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Biomedical subjects

Kimitoshi Kohno

Publications and source records attributed to Kimitoshi Kohno.

At least 19 recordsLinked to original sources
PubMed2007

Impaired activity of volume-sensitive Cl- channel is involved in cisplatin resistance of cancer cells.

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

YB-1 is important for an early stage embryonic development: neural tube formation and cell proliferation.

The eukaryotic Y-box-binding protein-1 (YB-1) is involved in the transcriptional and translational control of many biological processes, including cell proliferation. In clinical studies, the cellular level of YB-1 closely correlates with tumor growth and prognosis. To understand the role of YB-1 in vivo, especially in the developmental process, we generated YB-1 knock-out mice, which are embryonic lethal and exhibit exencephaly associated with abnormal patterns of cell proliferation within the neuroepithelium. beta-Actin expression and F-actin formation were reduced in the YB-1 null embryo and YB-1(-/-) mouse embryonic fibroblasts, suggesting that the neural tube defect is caused by abnormal cell morphology and actin assembly within the neuroepithelium. Fibroblasts derived from YB-1(-/-) embryos demonstrated reduced growth and cell density. A colony formation assay showed that YB-1(-/-) mouse embryonic fibroblasts failed to undergo morphological transformation and remained contact-inhibited in culture. These results demonstrate that YB-1 is involved in early mouse development, including neural tube closure and cell proliferation.

Animals↗
PubMed2006

Fragmented hyaluronan induces transcriptional up-regulation of the multidrug resistance-1 gene in CD4+ T cells.

P-glycoprotein, encoded by the multidrug resistance (MDR)-1 gene, expels various drugs from cells resulting in multidrug resistance. We found previously that interleukin-2, a lymphocyte-activation cytokine, induces P-glycoprotein expression on lymphocytes. Lymphocyte activation involves adhesion with the extracellular matrix, such as hyaluronan, through adhesion molecules on lymphocytes. We investigated the transcriptional regulation of MDR-1 in lymphocytes by fragmented hyaluronan. Fragmented hyaluronan (especially the 6.9-kDa form), not native high molecular hyaluronan, induced translocation of YB-1, a specific transcriptional factor for MDR-1, from the cytoplasm into the nucleus and resulted in the transcription of MDR-1 and the expression of P-glycoprotein on lymphocytes in a dose-dependent manner. Transfection of YB-1 antisense oligonucleotides inhibited P-glycoprotein expression induced by fragmented hyaluronan. The fragmented hyaluronan induced significant P-glycoprotein expression on only activated CD4+ T cells, which highly expressed CD69, and resulted in excretion of intracellular dexamethasone added in vitro. Cyclosporin A, a competitive P-glycoprotein inhibitor, restored intracellular dexamethasone levels in CD4+ T cells. Anti-CD44 monoclonal antibody (Hermes-1) inhibited fragmented hyaluronan-induced YB-1 activation and P-glycoprotein expression in CD4+ T cells. We provide the first evidence that binding of fragmented hyaluronan to CD44 induces YB-1 activation followed by P-glycoprotein expression in accordance with activation of CD4+ T cells. Our findings imply that CD4+ T cell activation by fragmented hyaluronan, induced by characteristic extracellular matrix changes in inflammation, tumors, and other conditions, results in P-glycoprotein-mediated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Expression and cellular localization of dbpC/Contrin in germ cell tumor cell lines.

The transcriptional regulation of the germ cell-specific cold-shock domain protein dbpC/Contrin was investigated, and the promoter region between -272 and -253 relative to the transcription start site was shown to be critical for the manifestation of cell-type specific transcription. In vivo footprint analysis demonstrated that the E-box located between -272 and -253 is protected in the dbpC/Contrin-positive germ cell tumor cell lines NEC8 and TERA1, but not in the dbpC/Contrin-negative bladder cancer cell line T24 or ovarian cancer cell line A2780. The promoter activity of the dbpC/Contrin gene was transactivated by co-transfection with c-Myc and the N-Myc expression plasmid. Western blotting analysis clearly showed that N-Myc is highly expressed in both NEC8 and TERA1 cells, and that c-Myc is expressed in both T24 and A2780 cells. These data demonstrate that cell-type specific dbpC/Contrin expression in germ cells is regulated by N-Myc. In addition, dbpC/Contrin is localized mainly in the cytoplasm of NEC8 and TERA1 cells, but is translocated to the nucleus when its C-terminal region is partially deleted. Our findings also suggest that dbpC/Contrin can be used as a molecular tool for the detection of germ cell tumors.

