenga LH Zafarana G Grygalewicz B Summersgill B Debiec-Rychter M Veltman J Schoenmakers EF Rodriguez S Jafer O Clark J van Kessel AG Shipley J van Gurp RJ Gillis AJ Oosterhuis JW Role of gain of 12p in germ cell tumour development.Apmis. 2003; 111 (discussion 172–163): 161-171Crossref PubMed Scopus (121) Google Scholar our knowledge of the genetic mechanisms in TGCT chemoresistance is still limited. In TGCTs, mutations of TP53 account for a very small subset of refractory cases.4Mayer F Honecker F Looijenga LH Bokemeyer C Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors.Ann Oncol. 2003; 14: 825-832Crossref PubMed Scopus (67) Google Scholar, 10Houldsworth J Xiao H Murty VV Chen W Ray B Reuter VE Bosl GJ Chaganti RS Human male germ cell tumor resistance to cisplatin is linked to TP53 gene mutation.Oncogene. 1998; 16: 2345-2349Crossref PubMed Scopus (135) Google Scholar, 11Kersemaekers AM Mayer F Molier M van Weeren PC Oosterhuis JW Bokemeyer C Looijenga LH Role of P53 and MDM2 in treatment response of human germ cell tumors.J Clin Oncol. 2002; 20: 1551-1561Crossref PubMed Scopus (110) Google Scholar Increased activity of DNA repair genes, such as ERCC1, XPA, XPD, and XPB, has also been reported to be associated with TGCT cisplatin resistance. However, their clinical significance has yet to be confirmed.4Mayer F Honecker F Looijenga LH Bokemeyer C Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors.Ann Oncol. 2003; 14: 825-832Crossref PubMed Scopus (67) Google Scholar, 5Borst P Rottenberg S Jonkers J How do real tumors become resistant to cisplatin?.Cell Cycle. 2008; 7: 1353-1359Crossref PubMed Scopus (179) Google Scholar, 8Masters JR Koberle B Curing metastatic cancer: lessons from testicular germ-cell tumours.Nat Rev Cancer. 2003; 3: 517-525Crossref PubMed Scopus (181) Google Scholar, 12Martin LP Hamilton TC Schilder RJ Platinum resistance: the role of DNA repair pathways.Clin Cancer Res. 2008; 14: 1291-1295Crossref PubMed Scopus (607) Google Scholar The association between microsatellite instability/mismatch repair and resistance has been extensively investigated. Although the results from different studies were controversial,12Martin LP Hamilton TC Schilder RJ Platinum resistance: the role of DNA repair pathways.Clin Cancer Res. 2008; 14: 1291-1295Crossref PubMed Scopus (607) Google Scholar, 13Mayer F Gillis AJ Dinjens W Oosterhuis JW Bokemeyer C Looijenga LH Microsatellite instability of germ cell tumors is associated with resistance to systemic treatment.Cancer Res. 2002; 62: 2758-2760PubMed Google Scholar, 14Velasco A Corvalan A Wistuba II Riquelme E Chuaqui R Majerson A Leach FS Mismatch repair expression in testicular cancer predicts recurrence and survival.Int J Cancer. 2008; 122: 1774-1777Crossref PubMed Scopus (42) Google Scholar, 15Helleman J van Staveren IL Dinjens WN van Kuijk PF Ritstier K Ewing PC van der Burg ME Stoter G Berns EM Mismatch repair and treatment resistance in ovarian cancer.BMC Cancer. 2006; 6: 201Crossref PubMed Scopus (79) Google Scholar, 16Olasz J Mandoky L Geczi L Bodrogi I Csuka O Bak M Influence of hMLH1 methylation, mismatch repair deficiency and microsatellite instability on chemoresistance of testicular germ-cell tumors.Anticancer Res. 2005; 25: 4319-4324PubMed Google Scholar a recent study of a large series of resistant TGCT samples generally confirmed the involvement of microsatellite instability/mismatch repair in cisplatin resistance, and also revealed the association between BRAF mutation and cisplatin resistance.17Honecker F Wermann H Mayer F Gillis AJ Stoop H van Gurp RJ Oechsle K Steyerberg E Hartmann JT Dinjens WN Oosterhuis JW Bokemeyer C Looijenga LH Microsatellite instability, mismatch repair deficiency, and BRAF mutation in treatment-resistant germ cell tumors.J Clin Oncol. 2009; 27: 2129-2136Crossref PubMed Scopus (138) Google Scholar Seladin-1, a multifunctional protein, has also recently been identified as a putative player in cisplatin sensitivity of TGCTs.18Nuti F Luciani P Marinari E Erdei E Bak M Deledda C Rosati F Mazzinghi B Danza G Stoop H Looijenga LH Peri A Serio M Krausz C Seladin-1 and testicular germ cell tumours: new insights into cisplatin responsiveness.J Pathol. 