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ABSTRACT: Hepatic portal venous gas (HPVG) is a rare entity commonly associated with intestinal necrosis and fatal outcome, and various underlying diseases have been reported. Pancreatic solitary metastasis without local extension is also rare in esophageal squamous cell carcinoma.
This report describes an interesting and unusual case of HPVG arising from pancreatic tumor. Autopsy revealed pathogenesis of HPVG and synchronous tumors of the esophagus and pancreas.
A 73-year-old man developed synchronous double tumor in the esophagus and pancreas several months before acute abdomen and his death, which were generated by HPVG. Autopsy revealed that HPVG was caused by gastric wall infarction owing to expansion of an isolated pancreatic metastasis from esophageal squamous cell carcinoma.
This is the first case of HPVG that was derived from pancreatic tumor infiltration. If he had been diagnosed with solitary pancreatic metastasis from esophageal squamous cell carcinoma in the first time, he might have an option for chemotherapy, which could let him live longer.
Hepatobiliary & pancreatic diseases international: HBPD INT 02/2013; 12(1):103-5. · 1.08 Impact Factor
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ABSTRACT: Ménétrier's disease (MD) is a rare, acquired, premalignant disorder of the stomach characterized by enlarged gastric folds with foveolar hyperplasia, the phenotype of antralization of gastric glands, hypochlorhydria and hypoproteinemia. The etiology of MD is unknown, but both increased signaling by transforming growth factor-α and infection with Helicobacter pylori (H. pylori) have been implicated. Here, a case involving 70-year-old man who lost weight after developing anorexia and diarrhea is reported. He was diagnosed as MD without H. pylori infection, and in spite of intensive care, he died 40 days after admission. An autopsy confirmed MD. Immunohistochemistry revealed overexpression of transforming growth factor-α in the foveolar region of the gastric mucosa. The autopsy also distinguished this H. pylori-negative MD from hyperplastic polyp of the stomach, which is important in clarifying the entity of H. pylori-negative MD.
Digestive Endoscopy 07/2012; 24(4):275-9. · 1.19 Impact Factor
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Lisa Kashima,
Masashi Idogawa, Hiroaki Mita,
Miki Shitashige,
Tesshi Yamada,
Kazuhiro Ogi,
Hiromu Suzuki,
Minoru Toyota,
Hiroyoshi Ariga,
Yasushi Sasaki,
Takashi Tokino
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ABSTRACT: The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.
Journal of Biological Chemistry 02/2012; 287(16):12975-84. · 4.77 Impact Factor
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Hiroaki Mita,
Minoru Toyota,
Fumio Aoki,
Hirofumi Akashi,
Reo Maruyama,
Yasushi Sasaki,
Hiromu Suzuki,
Masashi Idogawa,
Lisa Kashima,
Kazuyoshi Yanagihara,
Masahiro Fujita,
Masao Hosokawa,
Masanobu Kusano,
Sorin Vasile Sabau,
Haruyuki Tatsumi,
Kohzoh Imai,
Yasuhisa Shinomura,
Takashi Tokino
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ABSTRACT: Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.
DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.
DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8-18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8-50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.
Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.
BMC Cancer 07/2009; 9:198. · 3.01 Impact Factor
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ABSTRACT: Gene transfer involving p53 is viewed as a potentially effective cancer therapy, but does not result in a good therapeutic response in all human cancers. The activation of p53 induces either cell cycle arrest or apoptosis. Cell cycle arrest in response to p53 activation is mediated primarily through the induction of the cyclin-dependent kinase inhibitor p21. Because p21 also has an inhibitory effect on p53-mediated apoptosis, the suppression of p53-induced p21 expression would be expected to result in the preferential induction of apoptosis. However, p21 also has tumor-suppressive properties. In this study, we developed an adenovirus vector that expresses p53 and suppresses p21 simultaneously to enhance p53-mediated apoptosis.
We constructed a replication-deficient recombinant adenovirus (Ad-p53/miR-p21) that enabled cocistronic expression of the p53 protein and artificial microRNAs that targeted p21, and examined the therapeutic effectiveness of this vector in vitro and in vivo.
