Details of a Researcher - Kawakami, Yutaka

Papers - Kawakami, Yutaka

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A novel cancer testis antigen that is frequently expressed in pancreatic, lung, and endometrial cancers

Okada Takaho, Akada Masanori, Fujita Tomonobu, Iwata Takashi, Goto Yasufumi, Kido Kenji, Okada Tsutomu, Matsuzaki Yuriko, Kobayashi Kouichi, Matsuno Seiki, Sunamura Makoto, Kawakami Yutaka

Clinical Cancer Research 12 ( 1 ) 191 - 197 2006.01

ISSN 1078-0432

Purpose: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications. Experimental Design: To efficiently isolate cancer testis antigens, a testis cDNA library was immunoscreened (SEREX) with serum from a patient with pancreatic ductal adenocarcinoma. The expression of isolated antigens in various cancer cell lines and tissues was evaluated by reverse transcription-PCR and Northern blot analyses. The immunogenicity of the antigen in cancer patients was evaluated by detection of the IgG antibody in sera from patients with various cancers. Results: Of the three clones isolated through screening of a total of 2 × 10⁶ cDNA library clones, one clone (KU-CT-1) was found to be expressed in various cancers but only in testis among normal tissues, indicating that it was a novel cancer testis antigen. The KU-CT-1 gene is located on chromosome 10p12 and produces two splice variants, which encode proteins of 397 and 872 amino acids, respectively. KU-CT-1 was expressed in pancreatic cancer tissues (3 of 9, 33%), lung cancer tissues (9 of 24, 38%), and endometrial cancer tissues (7 of 11, 64%). Specific serum IgG antibodies were detected in 3 of 20 pancreatic cancer patients, 2 of 12 endometrial cancer patients, 1 of 18 colon cancer patients, and 1 of 10 prostate cancer patients but not detected in 30 healthy individuals. Conclusions: KU-CT-1 is a new cancer testis antigen that is expressed in pancreatic, lung, and endometrial cancers and may be useful for diagnosis and immunotherapy for patients with various cancers.

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Possible involvement of allogeneic antigens recognised by donor-derived CD4 ⁺ cytotoxic T cells in selective GVL effects after stem cell transplantation of patients with haematological malignancy

Matsushita Maiko, Yamazaki Rie, Ikeda Hideyuki, Mori Takehiko, Sumimoto Hidetoshi, Fujita Tomonobu, Okamoto Shinichiro, Ikeda Yasuo, Kawakami Yutaka

British Journal of Haematology 132 ( 1 ) 56 - 65 2006.01

ISSN 0007-1048

Cytotoxic T lymphocyte (CTL) lines specific for allogeneic antigens were generated by in vitro stimulation of donor-derived peripheral blood mononuclear cells obtained from patients who received human leucocyte antigen (HLA)-matched allogeneic haematopoietic stem cell transplantation (HSCT). One of the allogeneic antigen-specific CD4 ⁺ CTL lines, CTL-A, generated from a patient with T cell acute lymphoblastic leukaemia, recognised HLA-DPB1*0501-positive Epstein-Barr virus-immortalised human B cell line (EBV-B cells), phytohaemagglutinin blasts and leukaemia cells, but not interferon-γ (IFN-γ) treated HLA-DPB1*0501-positive fibroblasts, indicating that this CD4 ⁺ T-cell line recognised a minor histocompatibility antigen (mHa) that is preferentially expressed in haematopoietic cells in an HLA-DPB1*0501-restricted manner. The other CD4 ⁺ CTL line, CTL-B, generated from a patient with chronic myeloid leukaemia, recognised mismatched HLA-DQB1*0303 on EBV-B cells and phytohaemagglutinin (PHA) blasts. Interestingly, this CTL line did not recognise IFN-γ-treated recipient's skin fibroblasts, as HLA-DQ was merely upregulated even after IFN-γ stimulation in non-haematopoietic cells including fibroblasts, endothelial cells and hepatocytes. These results suggest that these CD4 positive CTLs, specific for mismatch HLA-DQ and mHa that are preferentially expressed on haematopoietic cells, may play an important role in induction of selective graft-versus-leukaemia effect without development of graft-versus-host disease after allogeneic HSCT.