2009; 219: 491-500Crossref PubMed Scopus (12) Google Scholar Since these resistance mechanisms only account for a limited proportion of the resistant cases, further studies are required. In this study, we performed a genome-wide gene expression analysis of three pairs of TGCT parental and resistant cell lines and identified CCND1 (Cyclin D1) overexpression in the three resistant lines. We confirmed the common involvement of CCND1 deregulation in cisplatin-resistant TGCT clinical cases, as well as in ovarian and prostate cancer samples. Three TGCT cell lines, 833K, Susa, and GCT27, nine ovarian cancer cell lines, A2780, CH1, 41M, OVCAR3, OVCAR4, OVCAR8, SKOV3, PXN94, and HX62, and four prostate cancer cell lines, PC3, DU-145, LNCaP, and 22RV1, were used for this study. The ovarian cancer lines OVCAR8, SKOV3, PXN94, and HX62 were cisplatin-resistant whereas A2780, CH1, 41M, and OVCAR3 were sensitive lines as previously reported.19Hills CA Kelland LR Abel G Siracky J Wilson AP Harrap KR Biological properties of ten human ovarian carcinoma cell lines: calibration in vitro against four platinum complexes.Br J Cancer. 1989; 59: 527-534Crossref PubMed Scopus (178) Google Scholar, 20Louie KG Behrens BC Kinsella TJ Hamilton TC Grotzinger KR McKoy WM Winker MA Ozols RF Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine.Cancer Res. 1985; 45: 2110-2115PubMed Google Scholar, 21Schilder RJ Hall L Monks A Handel LM Fornace Jr, AJ Ozols RF Fojo AT Hamilton TC Metallothionein gene expression and resistance to cisplatin in human ovarian cancer.Int J Cancer. 1990; 45: 416-422Crossref PubMed Scopus (160) Google Scholar Cisplatin-resistant derivative sublines were available for 833K, Susa, GCT27, A2780, CH1, and 41M, namely 833KR, SusaR, GCT27R, A2780R, CH1R, and 41MR. All of the parental cell lines were made resistant to cisplatin by long-term exposure to the drug. The TGCT cell lines have previously been analyzed by single nucleotide polymorphism arrays22Noel EE Perry J Chaplin T Mao X Cazier JB Joel SP Oliver RT Young BD Lu YJ Identification of genomic changes associated with cisplatin resistance in testicular germ cell tumor cell lines.Genes Chromosomes Cancer. 2008; 47: 604-613Crossref PubMed Scopus (17) Google Scholar, 23Lu YJ Yang J Noel E Skoulakis S Chaplin T Raghavan M Purkis T McIntyre A Kudahetti SC Naase M Berney D Shipley J Oliver RT Young BD Association between large-scale genomic homozygosity without chromosomal loss and nonseminomatous germ cell tumor development.Cancer Res. 2005; 65: 9137-9141Crossref PubMed Scopus (13) Google Scholar and karyotyped by 24-color fluorescence in situ hybridization (unpublished data). The genomic alterations were similar to those detected in the original cell lines using low resolution genetic analyses soon after they were established, including the gain of 12p which is specific for TGCTs. All of the cell lines were maintained by standard cell culture as previously described.22Noel EE Perry J Chaplin T Mao X Cazier JB Joel SP Oliver RT Young BD Lu YJ Identification of genomic changes associated with cisplatin resistance in testicular germ cell tumor cell lines.Genes Chromosomes Cancer. 2008; 47: 604-613Crossref PubMed Scopus (17) Google Scholar, 24Mao X James SY Yanez-Munoz RJ Chaplin T Molloy G Oliver RT Young BD Lu YJ Rapid high-resolution karyotyping with precise identification of chromosome breakpoints.Genes Chromosomes Cancer. 2007; 46: 675-683Crossref PubMed Scopus (16) Google Scholar Fourteen fresh-frozen and 25 formalin-fixed, paraffin-embedded (FFPE) TGCTs (see Supplemental Table S1 at http://ajp.amjpathol.org), and 15 fresh frozen prostate cancer samples were collected from Barts and The London Hospital and the Royal Marsden Hospital with ethical approval. For one FFPE TGCT platinum-resistant case, samples from two separate blocks (P24 and P25) were analyzed. All of the TGCT clinical samples were selected from cases treated with platinum-based regimens, and those with progression or partial remission tumors were defined as resistant cases. Samples were reviewed by a consultant pathologist (D.M.B.) and cancer lesions were macrodissected so that all TGCT samples contained at least 80% tumor material and the prostate cancer contained more than 50% cancer cells. ATP cell viability assays were performed by plating cells at a density of 5 × 104 cells per well in 96-well plates and subsequently treated with one of six serial dilutions of cisplatin (Sigma, St. Louis, MO), including a negative control. Cell viability for each drug concentration was measured using the HS Vialight Assay kit (Lonza, Conshohocken, PA) on a FluoroStar automated plate reader (BMG Labtechnologies, Durham, UK). Each experiment was performed in triplicate. Cell cycle distribution was established using a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Cells were harvested 24 hours after replating and fixed in 