Search PubMed⌕ Search

Biomedical subjects

S Nishimura

Publications and source records attributed to S Nishimura.

At least 307 records · Page 17Linked to original sources
PubMed1995

Trichostatin A inhibits both ras-induced neurite outgrowth of PC12 cells and morphological transformation of NIH3T3 cells.

During screening for inhibitors of ras-mediated differentiation of PC12 cells, trichostatin A (TSA) was isolated from the metabolites of Streptomyces as a potent inhibitor. TSA blocked both oncogenic ras- and NGF-induced neurite outgrowth from PC12 cells. However, addition of TSA 1 h after NGF-stimulation did not inhibit neuronal differentiation, suggesting that TSA affects an early step in the NGF-signaling pathway mediated by ras. Northern blotting analysis showed that TSA prolonged the maximum expression period of c=fos mRNA triggered by NGF and delayed its return to the basal level. TSA reduced c-jun mRNA induction by NGF but greatly enhanced c-myc mRNA induced by NGF. Yoshida et al. (J. Biol. Chem, 265, 17174-17179, 1990) showed that TSA inhibits histone deacetylation, which might influence the gene expression involved in cellular differentiation. In this study, we also found that TSA prevents histone deacetylation in PC12 cells as well as other cell lines, suggesting that inhibition of histone deacetylation by TSA might affect the expression of early-response genes. We also demonstrated that TSA induced reversion of oncogenic ras-transformed NIH3T3 cells to a normal morphology, suggesting that inhibitors of ras-mediated differentiation of PC12 cells may be effective as anticancer agents.

3T3 Cells↗
PubMed1995

Novel antitumor indolocarbazole compound 6-N-formylamino-12,13-dihydro-1,11- dihydroxy-13-(beta-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4- c]carbazole-5,7(6H)-dione (NB-506): induction of topoisomerase I-mediated DNA cleavage and mechanisms of cell line-selective cytotoxicity.

A new indolocarbazole antitumor agent, NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D-glucopyranosyl) -5H- indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione], enhanced the DNA cleavage catalyzed by HeLa S3 topoisomerase I at 0.01 microM but not the cleavage by topoisomerase II at 300 microM. It also caused single-strand DNA breakage in intact cells at 0.08 microM and more. Unlike the known topoisomerase I inhibitor camptothecin, NB-506 intercalated with DNA. However, the binding affinity to DNA and the inhibition against DNA polymerase alpha and RNA polymerase II were marginal compared with those of Adriamycin or actinomycin D. NB-506 inhibited the growth of various tumor cell lines at two micromoles or less, and its cytotoxicity was found to be cell line selective. This selective cytotoxicity of NB-506 was not fully explained by the differences in topoisomerase I activity in these cell lines, but there was some relationship between the amount of NB-506 accumulated in these cell lines and its cytotoxicity toward them. In conclusion, NB-506 is a potent topoisomerase I poison, acting selectively on tumor cell lines accumulating NB-506.

Animals↗
PubMed1995

Novel indolocarbazole compound 6-N-formylamino-12,13-dihydro-1,11-dihydroxy- 13-(beta-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo-[3,4-c]carbazole- 5,7(6H)-dione (NB-506): its potent antitumor activities in mice.

NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D- glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-di one d is a new antitumor indolocarbazole compound. The growths of murine M5076 and Ehrlich solid tumors were inhibited 76 and 96%, respectively, by i.v. injection at doses of 300 mg/m2. Furthermore, NB-506 caused regression of nodules of human PC-13 lung cancer and MKN-45 stomach cancer cells at i.v. doses of 90 mg/m2. Human HCT 116 and LS 180 colon cancers also regressed with injections of NB-506. Repeated injections of NB-506 had a stronger antitumor effect than intermittent injections in mice with MKN-45. The cumulative toxicity of NB-506 was low in terms of lethality in mice, i.e., the LD50s on single and 10 repeated i.v. injections into CDF1 mice were 990 and 810 mg/m2/injection, respectively. In conclusion, NB-506 is considered to be an interesting possible candidate as an anticancer drug for treatment of solid tumors in humans.

Animals↗
PubMed1995

A constitutive effector region on the C-terminal side of switch I of the Ras protein.