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Novel autoantibodies to a voltage-gated potassium channel K_V1.4 in a severe form of myasthenia gravis

Suzuki Shigeaki, Satoh Takashi, Yasuoka Hidekata, Hamaguchi Yasuhito, Tanaka Kortaro, Kawakami Yutaka, Suzuki Norihiro, Kuwana Masataka

Journal of Neuroimmunology 170 ( 1-2 ) 141 - 149 2005.12

ISSN 0165-5728

Sera from patients with myasthenia gravis (MG) were screened for autoantibodies to skeletal muscle-specific antigens by immunoprecipitation assay, using rhabdomyosarcoma and leukemia cell lines. Eleven of 61 MG sera immunoprecipitated a rhabdomyosarcoma-specific 70-kDa protein, which was identified as the voltage-gated K⁺ channel 1.4 (K_V1.4). This antibody specificity was not detected in 30 patients with polymyositis/dermatomyositis, 9 with thymoma alone, or 30 healthy controls. Clinical features associated with anti-K_V1.4 antibody included bulbar involvement, myasthenic crisis, thymoma, myocarditis, and QT prolongation on electrocardiogram. These findings suggest that anti-K_V1.4 antibody is a novel autoantibody associated with a severe MG subset and thymoma.

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Cardiomyogenic potential of mesenchymal progenitors derived from human circulating CD14⁺ monocytes

Kodama Hiroaki, Inoue Takafumi, Watanabe Ryuichi, Yasuoka Hidekata, Kawakami Yutaka, Ogawa Satoshi, Ikeda Yasuo, Mikoshiba Katsuhiko, Kuwana Masataka

Stem Cells and Development 14 ( 6 ) 676 - 686 2005.12

ISSN 1547-3287

Previously, we reported a unique CD14⁺CD45⁺CD34 ⁺type I collagen⁺ cell fraction derived from human circulating CD14⁺ monocytes, named monocyte-derived mesenchymal progenitors (MOMPs). These primitive cells differentiate along mesenchymal lineages, including bone, cartilage, fat, and skeletal muscle. Here, we demonstrate that CD14⁺ monocytes generate MOMPs that differentiate into cardiomyocytes. MOMPs labeled with a fluorescent marker and co-cultivated with rat cardiomyocytes for 4 weeks expressed the cardiomyocyte-specific transcription factors Nkx2.5, GATA-4, eHAND, and MEF2 and the hematopoietic/monocytic markers CD45 and CD14 within 10 days and retained their proliferative capacity for up to 16 days. A subpopulation of MOMPs subsequently expressed the cardiomyocyte-specific markers α-sarcomeric actinin, troponin I, and atrial natriuretic peptide on day 21. Furthermore, fluorescence-labeled, spontaneously beating cells that formed gap junctions with adjacent rat cardiomyocytes appeared in these cultures and these cells exhibited electrophysiological properties typical of ventricular myocytes. The co-cultivation of human MOMPs with rat GFP-tagged cardiomyocytes resulted in the generation of human cardiomyocytes lacking green fluorescent protein (GFP) staining, suggesting that our observations could not solely be explained by cell fusion. Our results demonstrate for the first time that human circulating CD14⁺ monocytes include progenitors capable of proliferating and differentiating along the cardiomyogenic lineage via their differentiation into MOMPs.

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Donor fibroblast chimerism in the pathogenic fibrotic lesion of human chronic graft-versus-host disease

Ogawa Yoko, Kodama Hiroaki, Kameyama Kaori, Yamazaki Kazuto, Yasuoka Hidekata, Okamoto Shinichiro, Inoko Hidetoshi, Kawakami Yutaka, Kuwana Masataka

