Biomedical subjects

Norihiko Kawamata

Publications and source records attributed to Norihiko Kawamata.

13 recordsLinked to original sources

PubMed2006

SAHA, a HDAC inhibitor, has profound anti-growth activity against non-small cell lung cancer cells.

Current chemotherapy of advanced non-small cell lung cancer (NSCLC) produces only a modest increase in survival time. New approaches are needed for this disease. The development of lung cancer is associated with silencing tumor suppressor genes that can occur not only by deletion or mutation, but also by epigenetic changes including histone deacetylation of key lysines. Histone deacetylase inhibitor (HDACI) increases histone acetylation, resulting in DNA with a more open chromatin that favors transcription. We found that the HDACI, suberoylanilide hydroxamic acid (SAHA), suppressed cell growth of five non-small cell lung cancer cell lines in a dose-dependent manner (50% growth inhibition approximately 2 microM). Cell cycle assay by fluorescence-activated cell sorting (FACS) demonstrated that SAHA induced a significant G0-G1 growth arrest of NSCLC cells. Protein assay by Western blot analysis showed that SAHA induced expression of p21WAF1. These results demonstrated that administration of SAHA may be a novel approach to the treatment of non-small cell lung cancer.

Acetylation↗

PubMed2005

2C4, a monoclonal antibody against HER2, disrupts the HER kinase signaling pathway and inhibits ovarian carcinoma cell growth.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is overexpressed in 25-30% of ovarian carcinoma cases and a correlation between increased HER2 expression and decreased survival has been demonstrated. HER2 is a ligand-less member of the HER family that functions as the preferred coreceptor for epidermal growth factor receptor (EGFR), HER3, and HER4. METHODS: An approach was developed to target HER2's role as a coreceptor using a monoclonal antibody, 2C4, which sterically hinders HER2's recruitment into a functional HER complex. RESULTS: HER2 was robustly expressed in all eight ovarian carcinoma cell lines; expression of EGFR and HER3 was variable. Even though four of the eight cell lines responded to EGF, 2C4 antibody moderately inhibited in vitro proliferation of only two cell lines (OVCA433 and SK-OV-3). Furthermore, ligand-stimulated p-MAPK expression was inhibited by 2C4 only in these two cell lines after exposure to EGF. Immunoprecipitation and eTag analysis revealed that OVCA433 expressed heterodimers of EGFR/HER2, and these heterodimers were disrupted after treatment with 2C4, whereas OVCA432 cells did not have these heterodimers. In murine xenograft experiments, the in vivo growth of OVCA433, but not of OVCA432, ovarian carcinoma cells was significantly inhibited by 2C4 treatment of the mice. CONCLUSION: 2C4 is able to disrupt the HER signaling pathway and inhibit the in vitro and in vivo growth of ovarian carcinoma cell lines. The response appears limited to lines in which HER2 heterodimers were able to transduce proliferative signals. Our findings suggest a strong rationale to conduct clinical trials of 2C4 in a subset of patients with ovarian tumors.

Animals↗

PubMed2005

Histone deacetylase inhibitors profoundly decrease proliferation of human lymphoid cancer cell lines.

Methylation of tumor suppressor genes is frequently observed in human cancers. These genes are silenced by histone deacetylase (HDAC) recruited by methylated DNA in their promoter regions. HDAC removes acetyl groups from histones and prevents the basic transcriptional machinary access to the target gene, leading to transcriptional repression. HDAC inhibitors (HDACIs) can restore the expression of the tumor suppressor and/or cell cycle regulatory genes in cancer cells and block the cellular proliferation of these cells. In this study, we investigated the in vitro antiproliferative activities of the HDACIs, suberoylanilide hydroxamic acid (SAHA), and valproic acid against 14 human lymphoid cancer cell lines. All of these cell lines were sensitive to the antiproliferative effects of the HDACI. SAHA induced either G1 or G2-M arrest as well as apoptosis. SAHA downregulated cyclin D1 and D2, and upregulated p53, p21, and p27. Chromatin immunoprecipitation analysis revealed a remarkable increase in the level of acetylated histones associated with the p21 promoter after SAHA treatment. In nude mice, SAHA significantly inhibited growth of a mantle cell lymphoma without major toxic side effects. In summary, HDACIs are promising therapeutic agents for human lymphoid cancers.

Animals↗

PubMed2005

Methylation status analysis of cell cycle regulatory genes (p16INK4A, p15INK4B, p21Waf1/Cip1, p27Kip1 and p73) in natural killer cell disorders.

