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Biomedical subjects

K Kohno

Publications and source records attributed to K Kohno.

At least 19 recordsLinked to original sources
PubMed2006

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells.

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

Animals↗
PubMed2006

Expression of Y-box-binding protein dbpC/contrin, a potentially new cancer/testis antigen.

Y-box-binding proteins are members of the human cold-shock domain protein superfamily, which includes dbpA, dbpB/YB-1, and dbpC/contrin. dbpC/contrin is a germ cell-specific Y-box-binding protein and is suggested to function as a nuclear transcription factor and RNA-binding protein in the cytoplasm. Whereas ubiquitous dbpB/YB-1 expression has been well studied in various types of human carcinomas as a prognostic or predictive marker, the dbpC/contrin expression in human tumour cells has not been reported. In this report, we provide the first evidence showing that dbpC was highly expressed in human testicular seminoma and ovarian dysgerminomas, and in carcinomas in other tissues and that its expression in normal tissues is nearly restricted to germ cells and placental trophoblasts. These results indicate that dbpC/contrin would be a potentially novel cancer/testis antigen.

Adult↗
PubMed2005

Roles of volume-sensitive Cl- channel in cisplatin-induced apoptosis in human epidermoid cancer cells.

The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl-/HCO3- exchangers and Cl- channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO3-, we investigated the relation between cisplatin-induced apoptosis and Cl- channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl- channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl- current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl- channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

Low expression of polypeptide GalNAc N-acetylgalactosaminyl transferase-3 in lung adenocarcinoma: impact on poor prognosis and early recurrence.

Initial glycosylation of mucin-type O-linked protein is catalysed by one of the UDP-GalNAc: polypeptide N-acetyl-galactosaminyl transferase-3 (GalNAc-T3). O-glycosylation is important in the binding of cell adhesion molecules, cell differentiation, invasion, and metastasis in tumours. This study was designed to detect GalNAc-T3 expression in lung adenocarcinoma by using immunohistochemical staining, and to evaluate the relationship between the GalNAc-T3 expression level and prognosis and recurrence in completely resected lung adenocarcinoma patients. A low expression of GalNAc-T3 was detected in the cytoplasm of tumour cells in 79 of 148 patients (53.4%) with lung adenocarcinoma. The low expression of GalNAc-T3 was associated with poorly differentiated tumour (P<0.0001), poor pathologic stage (P<0.0001), lymph node metastasis (P<0.0001), and tumour recurrence (P=0.016). The lung carcinoma patients with low GalNAc-T3 expression had a poorer prognosis than those with high GalNAc-T3 expression, using both univariate and multivariate analyses (overall survival: P<0.0001 and P=0.011, respectively). In addition, multivariate analysis of the clinicopathological characteristics of stage I lung adenocarcinoma indicated that the low expression of GalNAc-T3 was a significant independent factor for predicting poor prognosis and early recurrence (P=0.006, rr=2.87 and P=0.019, rr=3.05, respectively). The low expression of GalNAc-T3 may be a useful marker for predicting poor prognosis and early recurrence in completely resected lung carcinoma patients, particularly patients with stage I diseases.

Adenocarcinoma↗
PubMed2002

N-acetylgalactosaminyl transferase-3 is a potential new marker for non-small cell lung cancers.

N-acetylgalactosaminyl transferase-3 (GalNAc-T3) is an enzyme involved in the initial glycosylation of mucin-type O-linked proteins. In the present study, we used immunohistochemistry to examine GalNAc-T3 expression in 215 surgically resected non-small cell lung cancers. We analysed the biological and clinical importance of GalNAc-T3 expression, especially with regard to its potential as a prognostic factor. We found that normal bronchial epithelial cells, bronchial gland cells, and alveolar pneumocytes showed cytoplasmic immunostaining for GalNAc-T3. Low expression of GalNAc-T3, observed in 93 of 215 tumours (43.4%), was found more frequently in tumours from smokers than those from nonsmokers (P=0.001), in squamous cell carcinomas than nonsquamous cell carcinomas (P<0.0001), and in moderately and poorly differentiated tumours than well differentiated tumours (P=0.0002). Multivariate logistic regression analysis showed that an association of low GalNAc-T3 expression with squamous cell carcinomas was the only one significant relationship of GalNAc-T3 expression with various factors (P<0.0001). Moreover, tumours losing GalNAc-T3 expression had a significantly higher Ki-67 labelling index than tumours retaining GalNAc-T3 expression (P=0.0003). Patients with low GalNAc-T3 expression survived a significantly shorter time than patients with high GalNAc-T3 expression in 103 pStage I non-small cell lung cancers (5-year survival rates, 58% and 78%, respectively; P=0.02 by log-rank test) as well as in 61 pStage I nonsquamous cell carcinomas (5-year survival rates, 63% and 85%, respectively; P=0.03). Low GalNAc-T3 expression was an unfavourable prognostic factor in pStage I non-small cell lung cancers (hazards ratio, 2.04; P=0.03), and in pStage I nonsquamous cell carcinomas (hazards ratio, 2.70; P=0.03). These results suggest that GalNAc-T3 is a new marker of non-small cell lung cancers with specificity for histology and prognosis.

