Search PubMed⌕ Search

Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

At least 73 records · Page 4Linked to original sources
PubMed1998

Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias.

Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. Moreover, overexpression of the MDR1 gene has been shown to be associated closely with clinical outcome in various hematological malignancies, including acute myeloid leukemia (AML). However, the precise mechanism underlying overexpression of the MDR1 gene during acquisition of drug resistance remains unclear. We recently described an inverse correlation between the methylation status of CpG sites at the promoter region and expression of the MDR1 gene in malignant cell lines. In this study, we expanded this analysis to 42 clinical AML samples. We adapted a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for gene expression and a quantitative PCR after digestion by Hpa II for methylation status of the MDR1 gene. We observed a statistically significant inverse correlation between methylation and MDR1 expression in clinical samples. The hypomethylation status of the MDR1 promoter region might be a necessary condition for MDR1 gene overexpression and establishment of P-glycoprotein-mediated multidrug resistance in AML patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Isolation of a novel human canalicular multispecific organic anion transporter, cMOAT2/MRP3, and its expression in cisplatin-resistant cancer cells with decreased ATP-dependent drug transport.

The human multidrug resistance protein (MRP) gene encodes a membrane protein involved in the ATP-dependent transport of hydrophobic compounds. We previously isolated a canalicular multispecific organic anion transporter, cMOAT1/MRP2, that belongs to the ATP binding cassette (ABC) superfamily, which is specifically expressed in liver, and cMOAT1/MRP2 is responsible for the defects in hyperbilirubinemia II/Dubin-Johnson syndrome. In this study, we isolated a new cDNA of the ABC superfamily designated cMOAT2/MRP3 that is homologous to human MRP1 and cMOAT1/MRP2: cMOAT2/MRP3 is 56% identical to MRP1 and 45% identical to cMOAT1/MRP2, respectively. Fluorescence in situ hybridization demonstrated the chromosomal locus of this gene on chromosome 17q22. The human cMOAT2 cDNA hybridized to a 6.5-kb mRNA that was mainly expressed in liver and to a lesser extent in colon, small intestine, and prostate. The cMOAT2/MRP3 gene was not overexpressed in cisplatin-resistant cell lines with increased ATP-dependent transport of cisplatin over their parental counterparts derived from human head and neck cancer and human prostatic cancer cell lines. The human cMOAT2/MRP3, a novel member of the ABC superfamily, may function as a membrane transporter in liver, colon, and prostate.

Adenosine Triphosphate↗
PubMed1998

Novel biological functions of interleukin-4: formation of tube-like structures by vascular endothelial cells in vitro and angiogenesis in vivo.

The effect of interleukin-4 (IL-4) on angiogenesis was studied in vitro and in vivo. Human recombinant IL-4 significantly stimulated the formation of tube-like structures in collagen gels by bovine aortic endothelial cells as well as by human microvascular endothelial cells. Human recombinant IL-4 at 50-500 U/ml stimulated by about two- to threefold the formation of tubes by bovine aortic endothelial cells; the rate was comparable to that of basic fibroblast growth factor. Tube formation was almost completely inhibited by the addition of IL-4 receptor neutralizing antibody. Administration of rat recombinant IL-4 led to neovascularization when implanted in the rat cornea. Findings suggest that IL-4 may be a mediator of the immune system as well as an inducer of angiogenesis.

Animals↗
PubMed1998

Glutathione homeostasis in human hepatic cells: overexpression of gamma-glutamylcysteine synthetase gene in cell lines resistant to buthionine sulfoximine, an inhibitor of glutathione synthesis.

The synthesis of glutathione (GSH) and its conjugation to xenobiotics are essential for detoxification in liver cells. To understand how cellular levels of GSH are balanced in response to environmental stress, we cloned two cell lines, HLE/BSO1-1 and HLE/BSO1-2, from human hepatic HLE/WT cells resistant to buthionine sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase (GCS). HLE/BSO1-1 and HLE/BSO1-2 showed 35- and 40-fold higher resistance respectively, than the wild type to BSO. In the absence of BSO, cellular levels of GSH were 3.0-fold higher, whereas levels of Pi class glutathione thiol transferase, GSTP1, were 2-fold lower, in the subclones than in the wild type cells. GCS heavy subunit (GCSh) mRNA level were 2.5-fold higher in HLE/BSO1-1 and HLE/BSO1-2 as compared with HLE/WT. Sequences between -315 and -241 base pairs of the 5' region, which contain an AP1 site, were shown to be responsible for the enhanced expression of GCSh in HLE/BSO1-1 cells. The expression of a dominant-negative mutant of c-Jun was found to inhibit the AP1-dependent GCSh promoter activity in HLE/WT and HLE/BSO1-1. Both protein level of c-Jun and binding activity of AP-1 were increased in both HLE/BSO1-1 and HLE/BSO1-2 cells. The up-regulation of GCSh gene appeared to be due to enhanced GCSh promoter acting through AP-1 activation in BSO-resistant hepatic cells.