The levels of p21 were significantly attenuated following infection with Ad-p53/miR-p21. In colorectal and hepatocellular carcinoma cells, infection with Ad-p53/miR-p21 augmented apoptosis as compared with an adenovirus that expressed p53 alone (Ad-p53/miR-control). Ad-p53/miR-p21 also significantly increased the chemosensitivity of cancer cells to adriamycin (doxorubicin). In a xenograft tumor model in nude mice, tumor volume was significantly decreased following the direct injection of Ad-p53/miR-p21 into the tumor, as compared with the injection of Ad-p53/miR-control.
These results suggest that adenovirus-mediated transduction of p53 and p21-specific microRNAs may be useful for gene therapy of human cancers.
Clinical Cancer Research 06/2009; 15(11):3725-32. · 7.74 Impact Factor
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Yasushi Sasaki,
Hideaki Negishi,
Ryota Koyama,
Naoki Anbo,
Kanae Ohori,
Masashi Idogawa, Hiroaki Mita,
Minoru Toyota,
Kohzoh Imai,
Yasuhisa Shinomura,
Takashi Tokino
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ABSTRACT: p73 and p63 are members of the p53 gene family that play an important role in development and homeostasis, mainly by regulating transcription of a variety of genes. We report here that apolipoprotein D (apoD), a member of the lipocalin superfamily of lipid transport proteins, is a direct transcriptional target of the p53 family member genes. We found that the expression of apoD was specifically up-regulated by either TAp73 or TAp63 but not significantly by p53. In addition, apoD transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abolishes induction of apoD transcription following cisplatin treatment. We also identified a p73/p63-binding site in the promoter of the apoD gene that is responsive to the p53 family members. The ectopic expression of TAp73 as well as the addition of recombinant human apoD to culture medium induced the osteoblastic differentiation of the human osteosarcoma cell line Saos-2, as assessed by alkaline phosphatase activity. Importantly, apoD knockdown abrogated p73-mediated alkaline phosphatase induction. Moreover, TAp73-mediated apoD expression was able to induce morphological differentiation, as well as expression of neuronal markers, in the human neuroblastoma cell line SH-SY5Y. These results suggest that apoD induction may mediate the activity of p73 in normal development.
Journal of Biological Chemistry 12/2008; 284(2):872-83. · 4.77 Impact Factor
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Takashi Imai,
Minoru Toyota,
Hiromu Suzuki,
Kimishige Akino,
Kazuhiro Ogi,
Yohei Sogabe,
Lisa Kashima,
Reo Maruyama,
Masanori Nojima, Hiroaki Mita,
Yasushi Sasaki,
Fumio Itoh,
Kohzoh Imai,
Yasuhisa Shinomura,
Hiroyoshi Hiratsuka,
Takashi Tokino
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ABSTRACT: Genetic and epigenetic alterations in tumor-suppressor genes play important roles in human neoplasia. Ras signaling is often activated in oral squamous cell carcinoma (OSCC), although Ras mutations are rarely detected in Japanese OSCC patients, and the mechanisms underlying the gene's activation remain unclear. Here, we examined the expression of Ras association family (RASSF) genes in a panel of OSCC cell lines and found that RASSF2 is often downregulated by DNA methylation in OSCC cells. In addition, aberrant methylation of RASSF2 was detected in 12 of 46 (26%) primary OSCC, and 18 (39%) of those OSCC showed methylation of at least one RASSF gene. Ectopic expression of RASSF2 in OSCC cells suppressed cell growth and induced apoptosis. A RASSF2 deletion mutant lacking the Ras-association domain, which was therefore unable to interact with Ras, exhibited less pro-apoptotic activity than the full-length protein, indicating that the pro-apoptotic activity of RASSF2 is related to its association with Ras. Genomic screening of genes regulated by RASSF2 showed that genes involved in immune responses, angiogenesis, and metastasis are suppressed by RASSF2. Our results suggest that epigenetic inactivation of RASSF2 plays an important role in OSCC tumorigenesis, and that RASSF2 may be a useful molecular target for the diagnosis and treatment of OSCC.
Cancer Science 06/2008; 99(5):958-66. · 3.33 Impact Factor
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ABSTRACT: Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy.
