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Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

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↗
PubMed2005

Antimyeloma effects of a novel synthetic retinoid Am80 (Tamibarotene) through inhibition of angiogenesis.

In multiple myeloma (MM), the interaction between myeloma cells and bone marrow microenvironment has an important role in the pathogenesis of MM. We first examined the inducing effect of myeloma cells on migration of human umbilical vein vascular endothelial cells (HUVECs). Five myeloma cell lines produced varying amounts of VEGF, and migration of HUVECs was induced by coculture with myeloma cells. We next examined the inhibitory effect of a novel synthetic retinoid Am80 (Tamibarotene) on both myeloma cells and HUVECs. Am80 is specific for the retinoic-acid receptor-alpha/beta, and has therapeutic effects in all-trans retinoic acid resistant acute promyelocytic leukemia. Am80 slightly inhibited the growth of both myeloma cells and HUVECs, and remarkably inhibited the growth of HUVECs stimulated by VEGF. Am80 showed little growth inhibition of bone marrow stromal cells (BMSCs), but it markedly inhibited migration of HUVECs by cocultured myeloma cells. Am80 inhibited VEGF-induced phosphorylation of VEGF receptor. In addition, VEGF-induced formation of tube-like structures in vitro and neovascularization in mouse corneas were significantly inhibited by Am80. These findings clearly demonstrate that Am80 is a potential inhibitor of angiogenesis caused by the interaction between vascular endothelial cells and myeloma cells, and might be a useful therapeutic agent against MM.

Animals↗
PubMed2003

Basal membrane localization of MRP1 in human placental trophoblast.

The placental trophoblast is considered to act as a barrier between mother and fetus, mediating the exchange of various materials across the placenta. ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug-resistance protein (MRP) are expressed in the placenta and function as efflux transport systems for xenobiotics. In the present study, we aimed to determine the localization of MRP1 in the human placenta in comparison with that of P-gp. Western blotting analysis with human placental membrane vesicles indicated that P-gp and MRP1 are localized on the brush-border membranes and basal membranes, respectively. Immunohistochemical analysis with human normal full-term placenta showed that anti-P-gp monoclonal antibody F4 stained the brush-border side of the trophoblast cells, whereas anti-MRP1 monoclonal antibody MRPr1 stained the basal side. These results confirm that P-gp and MRP1 are located on the brush-border membranes and basal membranes, respectively, of human full-term placental trophoblast. MRP1 was also detected on the abluminal side of blood vessels in the villi. Accordingly, MRP1 may play a role distinct from that of P-gp, which is considered to restrict the influx of xenobiotics into the fetus.

ATP Binding Cassette Transporter, Subfamily B↗
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

Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein.

8-Oxoguanine, an oxidized form of guanine, has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms probably have a mechanism for recognizing RNA molecules carrying 8-oxoguanine and prevent them from entering into the cellular translational machinery. We now report that the Escherichia coli cell possesses proteins that bind specifically to RNA carrying 8-oxoguanine. On incubation with a cell-free extract, 8-oxoguanine-containing RNA is stable while normal RNA is degraded by cellular nucleases. The RNase protection assay and gel shift assay revealed that some proteins bind specifically to 8-oxoguanine-containing RNA, hence preventing nuclease attacks. Among the complexes that were detected, one with a 77 kDa protein exhibits tight binding between RNA and protein components. This protein was identified as polynucleotide phosphorylase, encoded by the pnp gene. pnp(-)() mutants are hyperresistant to paraquat, a drug that induces oxidative stress in the cell. Binding of Pnp protein to 8-oxoguanine-containing RNA would inhibit cell growth, probably due to withdrawal of such RNA from the translational machinery. The Pnp protein may, therefore, discriminate between an oxidized RNA molecule and a normal one, thus contributing a high fidelity of translation.

Binding, Competitive↗
PubMed2001

Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/MOAT).