Binding Sites↗
PubMed1998

The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIalpha gene by heat shock.

Expression of the DNA topoisomerase IIalpha (topoIIalpha) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIalpha mRNA was increased 1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 degreesC for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIalpha gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) -295 to +85) and of three deletion constructs (nt -197 to +85, -154 to +85, and -74 to +85) was increased approximately 3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt -20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt -74 and -21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3-24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIalpha gene promoter. Finally, in vivo footprint analysis of the topoIIalpha gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIalpha gene by heat shock requires the release of a negative regulatory factor from ICE1.

Antigens, Neoplasm↗
PubMed1998

Involvement of vascular endothelial growth factor and urokinase-type plasminogen activator receptor in microvessel invasion in human colorectal cancers.

To evaluate the association among known angiogenic growth factors or factors related to the plasminogen activation system and clinicopathological factors in patients with colorectal cancer, we examined the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF-alpha), urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PA-R) and plasminogen activator inhibitor-1 (PAI-1) in clinical specimens of colorectal cancers by Northern blot analysis. In comparison with the expression of these angiogenesis-related genes in 7 paired samples of colorectal cancers and the adjacent normal mucosa, VEGF mRNA level was significantly higher in the cancer tissues than in the adjacent normal mucosa (p < 0.05). We analyzed expression of these genes in 44 cases of primary colorectal cancers. Among the 3 angiogenic growth factors we examined, VEGF mRNA expression was significantly higher in the cancer tissues with blood vessel invasion or with lymphatic vessel invasion than in those without, respectively (p < 0.05). On the other hand, u-PA-R mRNA expression was significantly higher in the cancers with blood vessel invasion than in those without (p < 0.05). In addition, there was a correlation between the expression levels of VEGF and u-PA-R mRNA in the cancer tissues we have examined. Using immunohistochemistry, strong staining of VEGF or u-PA-R was observed in the cancer cells invading the microvessels. Our findings suggest that malignant transformation might accompany the upregulation of VEGF expression in colorectal cancers and that VEGF and u-PA-R might contribute cooperatively to increase angiogenesis around the tumor as well as the metastasis via microvessels.

Adult↗
PubMed1998

Localization of 67 exons on a YAC contig spanning 1.5 Mb around the multidrug resistance gene region of human chromosome 7q21.1.

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) was previously assembled across 1.5 Mb of the multidrug resistance (MDR) gene (PGY1 and PGY3) region of human chromosome 7q21.1. This region of the human genome has now been subjected to exon amplification to detect the presence of additional genes. Exon trapping was performed directly on the YACs. Sixty-seven gene fragments were isolated and characterized by sequence analysis and comparison with public databases. The localization of these exons in the 1.5-Mb region was determined by hybridization to YAC clones, and they were localized in 11 subregions of YAC contigs. The exon collection includes 21 exons that were identical to known cDNA sequences of PGY1, PGY3, sorcin (SRI), the cDNA similar to the delta subunit of the human amiloride-sensitive Na- channel (SCNED), and 4 cDNAs with unknown function; 43 exons that showed homology/similarity to known cDNA sequences of mouse DMP1, rat COT, mouse and human NADHD, human MDC, 3 cDNAs encoding possible membrane proteins, and 21 other cDNAs; and 3 exons that shared no homology/similarity with any sequence in public databases. The nucleotide sequences of all the PGY1 and PGY3 exons were identical to the corresponding cDNA sequences previously determined, and these exons were localized to the expected positions on the appropriate YAC clones. No other member of the MDR gene family thus appeared to be present in the 1.5-Mb region. The integrated physical and exon maps should prove valuable for both fine mapping and determination of a complete gene map of this segment of the genome.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene.