Molecular Cancer Therapeutics 05/2008; 7(4):779-87. · 5.23 Impact Factor
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ABSTRACT: p73 and p63 are members of the p53 gene family and have been shown to play an important role in development and homeostasis mainly by regulating the transcription of a variety of genes. A subset of these genes encodes secreted proteins and receptors that may be involved in the communication between adjacent cells. We report here that flotillin-2, a major hydrophobic protein on biomembrane microdomain lipid rafts, is a direct transcriptional target of the p53 family member genes. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. We found that the expression of flotillin-2 was specifically up-regulated by either TAp73beta or TAp63gamma, but not significantly by p53. In addition, flotillin-2 transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we showed that silencing endogenous p73 abolishes the induction of flotillin-2 transcription following cisplatin treatment. Furthermore, we identified a p73/p63-binding site located upstream of the flotillin-2 gene that is responsive to the p53 family members. This response element is highly conserved between humans and rodents. We also found that ectopic expression of TAp73 as well as TAp63 enhances signal transduction by assessing the interleukin-6-mediated phosphorylation of signal transducers and activators of transcription 3. Thus, in addition to direct transactivation, p53 family member genes enhance a set of cellular processes via lipid rafts.
Molecular Cancer Research 04/2008; 6(3):395-406. · 4.29 Impact Factor
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Toshihisa Kobayashi,
Yasushi Sasaki,
Yuichiro Oshima,
Hiroyuki Yamamoto, Hiroaki Mita,
Hiromu Suzuki,
Minoru Toyota,
Takashi Tokino,
Fumio Itoh,
Kohzoh Imai,
Yasuhisa Shinomura
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ABSTRACT: Although ribosomal proteins are major components of ribosomes, recent data have shown them to have extraribosomal functions apart from ribosome and protein biosynthesis. In our earlier study, we showed that ribosomal protein L13 mRNA was up-regulated in response to DNA damage in hamster cells. We report here that L13 expression is up-regulated in human gastrointestinal cancers. We also examined the biological role of L13 on human cancer cells. Knocking down L13 expression using small interfering RNA (siRNA) resulted in drastic attenuation of cancer cell growth with significant G1 and G2/M arrest of the cell cycle. Moreover, L13 siRNA significantly enhanced the cellular sensitivity to certain DNA damaging agents and, concordantly, L13-overexpressing cells demonstrated greater chemoresistance compared to parent cells, suggesting an inverse correlation between L13 expression and chemosensitivity. By using semiquantitative RT-PCR, we analyzed expression of L13 in freshly resected cancer tissue of the stomach, colorectum and liver. Up-regulation of L13 mRNA expression was observed in 10 (28%) of 36 gastric, 19 (41%) of 46 colorectal and 5 (20%) of 25 liver cancer tissue samples compared to adjacent normal tissue samples. We also found that increased expression of the L13 gene correlated with clinical staging in gastric cancers. The results of this study suggest that L13 plays an essential role in the progression of some gastrointestinal malignancies.
International Journal of Molecular Medicine 08/2006; 18(1):161-70. · 1.98 Impact Factor
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Reo Maruyama,
Fumio Aoki,
Minoru Toyota,
Yasushi Sasaki,
Hirofumi Akashi, Hiroaki Mita,
Hiromu Suzuki,
Kimishige Akino,
Mutsumi Ohe-Toyota,
Yumiko Maruyama,
Haruyuki Tatsumi,
Kohzoh Imai,
Yasuhisa Shinomura,
Takashi Tokino
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ABSTRACT: p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis.
Cancer Research 06/2006; 66(9):4574-83. · 7.86 Impact Factor
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ABSTRACT: Fimbrins (also known as plastins) are actin-binding proteins of the cortical cytoskeleton present in all cells and conserved from yeast to mammals. We previously reported that the up-regulation of T-fimbrin, a fimbrin isoform, in association with G2 arrest following DNA damage and that a lack of T-fimbrin expression shortens the radiation-induced G2 arrest in Chinese hamster ovarian cells. In this study, we further investigated the role of T-fimbrin in DNA-damage response using a panel of human liver cancer cell lines and small interfering RNA technology. T-fimbrin was differentially expressed in human liver cancer cell lines. Colony formation assays revealed that cell lines lacking T-fimbrin expression were highly sensitive to DNA damage compared to cell lines that express T-fimbrin. Using siRNA designed to target T-fimbrin, we demonstrated that silencing endogenous T-fimbrin causes a marked increase in the cellular sensitivity to VP-16 and UV irradiation. Moreover, T-fimbrin deletion abrogated UV-mediated cell cycle checkpoint, and consequently led to increased apoptotic cell death in resistant cells. These findings suggest that the status of T-fimbrin expression may be a useful molecular marker for predicting the responsiveness of cancer cells to treatment with chemotherapeutic drugs.
