Potentiation of N-methyl-N'-nitro-N-nitrosoguanidine-induced O6-methylguanine-DNA-methyltransferase activity in a rat hepatoma cell line by poly (ADP-ribose) synthesis inhibitors

The O6-methylguanine-DNA-methyltransferase (transferase) activity in a rat hepatoma cell line (H4 cells) is enhanced as a response to DNA damaging agents. To study whether poly (ADP-ribosylation) is involved in this induction, the cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) that induces the transferase activity and stimulates poly (ADP-ribose) synthesis. Addition of poly (ADP-ribose) polymerase inhibitors enhanced the transferase increase induced by MNNG. The influence of the inhibitors on the transferase induction was dose and time-dependent. The results suggest that poly (ADP-ribose) is involved in the induction of this protein.     

Downregulation of caveolin expression by cAMP signal

Recent data have demonstrated that caveolin, a major structural protein of caveolae, inhibits the function of molecules involved in cAMP signaling such as adenylyl cyclase. We examined the effect of cAMP signal on the expressions of caveolin subtypes using rat cardiac myoblasts (H9C2 cells) and smooth muscle cells (RASMC), which express caveolin subtypes. Treatment of RASMC and H9C2 cells with forskolin, an adenylyl cyclase stimulator, decreased caveolin-1 mRNA levels in a dose-dependent manner. Time course studies showed a time-dependent decrease of caveolin-1 mRNA levels in H9C2 cells (after 6 hours) while caveolin-1 mRNA levels in RASMC showed a biphasic response, i.e., an initial increase (within 3 hours) and a later decrease (after 3 hours). Similar biphasic changes were observed when RASMC was treated with IBMX, a phosphodiesterase inhibitor. The levels of caveolin-1 and -3 proteins were also decreased by forskolin treatment, but only after 60-72 hours in RASMC and 24-36 hours in H9C2 cells. In contrast, the expression of caveolin-2 remained similar in both cells and decreased to a small degree after prolonged treatment. Therefore, the expression of caveolin is downregulated by cAMP signal in a caveolin subtype-dependent manner.     

Regulation of the mesangial cell myofibroblast phenotype by actin polymerization

Mesangial cells in diverse glomerular diseases become myofibroblast-like, characterized by activation of smooth muscle alpha-actin (alpha-SMA) expression. In cultured mesangial cells, serum-deprivation markedly increases alpha-SMA expression, cell size, and stress fiber formation. Since stress fibers are assembled from actin monomers, we investigated the hypothesis that alterations in stress fiber formation regulate alpha-SMA expression and hypertrophy. Human mesangial cells were treated with agents that disrupt or stabilize actin stress fibers. Depolymerization of actin stress fibers in serum-deprived cells with actin-depolymerizing agents, cytochalasin B (CytB) and latrunculin B (LatB), or with inhibitors of Rho-kinase, Y-27632 and HA-1077 decreased alpha-SMA mRNA as judged by Northern blot analysis. Western blot analysis showed that CytB also reduced alpha-SMA protein levels. In serum-fed cells, agents that stabilized actin stress fibers, jasplakinolide (Jas) and phalloidin, increased alpha-SMA mRNA and protein. Treatment of human or rat mesangial cells with CytB, LatB, or Y-27632 decreased alpha-SMA promoter activity. In contrast, Jas increased promoter activity 5.6-fold in rat mesangial cells. The presence of an RNA polymerase inhibitor blocked degradation of alpha-SMA mRNA in cells treated with CytB suggesting that destabilization of this message is dependent on a newly transcribed or rapidly degraded factor. Inhibition of actin polymerization by CytB, LatB, Y-27623, and HA-1077 inhibited incorporation of (3)[H]-leucine into newly synthesized protein. Additionally, CytB and LatB decreased cell volume as determined by flow cytometry. Collectively, these results indicate that the state of polymerization of the actin cytoskeleton regulates alpha-SMA expression, hypertrophy, and myofibroblast differentiation in mesangial cells.     

Bone Gla protein messenger ribonucleic acid is regulated by both 1,25-dihydroxyvitamin D3 and 3',5'-cyclic adenosine monophosphate in rat osteosarcoma cells

