Lately, analyses of gene expression profiles of cancer and normal cells using cDNA microarray technologies have provided an effective approach for the identification of tumour-associated antigens (TAAs) (Nakatsuraet al, 2004b;Uchidaet al, 2004;Yoshitakeet al, 2004;Watanabeet al, 2005;Komoriet al, 2006;Sudaet al, 2006;Haraoet al, 2008;Imaiet al, 2008). from peripheral blood mononuclear cells of HLA-A2+healthy donors byin vitrostimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2. == Conclusion: == KIF20A is usually a novel promising candidate for anticancer immunotherapeutic Harpagoside target for pancreatic cancers. Keywords:anticancer immunotherapy, tumour-associated antigen, CTL, Harpagoside KIF20A/RAB6KIFL/MKlp2, HLA-transgenic mouse Pancreatic cancer is one of the highly lethal malignancies with an overall 5-year survival rate of 5% (Jemalet al, 2007). Although a surgical resection is the only treatment for long-term survival, patients with resectable pancreatic cancer are in the minority (922%) (Eloubeidiet al, 2006;Goonetilleke and Siriwardena, 2007). Furthermore, even the 5-12 months survival rate after a curative resection is usually reported to be 20% (Clearyet al, 2004;Smeenket al, 2005;Moonet al, 2006). Therefore, there is a strong Rabbit polyclonal to MET need for development of novel therapeutic modalities. Anticancer immunotherapy is considered to be the candidate modality for pancreatic cancer. Recently, analyses of gene expression profiles of cancer and normal cells using cDNA microarray technologies have provided an effective approach for the identification of tumour-associated antigens (TAAs) (Nakatsuraet al, 2004b;Uchidaet al, 2004;Yoshitakeet al, 2004;Watanabeet al, 2005;Komoriet al, 2006;Sudaet al, 2006;Haraoet al, 2008;Imaiet al, 2008). This study analysed the gene expression profiles of pancreatic cancer using a genome-wide cDNA microarray consisting of 27 648 genes, which revealed thatKIF20Awas overexpressed in pancreatic cancer tissues but not in many normal tissues. In this study, we examined whether KIF20A could be a potential target for anticancer immunotherapy. To this aim, human KIF20A-derived and HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes were identified using HLA-A2 transgenic mice (Tgm), and the ability of peptides to induce KIF20A-reactive human CTLs that kill cancer cells and the safety not to induce autoimmune responses in the mouse were investigated. == Materials and methods == == cDNA microarray analysis == A data set of genome-wide cDNA microarray analyses using cancerous and adjacent normal tissues obtained by a laser microbeam dissection (Nakamuraet al, 2004) was used in this study. The tissue samples were obtained from surgical specimens of pancreatic cancer patients. All patients provided their written informed consent to participate in this study. == Mice, cell lines, and HLA expression == The HLA-A2 Tgm, H-2Db/2m/double knockout mice introduced with a monochain gene construct of human2m-HLA-A2.1(1,2)-H-2Db(3, transmembrane, and cytoplasmic) (Pascoloet al, 1997;Firatet al, 1999), were kindly provided by Dr FA Lemonnier. Mice were maintained and handled in accordance with the animal care guidelines of the Kumamoto University. The human pancreatic cancer cell line PANC1, the colon cancer cell line CaCo-2, and a transporter associated with antigen processing (TAP)-deficient and HLA-A2 (A*02:01)-positive cell line T2 were purchased from the Riken Cell Lender (Tsukuba, Japan). The human liver malignancy cell line SKHep1 and the human pancreatic cancer cell line PK9 were provided by Harpagoside Dr Kyogo Itoh (Kurume University, Kurume, Japan) and the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan), respectively. The expression of HLA-A2 was examined by flow cytometry with an anti-HLA-A2 monoclonal antibody (mAb), BB7.2 (One Lambda Inc., Canoga Park, CA, USA) to select HLA-A2-positive blood donors and target malignancy cell lines. == Patients, blood samples, and tumour tissues == The clinical research using peripheral blood mononuclear cells (PBMCs) obtained from healthy donors was approved by the Institutional Review Board of the Kumamoto University. The cancer and adjacent non-cancerous tissues were obtained from 14 patients during routine diagnostic procedures from patients in the Kumamoto University Hospital. The tissues were subjected to either reverse transcriptionPCR (RTPCR) or immunohistochemical analyses as listed inTable 1. Blood and tissue samples were obtained from donors and patients, respectively, after receiving their written informed consent. == Table 1. Expression of theKIF20Agene or protein in pancreatic cancer tissues. == Abbreviations: IHC=immunohistochemical analysis; =negative; NT=not tested; +=positive; RTPCR=reverse transcriptionPCR. RTPCR data are shown inFigure 2. Immunohistochemical data are shown inFigure 3. == RTPCR == Reverse transcriptionPCR analyses were performed as described previously (Nakatsuraet al, 2004a). The primers used were:KIF20A, sense 5-CTACAAGCACCCAAGGACTCT-3 (788-808, the 4th exon) and antisense 5-AGATGGAGAAGCGAATGTTT-3 (1400-1381, the 8th exon) andACTB, sense 5-CATCCACGAAACTACCTTCAACT-3 (903-925, the 5th exon) Harpagoside and antisense 5-TCTCCTTAGAGAGAAGTGGGGTG-3 (1535-1513, the 6th exon). Primer positions are shown according to cDNA sequences presented in the Gene Lender Accession numbers ofNM_005733for humanKIF20Aand ofNM_001101for humanACTB. The products were 612 bp long forKIF20Aand 632 bp long forACTB. After normalisation by the intensity forACTBmRNA, the expression levels ofKIF20AmRNA were.