Investigative Ophthalmology and Visual Science 46 ( 12 ) 4519 - 4527 2005.12

ISSN 0146-0404

PURPOSE. Tissue atrophy and excessive fibrosis are prominent histologic features of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation, but the underlying mechanism remains unknown. The current study was undertaken to investigate whether the increase in fibroblasts at the site of pathogenic fibrosis originated from transplanted donor cells in patients with chronic GVHD. METHODS. Lacrimal gland biopsy specimens were obtained from nine patients with chronic GVHD. The male-specific sequences detected by fluorescein in situ hybridization (FISH) and in situ hybridization (ISH) were used as markers for the donor cells in seven female patients who had received a transplant from male donors. Primary fibroblast cultures were generated from lacrimal gland biopsy specimens and examined for mismatched genetic markers between recipients and donors. RESULTS. In lacrimal gland specimens obtained from seven female patients who received a sex-mismatched transplant, 13.4% to 26.7% of CD34⁺ fibroblasts that accumulated in the fibrotic lesion were donor derived, as determined by FISH for the Y-chromosome. The male-specific mRNA was also detected in the lacrimal gland fibroblasts by ISH. Primary lacrimal gland fibroblast cultures were generated from four patients with chronic GVHD and further examined for mismatched genetic markers between recipients and donors. As a result, the presence of donor origin of the fibroblasts was demonstrated by detecting the Y-chromosome sequence and donor-specific microsatellite genetic markers. CONCLUSIONS. These findings together indicate the chimeric status of accumulated CD34⁺ fibroblasts in the lacrimal gland of patients with chronic GVHD. Fibroblasts originating from circulating donor-derived precursors may participate in the excessive fibrosis in these patients.

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Carbonic anhydrase II is a tumor vessel endothelium-associated antigen targeted by dendritic cell therapy

Yoshiura Kenta, Nakaoka Takashi, Nishishita Toshihide, Sato Katsuaki, Yamamoto Akifumi, Shimada Shinji, Saida Toshiaki, Kawakami Yutaka, Takahashi Tsuneo A., Fukuda Hiroyuki, Imajoh-Ohmi Shinobu, Oyaizu Naoki, Yamashita Naohide

Clinical Cancer Research 11 ( 22 ) 8201 - 8207 2005.11

ISSN 1078-0432

Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium-associated antigen in melanoma and other cancers, and elicitation of serum anti-CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.

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Immune responses to DNA mismatch repair enzymes hMSH2 and hPMS1 in patients with pancreatic cancer, dermatomyositis and polymyositis

Okada Takaho, Noji Shinobu, Goto Yasufumi, Iwata Takashi, Fujita Tomonobu, Okada Tsutomu, Matsuzaki Yuriko, Kuwana Masataka, Hirakata Michito, Horii Akira, Matsuno Seiki, Sunamura Makoto, Kawakami Yutaka

International Journal of Cancer 116 ( 6 ) 925 - 933 2005.10

ISSN 0020-7136

To identify tumor antigens useful for diagnosis and immunotherapy of patients with pancreatic ductal adenocarcinoma, we applied a SEREX approach with a cDNA library made from 5 pancreatic cancer cell lines and sera obtained from 8 patients with pancreatic cancer, and isolated total 32 genes, including 14 previously characterized genes and 18 genes with unknown functions. Among these isolated antigens, serum IgG antibodies for 2 isolated DNA mismatch repair enzymes, Homo sapiens mutS homolog 2 (HMSH2) and Homo sapiens postmeiotic segregation increased 1 (hPMS1), were detected in patients with pancreatic ductal adenocarcinoma and dermatomyositis (DM), and polymyositis (PM), but not in sera from healthy individuals. Immunohistochemical study demonstrated that hMSH2 and hPMS1 were over-expressed in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. These results suggested that hMSH2 and hPMS1 may be useful as CD4+ helper T cell antigens for immunotherapy of pancreatic cancer patients and that serum IgG antibodies may be useful for diagnosis of patients with pancreatic ductal adenocarcinoma and DM/PM.

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Immunological detection of altered signaling molecules involved in melanoma development

Kawakami Yutaka, Sumimoto Hidetoshi, Fujita Tomonobu, Matsuzaki Yuriko

Cancer and Metastasis Reviews 24 ( 2 ) 357 - 366 2005.06

ISSN 0167-7659

To understand immune responses to human cancer and develop more effective immunotherapy, human tumor antigens has been isolated using various immunological methods with tumor reactive T cells or antibodies obtained from patients with melanoma. During the process of tumor antigen isolation, various molecules with genetic alterations or over-expression in tumor cells, which may be involved in proliferation, differentiation, or survival of various cancer cells, were identified. In melanoma, abnormal molecules with mutations including β -catenin, CDK4, and BRAF, and molecules with increased expression including Survivin, were immunologically detected. Therefore, immunological isolation of human tumor antigens contributes to the identification of important molecules including altered signaling molecules involved in melanoma formation.