70% ethanol. The cells were stained using a propidium iodide solution containing 50 μg/ml propidium iodide and 50 μg/ml RNase A (Sigma) before flow cytometric analysis. Five thousand cells were acquired and the proportion of cells in each of the cell cycle phases was quantified using the cell cycle analysis WinMDI v2.8 program. RNA was extracted from cell lines and fresh frozen samples using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). For each FFPE sample, RNA was extracted from a 7-μm section using the Paradise Whole Transcript RT Reagent System (MDS Analytical Technologies, Mountain View, CA) following the manufacturer’s instructions. The Affymetrix gene expression microarray profiling technology (Human Genome U133 plus 2.0 arrays) was applied using total RNA (5 μg) and a one-cycle eukaryotic target-labeling assay (Affymetrix, Santa Clara, CA) according to the manufacturer’s recommendations. Microarray data have been deposited in the Gene Expression Omnibus under Accession No. GSE14231. Unsupervised one-way cluster analysis with Spearman correlation was firstly applied to the data. Then data analysis was performed using GeneSpring software version 7.2 (Agilent Technologies). In vitro expression changes associated with cisplatin resistance in TGCTs were analyzed by comparing the cisplatin-sensitive and -resistant cell lines. Samples were processed using the Affymetrix Microarray Suite version 5.0 (MAS 5.0). Affymetrix default analysis settings and global scaling for normalization were used. Once imported onto GeneSpring software, data were further log-transformed, normalized per chip (50th percentile) and per gene (median). An additional “per gene to specific sample” normalization step was added. Absent calls were removed and statistical filters were applied. Different fold-change cut-off values were tested to identify differentially expressed genes/transcripts across the three pairs of cell lines when comparing resistant and parental cell lines. Finally, ontological analysis was undertaken using the Genecards database version 2.36 (http://www.genecards.org/) to determine the biological functions of candidate genes and their relevance to the mechanisms of chemoresistance. Comparative genomic hybridization (CGH) and comparative expressed sequence hybridization (CESH) analyses to profile genomic imbalances and differential expression patterns, respectively, on chromosomes, were performed as previously published.25Lu YJ Birdsall S Osin P Gusterson B Shipley J Phyllodes tumors of the breast analyzed by comparative genomic hybridization and association of increased 1q copy number with stromal overgrowth and recurrence.Genes Chromosomes Cancer. 1997; 20: 275-281Crossref PubMed Scopus (54) Google Scholar, 26Lu YJ Williamson D Clark J Wang R Tiffin N Skelton L Gordon T Williams R Allan B Jackman A Cooper C Pritchard-Jones K Shipley J Comparative expressed sequence hybridization to chromosomes for tumor classification and identification of genomic regions of differential gene expression.Proc Natl Acad Sci USA. 2001; 98: 9197-9202Crossref PubMed Scopus (52) Google Scholar For CGH analysis, DNA from each cell line was hybridized against a sample from a normal healthy female. For paired parental and cisplatin-resistant lines, samples were directly co-hybridized onto slides. The 0.8 and 1.2 cut-off values were used to identify chromosome copy number changes. For CESH analysis, each RNA sample was hybridized against a pooled RNA control from several individual muscle tissues. The paired parental and cisplatin-resistant cell lines were co-hybridized. Similar threshold values to the ones used for CGH analysis were used to identify expression variations. Total RNA (1 μg) from cell lines and fresh frozen tissues was reverse transcribed using random hexamers (50 μmol/L) (Sigma) and M-MLV RT RNase H Minus, point mutant enzyme (200 U/μL) (Promega, Madison, WI), and from FFPE tissues using the Paradise Whole Transcript RT reagent System (MDS Analytical Technologies), both following the manufacturer’s recommendations. The quality of reverse transcribed cDNA was checked by standard PCR using ß-actin primers (Forward sequence: 5′-GCGGGAAATCGTGCGTGCGTGACATT-3′; Reverse sequence: 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′) (Sigma) following standard PCR technique with an annealing temperature of 62°C. PCR products were visualized by electrophoresis and only samples with positive ß-actin products were used for CCND1 quantitative reverse transcription (qRT)-PCR analysis. Keeping the default settings