International Journal of Oncology 11/2005; 27(4):933-40. · 2.40 Impact Factor
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Kazuhiro Ogi,
Minoru Toyota, Hiroaki Mita,
Ayumi Satoh,
Lisa Kashima,
Yasushi Sasaki,
Hiromu Suzuki,
Kimishige Akino,
Noriko Nishikawa,
Makoto Noguchi,
Yasuhisa Shinomura,
Kohzoh Imai,
Hiroyoshi Hiratsuka,
Takashi Tokino
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ABSTRACT: Alterations in the function of cell cycle checkpoints are frequently detected in oral squamous cell carcinomas (OSCCs), and are often associated with the sensitivity of the cancer cells to chemotherapeutic drugs. Recently, a mitotic checkpoint gene, Chfr, was shown to be inactivated by promoter methylation and point mutations in various human tumors. Here we show that the absence of its product, CHFR, is associated with mitotic checkpoint dysfunction, and that cancer cells lacking CHFR are sensitive to microtubule inhibitors. Checkpoint impairment appears to be caused by a prophase defect in this case, as OSCC cells lacking CHFR showed phosphorylation of histone H3 on Ser10 and translocation of cyclin B1 to the nucleus. When CHFR-deficient OSCC cells were treated with a microtubule inhibitor (docetaxel or paclitaxel), significant numbers of apoptotic cells were observed. Moreover, disruption of CHFR using small interfering RNA (siRNA) impaired the mitotic checkpoint, thereby reducing the ability of OSCC cells to arrest at G2/M phase and making them more sensitive to microtubule inhibitors. Our results suggest that CHFR could be a useful molecular target for chemotherapy.
Cancer biology & therapy 08/2005; 4(7):773-80. · 2.64 Impact Factor
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ABSTRACT: Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells.
The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing.
Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas.
RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.
Gastroenterology 08/2005; 129(1):156-69. · 11.68 Impact Factor
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Masafumi Murai,
Minoru Toyota,
Hiromu Suzuki,
Ayumi Satoh,
Yasushi Sasaki,
Kimishige Akino,
Masako Ueno,
Fumihiko Takahashi,
Masanobu Kusano, Hiroaki Mita,
Kazuyoshi Yanagihara,
Takao Endo,
Yuji Hinoda,
Takashi Tokino,
Kohzoh Imai
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ABSTRACT: BNIP3 protein is a proapoptotic member of the Bcl-2 family that is expressed in hypoxic regions of tumors. To examine its role in the progression of gastrointestinal cancer, we examined the expression and DNA methylation status of BNIP3 gene in a panel of colorectal and gastric cancer cell lines. BNIP3 was not expressed in 14 of the 24 cell lines tested, and its absence was not caused by gene mutation or by altered expression of hypoxia inducible factor-1, a key transcription factor that regulates BNIP3 expression. On the other hand, methylation of the 5' CpG island of BNIP3 was closely correlated with silencing the gene. Moreover, treating methylated cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored hypoxia-induced expression of BNIP3 mRNA and protein, which in turn led to cell death. Aberrant methylation of BNIP3 was also detected in 66% of primary colorectal and 49% of primary gastric cancers, but not in normal tissue samples collected from areas adjacent to the tumors. Apparently, epigenetic alteration of BNIP3 is a frequent and cancer-specific event, which suggests that inactivation of BNIP3 likely plays a key role in the progression of some gastrointestinal cancers and that it may be a useful molecular target for therapy.