The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy. During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression. Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression. We have now established various cell lines from human head and neck cancer KB cells which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene driven by various MDR1 promoter deletion constructs. Transient transfection of antisense YB-1 expression constructs resulted in a decrease of both YB-1 protein levels and DNA binding activity to the inverted CCAAT box, as determined by Western blot and gel mobility shift assays. The limited expression and binding activity due to expression of antisense YB-1 constructs were also observed when cells were treated with UV. CAT activity of constructs containing the Y-box was enhanced after treatment with UV irradiation as well as genotoxic agents such as cisplatin and etoposide. Moreover, this activation was reduced by 50-80% by transfection of antisense YB-1 expression constructs. In contrast, transfection of antisense YB-1 expression constructs had no effect on CAT activity driven by MDR1 promoter constructs not containing the Y-box. These data indicate that YB-1 is directly involved in MDR1 gene activation in response to genotoxic stress.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Genomic organization of the human Y-box protein (YB-1) gene.

The human Y-box protein (YB-1) is a member of a family of DNA-binding proteins containing a highly conserved cold shock domain. The genomic organization of the human YB-1 gene was determined from five overlapping genomic clones that encompassed all exons of the gene. Sequence analysis of these clones revealed that human YB-1 spans approximately 19 kb of genomic DNA and contains eight exons. The cold shock domain is encoded by exons 1-5. Both exon-splitting and codon-splitting in the region of the gene encoding the cold shock domain are similar to those in the corresponding region of another Y-box binding protein, dbpA. Exon-intron structures and nucleotide sequences of the regions encoding the N-terminal and C-terminal domains of the two proteins differ markedly between YB-1 and dbpA. These observations suggest that YB-1 and dbpA arose by duplication of a common ancestral gene encoding all these domains.

Base Sequence↗
PubMed1998

Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma.

Cholangiocarcinoma (CCC) is relatively hypovascular, in contrast to hepatocellular carcinoma (HCC), which is often highly vascular. We investigated if the diminished vascularity of CCC is related to altered expression of thrombospondin-1 (TSP-1), an antiangiogenic factor, and/or vascular endothelial growth factor (VEGF), a potent angiogenic factor, comparing the relationships with those of high- and low-vascular HCC. We also investigated the relationship between the mutation of the p53 gene and TSP-1 expression or VEGF expression. Northern blot analysis and immunohistochemical staining were performed on surgically resected human CCC and HCC. The ratios of TSP-1 mRNA level in cancer cells versus adjacent noncancerous cells (T/N ratios) were significantly higher in CCC (n = 11) than in HCC with high vascularity (n = 15). In contrast, T/N ratios of VEGF mRNA level in CCC (n = 11) were comparable with those in HCC with low vascularity (n = 5). In CCC, the cancer cells and fibroblasts were positively stained with anti-TSP-1 antibody. We observed that T/N ratios of VEGF mRNA level, but not those of the TSP-1 mRNA level, were significantly correlated with vascularity in HCC. The relative increase in TSP-1 and the relative decrease in VEGF in tumors compared with normal tissue may underlie the limited angiogenesis of CCC. The p53 gene did not affect the expression of TSP-1 in CCC or VEGF in HCC.

Adult↗
PubMed1998

Newly synthesized dihydropyridine derivatives as modulators of P-glycoprotein-mediated multidrug resistance.

Newly synthesized 1,4-dihydropyridine derivatives possessing alkyl chains at the 4-position screened whether they could overcome P-glycoprotein-mediated multidrug resistance in cultured cancer cells and also leukemia-bearing animals. Of these derivatives, some could overcome drug resistance to doxorubicin and vincristine in multidrug resistant human cancer cell lines. Combined administration of vincristine and some of the derivatives significantly increased the life span of P-glycoprotein overexpressing multidrug-resistant P388 leukemia-bearing mice. The calcium antagonistic activities, an undesirable effects, were weaker than that of verapamil. These results suggested that the introduction of alkyl groups at the 4-position were effective for both overcoming multidrug resistance and reducing the calcium antagonistic activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Expression of multidrug resistance protein gene in patients with glioma after chemotherapy.

