Ogawa 16_8

Identification of the genes that are differentiallyexpressed between radiosensitive and radioresistant cancer cells is important to the ability to predict the clinical effectiveness of radiotherapy. We established radioresistant human pancreatic cancer cell lines using fractionated irradiation in order to identify genes that are differentially-expressed between parental lines and radioresistant cell sublines. Six pancreatic cancer cell lines (PK-1, PK-8, PK-9, T3M4, MiaPaCa2 and PANC-1) were treated with 10 Gy fractionated irradiation at approximately two-week intervals (total dose 150-180 Gy). Five radioresistant sublines (PK-1, PK-8, PK-9, T3M4, and MiaPaCa2) were successfully established. Using oligonucleotide microarrays containing 17,086 genes, we identified 73 up-regulated genes and 55 down-regulated genes common to radioresistant sublines. Subsequent analysis by quantitative RT-PCR confirmed the reliability of our microarray strategy. Up-regulated genes were associated with growth factor (example, amphiregulin), cell-cycle check point (MAPKAPK2), intracellular signaling pathway (regucalcin), and angiogenesis stimulation (angiopoietin 2). Down-regulated genes were associated with apoptosis (caspase 8), retinoid esterification (lecithin retinol acyltransferase), and electron transport (calcium-activated chloride channel 1). Some of these genes have known association with response to radiation, such as caspase 8 and MAPKAPK2, but others are novel. Global gene analysis of radioresistant sublines may provide new insights into the mechanisms underlying clinical radioresistance and to improving the efficacy of radiotherapy for pancreatic cancer. Introduction Radiotherapy is one of the major adjuvant treatments for many malignant tumors, and it has been frequently applied for patients with pancreatic cancer. The general rationale for radiotherapy is based on the findings that radiation can inhibit cell proliferation or induce apoptotic cell death in vitro and inhibit tumor growth in vivo (1). Pancreatic cancer has one of the poorest prognoses among malignant tumors, with the 5-year survival rate of patients treated with both surgery and chemoradiotherapy ranging from 1 to 2% (2,3). One of the reasons for this low survival rate is the insensitivity of pancreatic cancer to radiotherapy, which decreases the ability to cure or delay progression of disease in these patients (4-6). Therefore, it is necessary to elucidate the mechanisms underlying radioresistance to improve prognosis. Recently, a relationship between radioresistance and expression of several genes has been reported, with candidate genes including p53 (7), ras (8), raf-1 (9), bcl-2 (5), and survivin (6). Although such discoveries have helped develop a partial understanding of the molecular mechanisms responsible for cellular radiosensitivity, the entire process remains to be elucidated. The advent of microarray gene expression technology permits simultaneous analysis of the expression levels of thousands of genes (10-12). Therefore, a study on molecular genetic events related to radiosensitivity can be conducted (13-16). This research may also lead to identification of gene regulatory pathways that result in development of cell resistance to therapeutic procedures. To date, differential gene expression profiles of radioresistant cancer and cancer cell lines including breast cancer, esophageal cancer and uterine cervical cancer have been reported (13-16). However, to the authors' knowledge, there has been no information regarding the global gene analyses of radioresistant pancreatic cancer or cancer cell lines. In the current study, we tried to identify differentiallyexpressed genes between radiosensitive and radioresistant pancreatic cancer cell lines. Radioresistant sublines were established by fractionated irradiation, and we applied oligonucleotide microarrays containing 17,086 genes to parent and radioresistant sublines in order to investigate the differential expression of genes between parent and radioresistant cells. INTERNATIONAL JOURNAL OF ONCOLOGY 28: 705-713, 2006 705 Differential gene expression profiles of radioresistant pancreatic cancer cell lines established by fractionated irradiation KAZUHIKO OGAWA1,2, TOHRU UTSUNOMIYA1, KOSHI MIMORI1, FUMIAKI TANAKA1, NAOTSUGU HARAGUCHI1, HIROSHI INOUE1, SADAYUKI MURAYAMA2 and MASAKI MORI1 1Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Beppu; 2Department of Radiology, University of the Ryukyus, Okinawa, Japan Received August 16, 2005; Accepted October 14, 2005 _________________________________________ Correspondence to: Dr Masaki Mori, Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan E-mail: [email protected] Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction, MAPKAPK2, MAPK-activated protein kinase 2