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Biomedical subjects

F Kanai

Publications and source records attributed to F Kanai.

At least 19 recordsLinked to original sources
PubMed2001

Systematic analysis of the TGF-beta-Smad signaling pathway in gastrointestinal cancer cells.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway has an important role in carcinogenesis. To study the frequency and mechanism of functional impairment of this pathway in human gastrointestinal cancers, we used a reporter assay to examine the response of 38 cell lines (11 colorectal, 9 pancreatic, 10 gastric, and 8 hepatic cancers) to TGF-beta. We then analyzed TGF-beta type II receptor (T beta RII) gene, immunoblots of Smad4, and restoration of the pathway by rescuing T beta R or Smad. We observed impaired signaling in 91% of colorectal, 67% of pancreatic, and 40% of gastric cancer cell lines, but in none of the hepatic cancer cells. We suggest that this pathway does not function as a tumor suppressor in hepatic carcinogenesis. The impairment is due to inactivation of T beta RII and Smad4 in colorectal and pancreatic cancers. However, because the signal was not recovered by rescuing T beta R or Smad genes in TGF-beta-response-defective gastric cancer cell lines, we suggest that novel molecules or mechanisms are involved in the impaired pathway in some gastric cancers.

Blotting, Western↗
PubMed2001

LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest.

Inactivating mutations in the serine-threonine kinase LKB1 (STK11) are found in most patients with Peutz-Jeghers syndrome; however the function of LKB1 is unknown. We found that LKB1 binds to and regulates brahma-related gene 1 (Brg1), an essential component of chromatin remodeling complexes. The association requires the N terminus of LKB1 and the helicase domain of Brg1 and LKB1 stimulates the ATPase activity of Brg1. Brg1 expression in SW13 cells induces the formation of flat cells indicative of cell cycle arrest and senescence. Expression of a kinase-dead mutant of LKB1, SL26, in SW13 cells blocks the formation of Brg1-induced flat cells, indicating that LKB1 is required for Brg1-dependent growth arrest. The inability of mutants of LKB1 to mediate Brg1-dependent growth arrest may explain the manifestations of Peutz-Jeghers syndrome.

AMP-Activated Protein Kinase Kinases↗
PubMed2001

Target gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by E1B55k-attenuated adenovirus.

Gene therapy using replication-competent adenovirus that selectively propagates in tumor cells may be an effective treatment for cancer. We developed an adenovirus that would be replication specific for hepatocellular carcinoma (HCC). Based on our finding that the E1B55k-deficient adenovirus was able to replicate in human primary hepatocytes, we therefore designed an adenovirus carrying E1A and attenuated E1B gene driven by the alpha-fetoprotein promoter (Adv-AFP-E1AdB), thus restricting the replication specificity in AFP-producing HCC. Replication of Adv-AFP-E1AdB in primary hepatocytes was practically negligible 4 days after infection. Although Adv-AFP-E1AdB replicated slowly in AFP-producing HCC, it efficiently destroyed HCC cells independent of their p53 status. Experiments were conducted in vivo using systemic administration of Adv-AFP-E1AdB and we observed tumor size reduction in nude mice having liver cancer. The use of replication-competent adenovirus deficient of the E1B gene coupled to an AFP-targeting strategy may be a safe and efficacious treatment for HCC.

Adenoviridae↗
PubMed2001

The PX domains of p47phox and p40phox bind to lipid products of PI(3)K.

PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.

3T3 Cells↗
PubMed2001

Transcriptional targeted gene therapy for hepatocellular carcinoma by adenovirus vector.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis and is highly amenable to the development of novel therapeutic strategy. The human alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in HCC. In order to destroy AFP-producing HCC specifically, replication defective adenoviral vectors containing the transcriptional control elements of the AFP gene were designed. Expression of suicide genes by the AFP promoter/enhancer induced prodrug sensitivity in AFP (+) cells but not AFP (-) cells. The expression of suicide genes by ubiquitous promoter, however, showed no selectivity after prodrug treatment. Adenoviral vector transduced genes efficiently not only in vitro but also in vivo, and AFP-producing HCC xenografts regressed by transduction with transcriptionally targeted vectors and subsequent systemic administration of prodrug in animal model. Utilization of the transcriptional regulatory element to drive drug sensitive genes can be a promising strategy for cancer specific therapy.

Adenoviruses, Human↗
PubMed2000

TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.

The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.