Aged↗
PubMed2002

Prenatal course and pregnancy outcome of fetuses with a transient nuchal translucency.

OBJECTIVES: This study was designed to correlate fetuses in a normal prenatal population who exhibited transient nuchal translucency (NT) with both prenatal course and pregnancy outcome. METHODS: The fetuses with abnormal NT were followed by ultrasonography at 1-2-week intervals during their prenatal course. Fetuses with NT who exhibited no detectable congenital malformations were defined as the NTO group; fetuses with abnormal NT and structural malformations but no chromosomal abnormalities were placed in the SM group; and fetuses with abnormal NT, structural malformations, and chromosomal abnormalities were categorized as the CA group. The groups were compared by: maternal age, weeks of gestation by ultrasound and the NT value at the initial exam, the maximum NT value, and the duration of abnormal NT. RESULTS: During the study period, 92 fetuses with abnormal NT were found, monochorionic twins excluded. Of the 92 fetuses, 80 were in the NTO group, 10 were in the SM group, and two were in the CA group. The maternal age and the gestational weeks at the initial diagnosis were not significantly different in the NTO and the SM groups. However, the NT value at the initial diagnosis, maximum NT value, and the duration of abnormal NT were significantly greater in the SM group than those values in the NTO group. CONCLUSIONS: Fetuses with transient nuchal translucency commonly had structural malformations, particularly fetuses with significant and large persistent NT.

Adult↗
PubMed2001

Enhanced expression of the human multidrug resistance protein 3 by bile salt in human enterocytes. A transcriptional control of a plausible bile acid transporter.

The enterohepatic circulation is essential for the maintenance of bile acids and cholesterol homeostasis. The ileal bile acid transporter on the apical membrane of enterocytes mediates the intestinal uptake of bile salts, but little is known about the bile salt secretion from the basolateral membrane of enterocytes into blood. In the basolateral membrane of enterocytes, an ATP-binding cassette transporter, multidrug resistance protein 3 (MRP3), is expressed, which has the ability to transport bile salts. We hypothesized that MRP3 might play a role in the enterohepatic circulation of bile salts by transporting them from enterocytes into circulating blood through the up-regulation of MRP3 expression, so we investigated the transcriptional control of MRP3 in response to bile salts. MRP3 mRNA levels were increased about 3-fold in human colon cells by chenodeoxycholic acid (CDCA), in a dose- and time-dependent manner. In the promoter assay, the promoter activity of MRP3 was increased about 3-fold over the basal promoter activity when treated with CDCA, and the putative bile salt-responsive elements exist in the region -229/-138 including two alpha-1 fetoprotein transcription factor (FTF)-like elements. Constructs with a specific mutation in the consensus sequence of FTF elements showed no increase in basal transcriptional activity following CDCA treatment. In electrophoretic mobility shift assay with nuclear extracts, specific binding of FTF to FTF-like elements was observed when treated with CDCA. The expression of FTF mRNA levels were also markedly enhanced in response to CDCA, and overexpression of FTF specifically activated the MRP3 promoter activity about 4-fold over the basal promoter activity. FTF thus might play a key role not only in the bile salt synthetic pathway in hepatocytes but also in the bile salt excretion pathway in enterocytes through the regulation of MRP3 expression. MRP3 may contribute as a plausible bile salt-exporting transporter to the enterohepatic circulation of bile salts.

5' Untranslated Regions↗
PubMed2001

The role of heat shock protein 70 in vitamin D receptor function.

We previously demonstrated that the 1alpha,25-dihydroxyvitamin D(3) receptor (VDR) interacts with the constitutive heat shock protein, hsc70 in vitro, and with DnaK (Biochem. Biophys. Res. Commun. 260, 446-452, 1999). The biological significance of VDR-heat shock protein interactions, however, is unknown. To examine the role of such interactions in eukaryotic cells, we heterologously expressed VDR and RXRalpha together with a vitamin D-responsive reporter system in Saccharomyces cerevisiae and examined the consequences of heat shock protein 70 gene (SSA) deletion in these cells. We show that heterologously expressed VDR associates with the yeast cytosolic hsp70 protein, Ssa1p. Deletion of the SSA2, SSA3, and SSA4 genes and reduction of Ssa1p activity, reduces the intracellular concentrations of the VDR and its heterodimeric partner, RXRalpha and reduces the activity of a vitamin D-dependent gene. Hsp70-like chaperone proteins play a role in controlling concentrations of the VDR within the cell.

Adenosine Triphosphatases↗
PubMed2001

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Binding Sites↗
PubMed2001

Transcriptional activation of the human HMG1 gene in cisplatin-resistant human cancer cells.

