Biomedical subjects

A Hakura

Publications and source records attributed to A Hakura.

At least 37 records · Page 2Linked to original sources

PubMed1998

Suppression of v-src transformation by the drs gene.

Previously, we isolated a novel gene, drs, which was downregulated by retroviral oncogenes such as v-src and v-K-ras, from a cDNA library of primary rat embryo fibroblasts. Experiments using a temperature-sensitive mutant of the v-src gene indicated that downregulation of drs mRNA was dependent on functional expression of v-Src. In addition, expression of drs mRNA was also reduced by serum stimulation of G0-arrested normal rat fibroblast cells. To clarify the function of the drs gene in cell transformation and proliferation, we introduced drs linked to a potent promoter into a normal rat cell line, F2408, and examined the effect of ectopic expression of exogenous drs on the transformation by the v-src gene and growth properties. Cells expressing exogenous drs gene showed significantly decreased efficiency of transformation by v-src irrespective of functional expression of v-Src kinase, while the growth rate and G1/S progression of the cells were not suppressed by expression of exogenous drs gene, indicating that drs has the ability to suppress v-src transformation without disturbing cell proliferation.

Animals↗

PubMed1998

Dispensability of p53 degradation for tumorigenicity and decreased serum requirement of human papillomavirus type 16 E6.

Certain types of human papillomavirus (HPV), such as types 16 and 18, are etiological agents for carcinogenesis of the uterine cervix. These HPVs have two oncogenes, E6 and E7, that have transforming activities in established murine cells. Tumorigenicity and decreased serum requirement for cell growth are conferred by the E6 gene, whereas anchorage-independent growth is mainly governed by the E7 gene. To understand the mechanism of cellular transformation by the HPV16 E6 gene, we examined three mutant E6 proteins defective for p53 binding, p53 degradation, or transactivation of the adenovirus E2 promoter for the ability to induce tumorigenicity and decreased serum requirement. The results showed that tumorigenicity and decreased serum requirement were associated with the ability of E6 to bind to p53, although the subsequent degradation of p53 was not required for these functions.

Animals↗

PubMed1997

Suppression of tumor growth by the 3' untranslated region of mel-18 in 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18.

By introducing a cDNA library derived from rat embryonic fibroblast cells, we isolated several morphologically flat revertants of rat 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18 (HPV18). From one of the revertants, we recovered a 0.2-kb cDNA, N56, that suppresses the tumor growth of the transformed 3Y1 cells irrespective of the expression of the E6 and E7 genes. The nucleotide sequence of the cDNA was shown to be identical to that of the 3' untranslated region of a putative mammalian polycomb group gene, mel-18.

Animals↗

PubMed1997

Detection of Epstein-Barr virus transcripts in anaplastic large-cell lymphomas by mRNA in situ hybridization.

Anaplastic large-cell lymphoma (ALCL) is a recently proposed subset of non-Hodgkin's lymphoma. To determine whether Epstein-Barr virus (EBV) is associated with this lymphoma, we performed mRNA in situ hybridization on seven cases of ALCL using a probe consisting of an RNA sequence complementary to the transcripts of BamHIW fragment of the EBV genome. We detected BamHIW transcripts of EBV in the majority of atypical large cells of all cases of ALCL, but in none of three cases of lymphoblastic and small lymphocytic lymphomas. Furthermore, we detected latent membrane protein-1 (LMP1) in two cases of ALCL by means of immunofluorescence and immunoperoxidase stainings. These findings suggest that EBV is involved in the neoplastic transformation for ALCL as in the case of Hodgkin's disease, which shares several clinicopathologic features with ALCL.

Adolescent↗

PubMed1997

Human T-cell leukemia virus type 1 Tax protein transforms rat fibroblasts via two distinct pathways.