Active Transport, Cell Nucleus↗
PubMed2005

Increased nuclear localization of transcription factor Y-box binding protein 1 accompanied by up-regulation of P-glycoprotein in breast cancer pretreated with paclitaxel.

PURPOSE: The Y-box binding protein 1 (YB-1) regulates expression of P-glycoprotein encoded by the MDR1 gene. There have been no previous studies regarding the involvement of YB-1 in the development of resistance to paclitaxel. The present study was done to examine how paclitaxel affects the localization and expression of YB-1 in breast cancer. EXPERIMENTAL DESIGN: We evaluated the expression and localization of YB-1 and P-glycoprotein in breast cancer tissues obtained from 27 patients before and after treatment with paclitaxel. The effect of paclitaxel on localization of cellular YB-1 was examined by using GFP-YB-1. Interaction of YB-1 with the Y-box motif of the MDR1 promoters was studied by electrophoretic mobility shift assay. The effects of paclitaxel on MDR1 promoter activity were examined by luciferase assay. RESULTS: Of 27 breast cancer tissues treated with paclitaxel, nine (33%) showed translocation of YB-1 from the cytoplasm to the nucleus together with increased expression of P-glycoprotein during the course of treatment. Twelve breast cancer tissues (44%) showed neither translocation of YB-1 nor increased expression of P-glycoprotein. Nuclear translocation of YB-1 was correlated significantly with increased expression of P-glycoprotein (P=0.0037). Confocal analysis indicated that paclitaxel induced nuclear translocation of green fluorescent fused YB-1 in MCF7 cells. Furthermore, binding of YB-1 to the Y-box of MDR1 promoter was increased in response to treatment with paclitaxel. In addition, MDR1 promoter activity was significantly up-regulated by paclitaxel in MCF7 cells (P<0.001). CONCLUSIONS: The results of the present study suggested that YB-1 may be involved in the development of resistance to paclitaxel in breast cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Transcription factors and drug resistance.

Intrinsic or acquired resistance to anticancer agents is a major obstacle to the success of chemotherapy. Anticancer agents are known to modulate signal transduction pathways and alter expression of genes that play an important role in drug resistance. Emerging evidence suggests that the complexity of genomic response against anticancer agents arise from elaborate gene expression by multiple transcription factors. Here, we briefly describe the development of solid tumours and the appearance of drug-resistant cells. We also review what is known of the transcription factors that are involved in resistance to drugs, particularly cisplatin.

Antineoplastic Agents↗
PubMed2005

The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x(L) protein.

The broad-range cyclin-dependent kinase inhibitor 7-hydroxystaurosporine (UCN-01) is known to induce both a G1 cell cycle arrest and apoptosis. The mechanism of UCN-01-induced apoptosis is largely unknown. We analysed the mechanism of cytotoxicity of UCN-01 in four established colon carcinoma cell lines. The cell lines SW48 and LS513 responded to UCN-01 treatment by undergoing apoptosis in a concentration-dependent manner while the cell lines HT-29 and WiDr were completely resistant. Apoptosis in LS513 and SW48 cell lines was concomitant with the suppression of Bcl-x(L) on mRNA and protein level. In contrast, in the apoptosis-resistant cell lines, Bcl-x(L) expression was not affected by UCN-01. Stable overexpression of the Bcl-x(L) protein abrogated UCN-01-triggered apoptosis, but only partially restored growth, indicating that both cell cycle arrest and apoptosis exert the anticancer effect in a coordinated manner. The inhibition of Akt phosphorylation did not correlate with the apoptotic phenotype. UCN-01 inhibited the activating STAT3 phosphorylations on Ser727 and, notably, on Tyr705, but STAT3 did not contribute to Bcl-x(L) expression in colon carcinoma cells. Moreover, we show for the first time that UCN-01 induces apoptosis by suppression of Bcl-x(L) expression. The inhibition of this pathway is a new aspect of cytotoxic and modulatory potential of UCN-01.

Antineoplastic Agents↗
PubMed2005

The expression pattern of UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetyl-galactosaminyl transferase-3 in squamous cell carcinoma of the esophagus.