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] regulates the synthesis of bone gamma-carboxyglutamic acid (Gla) protein (BGP) by osteoblastic cells. In this study we examined the effect of cAMP, alone and in combination with 1,25-(OH)2D3, on the regulation of BGP mRNA levels in ROS 17/2 rat osteosarcoma cells. Elevation of intracellular cAMP levels by cAMP analogs or by isobutylmethylxanthine (IBMX), forskolin, or PTH, resulted in increased BGP mRNA levels and BGP secretion after 1 day of treatment. The effects of these agents were additive with 1,25-(OH)2D3 in stimulating BGP gene expression. After 4 days of treatment, pertussis toxin (PT) and 1,25-(OH)2D3 were synergistic in stimulating BGP mRNA, and the effect of PT could be mimicked by (Bu)2cAMP, IBMX, forskolin, cholera toxin, and to a lesser extent by PTH. The effect of 1-day treatment with cAMP alone and the synergistic effect with 1,25-(OH)2D3 on the stimulation of BGP mRNA were dependent on cell density, while basal and 1,25-(OH)2D3-stimulated synthesis were not. Cyclic AMP inhibited ROS 17/2 cell growth after 1 day of treatment, an effect that was also dependent on initial cell density. After 4 days of treatment, 1,25-(OH)2D3, cAMP, and PT all demonstrated inhibition of cell growth. When cells were treated with actinomycin D, both 1,25-(OH)2D3 and cAMP stimulation of BGP mRNA were blocked. In addition, neither agent was effective in enhancing BGP mRNA stability when prestimulated cells were exposed to actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of radiation killing of cultured mammalian cells by cordycepin.

Asynchronous populations of rat hepatoma cells (H4) in log-phase growth survived a 3-hour exposure to cordycepin (3'-deoxyadenosine), and RNA antimetabolite, in a simple exponential fashion with a 'DO' of 43.8 microM/l. When cordycepin-treated cells were exposed to X-irradiation, the resultant survival levels were much lower than one would expect were the agents simply additive. Patterns of X-ray survival of cells treated with cordycepin were dependent on drug concentration, the predominant effect being to decrease the DO of the X-ray survival curve. The increased sensitivity of cells exposed to cordycepin to subsequent X-ray treatment persists for longer than 4 hours after drug administration. Although immediate cordycepin post-treatment of X-irradiated cells is less effective than pre-treatment, the interaction is still significant. Cordycepin treatment did not appear to reduce split-dose recovery or to inhibit the rejoining of single-strand breaks as measured by DNA sedimentation in alkaline-sucrose gradients.     

Expression of the tumor necrosis factor locus is not necessary for the cytolytic activity of T lymphocytes

In order to test whether tumor necrosis factors alpha (TNF-alpha) or beta (TNF-beta, also known as lymphotoxin) are involved in the lysis of target cells by cytolytic T lymphocytes, we probed for the presence of the TNF mRNAs in several quiescent and activated CTL clones. No TNF mRNA could be found in constitutively cytolytic Lyt-2+ clones, and only two out of three clones tested accumulated TNF mRNA after stimulation with phorbol myristate acetate and ionomycin. Of two L3T4+ clones that can be induced to become cytolytic by a combination of antigen and IL-1, only one accumulated TNF-beta mRNA in the process. The PC60 rat X mouse T cell hybrid, which becomes cytolytic in response to a combination of IL-1 and IL-2, also failed to accumulate TNF mRNA after stimulation with these agents. Our results strongly suggest that TNF-alpha or -beta are not necessary agents of the cytolytic activity exhibited by antigen-specific T lymphocytes.     

Cell-based studies reveal differences in glutathione S-transferase induction between oltipraz and tert-butylhydroquinone

Selective induction of Phase II over Phase I drug-metabolizing enzymes has been proposed as a mechanism for reduction of chemical carcinogenesis. Enzymes likely to play a role in this amelioration include the glutathione S-transferases (GSTs) and among compounds that selectively induce key GSTs are tert-butylhydroquinone (tBHQ) and oltipraz [4-methyl-5-(2-pyrazinyl)-3H-1,2-dithiole-3-thione]. In vivo, and in hepatoma cells (H4IIE), these two agents induce rat GSTA2 mRNA to a similar extent. However, with a luciferase reporter construct containing 1651 bp of the proximal 5' flanking region of the rGSTA2 gene in the same cell line and under similar conditions, luciferase activity was induced to a much greater extent by tBHQ than by oltipraz. A similar large intercompound differential was seen with reporter constructs containing either the rGSTA2 ARE enhancer and HNF1 site (-872 to -582) or XRE enhancer and HNF1 site (-1110 to -812). In H4IIE cells, the rGSTA2 mRNA response to each agent was completely inhibited by 1 microM actinomycin-D cotreatment. With 1 microM cycloheximide cotreatment however, some induction by tBHQ remained, while induction by oltipraz was completely abolished. The induction response to tBHQ but not oltipraz was augmented by pretreatment with PD98059, a MEK1/2 specific inhibitor. Notwithstanding induction characteristics in common, oltipraz, and tBHQ have sufficient dissimilarities to indicate that rGSTA2 upregulation by the two agents is not identical.     

cDNA cloning of the rat O6-methylguanine-DNA-methyltransferase

A cDNA expression library was constructed from a rat hepatoma cell line ( H4 cells ) and introduced into an Escherichia coli strain ( BK2110 ) deficient in the repair of O6-methylguanine residues. Following three exposures to N-methyl-N'-nitro-N-nitrosoguanidine, a resistant colony harboring a plasmid named RMGMT was isolated. Extracts of BK2210 cells hosting the RMGMT plasmid expressed a O6-methylguanine-DNA-methyltransferase (transferase) activity and this protein had the same molecular weight as the transferase from H4 cells. The cDNA sequence of 763 bp contains an open reading frame of 630 bp encoding a protein of 209 amino acids with a calculated molecular weight of 22.2 kd. The rat protein shows 68% homology with the human transferase.     