14-3-3 Proteins↗
PubMed2000

Chemoprotective effects of KF41399, a derivative of carbazole compounds, on nimustine-induced thrombocytopenia.

We examined the chemoprotective effects of KF41399, a novel derivative of carbazole compounds, on severe thrombocytopenia induced by nimustine (ACNU, 45 mg/kg administered for 2 consecutive days intravenously) in mice. Administration schedule studies revealed that pretreatment of mice with KF41399 was necessary to improve thrombocytopenia. Oral administration of KF41399 ameliorated thrombocytopenia induced by ACNU and accelerated the rate of platelet recovery in a dose-dependent fashion. In addition, KF41399 pretreatment improved the decrease in body weight and spleen weight and in the colony-forming activity of bone marrow mononuclear cells (MNC). Oral administration of KF41399 to normal mice induced G(0)/G(1)-phase accumulation of MNC as well as hematopoietic progenitor cells (lineage negative cells [Lin(-)]) and reduced the colony-forming activity of MNC. In Lin(-) cells derived from KF41399-treated mice, up-regulation of Bcl-2 and down-regulation of cyclin E and cyclin A proteins were observed. In the same cells, a decrease in the phosphorylated form of Rb protein and an increase in the p130 protein were observed without changes in the protein level of cell cycle-dependent kinase 2 (Cdk2), Cdk4, and Cdk6. More important, KF41399 did not affect the antitumor activity of ACNU against mouse Sarcoma180 and human lung cancer LC-6. However, 25-mg/kg KF41399 treatment reduced the antitumor activity of ACNU against human lung cancer Lu-65, and 5 mg/kg KF41399 caused a slight reduction of the antitumor activity of ACNU without inducing thrombocytopenia. These results suggest that KF41399 might be useful as a chemoprotective agent to improve chemotherapy-induced thrombocytopenia and types of other toxicity. (Blood. 2000;95:3771-3780)

Adenocarcinoma↗
PubMed1999

Strategy of liver-directed gene therapy: present status and future prospects.

The liver is particularly amenable to gene therapy as it is the site of many metabolic diseases and malignancies. Thus, liver-directed gene therapy is being actively pursued and developed as a method of treatment for various liver diseases. Strategies of liver-directed gene therapy include drug delivery to the liver, compensation of the defective gene(s), anti-tumor activity, anti-viral therapy, and immunomodulation. The strategy chosen for liver-directed gene therapy depends on the genetic basis of the disease. Many aspects are key factors to the success of the chosen strategy: intervention of genes, efficient gene delivery system, stable transgene expression, transgene regulation, target cell transfection, and timing of transgene expression. Several tactics can be used to overcome problems in the above, and these include the use of a gene switch to exogenously regulate transgene expression, targeting at the transcriptional level, circumvention of the immune response (as in the use of adenovirus vector to achieve long-term correction of genetic diseases), and genetically engineered antibodies in gene transfer. At the present rate of research activity and development, gene therapies may soon be more efficient than current standard treatments for some liver diseases.

Animals↗
PubMed1999

Structure of cag pathogenicity island in Japanese Helicobacter pylori isolates.

BACKGROUND: cag pathogenicity island (PAI) is reported to be a major virulence factor of Helicobacter pylori. AIM: To characterise cagA and the cag PAI in Japanese H pylori strains. METHODS: H pylori isolates from Japanese patients were evaluated for CagA by immunoblot, for cagA transcription by northern blot, and for cagA and 13 other cag PAI genes by Southern blot. cagA negative strains from Western countries were also studied. Induction of interleukin-8 secretion from gastric epithelial cells was also investigated. RESULTS: All Japanese strains retained cagA. Fifty nine of 63 (94%) strains had all the cag PAI genes. In the remaining four, cag PAI was partially deleted, lacking cagA transcripts and not producing CagA protein. Details of the PAI of these strains were checked; three lacked cagB to cagQ (cagI) and continuously cagS to cag13 (cagII), and the remaining one lacked cagB to cag8. Western cagA negative strains completely lacked cag PAI including cagA. Nucleotide sequence analysis in one strain in which the cag PAI was partially deleted showed that the partial deletion contained 25 kb of cag PAI and the cagA promoter. Interleukin-8 induction was lower with the cag PAI partial deletion strains than with the intact ones. All Japanese cag PAI deleted strains were derived from patients with non-ulcer dyspepsia, whereas 41 of 59 (70%) CagA-producing strains were from patients with peptic ulcers or gastric cancer (p<0.05). CONCLUSIONS: Most Japanese H pylori strains had the intact cag PAI. However, some lacked most of the cag PAI in spite of the presence of cagA. Thus the presence of the cagA gene is not an invariable marker of cag PAI related virulence in Japanese strains.