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Frequent immune responses to a cancer/testis antigen, CAGE, in patients with microsatellite instability - Positive endometrial cancer

Iwata Takashi, Fujita Tomonobu, Hirao Nobumaru, Matsuzaki Yuriko, Okada Tsutomu, Mochimaru Hiroshi, Susumu Nobuyuki, Matsumoto Eri, Sugano Kokichi, Yamashita Naohide, Nozawa Shiro, Kawakami Yutaka

Clinical Cancer Research 11 ( 10 ) 3949 - 3957 2005.05

ISSN 1078-0432

Purpose: Identification of cancer/testis antigens useful for diagnosis or immunotherapy of cancers was attempted by cDNA expression cloning with patients' sera (SEREX). Experimental Design: cDNA expression libraries made from testis or endometrial cancer cell lines were screened using sera from patients with endometrial cancer or melanoma patients immunized with dendritic cells pulsed with autologous tumor lysates. Tissue-specific expression by RT-PCR and immunogenicity by Western blotting of the bacterial recombinant antigen with sera from cancer patients were evaluated. Results: A cancer/testis antigen, CAGE, was isolated by two independently performed SEREX. CAGE was expressed in various cancer cell lines including endometrial cancer, colon cancer, and melanoma in 7 of 10 endometrial cancer tissues and in 1 of 3 atypical endometrial hyperplasia, but not in normal tissues including the endometrium and testis. The protein expression on cancer cells was confirmed by Western blot analysis with the recombinant CAGE protein, anti-CAGE IgG antibody was detected in sera from 5 of 45 endometrial cancer, 2 of 24 melanoma, and 2 of 33 colon cancer patients, but not in sera from healthy individuals. By ELISA analysis, anti-CAGE antibody was detected in 12 of 45 endometrial cancer, 2 of 20 melanoma, and 4 of 33 colon cancer patients. Intriguingly, anti-CAGE antibody was highly positive in 7 of the 13 (53.8%) microsatellite instability (MSI)-H patients with endometrial cancer, but negative in 20 non-MSI-H patients (P = 0.001). Conclusion: CAGE may be useful for immunotherapy and diagnosis of various cancers particularly MSI-positive endometrial cancer.

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Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip Are immunogenic in multiple melanoma patients

Inozume Takashi, Matsuzaki Yuriko, Kurihara Sachiko, Fujita Tomonobu, Yamamoto Akifumi, Aburatani Hiroyuki, Shimada Shinji, Kawakami Yutaka

International Journal of Cancer 114 ( 2 ) 283 - 290 2005.03

ISSN 0020-7136

We applied a strategy that utilized a combination of systematic gene expression analysis with various tissues and immunological detection with sera from melanoma patients to identify melanoma antigens expressed preferentially in melanoma and melanocytes. We selected 101 genes by comparing cDNA profiles obtained by GeneChip analysis of a highly pigmented melanoma cell line, SKmel23, primary cultured melanocytes, HUVECs cultured endothelial cells, keratinocytes, liver and stomach. After the additional selection with criterion of high registered frequency of each cDNA in melanocyte-related cDNA libraries in the NCBI database, 15 genes including 12 known melanocyte specific genes were identified. One of the remaining 3 genes, FCRL/FREB, encoding a member of the Fc receptor family that was previously reported to express in germinal center B cells, was found to express preferentially in melanocytes and melanoma tissues by RT-PCR and Northern blot analysis. The FCRL/FREB protein was detected in the cytoplasm of melanoma cells by staining with the murine polyclonal antibody and by transfection with GFP-fused FCRL/FREB cDNA. The bacterial recombinant protein was recognized by serum IgG antibody obtained from some patients with melanoma. These results suggest that FCRL/FREB may function in melanocytes and melanoma and may be useful for development of diagnostic methods for various pigment disorders and immunotherapy of melanoma.