OBJECTIVE: UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyl transferase-3 (GalNAc-T3) regulates the initial glycosylation of mucin-type O-linked proteins. Although a different expression of GalNAc-T3 has been reported in various cancers, the expression has not been characterized in squamous cell carcinoma (SCC) of the esophagus. METHODS: We have also evaluated the expression of this enzyme in surgically resected esophageal mucosa. By immunohistochemical staining using a specific antibody, we evaluated the expression of GalNAc-T3 in 66 esophageal SCC and 28 dysplasia samples, and analyzed the relationship between the expression of GalNAc-T3 and clinicopathological features. RESULTS: GalNAc-T3 was positively detected in the majority of the cases of SCC, but not in dysplasia as well as the normal counterparts in resected esophagus. GalNAc-T3 was determined to be positive in 37 cases (68.5%) of differentiated carcinomas, but only in 4 cases (33.3%) of undifferentiated carcinomas (p < 0.05). Hematogeneous metastasis was observed in 13 of 41 (31.7%) GalNAc-T3-positive tumors, which was significantly more frequent than in negative tumors (2/25, 8%; p < 0.05). The number of metastatic nodes was significantly higher in tumors with GalNAc-T3-positive than GalNAc-negative expression (4.2 +/- 3.2 vs. 2.8 +/- 1.3, p < 0.05). The survival rate tended to be lower for patients with GalNAc-T3-positive tumors, although the difference was not statistically significant (p = 0.18). CONCLUSION: GalNAc-T3 may play a positive role in the process of carcinogenesis and progression in esophageal SCC. Functional inhibition of GalNAc-T3 may be effective for the prevention and treatment of esophageal SCC.

Carcinoma, Squamous Cell↗
PubMed2005

Cisplatin resistance and transcription factors.

Cisplatin is one of the most potent and widely used anti-cancer agents in the treatment of various solid tumors. However, the development of resistance to cisplatin is a major obstacle in clinical treatment. Several mechanisms are thought to be involved in cisplatin resistance, including decreased intracellular drug accumulation, increased levels of cellular thiols, increased nucleotide excision-repair activity and decreased mismatch-repair activity. In general, the molecules responsible for each mechanism are upregulated in cisplatin-resistant cells; this indicates that the transcription factors activated in response to cisplatin might play crucial roles in drug resistance. It is known that the tumor-suppressor proteins p53 and p73, and the oncoprotein c-Myc, which function as transcription factors, influence cellular sensitivity to cisplatin. So far, we have identified several transcription factors involved in cisplatin resistance, including Y-box binding protein-1 (YB-1), CCAAT-binding transcription factor 2 (CTF2), activating transcription factor 4 (ATF4), zinc-finger factor 143 (ZNF143) and mitochondrial transcription factor A (mtTFA). Two of these-YB-1 and ZNF143-lack the high-mobility group (HMG) domain and can bind preferentially to cisplatin-modified DNA in addition to HMG domain proteins or DNA repair proteins, indicating that these transcription factors may also participate in DNA repair. In this review, we summarize the mechanisms of cisplatin resistance and focus on transcription factors involved in the genomic response to cisplatin.

Activating Transcription Factor 4↗
PubMed2005

Mutant IkappaBalpha suppresses hypoxia-induced VEGF expression through downregulation of HIF-1alpha and COX-2 in human glioma cells.

Our previous study demonstrated that mutant IkappaBalpha (IkappaBalphaM) could inhibit glioma angiogenesis and tumorigenesis through the downregulation of vascular endothelial growth factor (VEGF) and IL-8. However, the pathways involved in VEGF expression are not well understood. Growing evidence indicates that hypoxia-inducible factor-1alpha (HIF-1alpha) and cyclooxygenases-2 (COX-2) play important roles in this progression. In this study, we first examined the expressions of hypoxia-induced genes in human glioma cells transfected with IkappaBalphaM (IN500deltaM) or control plasmid (IN500delta) in vitro. We found that hypoxic stress induced the expressions of HIF-1alpha, COX-2, and VEGF, and that IkappaBalphaM completely suppressed these expressions in vitro. Next, we injected these glioma cells into nude mice. After 3 weeks, the mice were moved to a hypoxic chamber (10% oxygen) for 3, 12, 24, 48, 96, or 144 h. The expressions of HIF-1alpha, COX-2, and VEGF in vivo were then analyzed by Northern blot and immunohistochemistry. IkappaBalphaM suppressed the expression of hypoxia-induced HIF-1alpha gene in vivo, but hypoxic stress induced the expression of COX-2 after 72 h. VEGF induction followed after 96 h of hypoxia in IN500deltaM cells. These findings suggest that VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 and COX-2 genes, and IkappaBalphaM could inhibit VEGF expression through these two pathways. Thus, IkappaBalphaM is identified as a pivotal factor in angiogenesis and is a potential target for neoplasm therapy.