The "switch I" region (Asp30-Asp38) of the Ras protein takes remarkably different conformations between the GDP- and GTP-bound forms and coincides with the so-called "effector region." As for a region on the C-terminal side of switch I, the V45E and G48C mutants of Ras failed to promote neurite outgrowth of PC12 cells (Fujita-Yoshigaki, J., Shirouzu, M., Koide, H., Nishimura, S., and Yokoyama, S. (1991) FEBS Lett. 294, 187-190). In the present study, we performed alanine-scanning mutagenesis within the region Lys42-Ile55 of Ras and found that the K42A, I46A, G48A, E49A, and L53A mutations significantly reduced the neurite-inducing activity. This is an effector region by definition, but its conformation is known to be unaffected by GDP-->GTP exchange. So, this region is referred to as a "constitutive" effector (Ec) region, distinguished from switch I, a "switch" effector (Es) region. The Ec region mutants exhibiting no neurite-inducing activity were found to be correlatably unable to activate mitogen-activated protein (MAP) kinase in PC12 cells. Therefore, the Ec region is essential for the MAP kinase activation in PC12 cells, whereas mutations in this region only negligibly affect the binding of Ras to Raf-1 (Shirouzu, M., Koide, H., Fujita-Yoshigaki, J., Oshio, H., Toyama, Y., Yamasaki, K., Fuhrman, S. A., Villafranca, E., Kaziro, Y., and Yokoyama, S. (1994) Oncogene 9, 2153-2157).

Animals↗
PubMed1995

Phosphorylation of E2F-1 by cyclin A-cdk2.

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.

Baculoviridae↗
PubMed1995

Lack of the expression of EBNA-2 and LMP-1 in T-cell neoplasms possessing Epstein-Barr virus.

We investigated 34 cases of T-cell neoplasm [15 cases of T-cell granular lymphocytic leukemia (T-GLL), 10 cases of T-cell non-Hodgkin's lymphoma (T-NHL), six cases of T-cell chronic lymphocytic leukemia (T-CLL), and three cases of cutaneous T-cell lymphoma] to study their association with Epstein-Barr virus (EBV). In 4 (three T-NHL and one T-GLL) of 34 cases, EBV genome was detected in a single episomal form, while polyclonal EBV-DNA was detected in one (T-NHL) of the remaining cases. All three cases of T-NHL having monoclonal EBV episome showed histologically diffuse large-cell lymphoma and developed leukemic conversion. Phenotypic analysis showed that two of these four cases were CD4+, CD8-, and the remaining two cases were CD4-, CD8+. The cells from all four cases were confirmed to be in T-cell lineage by detecting the rearrangement of T-cell receptor (TCR) beta or gamma chain gene. By reverse transcription-polymerase chain reaction (RT-PCR), EBNA-1 was detected at low levels, and neither EBNA-2 nor LMP-1 were found in any of the three cases examined. Lack of the expression of EBNA-2 and LMP-1 was also confirmed by immunocytochemical staining. The cells of these four cases did not show rearrangement or overexpression of c-myc and bcl-2 genes by Southern and Northern blots, and the mutation of p53 gene was detected in only one patient. These results suggest that other latent gene products of EBV or other cellular oncogenes are involved in the development of Japanese T-cell neoplasm after EBV infection.

Antigens, Viral↗
PubMed1995

Development of a sandwich enzyme-linked immunosorbent assay for the determination of human heart type fatty acid-binding protein in plasma and urine by using two different monoclonal antibodies specific for human heart fatty acid-binding protein.

We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human heart type fatty acid-binding protein (H-FABP) in human plasma and urine using the combination of two distinct monoclonal antibodies (MAbs) directed against human H-FABP purified from human heart muscle. The total assay time of the ELISA is practically much shorter than that of the competitive enzyme immunoassay (EIA) we previously reported. The immunoreactive mass of human H-FABP was specifically measured using a horseradish peroxidase (HRPO)-labeled anti-human H-FABP MAb as an enzyme-linked MAb, and anti-human H-FABP MAb immobilized on the polystyrene microtiter plate as a solid-phase MAb, and purified human H-FABP as standard materials. The assay range of the ELISA was 0-250 ng/ml of plasma and urine. The ELISA yielded a coefficient of variation of less than 10% in inter- and intra-assays, and the good linearity was obtained in dilution test using clinical samples. Anticoagulants, except sodium fluoride and a high concentration of hemoglobin and bilirubin, did not interfere with the assay of plasma samples. A high concentration of hemoglobin, bilirubin and immunoglobulin, and contamination with seminal plasma did not interfere with the assay of urine samples. The average recovery of purified human H-FABP added to human plasma and urine samples was 98.5% and 97.0%, respectively. Myoglobin and myosin did not crossreact in the ELISA. The minimum detection limit of the ELISA was 1.25 ng/ml. The immunoreactive masses of human H-FABP in plasma and urine samples, obtained from one hundred normal healthy subjects were quantified by the sandwich ELISA. The normal mean (+/- SD) level of human H-FABP mass in plasma was 3.65 +/- 1.81 ng/ml, and that in urine was 3.20 +/- 2.70 ng/ml. In conclusion, this sandwich ELISA is a useful tool for the sensitive and precise determination of human H-FABP in human plasma and urine, and it may be used specifically for clinical investigation and diagnosis of myocardial injury.