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates the transcription of several cellular genes. This function is thought to play a critical role in the Tax-dependent transformation step in HTLV-1 leukemogenesis. Tax activates transcription via three enhancers: the cyclic AMP response element (CRE)-like sequence, the kappaB element, and the CArG box. Their involvement in the transformation of rat fibroblasts by Tax was examined by colony formation of Rat-1 cells in soft agar and Ras cooperative focus formation of rat embryo fibroblasts (REF). Among Tax mutants, those retaining activity for the CArG box transformed REF like wild-type Tax, while those inactive for the CArG box did not. Thus, the activation of the CArG box pathway is essential for the transformation of REF by Tax. In contrast, activation of the kappaB element correlated with the transformation of Rat-1 by Tax. These results show that Tax transforms rat fibroblasts via two distinct pathways.

Animals↗

PubMed1997

Cytotoxicity of naphthoquinones toward cultured resting murine leukemia L1210 cells in the presence of glutathione, diethyl maleate, or iodoacetamide.

Sixteen naphthoquinones with various substituents were tested for cytotoxicity toward cultured resting leukemia L1210 cells. The cytotoxicity of all the naphthoquinones examined was not affected at all by the treatment in combination with diethyl maleate, a glutathione-specific SH-blocking agent in the cells. Depending on the characteristics of the response to the treatment in combination with iodoacetamide (IAA) and glutathione (GSH), these naphthoquinones were tentatively classified into three groups. The chemicals of Group I, which include naphthoquinones carrying an electron-donating group(s) other than an OH group on the quinone ring moiety, showed synergistic cytotoxicity with IAA and no reduction in cytotoxicity with GSH. Those of Group II, which include naphthoquinones without an electron-donating group on the quinone ring moiety, showed no synergy with IAA but appreciable reductions in cytotoxicity with GSH. Group III includes 2-hydroxylated naphthoquinones, which showed neither synergy with IAA nor cytotoxicity reduction with GSH. Cytotoxicity was discussed in terms of the electron-deficiency of the quinone ring moiety. It is suggested that the cytotoxicity of Group I quinones comes from the active oxygen generation which follows semiquinone formation, whereas that of the other groups of quinones is not likely caused simply by either SH-blocking of biomolecules or active oxygen-related mechanism.

Alkylating Agents↗

PubMed1997

Dual stimulatory and inhibitory effects of fluorine-substitution on mutagenicity: an extension of the enamine epoxide theory for activation of the quinoline nucleus.

Nineteen mono- and di-fluorinated derivatives of quinoline, 1,7-phenanthroline, 1,10-phenanthroline, benzo[h]quinoline, and benzo[f]quinoline were subjected to analysis of their structure-mutagenicity relationships. For this purpose, six new fluorinated derivatives were synthesized. The results support that the enamine epoxide structure of the pyridine moiety, as well as the bay-region epoxide structure, is responsible for mutagenicity. Formation of K-region epoxides might involve a detoxification process rather than mutagenic activation.

Benzo(a)pyrene↗

PubMed1996

Mutagenicity of dihydroxybenzenes and dihydroxynaphthalenes for Ames Salmonella tester strains.

The mutagenicity of 3 dihydroxybenzene (DHB) and 9 dihydroxynaphthalene (DHN) isomers was examined by using 5 different Ames Salmonella mutagenicity tester strains in the presence and absence of phenobarbital and 5,6-benzoflavone-treated rat liver S9-mix. Of the 3 DHB isomers, 1,4-DHB (hydroquinone) was mutagenic, and of the 9 DHN isomers, 1,3-DHN (naphthoresorcinol), 1,4-DHN (hydronaphthoquinone), 1,6-DHN and 1,7-DHN were mutagenic. Mutagenicity of all the compounds tested was observed in the absence of S9-mix, while 1,4-DHN and 1,6-DHN were also mutagenic in the presence of S9-mix. The mutagenicity of 1,4-DHB and 1,4-DHN for TA104, which is a strain sensitive to oxidative mutagens, was almost completely or partially inhibited by superoxide dismutase (SOD) and/or catalase, indicating the involvement of activated oxygen species in mutagenesis. Furthermore, from the finding that the 4 DHNs were mutagenic for TA2637, the strain sensitive to frameshift mutagens, it is possible that the mutagenicity of DHNs for S. typhimurium was also attributable to DNA adducts that form with quinones and/or semiquinones through oxidation of DHNs. The mutagenicity of 1,3-DHN, which showed the largest number of revertants in strains TA100, TA98, TA2637 and TA104, was greatly decreased, when their pKM101 plasmid-deficient strains, TA1535, TA1538, TA1537 and TA2659 were used. This observation suggests that an SOS repair system was involved in the mutagenesis of 1,3-DHN for S. typhimurium.