The nonhistone chromosomal protein, high mobility group 1 (HMG1), which is ubiquitously expressed in higher eukaryotic cells, preferentially binds to cisplatin-modified DNA. The observation that HMG1 is overexpressed in cisplatin-resistant human cancer cells suggests that cisplatin resistance may be closely associated with HMG1. To decipher the mechanism of HMG1 overexpression in cisplatin-resistant cells, we isolated two overlapping genomic DNA clones containing the entire human HMG1 gene. These clones, which span approximately 15 kb of contiguous DNA, include 5 kb of the 5' flanking region as well as the entire coding sequence. We sequenced 1500 bp upstream of the first exon. The segment proximal to the transcription initiation site did not contain a TATA box but did possess an activating transcription factor site, an activator protein-2 site, one CCAAT box, and two CCAAT-binding transcription factor/nuclear factor-1 (CTF/NF-1) sites. HMG1 promoter activity was 3-10-fold higher in cisplatin-resistant KB-CP20 cells than in parental KB cells. An in vivo footprint experiment showed several differences of dimethyl sulfate modifications between KB and KB-CP20 cells in the area around the CTF/NF-1 sites. In addition, electrophoretic gel mobility shift assays showed that binding of a nuclear factor from cisplatin-resistant cells to the CTF/NF-1 site was significantly higher than the binding of the same factor from parental cells. Semiquantitative reverse transcription-PCR and Western blot analysis also showed that expression of CTF/NF-1 was 3-20-fold higher in the resistant cell line than in its parental counterpart. These findings suggest that, in cisplatin-resistant cells, the expression of HMG1 gene product is enhanced at the transcriptional level and that this probably occurs through the enhanced expression of the CCAAT binding factor, CTF/NF-1.

Antineoplastic Agents↗
PubMed2001

Interferon gamma-dependent induction of thymidine phosphorylase/platelet-derived endothelial growth factor through gamma-activated sequence-like element in human macrophages.

Thymidine phosphorylase (TP), an enzyme involved in the reversible conversion of thymidine to thymine, is identical to platelet-derived endothelial cell growth factor. TP expression in cancer cells and/or infiltrated macrophages is associated with microvessel density and poor clinical prognosis in patients with various tumor types. However, how TP expression is up-regulated in human tumors is unclear. Of various inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1alpha), and interferon gamma (IFN-gamma), we observed that IFN-gamma most effectively increased the expression of TP in cultured human monocytic U937 cells. Transient transfection of the various deletion constructs of the TP promoter showed that the presence of the -474 to -355 sequence containing gamma-activated sequence-like element was essential for IFN-gamma-dependent activation of the TP gene. Furthermore, the IFN-gamma-dependent transcriptional activity of the promoter construct containing mutations in the gamma-activated sequence-like element was significantly decreased. An electrophoretic mobility shift assay showed that IFN-gamma increased signal transducers and activators of transcription 1 binding to gamma-activated sequence-like element in the TP promoter. IFN-gamma could be a mediator of TP expression in infiltrated monocyte/macrophages, and those monocyte/macrophages expressing TP might play an important role in malignancy and angiogenesis in various human tumors.

Base Sequence↗
PubMed2001

Elevated expression of vacuolar proton pump genes and cellular PH in cisplatin resistance.

V-ATPases are proton-translocating enzymes, which are found not only in numerous intracellular organelles but also in the plasma membranes of many eukaryotic cells. Using differential display, we have identified one of the proton pump subunit genes, ATP6C, as a cisplatin-inducible gene. Northern blot analysis demonstrated that expression of other members of the subunit is inducible by cisplatin treatment. Proton pump gene expression is also upregulated in 3 independent cisplatin-resistant cell lines but not in vincristine- or etoposide-resistant cell lines. Cellular pH was significantly higher in cisplatin-resistant cells than in sensitive parental cells. In vitro DNA-binding activity of cisplatin was markedly increased in acidic conditions, suggesting that the cytotoxicity of cisplatin is modulated by cellular pH. Furthermore, the proton pump inhibitor bafilomycin can synergistically potentiate the cytotoxicity of cisplatin but not of etoposide or camptothecin. These results indicate that cellular pH is one of the critical parameters for effective cancer chemotherapy with cisplatin.

Anti-Bacterial Agents↗
PubMed2001

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Catechol Oxidase↗
PubMed2001

Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress.

Under conditions of endoplasmic reticulum (ER) stress, mammalian cells induce both translational repression and the unfolded protein response that transcriptionally activates genes encoding ER-resident molecular chaperones. To date, the only known pathway for translational repression in response to ER stress has been the phosphorylation of eIF-2alpha by the double-stranded RNA-activated protein kinase (PKR) or the transmembrane PKR-like ER kinase (PERK). Here we report another pathway in which the ER transmembrane kinase/ribonuclease IRE1beta induces translational repression through 28S ribosomal RNA cleavage in response to ER stress. The evidence suggests that both pathways are important for efficient translational repression during the ER stress response.

Amino Acid Sequence↗