Antibodies, Monoclonal↗
PubMed1995

Reduced expression of c-Fos in female NOD mouse thymocytes and up-regulation with human lymphotoxin.

Previously we reported that the administration of human (h) lymphotoxin (h-LT) markedly protected NOD mice from insulin-dependent diabetes mellitus (IDDM) partly by affecting the generation phase of anti-islet effector cells, probably in the thymus. In this study, we investigated the effect of h-LT on the signal transduction of the mouse thymocytes by observing c-Fos expression in the thymocytes by using a flow cytometer. The intensity of c-Fos expression in whole thymocytes was significantly lower in the female NOD with a high incidence of diabetes than that in the male NOD mice with a low incidence of diabetes and than that in normal mice (P < 0.0001). The low c-Fos expression in the female NOD thymocytes was most prominent in CD3low thymocytes. c-Jun expression of the CD3low thymocytes was also lower in the female NOD mice. Administrations of h-LT, h-TNF, and h-IL-2, which has been reported to prevent IDDM in NOD mice by systemic administration, significantly up-regulated c-Fos expression in CD3low thymocytes. From these results, it is assumed that a relationship may exist between the high diabetes incidence and the defective c-Fos expression in female NOD mice and between the prevention of IDDM and the amelioration of the defective c-Fos expression with h-LT in female NOD mice.

Animals↗
PubMed1995

Analysis of action mechanism of lymphotoxin in prevention of cyclophosphamide-induced diabetes in NOD mice.

Recently we reported that lymphotoxin (LT) administration protected non-obese diabetic (NOD) mice and BB rats from insulin-dependent diabetes mellitus. In this study we analysed the protection mechanism of LT by using cyclophosphamide (CY)-induced autoimmune diabetes in NOD mice. Pre-administration of 500 or 1000 U of LT three times a week between the age of 4 and 11-13 weeks before CY-treatment strongly inhibited CY-induced diabetes. This inhibition was reproduced by LT pre-administration at an earlier age (4 to 7 weeks) but not at a later age (8 to 11 or 10 to 12 wks). LT post-administration (100 U daily or 500 U twice a week) after CY-treatment at 14 weeks of age also strongly inhibited CY-induced diabetes. Spleen cell transfer was carried out using various combinations of donors and recipients. Spleen cell transfer from the non-diabetic mice, which were LT pre-administered between the age of 4 and 13 wks, to CY-treated mice did not significantly inhibit CY-induced diabetes, while transfer of the cells from the similarly treated mice to irradiated recipients did induce diabetes although the onset of diabetes was significantly delayed. Diabetes was not transferred by spleen cells from diabetic mice to LT pre-administered and CY-treated mice. LT administration did not change subpopulations and adhesion molecule expressions of the spleen lymphocyte. Taken together, these results suggest that LT protects NOD mice from CY-induced diabetes by making the mice resistant to autoimmune diabetes and possibly by suppressing anti-islet effector cells, but not by inducing adoptively transferable suppressor cells, although the precise mechanisms still remain to be elucidated.

Animals↗
PubMed1995

Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling.

Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and transverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules. CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the alpha 2, beta and gamma subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Animals↗
PubMed1995

Preventive effect of simultaneously infused lipid emulsion against thrombophlebitis during postoperative peripheral parenteral nutrition.

A prospective, randomized study was conducted to determine whether simultaneous infusion of lipid emulsion with an amino acid-dextrose-electrolyte solution would reduce the incidence of thrombophlebitis (TP) during postoperative peripheral parenteral nutrition (PPN). Thirty patients who had undergone gastric resection for adenocarcinoma were randomly divided into two groups according to whether they were infused with 10% lipid emulsion (group A) or 5% glucose solution (group B) simultaneously with the amino acid-glucose solutions. The total osmolarity of the infusion solutions in each group was 853 mOsm/l. The incidence of complications due to TP, namely, redness and/or edema beneath the cannula insertion site and/or pain, was investigated. There were no differences in the background characteristics of the patients in groups A and B, except regarding concurrent resection of other organs (P = 0.03). The incidence of edema in group A was significantly lower than in group B on postoperative days 2 and 4, although there was no difference in the incidence of redness and pain between the two groups. These findings suggest that the simultaneous infusion of lipid emulsion has a preventive effect against TP during postoperative PPN, and may be a practical means of providing PPN after gastrointestinal surgery.