Natural killer (NK) cell disorders are rare diseases. Genetic abnormalities of the several tumor suppressor genes, including p15INK4B, p16INK4A/p14ARF, p53, p73, and Rb genes have been reported. Deletions and point mutations of these genes are frequently detected in these diseases. It has been reported that tumor suppressor genes are inactivated by DNA methylation of the promoter region and/or first exon of the genes in a variety of human cancers. In this study we analyze the methylation status of the genes associated with cell cycle regulation, including p16INK4A, p15INK4B, p21/Waf1/Cip1, p27/Kip1, p73, and p14ARF, by methylation specific (MS) PCR and/or bisulfite sequencing. We examined 29 cases of NK cell disorders (five aggressive NK cell leukemia/lymphoma, three blastic NK cell lymphoma/leukemia, five nasal NK cell lymphoma, three myeloid/NK cell precursor acute leukemia, 13 chronic NK lymphocytosis). We found methylation of the first exon of the p16INK4A gene in two cases (one aggressive, one blastic), and methylation of the p14ARF gene in one aggressive NK cell leukemia. Bisulfite sequencing revealed that methylation of the p15 and p27 genes was rare in these disorders. MS-PCR suggested that the p73 and p21 genes were methylated in seven cases, respectively (p73: one blastic, one nasal, five chronic; p21: one myeloid/NK, one aggressive, one nasal, and four chronic); bisulfite sequencing confirmed that methylated alleles of these genes were dominant in the samples except three cases (one myeloid/NK, one aggressive, and one chronic) in which methylated alleles of the p21 genes were less than 34% of all alleles. These results suggested that inactivation of the cell cycle regulatory genes by DNA methylation could be associated with tumorigenesis in NK cell disorders, not only aggressive subtypes but also chronic subtype.

Base Sequence↗

PubMed2005

Discovery of epigenetically masked tumor suppressor genes in endometrial cancer.

Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated the discovery of novel tumor suppressor genes. We used a variety of research tools to search for genes that are epigenetically silenced in human endometrial cancers. Changes in global gene expression of the endometrial cancer cell line Ishikawa was analyzed after treatment with the demethylating agent 5-aza-2'-deoxycytidine combined with the histone deacetylase inhibitor suberoylanilide bishydroxamide. By screening over 22,000 genes, candidate tumor suppressor genes were identified. Additional microarray analysis and real-time reverse transcription-PCR of normal and cancerous endometrial samples and search for CpG islands further refined the list. Tazarotene-induced gene-1 (Tig1) and CCAAT/enhancer binding protein-alpha (C/ebpalpha) were chosen for further study. Expression of both genes was low in endometrial cancer cell lines and clinical samples but high in normal endometrial tissues. Bisulfite sequencing, restriction analysis, and/or methylation-specific PCR revealed aberrant methylation of the CpG island in the Tig1 gene of all 6 endometrial cancer cell lines examined and 4 of 18 clinical endometrial cancers, whereas the C/ebpalpha promoter remained unmethylated in endometrial cancers. Chromatin immunoprecipitation showed increased acetylated histone H3 bound to both Tig1 and C/ebpalpha genes after treatment with 5-aza-2'-deoxycytidine and/or suberoylanilide bishydroxamide. Forced expression of either TIG1 or C/EBPalpha led to significant growth reduction of Ishikawa cells. Our data suggest that C/ebpalpha and Tig1 function as tumor suppressor proteins in endometrial cancers and that their reexpression may be a therapeutic target.

Aged↗

PubMed2005

Mxi1 isoforms are expressed in hematological cell lines and normal bone marrow.

Mxi1 protein is a basic helix-loop-helix, leucine zipper (bHLHZIP) transcriptional factor, which dimerizes with the Max protein. This heterodimer binds a specific DNA sequence (E-box) and suppresses transcription of target genes. On the other hand, c-Myc protein is also a bHLHZIP protein that dimerizes with Max, binds the identical E-box sequence but activates transcription of the target genes. We report that hematopoietic cells have three novel Mxi1 transcripts: Mxi-D lacks exon 3, which encodes the basic region; Mxi-ND lacks the N-terminal mSin3 binding region and the DNA binding region; and Mxi-NF lacks the N-terminal mSin3 binding region. In normal bone marrow and hematological cell lines, the dominantly expressed isoforms are Mxi-D and full-length Mxi1 (Mxi-F). In GST-pull down assays, the Mxi-D protein binds the Max protein and the PHA2 regions of mSin3 proteins. Whereas the Mxi-F/Max heterodimer binds the E-box sequence, Mxi-D/Max heterodimer can not bind this sequence in electrophoretic mobility shift assays. In reporter gene assay, Mxi-D suppresses transcription as strongly as Mxi-F. In colony formation assay using Rat1 fibroblast cells, Mxi-D cannot suppress clonal growth stimulated by c-Myc as strongly as Mxi-F. In summary, Mxi-D may play an important role in the c-Myc family protein network acting as a dominant negative isoform of Mxi-F since: i) Mxi-D can dimerize with Max in vitro; ii) Mxi-D/Max heterodimers cannot bind E-box in vitro, iii) Mxi-D cannot suppress clonal growth stimulated by c-Myc.

