172 J Nippon Med Sch 2004; 71（3） ―Original― Effect of Ionizing Irradiation on Human Esophageal Cancer Cell Lines by cDNA Microarray Gene Expression Analysis Hideki Bo1,2, Mohammad Ghazizadeh1, Hajime Shimizu1, Yuji Kurihara2, Seiko Egawa1, Yukichi Moriyama2, Takashi Tajiri3, Oichi Kawanami1 1Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School 2Center for Digestive Diseases, Nippon Medical School Second Hospital 3Surgery for Organ Function and Biological Regulation, Nippon Medical School, Graduate School of Medicine Abstract To provide new insights into the molecular mechanisms underlying the effect of irradiation on esophageal squamous cell carcinomas（ESCCs）, we used a cDNA microarray screening of more than 4,000 genes with known functions to identify genes involved in the early response to ionizing irradiation. Two human ESCC cell lines, one eachofwell（TE-1）and poorly（TE-2）differentiated phenotypes were screened. Subconfluent cells of each phenotype were treated with single doses of 2.0 Gy or 8.0 Gy irradiations. After a 15 min incubation time- point, the cells were collected and analyzed. Compared with non-irradiated cells, many genes revealed at least 2-fold upregulation or downregulation at both doses in well or poorly differentiated ESCC cells. The common upregulated genes in well and poorlydifferentiatedcell types at both irradiation doses included SCYA5, CYP51, SMARCD2, COX6C, MAPK8, FOS, UBE2M, RPL6, PDGFRL, TRAF2, TNFAIP6, ITGB4, GSTM3, and SP3 and common downregulated genes involved NFIL3, SMARCA2, CAPZA1, MetAP2, CITED2, DAP3, MGAT2, ATRX, CIAO1, and STAT6. Several of these genes were novel and not previously known to be associated with irradiation. Functional annotations of the modulated genes suggested that at the molecular level, irradiation appears to induce a regularizing balance in ESCC cell function. The genes modulated in the early response to irradiation may be useful in our understanding of the molecular basis of radiotherapy and in developing strategies to augment its effect or establish novel less hazardous alternative adjuvant therapies. （J Nippon Med Sch 2004; 71: 172―180） Key words: esophageal squamous cell carcinoma（ESCC）, cell line, radiation, gene expression, cDNA microarray Preoperative radiotherapy is an acceptable modality Introduction in the treatment of ESCCs. This mode of therapy is thought to improve resection rates, increase survival Esophageal squamous cell carcinomas（ESCCs）are times , and decrease lymph metastases2 . mostly associated with poor prognosis and the Furthermore, several studies have demonstrated majority of patients die within 1 year of diagnosis1. that the inclusion of chemotherapy in this treatment Correspondence to Mohammad Ghazizadeh, MD, Ph D, Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School, 1―396 Kosugi-cho, Nakahara-ku, Kawasaki, 211―8533, Japan E-mail: [email protected] Journal Website（http: !!www.nms.ac.jp!jnms!） J Nippon Med Sch 2004; 71（3） 173 modality improves a patient’soutcome3－5.Hence modulated genes was classified by clustering and chemoradiotherapy is now considered standard biological functions were assigned to the genes therapy in the non-surgical management of locally whichwerethencorrelatedwithexpression advanced ESCCs. This combination therapy has patterns. Functional annotations of the modulated significantly improved median survival and reduced genes could provide clues as to the effect of late relapses in patients with ESCCs3.Ina irradiation as well as suggesting putative genes as randomized trial in patients with ESCCs , molecular targets for developing strategies to preoperative chemotherapy and concurrent augment the irradiation effect or establish novel less radiotherapy significantly improved survival hazardous alternative adjuvant therapies for ESCCs. compared with surgery alone4 .Inanother These approaches may provide new insights into randomized trial in which the preoperative the molecular mechanisms involved in the chemoradiotherapy was applied, a significant irradiation response. survival benefit for combined therapy was observed5. Materials and Methods In addition to the preoperative radiotherapy, postoperative prophylactic radiotherapy has also Cell Lines and Culture been used in the treatment of ESCCs, but with Two human ESCC cell lines（obtained from Cancer contradictory results. In a multicenter controlled Cell Repository, Institute of Development, Aging and trial, postoperative radiotherapy did not increase Cancer, Tohoku University, Sendai, Japan）, one each survival after curative resection of ESCCs6.In of well（TE-1）and poorly（TE-2）differentiated contrast, a recent randomized prospective study on phenotypes were studied. The cell lines had been 495 ESCC patients showed that postoperative established from the resected primary esophageal radiotherapy improved 5-year survival rate in tumors of male patients7. Cells were cultured in patients with positive lymph node metastases and in RPMI-1640 supplemented with 5％ FBS , patients with stage III disease compared with streptomycin（100 µg!ml ）, and penicillin（100 units! 