Animals↗
PubMed2004

DNA topoisomerase II poison TAS-103 transactivates GC-box-dependent transcription via acetylation of Sp1.

Drug-induced modifications of transcription factors play important roles in both apoptosis and survival signaling. The data presented here show that the DNA topoisomerase II poison TAS-103 transactivated the SV40 promoter in a GC-box-dependent manner and induced Sp1 acetylation in cells expressing p300. This activity was not observed in cells lacking p300. TAS-103 treatment also enhanced the p300 content of the nucleus and the interaction of p300 with Sp1. Cellular susceptibility to TAS-103 was correlated with p300 expression but not with topoisomerase II expression. Furthermore, the presence of p300 significantly sensitized cancer cells to TAS-103 but not to cisplatin. Taken together, these findings demonstrate novel genomic responses to anticancer agents that modulate Sp1 acetylation and Sp1-dependent transcription in an apoptotic pathway.

Acetylation↗
PubMed2004

ZNF143 activates gene expression in response to DNA damage and binds to cisplatin-modified DNA.

We have identified a cisplatin-inducible gene, the mitochondrial ribosomal protein S11 (MRP S11) gene, by means of mRNA differential display. Functional analysis of the MRP S11 promoter showed that a Staf binding site in the promoter is required for both basal promoter activity and cisplatin-inducible activity. We also found that Staf binding activity is significantly increased in nuclear extracts from cells treated with cisplatin. ZNF 143 and ZNF 76 are human homologues of the Xenopus transcriptional activator, Staf. ZNF 143 expression is induced by cisplatin but ZNF 76 expression is not. However, ZNF 143 expression is not induced by transplatin, which is clinically ineffective. ZNF143 is an inducible gene by other DNA damaging agents such as gamma-irradiation, etoposide and adriamycin. ZNF 143 also binds preferentially to cisplatin-modified DNA. These results suggest that ZNF 143 participates in cellular responses to DNA damage.

Antineoplastic Agents↗
PubMed2004

Binding of RNA to p53 regulates its oligomerization and DNA-binding activity.

The C-terminus of p53 is responsible for maintaining the latent, non-DNA-binding form of p53. However, the mechanism by which the C-terminus regulates DNA binding is not yet fully understood. We show here that p53 interacts with RNA via its C-terminal domain and that disruption of this interaction, by RNase A treatment, truncation or phosphorylation of the C-terminus, restores DNA-binding activity. Furthermore, the oligomerization of p53 is significantly enhanced by disrupting the interaction between p53 and RNA. These findings suggest that binding of RNA to p53 is involved in the mechanism of p53 latency.

Biopolymers↗
PubMed2004

The expression pattern of UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 in early gastric carcinoma.

BACKGROUND AND OBJECTIVES: Tumor development usually is accompanied by alterations of O-glycosylation. Initial glycosylation of mucin-type O-linked proteins is regulated by UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 (GalNAc-T3). Although the expression of GalNAc-T3 has been examined in various cancers, the expression has not been characterized in early stages of cancer. METHODS: Using the specific antibody, we evaluated the expression of GalNAc-T3 in 125 early gastric cancers that were treated as classical gastrectomy with lymphadenectomy, and analyzed the relationship between the expression of GalNAc-T3 and clinicopathological features. RESULTS: GalNac-T3 was positively expressed in 40 cases (76%) in differentiated carcinomas, whereas in only six cases (8%) in undifferentiated carcinomas (P < 0.001). Positive staining was observed in 17 (26%) intramucosal and in 29 (48%) submucosal carcinomas, indicating that GalNac-T3 tended to be highly expressed as the depth of invasion increased (P < 0.05). Lymph node metastasis tended to be observed more frequently in GalNac-T3 positive than negative cases, and the difference was significant in undifferentiated type cancer (P < 0.05). CONCLUSIONS: GalNAc-T3 expression was a useful indicator of tumor differentiation in early gastric cancer, and the expression had positive correlation with depth of tumor invasion and lymph node metastasis. This suggests that the overexpression of GalNAc-T3 may have a role in invasion and metastasis in early stages of gastric cancer.

Adenocarcinoma↗
PubMed2004

Expression of UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase isozyme 3 in the subserosal layer correlates with postsurgical survival of pathological tumor stage 2 carcinoma of the gallbladder.