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Binding of β₂-glycoprotein I to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells

Kuwana Masataka, Matsuura Eiji, Kobayashi Kazuko, Okazaki Yuka, Kaburaki Junichi, Ikeda Yasuo, Kawakami Yutaka

Blood 105 ( 4 ) 1552 - 1557 2005.02

ISSN 0006-4971

Antiphospholipid syndrome (APS) is an autoimmune prothrombotic disorder in association with autoantibodies to phospholipid (PL)-binding plasms proteins, such as β₂-glycoprotein I (β₂GPI). We have recently found that CD4⁺ T cells autoreactive to β ₂GPI in patients with APS preferentially recognize a cryptic peptide encompassing amino acid residues 276-290 (p276-290), which contains the major PL-binding site, in the context of DR53. However, it is not clear how previously cryptic p276-290 becomes visible to the immune system and elicits a pathogenics autoimmune response to β₂GPI. Here we show that presentation of a disease-relevant cryptic T-cell determinant in β₂GPI is induced as a direct consequence of antigen processing from β₂GPI bound to anionic PL. Dendritic cells or macrophages pulsed with PL-bound β₂GPI induced a response of p276-290-specific CD4⁺ T-cell lines generated from the patients in an HLA-DR-restricted and antigen-processing-dependent manner but those with β₂GPI or PL alone did not. In addition, the p276-290-reactive T-cell response was primed by stimulating peripheral blood T cells from DR53-carrying healthy individuals with dendritic cells bearing PL-bound β₂GPI in vitro. Our finding is the first demonstration of an in vitro mechanism eliciting pathogenic autoreactive T-cell responses to β₂GPI and should be useful in clarifying the pathogenesis of APS.

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Genomic alterations in primary cutaneous melanomas detected by metaphase comparative genomic hybridization with laser capture or manual microdissection

Namiki Takeshi, Yanagawa Shigeru, Izumo Toshiyuki, Ishikawa Masashi, Tachibana Masayoshi, Kawakami Yutaka, Yokozeki Hiroo, Nishioka Kiyoshi, Kaneko Yasuhiko

Cancer Genetics and Cytogenetics 157 ( 1 ) 1 - 11 2005.02

ISSN 0165-4608

To clarify the correlation of genomic alterations with clinical and histological features, we performed metaphase comparative genomic hybridization analysis on 20 primary cutaneous melanomas, which were obtained by laser capture or manual microdissection, and 16 melanoma cell lines. There were no differences in the average number of aberrations between acral melanomas (AM) and non-AM, although gains of 5q and 11q13 were more frequent (P = 0.05) and 10q loss was less frequent (P = 0.01) in AM than in non-AM. Although tumor thickness is considered a measurable estimate of clinical expression, there were no differences in the average number of aberrations among 4 groups, classified by thickness of the tumor. While the majority of aberrations were equally distributed among the 4 groups, 6p gains were found only in the thickest tumors. Patients with 6p or 1q gains had a lower overall survival rate than those without them (P = 0.0002 or P = 0.013). While gains of 1q, 2q, 3p, 3q, 7q, 20p, and 20q were more frequent in the cell lines than in the primary tumors (P < 0.01), losses of 6q, 9p, 10p, and 10q were equally found in both cell lines and primary tumors. The present study showed that chromosomal aberrations had already occurred in the thinner tumors, and that 6p and 1q gains may be a prognostic factor.

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Lack of circulating autoantibodies to bone morphogenetic protein receptor-II or activin receptor-like kinase 1 in mixed connective tissue disease patients with pulmonary arterial hypertension

Satoh T., Kimura K., Okano Y., Hirakata M., Kawakami Y., Kuwana M.