The human canalicular multispecific organic anion transporter (cMOAT), known as the multidrug resistance-associated protein 2 (MRP2), is normally expressed in the liver and to a lesser extent in the kidney proximal tubules. In these tissues MRP2 specifically localizes to the apical membrane. The construction of MRP2 fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2 that is responsible for its apical localization in polarized cells. The specific apical localization of MRP2 is due to a C-terminal tail that is not present in the basolaterally targeted MRP1. Deletion of three amino acids from the C-terminal of MRP2 (DeltaMRP2) causes the protein to be localized predominantly in the basolateral membrane in polarized Madin-Darby canine kidney cells. Interestingly, MRP2 expressed in a mouse leukemia cell line (L1210 cells) predominantly accumulates intracellularly with minimal cell membrane localization. In contrast, DeltaMRP2 was shown to predominantly localize in the cell membrane in L1210 cells. Increased transport of 2,4-dinitrophenyl glutathione from L1210 cells expressing DeltaMRP2 showed that the re-targeted protein retains its normal function.

ATP Binding Cassette Transporter, Subfamily B↗
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

Retrovirus insertion and transcriptional activation of the multidrug-resistance gene in leukemias treated by a chemotherapeutic agent in vivo.

To understand the molecular basis for multidrug-resistant (MDR) cancer cells in vivo, this study analyzed molecular changes of the mdr1a gene region in leukemia cells in mice during continuous treatment with vincristine. An inverse insertion of murine leukemia retrovirus (MuLV) into the 5'-flanking region of the mdr1a gene was found. This insertion was concomitantly accompanied by up-regulation of the mdr1a gene and the loss of chemosensitivity. Deletion of long-terminal repeat (LTR) sequences dramatically decreased the mdr1a promoter-driven reporter activity. The MuLV LTR insertion appears to exert its enhancer activity on mdr1a transcription during the appearance of MDR leukemia cells. Two mechanisms were postulated to explain the mdr1a gene activation by retrovirus insertion during in vivo chemotreatment: de novo insertion of MuLV induced by vincristine treatment and selection of a small fraction of pre-existing cells carrying MuLV insertion during vincristine treatment. No rearranged sequence was detected by polymerase chain reaction in parental cells. This result argued for the first mechanism. The randomly altered distribution of MuLV during repetitive chemotreatment might also be consistent with this hypothesis. On the other hand, the retrovirus insertion was detected at the same site of the mdr1a promoter region in 2 independent experiments, which suggests the second mechanism. It should be noted that in vivo chemotreatment using vincristine could generate the mdr1a-overexpressing cells through retrovirus insertion and the enhancer effect of the LTR.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Synthesis and structure--activity analysis of novel dihydropyridine derivatives to overcome multidrug resistance.

The structure activity relationships were studied on newly synthesized 1,4-dihydropyridine derivatives possessing a 1-pentyl group at the 4-position, and 3-pyridylpropylester was found to be one of the effective fragments for overcoming P-glycoprotein mediated multidrug-resistance (MDR) in cultured human cancer cells, in vitro. 3-Pyridylpropylester was also found to be one of the effective fragments for increasing the life span of P-glycoprotein overexpressing MDR P388 leukemia-bearing mice, in vivo. All compounds had weak calcium antagonistic activities, but there appeared no relationship between MDR reversing effect and calcium antagonistic activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
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

Decreased expression of an ATP-binding cassette transporter, MRP2, in human livers with hepatitis C virus infection.

BACKGROUND/AIMS: To understand hepatic injury during the process of hepatitis viral infection, determination of liver-specific functions at molecular levels is critical. Because the transport of endogenous/exogenous toxic substances is an intrinsically important hepatic function, we examined whether expression of the ATP-binding cassette (ABC) transporter gene was affected in patients with hepatitis viral infection. METHODS: To determine which ABC transporter was expressed differently in patients with hepatic viral infection, we assayed the expression of MDR1, MDR3, MRP1, MRP2, and MRP3 in non-cancerous regions in the liver of 42 patients with hepatic tumors using both quantitative RT-PCR and immunological staining analysis, and compared the hepatic expression levels between patients with hepatitis viral infection and non-infected controls. RESULTS: Of the five ABC transporter genes studied, the mRNAs of MRP2 and MRP3 were highly expressed in the human liver. There was a significant reduction in MRP2 expression to 29% in the virus-infected liver. Treatment of hepatic cells with inflammatory cytokines resulted in decreased mRNA levels of MRP2 and decreased MRP2 promoter activity. CONCLUSIONS: The down-regulation of MRP2 might induce a failure in the transport of various genotoxic substances in the liver with hepatitis virus infection.