Clinical Cancer Research 03/2005; 11(3):1021-7. · 7.74 Impact Factor
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Ayumi Satoh,
Minoru Toyota,
Hideyuki Ikeda,
Yoshikazu Morimoto,
Kimishige Akino, Hiroaki Mita,
Hiromu Suzuki,
Yasushi Sasaki,
Takayuki Kanaseki,
Yukio Takamura,
Hidenobu Soejima,
Takeshi Urano,
Kazuyoshi Yanagihara,
Takao Endo,
Yuji Hinoda,
Masahiro Fujita,
Masao Hosokawa,
Noriyuki Sato,
Takashi Tokino,
Kohzoh Imai
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ABSTRACT: Tightly regulated at the level of transcription, expression of MHC class II molecules varies significantly among gastrointestinal cancers. High levels of MHC class II expression are often associated with a better prognosis, which is indicative of the involvement of CD4+ lymphocytes in tumor suppression, but the molecular mechanism by which MHC class II expression is regulated remains unclear. In the present study, we investigated the expression of one inducible MHC class II molecule, HLA-DR, and its coactivators in a panel of colorectal and gastric cancer cell lines. Interferon-gamma induced expression of HLA-DR in 14 of 20 cell lines tested; the remaining six cell lines did not express HLA-DR. Analysis of the expression of transcription factors and coactivators associated with HLA-DR revealed that the loss of CIITA expression was closely associated with the absence of HLA-DR induction. Moreover, DNA methylation of the 5' CpG island of CIITA-PIV was detected in all cancer cells that lacked CIITA. The methylation and resultant silencing of CIITA-PIV depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3B, and their genetic inactivation restored CIITA-PIV expression. It thus appears that CIITA methylation is a key mechanism that enables some gastrointestinal cancer cells to escape immune surveillance.
Oncogene 12/2004; 23(55):8876-86. · 6.37 Impact Factor
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ABSTRACT: The purpose of this work was to explore the role of epigenetic inactivation of apoptotic pathways in ovarian cancer by examining the DNA methylation and expression status of four proapoptotic genes in primary ovarian cancers and cancer cell lines and to correlate those findings with the clinicopathological features of ovarian cancer patients.
Genomic DNA was isolated from 15 ovarian cancer cell lines, 80 primary ovarian cancer specimens, and 4 normal ovary specimens using phenol-chloroform extraction. The methylation status of the DNA was evaluated using combined bisulfite restriction analysis, gene expression was evaluated using reverse transcription-PCR, and histone acetylation was evaluated using chromatin immunoprecipitation.
Of the four proapoptotic genes studied, expression of TMS1/ASC was absent in six ovarian cancer cell lines. Dense methylation of the 5' region of TMS1/ASC was detected in cells not expressing TMS1/ASC. Treating methylated cells with 5-aza-deoxycytidine restored gene expression, confirming the role of methylation in silencing the gene. Chromatin immunoprecipitation revealed histone to be deacetylated in cells not expressing TMS1/ASC, indicating that histone deacetylation is also involved in silencing TMS1/ASC. Aberrant methylation of TMS1/ASC was detected in 15 of 80 ovarian cancer tissues (19%) but in none of the normal ovary specimens. Aberrant methylation of TMS1/ASC was observed significantly more often in clear cell-type ovarian cancers than in other tumor types (P < 0.0001).
Methylation-mediated silencing of TMS1/ASC confers a survival advantage to tumor cells by enabling them to escape apoptosis. The role for aberrant methylation in human ovarian tumorigenesis may be particularly important for ovarian cancers with the clear cell phenotype.
Clinical Cancer Research 04/2004; 10(6):2000-6. · 7.74 Impact Factor
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Ayumi Satoh,
Minoru Toyota,
Fumio Itoh,
Yasushi Sasaki,
Hiromu Suzuki,
Kazuhiro Ogi,
Takefumi Kikuchi, Hiroaki Mita,
Toshiharu Yamashita,
Takashi Kojima,
Masanobu Kusano,
Masahiro Fujita,
Masao Hosokawa,
Takao Endo,
Takashi Tokino,
Kohzoh Imai
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ABSTRACT: Mitotic checkpoints prevent errors in chromosome segregation that can lead to neoplasia. Therefore, it is notable that gastric cancers often show impaired checkpoint function. In the present study, we examined the functional consequences of epigenetic inactivation of the mitotic checkpoint gene CHFR in gastric cancers. CHFR expression was silenced by DNA methylation of the 5' region of the gene in 20% of the gastric cancer cell lines tested and in 39% of primary gastric cancers; expression could be restored by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, histones H3 and H4 were found to be deacetylated in cell lines showing aberrant methylation, indicating a role for histone deacetylation in the methylation-dependent gene silencing. Cells not expressing CHFR showed impaired checkpoint function, which led to nuclear localization of cyclin B1 after treatment with docetaxel or paclitaxel, two microtubule inhibitors. Apparently, the absence of CHFR is associated with sensitivity of cells to mitotic stress caused by microtubule inhibition, and restoration of CHFR expression by 5-aza-2'-deoxycytidine or adenoviral gene transfer restored the checkpoint. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in gastric cancer. Moreover, the aberrant methylation of CHFR appears to be a good molecular marker with which to predict the sensitivity of gastric cancers to microtubule inhibitors.