Rheumatology 44 ( 2 ) 192 - 196 2005.02

ISSN 1462-0324

Objectives. To examine whether autoantibodies against bone morphogenetic protein receptor-II (BMPR-II) or activin receptor-like kinase 1 (ALK-1) are associated with pulmonary arterial hypertension (PAH) in patients with mixed connective tissue disease (MCTD). Methods. We studied sera from 37 MCTD patients with or without PAH, six patients with idiopathic PAH, and 30 healthy controls. Circulating anti-BMPR-II and anti-ALK-1 antibodies were detected using immunoprecipitation of recombinant antigens generated by in vitro transcription/translation and indirect immunofluorescence of cultured cells that were induced to express these antigens by gene transfer. Anti-BMPR-II antibodies were further examined by immunoprecipitation and immunoblotting using a recombinant fragment of the extracellular domain of BMPR-II. Results. Serum anti-BMPR-II and anti-ALK-1 autoantibodies were not detected in MCTD patients irrespective of the presence or absence of PAH, or in patients with idiopathic PAH. Conclusions. Our finding does not support the hypothesis that autoantibody-mediated dysregulation of signals through BMPR-II or ALK-1 contributes to the development of PAH in patients with connective tissue diseases.

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Systematic identification of human melanoma antigens using Serial Analysis of Gene Expression (SAGE)

Matsuzaki Yuriko, Hashimoto Shin Ichi, Fujita Tomonobu, Suzuki Takuji, Sakurai Toshiharu, Matsushima Kouji, Kawakami Yutaka

Journal of Immunotherapy 28 ( 1 ) 10 - 19 2005.01

ISSN 1524-9557

To identify new melanoma antigens using systematic gene expression analysis combined with rapid screening of patient sera for immunogenicity, a serial analysis of gene expression (SAGE) method was applied to profile transcripts in a highly pigmented melanoma cell line SKmel23. 25,997 SAGE tags consisting of 10,382 unique transcripts were sequenced. This melanoma SAGE library was compared with a testis SAGE library and the colon SAGE database, and to the cDNA database obtained by random sequencing of a melanocyte cDNA library. Among the 15 tags finally selected with criteria of preferential expression on melanoma and melanocytes at relatively high frequency, two tags were further analyzed for their structure and immunogenicity. One was identified as PAX3, and its isoform, PAX3d, was found to be dominantly expressed in melanoma and melanocytes. The other was derived from a novel gene and its full-length cDNA clone was isolated. Preferential expression of these genes in melanoma and melanocytes was confirmed by RT-PCR and Northern blot analysis. The recombinant bacterial PAX3d protein was recognized by serum IgG from some patients with melanoma and Vogt-Koyanagi-Harada (VKH) disease but not from healthy individuals, indicating that PAX3d is a new melanocyte-specific antigen immunogenic in patients with melanoma or VKH disease. The authors report two melanocyte/melanoma-specific molecules, which may be useful for development of diagnosis or treatment of these pigment disorders. In addition, a system using SAGE and immunoscreening with patients' sera is shown to be an efficient method for the systematic identification of tumor antigens.

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference

Sumimoto H., Yamagata S., Shimizu A., Miyoshi H., Mizuguchi H., Hayakawa T., Miyagishi M., Taira K., Kawakami Y.

Gene Therapy 12 ( 1 ) 95 - 100 2005.01

ISSN 0969-7128

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27^Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27^Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

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Identification of human tumor antigens recognized by T cells

Kawakami Yutaka

Nippon rinsho. Japanese journal of clinical medicine 63 Suppl 4 562 - 567 2005

ISSN 0047-1852

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines

Tanami Hideaki, Imoto Issei, Hirasawa Akira, Yuki Yasuhiro, Sonoda Itaru, Inoue Jim, Yasui Kohichiro, Misawa-Furihata Akiko, Kawakami Yutaka, Inazawa Johji

Oncogene 23 ( 54 ) 8796 - 8804 2004.11

ISSN 0950-9232

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive over-expression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

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Autoreactive CD8+ cytotoxic T lymphocytes to major histocompatibility complex class I chain-related gene A in patients with Behçet's disease

Yasuoka Hidekata, Okazaki Yuka, Kawakami Yutaka, Hirakata Michito, Inoko Hidetoshi, Ikeda Yasuo, Kuwana Masataka