Adult↗
PubMed1999

Adenovirus mediated p53 tumour suppressor gene therapy for human gastric cancer cells in vitro and in vivo.

BACKGROUND/AIMS: Gastric cancer is one of the most prevalent forms of cancer in East Asia. Point mutation of the p53 gene has been reported in more than 60% of cases of gastric cancer and can lead to genetic instability and uncontrolled cell proliferation. The purpose of this investigation was to evaluate the potential of p53 gene therapy for gastric cancer. METHODS: The responses of human gastric cancer cell lines, MKN1, MKN7, MKN28, MKN45, and TMK-1, to recombinant adenoviruses encoding wild type p53 (AdCAp53) were analysed in vitro. The efficacy of the AdCAp53 treatment for MKN1 and MKN45 subcutaneous tumours in nude mice was assessed in vivo. RESULTS: p53-specific growth inhibition was observed in vitro in two of four gastric cancer cell lines with mutated p53, but not in the wild type p53 cell line. The mechanism of the killing of gastric cancer cells by AdCAp53 was found, by flow cytometric analysis and detection of DNA fragmentation, to be apoptosis. In vivo studies showed that the growth of subcutaneous tumours of p53 mutant MKN1 cells was significantly inhibited by direct injection of AdCAp53, but no significant growth inhibition was detected in the growth of p53 wild type MKN45 tumours. CONCLUSIONS: Adenovirus mediated reintroduction of wild type p53 is a potential clinical utility in gene therapy for gastric cancers.

Adenoviridae↗
PubMed1998

Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5-fluorouracil.

5-fluorouracil (5-FU), although a widely used chemotherapeutic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate. The present study demonstrates that adenovirus-mediated transduction of E. coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro. The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus. In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects. The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.

Adenoviridae↗
PubMed1998

Prostaglandin D2 inhibits inducible nitric oxide synthase expression in rat vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) as well as macrophages have been shown to generate a substantial amount of NO in inflammatory vascular lesions. Prostaglandin (PG) D2 (PGD2) is produced by inflammatory cells, including mast cells and macrophages. We investigated whether PGD2 modulates NO metabolism in rat VSMCs. PGD2 at a concentration of 10(-7) mol/L or greater dose-dependently inhibited nitrite accumulation in the medium of cultured VSMCs stimulated with interleukin 1beta (IL-1beta). In a dose-response analysis of IL-1beta and nitrite accumulation, PGD2 was seen to decrease the maximal ability of VSMCs to generate NO, arguing against competition by PGD2 at cytokine receptors. Northern analysis showed that PGD2 suppresses induction of inducible NO synthase (iNOS) mRNA in IL-1beta-stimulated VSMCs, with consequent inhibition of iNOS protein expression in Western analysis. A thromboxane A2 (TXA2) analogue, U46619 (10(-5) mol/L), produced less inhibition of NO generation than did PGD2. Neither the PGI2 analog carbaprostacyclin nor PGE1 showed any inhibition. PGD2 dose-dependently inhibited NO generation despite the addition of the TXA2 antagonist SQ29548. PGJ2, delta12-PGJ2, and 15-deoxy-delta12,14-PGJ2, all metabolites of PGD2, were as potent as or slightly stronger than PGD2 in the inhibition of NO generation. These data suggest that PGD2 suppresses NO generation in VSMCs by inhibiting iNOS mRNA expression, most likely through the cascade of the PGJ2 series rather than through the TX receptor or cAMP upregulation. Such action makes it likely that PGD2 regulates NO metabolism in vascular lesions.

Animals↗
PubMed1998

pp60c-src activation in hepatocellular carcinoma of humans and LEC rats.