Two different ATP-binding membrane glycoproteins, the 170 kDa P-glycoprotein (P-gp) and the 190 kDa multidrug resistance protein (MRP), are involved in the acquisition of multidrug resistance phenotypes in cancer cells. Overexpression of P-gp is often observed in various human tumors when treated with anticancer agents. In this study, we asked whether MRP was overexpressed in human gliomas after cancer chemotherapy. We investigated expression of MRP and P-gp before and after chemotherapy in tumor samples from patients with glioma. MRP expression was observed in 16 (70%) of 23 untreated patients, and the proportion of MRP-positive cells in the whole cell population ranged from 3 to 32% in the 16 MRP-positive patients. P-gp-positive tumors were observed in 4 (18%) of 23 patients, and the proportional rates of P-gp-positive cells in the whole cell population ranged from 4 to 23%. The proportional rate of MRP-positive or P-gp-positive glioma cells increased after chemotherapy when compared with that before chemotherapy in all patients examined. We could observe no statistically significant correlation between expression of MRP or P-gp and tumor grade. These results suggest that MRP as well as P-gp may be involved in acquired or intrinsic drug resistance in human glioma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Alterative expression of the collagenase and adhesion molecules in the highly metastatic clones of human colonic cancer cell lines.

Human colonic carcinoma cell lines, KM12C, KM12SM and KM12L4, were previously established and their in vivo metastatic potentials have been well evaluated. The highly metastatic cell lines KM12SM and KM12L4 were derived from the parental low metastatic cell line KM12C in vivo. To evaluate the metastatic behavior of these cell lines in vitro, we examined colony formation on monolayers of the pulmonary arterial endothelial (CPAE) cells. On day 4, the highly metastatic cell lines showed an approximately 2-fold increase in number of colonies on CPAE cell monolayers relative to the parental KM12C cell line. To investigate what evidence is correlated with their metastatic and invasive abilities, Northern blot analysis and flow cytometry were performed in all cell lines. According to the results of Northern blot analysis, the levels of matrix metalloproteinase (MMP)-2 and c-met mRNA expression were increased in highly metastatic cell lines as compared with the parental cell line. We also examined the cell-surface expression of several adhesion molecules by flow cytometry. The levels of expression of sialyl Lewisa antigen (sLe(a)) in KM12SM and KM12L4 were twice higher than that in KM12C. However, the levels of expression of E-cadherin in KM12SM and KM12L4 were decreased to half that in KM12C. The alterative expression of the collagenase and adhesion molecules might contribute to their metastatic/invasive abilities of these cell lines both in vivo and in vitro.

Antigens, CD↗
PubMed1998

A new synthetic matrix metalloproteinase inhibitor modulates both angiogenesis and urokinase type plasminogen activator activity.

Proteolytic degradation of the extracellular matrix is essential to angiogenesis. Two families of proteases, the serine proteases of plasminogen activator/plasmin system and the matrix metalloproteinases (MMPs) are closely involved in these processes. The treatment of mice with a diet containing a new synthetic MMP inhibitor, OPB-3206: 3S-[4-(N-hydroxyamino)-2R-isobutylsuccinyl] amino-1methoxy-3, 4-dihydrocarbostyril, abrogated the development of new vessels in a rat corneal assay, and in a mouse Matrigel assay. In an in vitro angiogenesis model, OPB-3206 inhibited the migration and the tube formation of bovine aortic endothelial cells at 10-100 times lower concentrations than those required to inhibit the growth of these cells. OPB-3206 as well as other MMP inhibitory drugs, batimastat/BB-94 and marimastat/BB-2516, also selectively inhibited tubular morphogenesis in vitro. OPB-3206 reduced the activities of interstitial collagenase and type IV collagenase, but the concentrations of 50% inhibition against these MMPs were much higher than those of BB-94 and BB-2516. However, this new compound also inhibited urokinase type plasminogen activator activity on fibrin zymogram, while BB-94 and BB-2516 did not. Furthermore, the addition of urokinase type plasminogen activator reduced the inhibitory effect of the tubular morphogenesis of vascular endothelial cells by OPB-3206. The treatment of mice with a diet containing this new compound also reduced the growth of implanted mammary carcinomas as well as the lung metastasis of colon carcinoma. The anti-angiogenic effect of OPB-3206 appeared to be associated with its inhibition of tumor growth and metastasis.