Animals↗

PubMed1996

Increased sensitivity of EBNA2-transformed rat fibroblasts to ionizing radiation.

To clarify whether expression of Epstein-Barr virus(EBV) nuclear antigen 2 (EBNA2) correlates with sensitivity to ionizing radiation, we tested EBNA2-transformed rat fibroblast clones for their radiosensitivity to X-rays. These transformed clones reproducibly generated tumors in mice. X-irradiation suppressed the growth of the tumors, and irradiated mice survived longer than non-irradiated ones. In contrast, tumors formed by activated H-ras or E6-E7 genes of human papillomavirus type 16 (HPV16) were strongly resistant to the same dose of X-irradiation. In in vitro culture, these EBNA2-expressing clones also showed higher radiosensitivity than cell lines transformed by activated H-ras and E6E7 genes. The averaged D(o) of EBNA2-expressing clones was 2.3 times lower than that of non-expressing and control clones. These results suggest that expression of EBNA2 is responsible for the radiosensitivity.

Animals↗

PubMed1996

Adhesion-dependency of serum-induced p42/p44 MAP kinase activation is released by retroviral oncogenes.

G1/S transition of the cell cycle is blocked when cells are cultured without cell adhesion, even if nutrients and serum are present. Shift experiments involving transfer from adhesion to suspension cultures have revealed that cell adhesion in the early half of G1 phase is required for induction of DNA synthesis. We found that activation of p42/p44 mitogen-activated protein (MAP) kinases, one of the earliest responses induced by serum stimulation, was also dependent on cell adhesion in rat F2408 and mouse Swiss3T3 fibroblast cell lines, suggesting that MAP kinase activation is a critical step in adhesion-dependent G1 progression. F2408 cell lines transformed by the v-src, v-K-ras, and v-mos oncogenes showed constitutively high MAP kinase activity, even in the absence of serum and cell adhesion, while both serum stimulation and cell adhesion were necessary to induce intensive activation of MAP kinase in a F2408 cell line transformed by the E6 and E7 genes of human papillomavirus type 16, as well as in untransformed F2408.

3T3 Cells↗

PubMed1996

Abrogation of c-kit/Steel factor-dependent tumorigenesis by kinase defective mutants of the c-kit receptor: c-kit kinase defective mutants as candidate tools for cancer gene therapy.

The growth and survival of many types of cancer cells are known to be supported by specific growth factor/cytokine systems. Among these, the activation of c-kit receptor and its ligand steel factor participates in several types of human carcinogenesis. W mutations of laboratory mouse strains are loss of functional mutations of the c-kit receptor. To examine the validity of these mutants in investigating c-kit-mediated carcinogenesis and in the treatment of c-kit-dependent tumors, we introduced various W mutations (W, Wv, and W42) into a transgenic mouse strain carrying human papillomavirus oncogenes, in which c-kit/Steel-mediated tumorigenesis occurs with a very high incidence. In all transgenic strains carrying a W mutation, the c-kit deficiency affected the tumorgenic process to various degrees. Tumor development was markedly suppressed in transgenic strains carrying kinase defective mutations (Wv and W42) in a heterozygous condition. In null-type (W) heterozygous transgenic mice, tumorigenesis was suppressed at a lower level. Moreover, minimal focal legions or, in some cases, no focal legions were found in the testes of W/Wv heterozygous transgenic mice, showing a close relationship between tumor cell growth and the degree of c-kit inactivation. These results indicated that c-kit activity is a pivotal determinant of testicular tumor development and that the kinase defective mutants of c-kit are valuable for treating c-kit-dependent cancer, as well as for clarifying the c-kit-mediated carcinogenesis.

