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ABSTRACT: In vivo cell cycle analysis in higher eukaryotes has been limited by the challenge of preserving the integrity of the living organism while visualizing dividing cells. Here, we propose a new model, which uses the unique combination of features of the Japanese medaka in order to visualize and manipulate the cell cycle progression in a live vertebrate. Our stable transgenic histone H2B-GFP medaka line allows fluorescence-based monitoring of the chromosomes. The system has a high specificity, with a strong GFP signal labeling the chromatin architecture. The subcellular resolution ensures detection of both normal and abnormal divisions in live recordings. This translates into the possibility to quantify temporal and spatial aspects of the cell cycle, such as length or nuclear size, as well as to expose drug toxicity at the earliest stage. We also show that acclimation to cold, a prominent feature of the eurytherm medaka, is a valuable natural way of inducing a reversible cell cycle arrest in the entire living organism. Our results suggest that this manipulation can be performed from the early stages of development, has no toxicity and does not alter the cell cycle profile of the embryo.
Cell cycle (Georgetown, Tex.) 03/2009; 8(4):620-7. · 5.36 Impact Factor
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Yutaka Kawakami,
Tomonobu Fujita,
Chie Kudo,
Toshiharu Sakurai,
Masaru Udagawa,
Tomonori Yaguchi,
Go Hasegawa,
Emiko Hayashi,
Yoko Ueda,
Tomoko Iwata,
Qinfu Wang,
Starlyn Okada,
Nobuo Tsukamoto, Yuriko Matsuzaki,
Hidetoshi Sumimoto
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ABSTRACT: Human tumor antigens were identified using various immunological and genetic methods, and immune responses to the identified antigens were evaluated in cancer patients. Autologous tumor specific unique antigens derived from genetic alterations in cancer cells were isolated from patients with favorable prognosis after immunotherapy, indicating that they are attractive targets for immunotherapy. Immunogenicity of shared antigens was found to differ among patients due to antigen expression in cancer cells and patients' immunoreactivity. These observations suggest that personalization may be applied for cancer immunotherapy. We therefore developed intratumoral DC administration protocols that are able to induce immune responses to both unique and shared tumor antigens expressed in each individual cancer. By combining cryoablative tumor pretreatment and TLR stimulated DC, the anti-tumor effect of the intratumoral DC administration was significantly augmented in a murine tumor model. This improved protocol enhanced systemic induction of anti-tumor CD8+ CTL, and was able to regress relatively large remote untreated tumors. In clinical trials, systemic immune induction was observed by intratumoral DC administration following cryoablative tumor treatment, although anti-tumor effects are relatively weak, indicating that additional interventions are required for more effective immunotherapy.
Frontiers in Bioscience 02/2008; 13:1952-8. · 3.52 Impact Factor
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ABSTRACT: We previously reported early evidence of the human Fc receptor-like A (hFCRLA), an antigen (Ag) that was specifically expressed in melanocytes, melanoma cells, and some B-cell states, and was recognized by IgG antibodies from melanoma patients. Recently, it has been demonstrated that hFCRLA is expressed in most human B-cell lymphoma tissues. In this report, we investigated the potential of FCRLA as a tumor-associated Ag of B-cell lymphoma for immunotherapy. We confirmed that murine FCRLA (mFCRLA) was expressed and distributed in murine tissues similar to hFCRLA. Recombinant mFCRLA fusion protein was constructed with a polyarginine (R9)-protein-transduction domain (PTD) (rR9-HA-mFCRL), and was transduced into bone marrow-derived dendritic cells (DC) ex vivo. Mice immunized with rR9-HA-mFCRL-treated DC primed cytotoxic T-lymphocyte (CTL) that killed the B-cell lymphoma cell line (A20), which express mFCRLA abundantly. In a tumor challenging study, A20 tumor growth inoculated in skin was significantly suppressed in mice vaccinated with rR9-HA-mFCRL-treated DC, compared with control mice. These results indicated that FCRLA is a potential target Ag in immunotherapy for B-cell lymphoma. In addition, our experimental system using R9-PTD-containing full-length proteins might be a useful method to analyze the immunogenicity of novel candidates of tumor-associated Ags in vivo.
Journal of Investigative Dermatology 01/2008; 127(12):2818-22. · 6.31 Impact Factor
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ABSTRACT: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation.
cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis-like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes.
CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs.
CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.
Clinical Cancer Research 12/2007; 13(21):6267-74. · 7.74 Impact Factor
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Yasufumi Goto, Yuriko Matsuzaki,
Sachiko Kurihara,
Ayako Shimizu,
Tsutomu Okada,
Kazuhiko Yamamoto,
Hiroshi Murata,
Minoru Takata,
Hiroyuki Aburatani,
Dave S B Hoon,
Toshiaki Saida,
Yutaka Kawakami
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ABSTRACT: The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.