2 similar patients who did not receive radiotherapy . ml ）at 37℃ in a humidified 5％ CO 2 incubator. These results signify the need for defining the molecular mechanism（s）of cellular response to Irradiation and Isolation of RNA ionizing irradiation that may provide clues for the Subconfluent monolayer from each cell line was development of strategies to augment its effect or treated with 2.0 Gy or 8.0 Gy irradiation at 200 kV establish novel less hazardous alternative adjuvant and 10 mA with 0.5 mm Cu and 1.0 mm A1 filters. therapies. Non-irradiated paired preparations served as In this study, we sought to obtain the gene controls. After 15 min incubation time, the cells were expression profile of ESCC cells early after collected for analyses. Total cellular RNA was irradiation. Early alterations in gene expression prepared using the RNeasy Mini Kit（QIAGEN, levels appear more likely to involve genes Hilden, Germany）. The RNA was treated with participating in signaling pathways that determine DNase and precipitated using 95％ ethanol prior to the cell destiny. We cultured human ESCC cell lines cDNA synthesis. Isolated RNA was electrophoresed to reach subconfluence and treated them with 2.0 Gy through 1.0％ agarose-formaldehyde gels to verify （a conventional daily dose during fractionated the quality of the RNA, and RNA concentrations radiotherapy）or 8.0 Gy（usually applied for total-body were determined by UV spectrophotometry. irradiation）irradiation. These conditions approach the situation in clinical practice. After a 15 min cDNA Microarrays incubation time, the cells were collected and The GeneFilters cDNA microarrays（Named analyzed using a cDNA microarray screening Genes; GF211）were purchased from Research against more than 4,000 known genes. The subset of Genetics（Huntsville, AL）. Each filter is a 5×7-cm 174 J Nippon Med Sch 2004; 71（3） charged nylon membrane on which more than 4,000 One µl of the diluted cDNA was used for each PCR sequence-verified human genes, all of which have reaction. The PCR primer set used for each gene; known functions , are printed as individual SCYA 5 was : forward, 5’-CGCTGTCATCC immobilized spots. TCATTGCTA-3’and reverse, 5’-GCTGTCTCGAA CTCCTGACC-3’;the product size, 498 bp ; Hybridization and Analysis of Microarrays SMARCD 2 was : forward, 5’-CTCGTCATTG cDNA microarray filters were hybridized AGCTGGACAA-3’and reverse, 5’-TCTCTGGGTC according to a protocol developed by the TTCAGCTGGT-3’; the product size, 597 bp; COX6C manufacturer （ Research Genetics; http: !!www . was : forward, 5’-GTCAGGAAGGACGTT resgen.com）.Oneµg of total RNA was utilized as a GGTGT-3’ and reverse, 5’-ACCAGCCTTCCTCA template for a reverse transcriptase reaction TCTCCT-3’; the product size, 262 bp ; CAPZA 1 （Superscript II, Invitrogen, Carlsbad, CA）to create was : forward, 5’-TCGGATGAGGAGAAGG ［33P］dCTP labeled cDNA probes. Reactions were TACG-3’ and reverse, 5’-ATCTTAAGCACGCCA purified by chromatography-columns（Bio-Spin 6, Bio- ACCAC-3’; the product size, 557 bp ; SMARCA 2 Rad Labs., Hercules, CA）. The microarray filters was : forward, 5’-AGACGGCTCTCAACTCCA were pre-washed in boiling 0.5％ SDS for 5 min, AA-3’ and reverse, 5’-AGGTCACTCATCTGGCT placed individually in hybridization roller bottles GCT-3’; the product size, 559 bp ; MetAP 2 was : with 5 ml MicroHyb solution, pre-hybridized with 5 forward, 5’-ATG-GCGGGTGTGGAGGAGGT µg denatured poly-dA and Cot-1 DNA（Research AGCGGCCT-3’and reverse, 5’-TTAATAGTCATC Genetics）for 2 h at 42℃, and then hybridized TCCTCTGCTGACAACT-3’; the product size, 1,440 overnight with individual［33P］labeled cDNA probes. bp ; and DAP 3 was : forward, 5’-TGCCTGATGGT The filters were washed for 20 min in hybridization AAGGAAACC-3’and reverse, 5’-TCCCCAAAGAG bottles at 50℃ each with 2×SCC 3 times, 1％ SDS 2 CATTGATTC-3’;the product size, 519 bp. times, and 0.5×SCC!1％ SDS 1 time. Moist filters Glyceraldehyde-3-phosphate dehydrogenase（GAPDH） were wrapped individually with plastic wrap, was used as an internal control in RT-PCR reaction carefully oriented and exposed to phosphor-storage with primers : forward, 5’-TCCACCACC screens（Imaging Screen K, BioRad）in photographic CTGTTGCTGTA-3’ and reverse, 5’-ACCACAGT cassettes for 16 h. Exposed screens were imaged CCATGCCATCAC-3’; the product size, 450 bp. PCR using Molecular Imager FX（Bio-Rad）and tiff files was carried out for 24 cycles consisting of heat were imported into Pathways 4.0 software（Research