Cancer Research 01/2004; 63(24):8606-13. · 7.86 Impact Factor
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Toshiro Obata,
Minoru Toyota,
Ayumi Satoh,
Yasushi Sasaki,
Kazuhiro Ogi,
Kimishige Akino,
Hiromu Suzuki,
Masafumi Murai,
Takefumi Kikuchi, Hiroaki Mita,
Fumio Itoh,
Jean-Pierre J Issa,
Takashi Tokino,
Kohzoh Imai
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ABSTRACT: Aberrant methylation of CpG islands can be a good molecular marker for identifying genes inactivated in cancer. We found the proapoptotic gene HRK to be a target for hypermethylation in human cancers and examined the role of such methylation in silencing the gene's expression.
Methylation of HRK was evaluated by bisulfite-PCR and bisulfite sequencing in a group of colorectal and gastric cancer cell lines and primary cancers. Gene expression and histone acetylation were examined by reverse transcription-PCR and chromatin immunoprecipitation analyses, respectively. Apoptosis of cancer cells after treatment with a DNA methyltransferase inhibitor and/or histone deacetylase inhibitor was examined with fluorescence-activated cell-sorting analysis.
The region around the HRK transcription start site was methylated in 36% of colorectal and 32% of gastric cancer cell lines and was closely associated with loss of expression in those cell types. HRK expression was restored by treatment with a methyltransferase inhibitor, 5-aza-deoxycytidine, and enhanced further by addition of histone deacetylase inhibitor trichostatin A or depsipeptide. Such restoration of HRK expression was well correlated with induction of apoptosis and enhancement of Adriamycin-induced apoptosis. Expression of other proapoptotic genes, including BAX, BAD, BID, and PUMA, was unaffected by treatment with 5-aza-deoxycytidine. Aberrant methylation of HRK was also frequently detected in primary colorectal cancers that showed methylation of multiple genes, including p16INK4A and hMLH1, and was associated with wild-type p53.
HRK methylation can be a useful molecular target for cancer therapy in a subset of colorectal and gastric cancers.
Clinical Cancer Research 01/2004; 9(17):6410-8. · 7.74 Impact Factor
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ABSTRACT: p73 has a high degree of structural homology to p53 and can activate transcription of p53-responsive genes. However, analysis of p73-deficient mice revealed a marked divergence in the physiological activities of p53 family genes and distinguishes p73 from p53. Mice deficient for p73 exhibit profound defects, including hippocampal dysgenesis, chronic infection, and inflammation, as well as abnormalities in pheromone sensory pathways. p73 plays important roles in neurogenesis, sensory pathways, and homeostatic regulation. Here, we found that the interleukin 4 receptor alpha (IL-4Ralpha) gene is up-regulated by p73 but not significantly by p53 in several human cancer cell lines. IL-4Ralphatranscription is also activated in response to cisplatin, a DNA-damaging agent known to induce p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abrogates the induction of the IL-4Ralpha gene after cisplatin treatment. Furthermore, we identified a p73-binding site in the first intron of the IL-4Ralpha gene that can directly interact with the p73 protein in vivo. This p73-binding site consists of eight copies of a 10-bp consensus p53-binding motif and is a functional response element that is relatively specific for p73 among the p53 family. p73beta promoted localized nucleosomal acetylation through recruitment of coactivator p300, indicating that p73 regulates transcription of IL-4Ralpha through the unique p73-binding site. We also found that p73beta-transfected tumor cells are sensitive to IL-4-mediated apoptosis. Our data suggest that IL-4Ralpha could mediate, in part, certain immune responses and p73-dependent cell death.
Cancer Research 01/2004; 63(23):8145-52. · 7.86 Impact Factor