for baselines and thresholds, qRT-PCR was performed using the ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA). Pre-designed Taqman gene expression assays targeting CCND1 (product number: Hs99999004_m1, exon boundary 4 to 5) and the endogenously expressed GAPDH gene (product number: Hs99999905_m1) were purchased (Applied Biosystems). Each reaction was performed in triplicate. CCND1 was detected using mouse monoclonal antibodies against CCND1 (sc-20044, Santa Cruz Biotechnology, Santa Cruz, CA) and the standard Western Blotting analysis, as previously described.27Yeste-Velasco M Folch J Casadesus G Smith MA Pallas M Camins A Neuroprotection by c-Jun NH2-terminal kinase inhibitor SP600125 against potassium deprivation-induced apoptosis involves the Akt pathway and inhibition of cell cycle reentry.Neuroscience. 2009; 159: 1135-1147Crossref PubMed Scopus (29) Google Scholar Whole cell extracts of protein were obtained by lysing cells using PBS-1% Triton X-100 (T8532, Sigma) and 25 μg were loaded onto 12% acrylamide gels and were separated by SDS polyacrylamide gel electrophoresis, keeping the default settings for baselines and thresholds electrophoresis. Proteins were subsequently transferred to polyvinylidene difluoride membranes (Immobilon-P, Milllipore, Billerica, MA) and incubated with the monoclonal antibodies against CCND1 and β-Actin (A5441, Sigma). Bands were detected using horseradish peroxidase chemiluminescence-based detection kit (Millipore). The standard avidin-biotin complex method (Vector ABC kit, Vector Laboratories Inc, Burlingame, CA) was used for immunostaining. Briefly, 4-μm tissue sections were dewaxed in xylene and rehydrated. Antigen retrieval was then performed by pressure cooking (10 minutes) in citrate buffer, pH 6.0 using Vector laboratories antigen unmasking solution. Endogenous peroxidase activity was blocked by hydrogen peroxide and nonspecific staining was prevented using diluted (1/50) normal horse serum. Sections were then incubated with diluted (1/50) CCND1 Clone SP4, VP-RMO3 (Vector Laboratories Inc) primary antibody for 40 minutes at room temperature. The bound antibody was detected with biotinylated universal secondary antibody for 30 minutes and avidin-biotin complex for 20 minutes, and 3, 3′-diaminobenzidine (BioGenex, San Ramon, CA) was used as the chromogen. Finally, slides were counterstained with hematoxylin. In control experiments, the primary antibody was replaced by bovine serum albumin phosphate buffer solution. The basal epithelial cells of the normal tonsil were used as positive controls. CCND1 expression was scored as negative, weakly positive, and strongly positive stain and the percentage of positively stained cells was counted for each sample. Cells from SusaR and PC3 cell lines were transfected with 40 nmol/L and 133.3 nmol/L CCND1 ON-TARGET plus SMARTpool small-interfering RNA (siRNA) respectively using Oligofectamine transfection reagent (Invitrogen). Negative controls were added that included untreated and nontargeting siRNA-transfected cells. Following a 48-hour transfection, cells were treated with 0.5 μmol/L and 8.65 μmol/L cisplatin respectively. Both floating and attached cells were harvested for cell count analysis using a Beckman Coulter Vi-CELL XR cell counter (Beckman Coulter, Fullerton, CA). For SusaR cells, ATP assay was also used as described above to determine cell viability. For the gene expression microarray profiling study, a one-way parametric non-equal variance analysis of variance (between groups) test (P < 0.05) was applied to the normalized data (Welch analysis of variance test). Genes (probes) with statistically significant differences in mean expression value were identified. No further multiple testing correction was applied. The gene (probe) list generated after Welch analysis of variance test was then filtered on confidence with “t-test P value” as the measurement. The final statistically significant genes (probes) were retained for further clustering and ontology analysis. We applied two-tailed Student’s t-tests for the analysis of cell cycle distribution and qRT-PCR CCND1 expression; and one-tailed Student’s t-tests for the analysis of cell viability and other functional analyses. No apparent change in growth rate or morphology associated with the resistant phenotype was observed when comparing the parental and resistant TGCT cell lines. By comparing the EC50 of resistant and parental cells, a 1.78(0.27 vs. 0.48 μmol/L)-, 2.67(1.25 vs. 3.34 μmol/L)-, and 3.80 (0.51 vs. 1.94 μmol/L)-fold resistance to cisplatin treatment was observed in the 833K, GCT27, and Susa cell lines, respectively. The cell response curves to cisplatin from which the EC50s were derived have been published separately.28Perry J Powles T Shamash J Veerupillai A McGrowder E Noel E Lu YJ Oliver T Joel S The relative activity of cisplatin, oxaliplatin and satraplatin in testicular germ cell tumour sensitive and resistant cell lines.Cancer Chemother Pharmacol. 