Animals↗

PubMed1996

Isolation of a novel gene down-regulated by v-src.

We have isolated a novel gene which was expressed in normal rat cells, but completely suppressed in cells transformed by v-src. The molecularly cloned cDNA was about 1.8 kb in size, containing an open reading frame composed of 464 amino acid residues. DNA sequence analysis showed that there was no corresponding gene in the data bases. Besides the suppression of gene expression in the v-src transformed cells, its expression was also strongly suppressed in cells transformed by other oncogenes such as v-abl, v-fps, v-mos, v-sis, v-K-ras, and polyomavirus middle T, but not affected in cells transformed by human papillomavirus type 16 E6E7 and polyomavirus large T. We named the gene drs for a gene down-regulated by v-src.

Amino Acid Sequence↗

PubMed1995

Cloning of a new cytokine that induces IFN-gamma production by T cells.

The mechanism underlying the differentiation of CD4+ T cells into functionally distinct subsets (Th1 and Th2) is incompletely understood, and hitherto unidentified cytokines may be required for the functional maturation of these cells. Here we report the cloning of a recently identified IFN-gamma-inducing factor (IGIF) that augments natural killer (NK) activity in spleen cells. The gene encodes a precursor protein of 192 amino acids and a mature protein of 157 amino acids, which have no obvious similarities to any peptide in the databases. Messenger RNAs for IGIF and interleukin-12 (IL-12) are readily detected in Kupffer cells and activated macrophages. Recombinant IGIF induces IFN-gamma more potently than does IL-12, apparently through a separate pathway. Administration of anti-IGIF antibodies prevents liver damage in mice inoculated with Propionibacterium acnes and challenged with lipopolysaccharide, which induces toxic shock. IGIF may be involved in the development of Th1 cells and also in mechanisms of tissue injury in inflammatory reactions.

Amino Acid Sequence↗

PubMed1995

Tumorigenicity of EBNA2-transfected cells.

The Epstein-Barr virus nuclear antigen 2 (EBNA2) gene is thought to be important for transformation by Epstein-Barr virus (EBV), but the mechanism of this transformation is little understood. Here, to examine the transforming ability of EBNA2, we transfected a rat fibroblast cell line F2408 with a recombinant EBNA2 expression plasmid and examined cell morphology, colony formation in soft agar, and tumorigenicity in nude mice. The morphology of transfected clones was similar to those of untransfected cells, but two of seven clones grew in soft agar, and four clones of seven clones reproducibly formed tumors in nude mice. These four clones showed EBNA2 expression, but non-tumorigenic clones did not. These results indicate that the expression of EBNA2 is correlated with tumorigenicity.

Animals↗

PubMed1995

An in vivo model for receptor tyrosine kinase autocrine/paracrine activation: auto-stimulated KIT receptor acts as a tumor promoting factor in papillomavirus-induced tumorigenesis.

Constitutive overactivation of growth factor receptors through autocrine/paracrine mechanisms occurs frequently in cancer cells and are thought to play a critical role in carcinogenesis. In the present report, we propose a refined in vivo model which explains the significance of these mechanisms in tumour development. We have previously established transgenic mouse lines containing human papillomavirus type 16 (HPV16) E6E7 oncogenes, in male mice of which a Leydig cell tumor developes with a very high incidence. Not only HPV transgene but also the c-kit proto-oncogene receptor tyrosine kinase and its ligand Steel Factor (SLF) were coexpressed in all tumors analysed. This coexpression of c-kit/SLF was also found in two other Leydig cell tumor lines. Moreover, the proliferation of transgenic tumor cells was attenuated by treatment with a c-kit neutralizing antibody in vitro, strongly suggesting that tumorigenesis is closely related to stimulation of receptors through ligand induction. To confirm the significance of these findings, a defective mutation of the SLF gene in a laboratory mouse, the Steel-Dickey (Sld) mutation, was introduced into a line of transgenic mice showing 100% incidence of the tumor. In Sld-E6E7 transgenic mice, tumorigenesis was initiated but numbers of tumor cells were markedly reduced compared with transgenic mice carrying both wild type SLF allele, showing that c-kit activation through the induction of SLF is essential for testicular tumorigenesis, especially in tumour promotion. This transgenic mice system should be a useful in vivo model for clarifying the implication of growth factor autostimulation in carcinogenesis.