Journal Article↗
PubMed1998

Tubular morphogenesis by genotoxic therapeutic agents that induce NF-kappaB activation in human vascular endothelial cells.

Angiogenic stimuli induce tubular morphogenesis and angiogenesis in vascular endothelial cells, but these cells are highly vulnerable to cytokines, oxidative stress, and genotoxic anticancer agents. A transcription factor, NF-kappaB, is involved in the protection against apoptosis and in angiogenesis in response to stimuli that could induce cell death. NF-kappaB was specifically activated by the genotoxic anticancer therapeutic agents etoposide and doxorubicin, but not by bleomycin, mitomycin C and cisplatin, in human vascular endothelial cells in three independent assay systems: nuclear translocation of NF-kappaB, binding of NF-kappaB to its consensus sequence, and NF-kappaB -dependent transcription. Exposure to etoposide and doxorubicin induced tubular morphogenesis by vascular endothelial cells in type I collagen gel at rates comparable to tumor necrosis factor-alpha. Co-administration of NF-kappaB antisense oligonucleotides inhibited the angiogenesis by doxorubicin and etoposide. In contrast, bleomycin, mitomycin C, and cisplatin did not induce angiogenesis. An angiogenic factor, interleukin 8, was dramatically induced in vascular endothelial cells treated with doxorubicin, but not in cells treated with cisplatin. Co-administration of anti-interleukin 8 antibody almost completely blocked the doxorubicin-induced angiogenesis in vitro, suggesting a paracrine/autocrine control through drug-induced angiogenic factor(s). The presence or absence of NF-kappaB activation may have an essential role in tubular morphogenesis by vascular endothelial cells during chemotherapeutic treatment, possibly through interleukin 8.

Journal Article↗
PubMed1998

Mutations in the canilicular multispecific organic anion transporter (cMOAT) gene, a novel ABC transporter, in patients with hyperbilirubinemia II/Dubin-Johnson syndrome.

Members of the ATP-binding cassette (ABC) transporter superfamily are mutated to cause diseases that include cystic fibrosis, hyperinsulinemia, adrenoleukodystrophy, Stargardt disease and multidrug resistance. We recently isolated a novel human member of ABC transporter superfamily as the candidate transporter for the glucuronide and glutathione-conjugated antitumor agents, and found it highly homologous to the rat cmoat gene. consistent with recent findings of defects in the homologous cmoat gene in two rat models of hyperbilirubinemia (TR- and Eisai), we report two deletions and a missense mutation in the active transport family signature region in the gene in patients with hyperbilirubinemia II/Dubin-Johnson syndrome (DJS; MIM 237500), respectively. These results strongly implicate the cMOAT gene as responsible for the defects in DJS patients.

Amino Acid Sequence↗
PubMed1998

Effect of retinoic acid on morphological changes of human pancreatic cancer cells on collagen gels: a possible association with the metastatic potentials.

Pancreatic carcinoma is an invasive and metastasizing type of malignancy. We established six pancreatic cancer cell lines from human pancreatic carcinomas, three highly metastatic lines (KP-1NL, KP-4, and SUIT-2) and three minimally metastatic lines (KP-2, KP-3, and BxPC-3). The three highly metastatic cell lines grew in a fibroblastoid pattern on collagen gels, whereas the three minimally metastatic cell lines grew in an epithelioid pattern under similar conditions. Western blot and Northern blot analyses indicated much higher levels of E-cadherin in the three minimally metastatic cell lines relative to the three highly metastatic cell lines. When the effect of all-trans-retinoic acid on the growth patterns of the three highly metastatic lines was examined, we observed a dramatic change from fibroblastoid to epithelioid growth in SUIT-2 cells. Although all six cell lines had comparable levels of retinoic acid receptor-gamma, retinoic acid receptor-beta was expressed only in SUIT-2 cells. Treating SUIT-2 cells with retinoic acid also induced the upregulation of E-cadherin expression. When SUIT-2 cells were treated with retinoic acid receptor-specific agonists, 13-cis-retinoic acid and Am555S, a morphological change from fibroblastoid to epithelioid growth was induced. Retinoic acid receptor-specific antagonists, LE135 and LE540, inhibited retinoic acid-induced change of the growth patterns. The effect of retinoic acid and its derivatives on the growth pattern was discussed in a possible association with their antimetastatic activities of pancreatic cancer.

Cadherins↗