Adult↗
PubMed2001

Involvement of the multidrug resistance protein 3 in drug sensitivity and its expression in human glioma.

The multidrug resistance protein (MRP) family belongs to the ATP-binding cassette superfamily (ABC) of transporters, which are involved in ATP-dependent transport of hydrophobic compounds. One of the MRP family, MRP1, is partially associated with the multidrug resistance phenotype in brain tumors. In this study, we asked whether another MRP family gene, MRP3, could affect drug sensitivity to anticancer agents in human glioma cell lines and clinical glioma specimens. We first produced two antisense transfectants by introduction of antisense MRP3 cDNA into the glioma cell line NHG2, which endogenously expresses MRP3. The two MRP3 antisense transfectants showed 2- to 5-fold increases in drug sensitivity to etoposide and cisplatin compared with NHG2 cells, but their sensitivity to vincristine or nitrosourea was not changed. Two MRP3 cDNA sense transfectants of pig kidney cell lines showed 4- to 6-fold drug resistance to etoposide, but only 1.4- to 1.5-fold to cisplatin. We next compared the mRNA levels of four ABC transporters, multidrug resistance 1 (MDR1), MRP1, MRP2 and MRP3 in clinical samples, including 34 patients with gliomas, by quantitative RT-PCR analysis. In some of the clinical samples, increased expression of MRP1 and MRP3 was apparent in malignant gliomas. In situ hybridization revealed that glioma cells were stained with MRP3 probe. MRP3 may modulate drug sensitivity to certain anticancer agents in human gliomas.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Angiogenesis factors.

Angiogenesis is a recent highlight in the medical field; the developmental process and pathological conditions for various diseases can be understood from the novel aspect of "angiogenesis". Many angiogenesis-related factors are involved in the development of vessels during embryogenesis (vasculogenesis), as well as the induction of new vessels in response to physiological or pathological stimuli. In particular, the appearance of hemangioblasts, precursor cells for vascular endothelial cells and blood cells, and blood islands are expected to play a "prelude" role in tubulogenesis. Gene knock out mice of vascular endothelial growth factor (VEGF)/VEGF receptor, ephrin-B2, and angiopoietin-1 results in a failure of normal vessels production. Dormant factors derived from proteolytic cleavage of various physiological substrates are expected to balance a homeostasis of "angiogenic states" in the host. Furthermore, angiogenesis under various pathological conditions of malignant tumors, ocular diseases, psoriasis, rheumatoid arthritis, atherosclerosis and other diseases is associated with complex angiogenesis networks, suggesting pleiotropic mechanisms for angiogenesis.

Angiogenesis Inducing Agents↗
PubMed2001

[Tumor angiogenesis and tumor angiogenesis inhibitors].

It is important to survey the molecular targets which are involved in tumor angiogenesis for the development of antiangiogenic agents as one of the cancer therapy. This article is meant to review the recent molecular targets of tumor angiogenesis and the molecular mechanism of antiangiogenic agents in human clinical trials.

Angiogenesis Inhibitors↗
PubMed2001

High expression of the Cap43 gene in infiltrating macrophages of human renal cell carcinomas.