Basic Helix-Loop-Helix Proteins↗

PubMed2004

Human ovarian carcinoma cells: histone deacetylase inhibitors exhibit antiproliferative activity and potently induce apoptosis.

BACKGROUND: Histone deacetylase inhibitors (HDACIs) can inhibit proliferation, stimulate apoptosis, and induce cell cycle arrest in malignant cells. METHODS: The authors investigated the effects of four HDACIs on nine ovarian carcinoma cell lines in vitro and in vivo. Ovarian carcinoma cells were treated with a variety of HDACIs, and their effects on cell growth, the cell cycle, apoptosis, and related events were investigated. The ability of valproic acid (VPA) to inhibit the growth of ovarian tumors in immunodeficient mice was also assessed. RESULTS: Clonogenic assays showed that all ovarian carcinoma cell lines were sensitive to the growth-inhibitory effects of the HDACIs. Cell cycle analysis indicated that their exposure to HDACIs decreased the proportion of cells in S phase and increased the proportion of cells in the G0/G1 and/or G2/M phases of the cell cycle. Terminal deoxynucleotidyltransferase-mediated uridine triphosphate end-labeling assays demonstrated that HDACIs induced apoptosis, which occurred in concert with alterations in the expression of genes related to apoptosis, cell growth, and malignant phenotype, including the activation of caspase-9 and caspase-3. Chromatin immunoprecipitation analysis revealed a notable increase in levels of acetylated histones associated with the p21 promoter after treatment with suberoylanilide bishydroxamine. In addition, in experiments involving nude mice, VPA significantly inhibited human ovarian tumor growth without toxic side effects. CONCLUSIONS: The results of the current study suggest that HDACIs may be particularly effective in the treatment of ovarian tumors.

Acetylation↗

PubMed2003

Absence of R24C mutation of the CDK4 gene in leukemias and solid tumors.

A mutation of the p16(INK4a)-binding domain of the cyclin dependent kinase 4 (CDK4) gene, R24C, has been reported in some cases of melanoma. This mutation prevented binding of the CDK4 inhibitor p16(INK48) to CDK4. To determine the relevance of the mutation, we performed polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis in diverse types of human leukemias and solid tumors. No mobility shifts indicating sequence alterations were observed in 273 tumors and 49 cell lines from diverse kinds of tumors These results suggest that in contrast to melanoma, in many other types of human neoplasms the mutation of the CDK4 gene is very rare. To better understand these findings, we randomly mutagenized the CDK4 gene and used the yeast two-hybrid method to screen for CDK4 mutants that had lost the ability to bind to p16(INK4a). Sequence analysis and in vitro kinase assays showed that most of the mutations that disrupted interactions with p16(INK4) also knocked out the activity of CDK4. This result may explain the rareness of CDK4 mutations in human tumors.

Cell Line, Tumor↗

PubMed2003

[Zygomycosis: two case reports and review of reported cases in the literature in Japan].

This article reports two cases of zygomycosis and analyzes the zygomycosis cases reported in the literature in Japan. Case 1 was a 43-year-old male with malignant lymphoma who presented complications of pneumonia and cerebral bleeding, leading to his death. Autopsy findings showed pulmonary lesions were due to zygomycosis. Cerebral lesion was presumed to be due to zygomycosis without pathological examination. Case 2 was a 52-year-old male with acute lymphocytic leukemia from whom 4 sputum cultures were taken that were positive for Cunninghamella elegans. Combination therapy of itraconazole and amphotericin B (AMPH) was begun, and AMPH was changed to liposomal amphotericin B. During the neutropenic period after receiving premedication for a peripheral blood stem cell transplantation performed for his underlying disease, high fever was recognized and Staphylococcus epidermidis was isolated from the blood culture. Despite the change in antibiotics administered, pneumonia also developed as a complication, causing his death. Two hundred four cases of zygomycosis have been reported in the literature in Japan: 55 cases were rhinocerebral zygomycosis, including 29 cases with no underlying disease. A premortem diagnosis was made in 34 cases by pathological findings of operation materials or drainage samples, and 24 cases were postmortem. Pulmonary, disseminated, cardiovascular, gastrointestinal and thyroidal zygomycoses were found in 144 cases, including 66 cases with leukemia. A premortem diagnosis was made in 39 cases and 120 cases were postmortem. Prognosis of rhinocerebral type was better in operated or drainage cases, and for resected cases in all other types. Five cases with allergic zygomycosis were all alive. There were only 14 cases in which isolated fungi were identified (Cunninghamella spp. from 5 cases, Mucor spp. from 2, Rhizomucor spp. from 2, and Rhizopus spp. from 5).