Adenocarcinoma↗
PubMed1995

Effects of free radical scavengers on indomethacin-induced aggravation of gastric ulcer in rats.

Effects of treatment with free radical scavengers in the healing process of acetic acid-induced gastric ulcer on the ulcer aggravation induced by indomethacin were investigated. Gastric ulcers were produced on the anterior wall of the stomach of male Sprague-Dawley rats by submucosal injection of 20% acetic acid. To investigate the role of oxygen radicals, rats with gastric ulcer were treated with scavengers for six weeks and then treated with indomethacin (1 mg/kg/day). While superoxide dismutase (10,000 units/kg/day) did not affect the ulcer area after indomethacin treatment, allopurinol (50 mg/kg/day) slightly inhibited the increase in ulcer area. Dimethyl sulfoxide (1% solution, ad libitum) produced a significant decrease in size of the ulcer after indomethacin treatment. Increased lipid peroxides in the gastric mucosa after indomethacin treatment decreased significantly in the rats of the dimethyl sulfoxide and allopurinol groups. These results indicate that lipid peroxidation mediated by oxygen radicals plays an important role in the mechanism of ulcer aggravation induced by indomethacin.

Acetates↗
PubMed1995

Impaired myocardial fatty acid metabolism detected by 123I-BMIPP in patients with unstable angina pectoris: comparison with perfusion imaging by 99mTc-sestamibi.

The present study was undertaken to determine the potential diagnostic value of 123I-BMIPP scintigraphy for the detection of altered myocardial fatty acid metabolism in patients with unstable angina. Both myocardial metabolic imaging with 123I-BMIPP and perfusion imaging with 99mTc-sestamibi were performed at rest in 28 patients with unstable angina in the pain-free state. The regional uptakes of 123I-BMIPP or 99mTc-sestamibi were scored semiquantitatively (0 = normal, 4 = no activity) and compared with the coronary arteriographic findings. Decreased uptakes of 123I-BMIPP were observed in 18 patients, and 11 patients had abnormal 99mTc-sestamibi images. Defect scores of 123I-BMIPP were larger than those of 99mTc-sestamibi (7.8 +/- 2.1 vs. 5.2 +/- 1.9, p < 0.01). The sensitivity for the detection of patients with unstable angina was higher in 123I-BMIPP than in 99mTc-sestamibi (77% vs. 45%, p < 0.01). The site of the decreased 123I-BMIPP uptake corresponded to the most stenotic coronary artery lesion in all patients. Fatty acid metabolic imaging with 123I-BMIPP was more sensitive for detecting myocardial ischemia than perfusion imaging with 99mTc-sestamibi. 123I-BMIPP may be a clue to define the culprit lesion in unstable angina and be helpful to decide the best treatment and guide coronary angioplasty.

Aged↗
PubMed1995

Establishment of two new scirrhous gastric cancer cell lines: analysis of factors associated with disseminated metastasis.

Determination of the differences between cell lines which are derived from a primary tumour and a disseminated metastatic lesion from the same patient may aid in elucidating the factors associated with disseminated metastases. We report on the establishment and characterisation of two new scirrhous gastric cancer cell lines, designated OCUM-2M and OCUM-2D, derived from a 49-year-old female. OCUM-2M was derived from a primary gastric tumour, and OCUM-2D was derived from a sample of disseminated metastasis. The two cell lines were derived from the same patient. We investigated biological differences between the two cell lines to study mechanisms involved in disseminated metastasis. The growth activity of OCUM-2D cells as determined by doubling time and tumorigenicity was greater than that of OCUM-2M cells. The level of epidermal growth factor receptor (EGFR) expression in OCUM-2D cells was about twice that of OCUM-2M cells and the growth of OCUM-2D cells was stimulated more by epidermal growth factor (EGF) than that of OCUM-2M cells. The invasive activity of OCUM-2D cells was higher than that of OCUM-2M cells and was increased after addition of transforming growth factor-beta 1 (TGF-beta 1). An increase in the number of attached and spreading cells was found following the addition of 10 ng ml-1 TGF-beta 1. These findings suggest that high growth and invasive activity may play an important role in disseminated metastasis and that EGF and TGF-beta 1, which affect the growth and invasive activity of OCUM-2D cells, might be factors associated with metastasis in scirrhous gastric carcinoma. The two cell lines OCUM-2M and OCUM-2D should be beneficial for analysing mechanisms of tumour progression.