PURPOSE AND EXPERIMENTAL DESIGN: Little is known about the molecular events leading to the development and progression of pathological tumor stage 2 (pT(2)) gallbladder carcinoma. An alteration in the site of O-glycosylation may be associated with malignant behavior of carcinoma cells by modulation of the biological properties of the target mucin. The UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase isozyme 3 (GalNAc-T3) has the epithelial gland-specific expression and catalyzes mucin-type O-glycosylation. In this study, immunohistochemistry was performed to determine the expression level of GalNAc-T3 in 34 cases of pT(2) gallbladder carcinoma to determine the correlation of the GalNAc-T3 expression level with mode of recurrence and postsurgical survival. RESULTS: The expression levels of GalNAc-T3 protein and mRNA were increased in gallbladder carcinomas compared with the levels in adjacent noncancerous tissues and in intact gallbladders. Immunostaining of GalNAc-T3 was recognized in the cancerous epithelia, and the subcellular localization was classified into granular and diffuse types. In the 34 cases of pT(2) carcinoma, the localization of GalNAc-T3 was granular type in 50% and diffuse type in 50% of the cases at the deepest invading sites in the subserosal layer. Postsurgical recurrence was significantly more frequent in cases showing diffuse-type localization of GalNAc-T3 at the deepest invading sites (65%) than in those showing granular-type localization (23%; P < 0.05). Postsurgical survival was significantly poorer in cases showing diffuse-type localization than in those showing granular-type localization (P = 0.033) CONCLUSIONS: In pT(2) gallbladder carcinoma, the presence of diffuse-type localization of GalNAc-T3 in the subserosal layer is correlated with aggressiveness of the disease. This phenotype may serve as a unique biological feature associated with the malignant behavior.

Biomarkers↗
PubMed2004

Characterization of the 5'-untranslated region of YB-1 mRNA and autoregulation of translation by YB-1 protein.

The eukaryotic Y-box binding protein YB-1 is involved in various biological processes, including DNA repair, cell proliferation and the regulation of transcription and translation. YB-1 protein is abundant and expressed ubiquitously in human cells, functioning in cell proliferation and transformation. Its concentration is thought to be highly regulated at both the levels of transcription and translation. Therefore, we investigated whether or not the 5'-UTR of YB-1 mRNA affects the translation of YB-1 protein, thus influencing expression levels. Luciferase mRNA ligated to the YB-1 mRNA 5'-UTR was used as a reporter construct. Ligation of the full-length YB-1 5'-UTR (331 bases) enhanced translation as assessed by in vitro and in vivo translation assays. Deletion constructs of the YB-1 5'-UTR also resulted in a higher efficiency of translation, especially in the region mapped to +197 to +331 from the major transcription start site. RNA gel shift assays revealed that the affinity of YB-1 for various 5'-UTR probe sequences was higher for the full-length 5'-UTR than for deleted 5'-UTR sequences. An in vitro translation assay was used to demonstrate that recombinant YB-1 protein inhibited translation of the full-length 5'-UTR of YB-1 mRNA. Thus, our findings provide evidence for the autoregulation of YB-1 mRNA translation via the 5'-UTR.

5' Untranslated Regions↗
PubMed2004

Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

The human multidrug resistance protein 2 (MRP2/ABCC2), expressed on the bile canalicular membrane, mediates the multispecific efflux of several organic anions, including conjugates of glucuronate, sulfate, and glutathione. Expression of MRP2 can be altered in response to environmental stimuli such as cholestasis and jaundice. We previously reported that MRP2 mRNA expression levels are decreased in the nontumorous part of hepatitis C virus-infected human liver tissues, and that inflammatory cytokines inhibit MRP2 expression in human hepatic (HepG2) cells. We investigated the molecular mechanisms by which inflammatory cytokines modulate MRP2 gene expression in hepatic cells. Treatment of human hepatic cells with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha resulted in a decrease in the protein and mRNA levels of MRP2. IL-1beta inhibited the transcriptional activity of MRP2 promoter constructs by 40%, and this inhibition of MRP2 promoter activity was mediated through the interferon stimulatory response element (ISRE). Electrophoretic mobility shift assays with IL-1beta-treated nuclear extracts showed a decrease in the formation of DNA protein complexes, specifically those including interferon regulatory factor 3 (IRF3). Expression of recombinant human IRF3 increased MRP2 promoter activity. Treatment with a specific extracellular signal-regulated kinase inhibitor relieved IL-1beta-induced MRP2 mRNA downregulation and abrogated the binding of IRF3 to the ISRE element. In conclusion, IL-1beta induces downregulation of the MRP2 gene by inactivating IRF3 binding to ISRE on the MRP2 promoter in human hepatic cells; this inactivation is accomplished via interference with the extracellular signal-regulated kinase pathway.

DNA-Binding Proteins↗