For the related Src kinases, a close correlation exists between elevated tyrosine kinase activity and cell transformation. However, the involvement of pp60c-src in hepatocellular carcinoma (HCC) remains obscure. The aim of this study was to evaluate whether pp60c-src tyrosine kinase activity is elevated in HCC. We analyzed the kinase activity of pp60c-src in normal liver tissue, chronic hepatitis liver tissue, and tumorous and adjacent nontumorous portions of HCC tissue from patients and Long-Evans cinnamon (LEC) rats that are known to develop liver cancer spontaneously. The kinase activity of pp60c-src was rarely detected in the normal human liver tissue and chronic hepatitis liver tissue, but it was elevated in tumorous and nontumorous portions of HCC tissue. Furthermore, the kinase activity of pp60c-src was significantly elevated in tumorous tissues compared with nontumorous tissues. The kinase activity of pp60c-src was also higher in poorly differentiated HCC. In addition, the kinase activity of pp60c-src increased proportionately with the development of HCC of LEC rats. Our results suggest that activation of the protooncogene product pp60c-src may play an important role in the malignant transformation of hepatocytes in human and LEC rats, and that it may be closely related to the histopathological grading of human HCC.

Animals↗
PubMed1998

Gene therapy for hepatic micrometastasis of murine colon carcinoma.

BACKGROUNDS/AIMS: Pit cells are located in the hepatic sinusoids and are organ-associated natural killer cells that contribute to immune surveillance in the liver. In the present study, the interleukin-2 gene was introduced into hepatocytes using an adenovirus vector to induce interleukin-2 production in an attempt to enhance the natural killer activity of pit cells, leading to inhibition of metastasis of colon carcinoma. METHODS: The recombinant adenovirus vector "Adex1CAmIL2" was constructed by inserting an expression unit which was composed of the CAG promotor (cytomegalovirus enhancer plus chicken beta-actin promotor), murine interleukin-2 cDNA, and a rabbit beta-globin polyadenylation signal. After administration of Adex1CAmIL2 to mice (4x10(7) pfu per animal), the expression of murine interleukin-2 in hepatocytes was examined by immunostaining and in situ hybridization, and the natural killer activity of hepatic mononuclear cells was measured. Inhibition of hepatic metastasis of colon carcinoma was examined after infusion of colon 38 tumor cells into the superior mesenteric vein. RESULTS: After administration of Adex1CAmIL2, interleukin-2 mRNA expression was demonstrated in hepatocytes until day 7, and the serum interleukin-2 level was increased. The natural killer activity of hepatic mononuclear cells was markedly enhanced for 7-10 days. Hepatic metastasis was inhibited by administration of Adex1CAmIL2 until day 7 after tumor cell inoculation. CONCLUSION: These results suggest that gene therapy using Adex1CAmIL2 could be potentially useful for inhibiting hepatic micrometastasis by enhancing the natural killer activity of pit cells.

Actins↗
PubMed1998

Interleukin-8 production in primary cultures of human gastric epithelial cells induced by Helicobacter pylori.

Interleukin-8 (IL-8) production by the gastric mucosa is increased in Helicobacter pylori infection. Previous studies indicated that H. pylori induces IL-8 synthesis in cancer cell lines, and the ability of H. pylori to stimulate IL-8 production is supposed to be associated with cagA and other cag pathogenicity island genes, including picB gene. In the present study, we investigated the induction of IL-8 in primary cultures of normal human gastric epithelial cells to elucidate the IL-8 induction by wild type strains and by the picB knockout strain. Human gastric epithelial cells were obtained from surgically resected specimens from four patients. Three H. pylori strains (TN2F4; type 1 clinical isolate, TN2F4m1; isogenic picB mutant of TN2F4, Tx30a; type 2 strain) were cocultured with the normal gastric epithelial cells or the transformed MKN-28. IL-8 levels in culture medium were determined by enzyme immunoassay. Human gastric epithelial cells produced IL-8 at a 10-50 times higher level than MKN-28 did when cocultured with TN2F4. The mutant TN2F4m1 induced IL-8 at significantly lower levels than the parent strain. Cells from four patients behaved similarly on IL-8 production. The results of the present study demonstrated the induction of IL-8 in normal gastric epithelial cells, suggesting that picB gene product may play an essential role in vivo.

Bacterial Proteins↗
PubMed1998

In vivo adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing pancreatic cancer.

In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)-ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus-carrying HSVtk gene under the control of a carcinoembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA-producing Su.86.86. BxPC-3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA-nonproducing HeLa, were used for analysis of GCV sensitivity induced by adenoviral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA-positive pancreatic cancer in vivo, a subcutaneously implanted tumor-bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA-production. In contrast, when the cells were transduced with a CEA promoter, the cell-killing effect of GCV was increased in only CEA-producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus-mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA-producing pancreatic cancer cells in vitro and in vivo. This strategy may provide a useful tool for treating pancreatic cancer, especially CEA-producing tumor cells.

Adenoviridae↗