Adenocarcinoma, Scirrhous↗
PubMed1995

Effects of docosahexaenoic acid (DHA) on intestinal polyp development in Apc delta 716 knockout mice.

Epidemiological studies and animal experiments show an association of dietary intake of fish oils and low incidence of several types of cancers. The active ingredients of fish oils appear to be polyunsaturated fatty acids of omega-3 type such as eicosapentaenoic acid and docosahexaenoic acid (DHA). We have investigated chemopreventive effects of DHA on mouse intestinal polyposis using adenomatous polyposis coli (Apc) gene knockout mice. Damage to the human APC gene is responsible for not only familial adenomatous polyposis but also many sporadic cancers of the entire digestive tract. Using homologous recombination in embryonic stem cells, we recently constructed gene knockout mice containing a truncation mutation in the Apc gene at codon 716 (Apc delta 716). The heterozygous mice developed numerous intestinal polyps, and all microadenomas dissected from the earliest polyps had already lost the wild-type allele, indicating the loss of heterozygosity [Oshima et al. (1995), Proc. Natl Acad. Sci. USA, 92, 4482-4486]. We fed Apc delta 716 heterozygotes with AIN-76A purified diet containing 3% DHA for 7 weeks, and scored the number and size of intestinal polyps. Average DHA intakes per day were 4.1 and 4.3 g/kg body wt for males and females, respectively. DHA-fed females had only 31% of polyps compared with the control females that developed about 220 polyps, whereas DHA-fed females showed no significant decrease in the polyp number. As for the polyp size, the proportion of larger polyps decreased more significantly in females than in males. This is the first demonstration that DHA inhibits intestinal polyposis induced by an Apc mutation at both its formation and growth.

Animals↗
PubMed1995

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene: effects of sequence contexts on mutation spectra.

We previously reported that 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) at the second position of codon 12 of the c-Ha-ras gene induces many types of mutations in NIH3T3 cells. In this study we incorporated the modified base into the first and second positions of codon 12 in the coding strand and into the first position of codon 61 in the non-coding strand of the gene using a new 8-hydroxyguanine phosphoramidite as a building block during oligonucleotide synthesis. The ras genes with 8-hydroxyguanine were transfected into NIH3T3 cells and the mutations induced were analyzed. 8-Hydroxyguanine residues at the first positions of codons 12 and 61 induced mutations to T at the modified sites almost exclusively. On the other hand, the DNA lesion at the second position of codon 12 induced a G-->A transition in addition to a G-->T transversion, confirming our previous results. Mutations in 5'-flanking sites were observed with 8-hydroxyguanine at the second position of codon 12 or the first position of codon 61. These results indicate that 8-hydroxyguanine in mammalian cells mainly induces a G-->T transversion at the modified site, but that other types of mutations are also elicited.

3T3 Cells↗
PubMed1995

Decreased expression of glutathione S-transferases and increased fatty change in peroxisomal enzyme-negative foci induced by clofibrate in rat livers.

Alteration in glutathione S-transferase (GST) isoenzymes was compared with that of a peroxisomal enzyme, enoyl-CoA hydratase (ECH), during hepatocarcinogenesis caused by clofibrate (CF) administration in male Sprague-Dawley rats. The amount of alpha class GST forms, determined by single radial immunodiffusion using anti-GST 1-2 antibody, was inversely correlated with that of ECH and was decreased at week 2 of CF administration to approximately 50% of the value prior to treatment and then slightly increased to 70% of the control value by week 15, without change thereafter up to 93 weeks. Resolution of GST subunits by high performance liquid chromatography revealed an approximately 60% decrease in the amounts of subunits 1 and 3 at week 93 and a 25% decrease in the amounts of subunits 2 and 4. Immunohistochemical staining of rat livers revealed hepatic foci and minifoci negative for ECH at week 60 and thereafter. Almost all ECH-negative foci (95.1-97.9%) were clear cell in character, along with a somewhat lower proportion (68.0-73.8%) of ECH-negative minifoci. Numerous fat-positive granules were detected in 78.3% of those lesions exhibiting a clear cell change. Although the amounts of GST and ECH exhibited contrasting patterns of alteration in whole livers following CF administration, the expression of both alpha and mu class GST forms was decreased in the majority of ECH-negative foci at week 93, but were not altered in minifoci. The repression of GST forms appeared to be a later event than the loss of ECH or the clear cell change in CF-associated hepatic lesions and was in clear contrast to the enhanced expression reported for preneoplastic lesions induced by mutagenic carcinogens.

Animals↗