Cancer Research 05/2006; 66(8):4443-9. · 7.86 Impact Factor
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Takaho Okada,
Masanori Akada,
Tomonobu Fujita,
Takashi Iwata,
Yasufumi Goto,
Kenji Kido,
Tsutomu Okada, Yuriko Matsuzaki,
Kouichi Kobayashi,
Seiki Matsuno,
Makoto Sunamura,
Yutaka Kawakami
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ABSTRACT: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications.
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.
Of the three clones isolated through screening of a total of 2 x 10(6) 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.
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.
Clinical Cancer Research 02/2006; 12(1):191-7. · 7.74 Impact Factor
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Takaho Okada,
Shinobu Noji,
Yasufumi Goto,
Takashi Iwata,
Tomonobu Fujita,
Tsutomu Okada, Yuriko Matsuzaki,
Masataka Kuwana,
Michito Hirakata,
Akira Horii,
Seiki Matsuno,
Makoto Sunamura,
Yutaka Kawakami
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ABSTRACT: 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.
International Journal of Cancer 11/2005; 116(6):925-33. · 5.44 Impact Factor
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ABSTRACT: 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 beta -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.
Cancer and metastasis reviews 07/2005; 24(2):357-66. · 10.57 Impact Factor
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Takashi Iwata,
Tomonobu Fujita,
Nobumaru Hirao, Yuriko Matsuzaki,
Tsutomu Okada,
Hiroshi Mochimaru,
Nobuyuki Susumu,
Eri Matsumoto,
Kokichi Sugano,
Naohide Yamashita,
Shiro Nozawa,
Yutaka Kawakami
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ABSTRACT: Identification of cancer/testis antigens useful for diagnosis or immunotherapy of cancers was attempted by cDNA expression cloning with patients' sera (SEREX).
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 tum or lysates. Tissue-specific expression by RT-PCR and immunogenicity by Western blotting of the bacterial recombinant antigen with sera from cancer patients were evaluated.
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).
CAGE may be useful for immunotherapy and diagnosis of various cancers particularly MSI-positive endometrial cancer.
Clinical Cancer Research 06/2005; 11(10):3949-57. · 7.74 Impact Factor
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ABSTRACT: 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.
International Journal of Cancer 04/2005; 114(2):283-90. · 5.44 Impact Factor
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ABSTRACT: Human tumor antigens recognized by T cells have been identified by means of various molecular biological and immunological methods, including cDNA expression cloning with patients' T cells and antibodies, cDNA subtraction using RDA and PCR differential display, systematic gene analysis such as DNA sequencing, CGH, DNA chip/microarray and SAGE, in vitro T cell induction and immunization of HLA transgenic mice. The identification of human tumor antigens has led to a better understanding of the nature of tumor antigens, anti-tumor immune responses in patients before and after immunotherapy, and tumor escape mechanisms. The information obtained from these researches has enabled us to develop and improve immunotherapy by attempting to overcome the identified problems, including intrinsically low immunogenicity of tumor antigens and several escape mechanisms, such as regulatory T cell induction. The existence of immunogenic unique antigens derived from genetic alterations in tumor cells, and the varied immunogenicity of shared tumor antigens among patients due to differing expression in tumor cells and immunoreactivity of patients, indicates that individualized immunotherapy should ideally be performed. The identified antigens will also be useful for development of diagnostic methods and molecular targeting therapy for cancer.
Cancer Science 11/2004; 95(10):784-91. · 3.33 Impact Factor
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Keiichi Ito,
Tomonobu Fujita,
Masanori Akada,
Yukiko Kiniwa,
Makoto Tsukamoto,
Aiko Yamamoto, Yuriko Matsuzaki,
Maiko Matsushita,
Takako Asano,
Jun Nakashima,
Masaaki Tachibana,
Masamichi Hayakawa,
Hideyuki Ikeda,
Masaru Murai,
Yutaka Kawakami
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ABSTRACT: To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high-grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty-eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue-specific expression, KU-BL-1 and KU-BL-2 were identified as immunogenic antigens with restricted tissue expression. KU-BL-1 was found to be a putative human lipoic acid synthetase with a metal-binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU-BL-1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU-BL-2 was found to be a putative human kelch-like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU-BL-2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU-BL-2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA-A24 binding KU-BL-2 peptides. Therefore, KU-BL-1 and KU-BL-2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.
International Journal of Cancer 03/2004; 108(5):712-24. · 5.44 Impact Factor
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ABSTRACT: 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.
Journla of Immunotherapy 28(1):10-9. · 3.27 Impact Factor