Arthritis and Rheumatism 50 ( 11 ) 3658 - 3662 2004.11

ISSN 0004-3591

Objective. To detect and characterize the autoreactive CD8+ T cells to major histocompatibility complex class 1 chain-related gene A (MICA), a stress-inducible antigen preferentially expressed on the epithelium and endothelium, in patients with Behçet's disease (BD). Methods. A candidate for the antigenic MICA peptide was selected based on its predicted binding affinity for HLA-B51 and proteasomal cleavage sites. Peripheral blood T cells from 14 patients with BD and 15 healthy controls were repeatedly stimulated with the MICA peptide, and the specific T cell response was measured by peptide-induced interferon-γ. Cytotoxic T lymphocyte activity was examined by chromium-51 release from an BLA-151-transfected B cell line in the presence of the MICA peptide. Results. A 9-mer peptide AAAAAIFVI (termed MICA transmembrane [MICA-TM]) was selected as a candidate for the antigenic peptide presented by HLA-B51. A specific T cell response to MICA-TM was detected in 4 patients with BD (29%) but in none of the 15 healthy donors. All 4 responders had HLA-B51 and active disease, and the specific T cell response was lost after the BD-related symptoms disappeared. The MICA-induced T cell response was specifically inhibited by anti-HLA class I antibody or by CD8+ cell depletion. MICA-reactive T cells recognized an HLA-B51-transfected B cell line pulsed with MICA-TM or a B cell line transfected with both HLA-B51 and MICA in the absence of exogenous peptides. Finally, MICA-stimulated T cell lines lysed the HLA-B51-expressing B cell line in the presence of MICA-TM. Conclusion. HLA-B51-restricted cytotoxic T lymphocytes autoreactive to MICA may be involved in the pathogenesis of BD.

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Recent progress in identification methods for human tumor antigens recognized by T cells

Hayashi Emiko, Matsuzaki Yuriko, Kawakami Yutaka

Biotherapy 18 ( 6 ) 497 - 502 2004.11

ISSN 0914-2223

Recent identification of human tumor antigens recognized by T cells made it possible to analyze anti-tumor immune responses quantitatively and qualitatively in human and to develop immunotherapy in a more scientific way. The identification methods have been being improved during the past 10 years. When tumor reactive T-cells are available, DNA expression cloning or direct peptide sequencing from HPLC fractions extracted from MHC class I/peptide complex can be performed. However, it is difficult to establish tumor reactive T cells in many tumors. In those cases, we can apply the reverse immunology approach, in which tumor antigen candidates are first identified by various genetic or immunological methods, including cDNA subtraction between cancers and various tissues with cDNA profiles obtained through systematic gene analysis such as DNA chip/microarrays, SAGE, or public gene databases, or cDNA expression cloning with patients' sera (SEREX). Then antigenicity of the identified candidates is confirmed by demonstration of T cell induction against candidates through in vitro T cell induction or immunization of HLA transgenic mice. We have made various modifications on these techniques. Identification of human tumor antigens in various cancers will lead to development of more effective immunotherapy in the future.

Identification of an epigenetically silenced gene, RFX1, in human glioma cells using restriction landmark genomic scanning

Ohashi Yohei, Ueda Masakazu, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

Oncogene 23 ( 47 ) 7772 - 7779 2004.10

ISSN 0950-9232

To identify the CpG islands differentially methylated in human glioma, we performed restriction landmark genomic scanning with a CpG methylation-sensitive enzyme. We found 12 spots, the intensity of which was entirely lost or decreased in both the human glioma tissues examined as compared with that in matched normal lymphocytes, indicating aberrant methylation of these CpG islands in gliomas. The expression of RFX1, one of the genes associated with the methylated CpG islands, was frequently decreased in human glioma cell lines and tissues. We also demonstrated that the isolated CpG island located in the seventh intron of the RFX1 gene had enhancer activity and was hypermethylated in all of the glioma tissues and cell lines analysed, but not in normal brains or lymphocytes. Treatment of glioma cells with a demethylating agent, 5-azacytidine, resulted in the expression of RFX1, indicating that the silencing of the RFX1 gene may be attributable to its methylation. RFX1 has been implicated in transcriptional downregulation of the proto-oncogene c-myc. By expression of the RFX1 gene, the cellular proliferative activity of glioma cells was suppressed. Taken together, these results suggest that the RFX1 gene may be epigenetically silenced in human gliomas and involved in glioma tumorigenesis.

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