Adult↗

PubMed2002

A novel chromosomal translocation t(1;14)(q25;q32) in pre-B acute lymphoblastic leukemia involves the LIM homeodomain protein gene, Lhx4.

Chromosome 1q21-25 is one of the hotspots of chromosomal abnormalities including translocations and duplications in hematological malignancies. This would suggest that oncogene(s) reside in this region. We have cloned the junctional sequence of t(1;14)(q25;q32) in pre-B acute lymphoblastic leukemia cells by an inverse PCR method. A novel sequence was fused to the joining region of the immunoglobulin heavy chain gene. We confirmed this rearrangement by Southern blot analysis, genomic PCR and fluorescence in situ hybridization. We found a coding sequence which is homologous to the mouse Lhx4 cDNA sequence 17 kb from the breakpoint. The human Lhx4 gene encodes 390 amino-acids, including one tandem pair of LIM domains and one homeodomain. The human Lhx4 gene consists of six exons. Lhx4 protein is very homologous to human Lhx3 protein except in the N-terminal region. The transcripts of the Lhx4 gene were not detected in adult multiple tissues analysed by Northern blotting, but were detected in the leukemic cells carrying t(1;14)(q25;q32) by reverse-transcription PCR. The protein expression of Lhx4 in these leukemic cells was confirmed by Western blot analysis. Lhx4 activated the reporter gene carrying the mouse alpha-glycoprotein subunit promoter region, which is regulated by Lhx3. LIM protein and homeodomain protein genes are frequently involved in translocations of hematological malignancies. The Lhx4 gene is deregulated in the leukemic cells and Lhx4 protein may play an important role, possibly as an activator, in leukemogenesis.

Acute Disease↗

PubMed2002

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3.

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.

Amino Acid Sequence↗

PubMed2002

Epstein-Barr virus-associated T-cell acute lymphoblastic leukaemia.

Epstein-Barr virus (EBV) is frequently detected in haematological malignancies, including Burkitt's lymphomas/leukaemias, Hodgkin's diseases and non-Hodgkin's lymphomas. However, immature T-cell malignancies associated with EBV have not been reported previously. We report a patient with T-cell acute lymphoblastic leukaemia (T-ALL), whose leukaemic cells had EBV, confirmed by Southern blotting and in situ hybridization. The EBV existed in episomal form and was detected in most leukaemic cells, but not in bystander normal B-cells. The leukaemic cells, massively infiltrated into the liver and spleen, were resistant to chemotherapy. EBV might be associated with tumorigenesis of T-ALL, and characteristic clinical features of the patient.

Adult↗

PubMed2002

Molecular analysis of oncogenes, ras family genes (N-ras, K-ras, H-ras), myc family genes (c-myc, N-myc) and mdm2 in natural killer cell neoplasms.

Natural killer (NK) cell neoplasms are rare diseases. Frequent abnormalities of the tumor suppressor genes Rb, p53, p15INK4B, p16INK4A and p14ARF have been reported. However, no oncogenes associated with tumorigenesis of NK cell neoplasms have been reported so far. We analyzed the status of oncogenes including N-ras, K-ras, H-ras, c-myc, N-myc and mdm2 by Southern blot, PCR-SSCP, western blot analysis and immunohistochemical staining. We analyzed four cell lines derived from NK cell neoplasms and 31 clinical samples with five subclasses of NK cell neoplasms. We found no point mutations of the ras family genes. We detected no mutations in the c-myc and N-myc genes. No overexpression of c-Myc protein was detected by western blot analysis. Although we found neither amplification nor rearrangement of the mdm2 gene, we found high expression of MDM2 protein in some cases by western blot analysis. Immunohistochemical staining confirmed the overexpression of MDM2 protein. We found 14 cases with overexpression of MDM2 protein out of 15 cases (93%) with four subclasses of NK cell neoplasms except chronic NK lymphocytosis. Our previous and these results suggested that the expression level of MDM2 protein is independent of the status of the p14ARF, p53, Rb genes. MDM2 protein might independently contribute to carcinogenesis of NK cell neoplasms. Although the number of the cases we analyzed was not large, alterations of ras and myc family genes may rarely contribute to tumorigenesis in NK cell neoplasms. In contrast, overexpression of MDM2 might be associated with tumorigenesis of NK cell neoplasms, especially aggressive subclasses.

Blotting, Southern↗