Animals↗

PubMed1995

Mutagenicity of benzoquinones for Ames Salmonella tester strains.

The mutagenicity of 12 simple benzoquinone (BQ) derivatives was studied using five different Ames Salmonella mutagenicity tester strains in the presence and absence of S9 mix. Seven of the BQs used displayed mutagenicity with and/or without S9 mix, and most of them produced a marginal increase in revertants. p-Benzoquinone (p-BQ) showed the most potent mutagenic activity (17 induced revertants/nmol/plate for strain TA104 without S9 mix) among the BQs tested. TA104, which is sensitive to oxidative mutagens, was the most sensitive to the mutagenicity of the BQs of the five strains used, while the second most sensitive strain was TA2637, which detects bulky DNA adducts. Significant reductions in the mutagenicity of p-BQ, and 2,3-diCl-5,6-diCN-BQ without S9 mix were observed in the presence of catalase. These findings suggest that the mutagenicity of BQs for S. typhimurium is attributable to oxidative injury after BQ reduction and to DNA adducts that form with BQs that have electrophilic substituents.

Benzoquinones↗

PubMed1995

Combination effects of clastogens in the mouse peripheral blood micronucleus assay.

In order to study how two chemicals interact to induce micronuclei, simple ethylating agents [ethyl methanesulfonate (EMS), ethyl ethanesulfonate (EES) and N-ethyl-N-nitrosourea (ENU)], spindle poisons [vincristine sulfate (VINC) and colchicine (COL)] and an oxidizing agent [potassium bromate (KBrO3)] were used as model chemicals for combination treatments. The frequency of micronucleated reticulocytes (MNRETs) was evaluated in mice treated with two of these chemicals at a time. The combinations of ethylating agents (EMS and EES; EMS and ENU) and of spindle poisons (VINC and COL) induced more micronuclei than those expected on an additive basis. The apparent synergism was due to a 'combined dose' which could be calculated by the dosimetric conversion of one chemical to the other, when damage induced by each chemical was 'equivalent' in the induction of MNRETs. In contrast, no apparent synergism in induction of micronuclei was observed when two chemicals with different modes of clastogenic action (EMS and KBrO3 or EMS and VINC) were combined.

Alkylating Agents↗

PubMed1995

Transgenic models for papillomavirus-associated multistep carcinogenesis.

To investigate the physiological and pathological in vivo functions of molecularly cloned genes, the transgenic mouse is one of the most useful experimental animal systems. Many kinds of transgenic mice carrying papillomavirus genes have been produced, and the studies have revealed several new aspects in the field of papillomaviral oncology. Among these transgenic mice, the mechanism of skin carcinogenis in the bovine papillomavirus (BPV) transgenic mouse has been well characterized, demonstrating that numbers of genetic alterations in specific cellular genes were deeply involved in tumor progression as well as in the expression of transforming genes encoded by the viral genome. Comparable mechanisms were found in testicular tumorigenesis in the HPV 16 E6E7 transgenic mouse. Here we discuss the mechanism of testicular tumorigenesis in the HPV 16 E6E7 transgenic mouse together with that of skin carcinogenesis in the BPV transgenic mouse in order to clarify some part of papillomavirus-associated carcinogenesis.

Animals↗