We used suppression subtractive hybridization to identify highly expressed genes in the cancerous region of human renal cell carcinoma (RCC) compared with noncancerous tissue. Nine genes were identified to show increased expression in the cancerous region compared with the noncancerous region. The nine genes included thymosin beta4, secreted protein acidic and rich in cysteine (SPARC), Cap43, ceruloplasmin, serum amyloid A, osteopontin, heat shock protein 90 (HSP90), LOT1, and casein kinase I. Of these 9 genes, in situ hybridization with 10 clinical samples consistently showed a strong expression of Cap43 mRNA in infiltrating macrophages in RCCs, but not in cancer cells proliferating in an alveolar pattern. However, Cap43 mRNA was also apparently detected in epithelial cells of the renal proximal tubuli in noncancerous tissue. The higher expression of the Cap43 gene in the cancerous region of RCCs appears to depend on macrophage infiltration. Moreover, treatment with phorbol ester resulted in enhanced expression of the Cap43 gene in human monocytic cells in vitro. The expression of the Cap43 gene in infiltrating macrophages is discussed in association with the differentiated or activated status of monocyte/macrophage.

Adult↗
PubMed2001

The accumulation of angiostatin-like fragments in human prostate carcinoma.

PURPOSE: Angiostatin, a potent inhibitor of angiogenesis and, hence, the growth of tumor cell metastasis, is generated by a proteolytic enzyme from plasminogen. However, its localization and specific enzymes have yet to be ascertained in human tissue. EXPERIMENTAL DESIGN: To elucidate the generation and the localization of angiostatin in prostate carcinoma, we examined angiostatin generation in a panel of human prostate cancer cell lines and performed immunohistochemistry with the antibodies to angiostatin and prostate-specific antigen (PSA), a potent proteolytic enzyme of angiostatin in 55 cases of prostate carcinoma. RESULTS: We demonstrated that the lysates of human prostate carcinoma cell lines could generate angiostatin-like fragments from purified human plasminogen but could not generate angiostatin in the absence of exogenous plasminogen. The fragmented proteins were reacted with the monoclonal antibody specific for plasminogen lysine-binding site 1 (LBS-1). Immunohistochemically, the intracytoplasmic immunostaining of LBS-1 was positive in 87.3% (48 of 55) of prostate carcinoma cases, and the immunostaining of miniplasminogen was negative in all cases. There was a significant relationship between the positive immunostaining of LBS-1 and Gleason score (P = 0.0007). The intracytoplasmic immunostaining of PSA was positive in 37.0% (20 of 54) of prostate carcinoma cases, but there was no significant relationship between the expression of PSA and Gleason score, or between the positive immunostaining of LBS-1 and PSA. CONCLUSIONS: These findings suggest that angiostatin is generated by prostate carcinoma cells and is accumulated within the cytoplasm. In addition, the generation of angiostatin-like fragments was correlated with tumor grade; however, PSA may not be the only enzyme for angiostatin generation in human prostate carcinoma.

Aged↗
PubMed2001

Nuclear expression of the Y-box binding protein, YB-1, as a novel marker of disease progression in non-small cell lung cancer.

Transcription factor Y-box binding protein 1 (YB-1) that binds to the inverted CCAAT box is involved not only in transcription of various genes but also in cell proliferation and DNA repair. We determined whether localization of YB-1 in either the nucleus or cytoplasm could serve as a prognostic marker for patients with non-small cell lung cancer (NSCLC). In 196 NSCLC patients, expression of YB-1 protein in the nucleus or cytoplasm was immunohistochemically evaluated. Of the 196 tumors examined, 88 (44.9%) were positive for YB-1 expression in the nucleus. Nuclear YB-1 expression significantly correlated with T factor, lymph node metastasis, and stage of the disease. Patients with a nuclear YB-1 tumor had a poorer prognosis than did those with a cytoplasmic YB-1 tumor in all of the NSCLC patients (P = 0.0494) and in patients with squamous cell carcinoma (P = 0.0313) but not in patients with adenocarcinomas. Nuclear localization of the YB-1 protein may prove to be an important factor of disease progression for patients with NSCLC, in particular, in cases of squamous cell carcinoma.

Adenocarcinoma↗