2009; 64: 925-933Crossref PubMed Scopus (9) Google Scholar Cell cycle distribution was monitored across the three pairs of TGCT cell lines (see Supplemental Table S1 at http://ajp.amjpathol.org). When comparing the parental lines to their corresponding resistant derivatives, a significantly higher number of 833KR and GCT27R cells were found to accumulate in G1/0 (P = 0.0356 and 0.006, respectively), as compared with the parental lines. Apoptosis was significantly reduced in SusaR (P = 0.012) and GCT27R (P = 0.002), but was at a similar level in 833K sensitive and resistant cells. Unsupervised hierarchical clustering analysis of Affymetrix gene expression microarray data showed a clear grouping of the biological duplicates together, indicating good quality array data. Interestingly, sublines derived from the same cell line clustered together, rather segregating by sensitive and resistant cell lines into two distinct groups. This suggested fewer gene expression changes between parental and resistant sublines than between different cell lines. Using GeneSpring supervised analysis, we identified a number of differentially expressed genes/transcripts between individual cell line pairs when comparing the corresponding parental and resistant cell lines. In this study, more importance was given to genes/transcripts that were consistently differentially expressed across all of the three pairs of cell lines, representing genes that are more likely to be found in a large proportion of resistant samples. To achieve this, we determined the transcript probes with a fold-change between resistant and sensitive cells larger than certain cut-off value in each cell line pair. Applying a 1.5-fold cut-off value, only seven up-regulated transcript probes, including three well-characterized genes, and no down-regulated transcript probes, were identified in all three resistant cell lines (Table 1). The CCND1 probe (accession number 208712_at) showed the most dramatic changes across the three pairs of cell lines with a greater than sevenfold overexpression in the SusaR line compared with its parental counterpart. Two transcript probes of CCND1 are present on the U133 plus 2.0 chips (accession number 208712_at and 208711_s_at). We manually checked the expression profile for the second probe (208711_s_at) and found a 1.4-, 1.6-, and 3.9-fold change in 883K, GCT27, and Susa pair of cell lines, respectively. Therefore, although just missing the 1.5-fold change cut-off in 833K, the second probe confirmed that CCND1 is consistently differentially expressed in all of the cell line pairs. The differential expression of CCND1 between parental and resistant cells was confirmed using qRT-PCR in Susa and GCT27 cell lines, although not in the least resistant line 833K28Perry J Powles T Shamash J Veerupillai A McGrowder E Noel E Lu YJ Oliver T Joel S The relative activity of cisplatin, oxaliplatin and satraplatin in testicular germ cell tumour sensitive and resistant cell lines.Cancer Chemother Pharmacol. 2009; 64: 925-933Crossref PubMed Scopus (9) Google Scholar(Figure 1A). Differences in expression of CCND1 protein were less obvious than that at RNA level, possibly due to the lower dynamic range of the approach, such that only the Susa pair of cell lines showed apparent difference between sensitive and resistant sublines (2.5 fold) (see Supplemental Figure S1 at http://ajp.amjpathol.org.).Table 1Consistently Up-regulated Transcripts (1.5-fold) in the Three Resistant TGCT Cell Lines, as Compared with Their Parental Sensitive LinesHybridization value (normalized)ID NumberNameLocationDescription833K833KRGCT27GCT27RSusaSusaR1560133_atGIGYF22q37GRB10 interacting GYF protein 20.9991.6140.9991.7920.9381.647242593_at—Transcribed sequence with weak similarity to protein NP_112159.1 (Hs)0.9771.5031.0001.7150.7091.8881562169_at—mRNA full length insert cDNA clone EUROIMAGE 1317750.8951.4350.9811.9590.9972.840212240_s_atPIK3R15q13.1phosphoinositide-3-kinase, regulatory subunit 1 (alpha)0.9951.5640.9982.1681.0002.048235274_at—Transcribed sequence with w
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