Expression of protein disulfide isomerase in cultured rat liver epithelial cells is unrelated to the growth inhibitory effect of TGF-beta 1.

The effect of TGF-beta 1 treatment on the level of protein disulfide isomerase (PDI) mRNA in normal and chemically or spontaneously transformed rat liver epithelial cell lines was investigated. TGF-beta 1 at 1 or 10 ng/ml concentrations did not significantly decrease the mRNA level of PDI at 4 or 24 hours after exposure to TGF-beta 1, irrespective whether the cell line was sensitive or resistant to the growth-inhibitory effect of TGF-beta 1 at these concentrations. The results indicate that in normal or neoplastic rat liver epithelial cells, the expression of PDI is unrelated to the growth inhibitory effect of TGF-beta 1.     

Decrease in cytosolic phospholipase A2alpha mRNA levels by reactive oxygen species via MAP kinase pathways in PC12 cells: effects of dopaminergic neurotoxins

Excess production of reactive oxygen species (ROS), including H2O2, leads to neuronal death in pathological conditions. Although ROS stimulates alpha-type cytosolic phospholipase A2 (cPLA2alpha) activity, their role in cPLA2alpha expression has not been elucidated. We investigated the effect of ROS on cPLA2alpha mRNA levels and signaling pathways in rat pheochromocytoma PC12 cells. Treatment with H2O2 and xanthine-xanthine oxidase (X/XO) for 4 h decreased cPLA2alpha mRNA levels without changing the mRNA levels of other tested proteins. H2O2 and X/XO caused cell toxicity not after 4 h but 24 h after their addition. The H2O2-induced decrease in cPLA2alpha mRNA levels was inhibited in cells treated with N-acetyl-cysteine and selective inhibitors of mitogen-activated protein kinase (MAPK) pathways (extracellular signal-regulated kinase and p38 MAPK). Treatment with dopaminergic neurotoxins, including 1,2,3,4-tetrahydroisoquinoline (TIQ)-inducing ROS formation, decreased cPLA2alpha mRNA levels. These findings suggest that ROS decreases cPLA2alpha mRNA levels via MAPK pathways in PC12 cells.     

Annexin A3-expressing cellular phenotypes emerge from necrotic lesion in the pericentral area in 2-acetylaminofluoren/carbon tetrachloride-treated rat livers

Recently we found a small hepatocyte-specific protein, annexin A3 (AnxA3), in fractionated adult rat hepatocytes. Here we describe the results of an in vivo demonstration of AnxA3-expressing cellular phenotypes in the liver with 2-acetylaminofluoren (2-AAF)/carbon tetrachloride (CCl(4))-injury. In association with an elevation of alanine amino transferase (ALT) and aspartic acid amino transferase (AST) activities, hepatic AnxA3 mRNA increased markedly. AnxA3-positive cells were detected in clustered cells present in or emerging from the pericentral region. These albumin-expressed cells were histologically similar to cells expressing CD34, a hematopoietic cell marker protein. The number of clusters decreased in the days following CCl(4) treatment, and annexin-negative, but albumin-positive, oval cells appeared. We concluded that the agent-induced liver defect initially recruits bone marrow-derived cells, and that it promotes differentiation of these cells into AnxA3-positive cells, followed by emergence of the oval cells, which might have a role in the restitution of the damaged liver.     

Requirement of down-regulation of NAD+ ADP-ribosyltransferase for the interferon-gamma-induced activation process of murine macrophage tumor cells.

Expression of the NAD+ ADP-ribosyltransferase gene is depressed during interferon-gamma-induced activation of murine macrophage P388D1 tumor cells [Taniguchi, T., Yamauchi, K., Yamamoto, T., Tokushima, K., Harada, N., Tanaka, H., Takahashi, S., Yamamoto, H. & Fujimoto, S. (1988) Eur. J. Biochem. 171, 571-575]. In order to study the role(s) of NAD+ ADP-ribosyltransferase in interferon-gamma-induced activation of P388D1 cells, we transfected an cloned synthetase gene into P388D1 cells and examined the effect of exogenous transferase gene expression on the induction of the Ia antigen, one of the major histocompatibility gene products, by interferon-gamma. The transferase activity of the transfected cells was twice that of control cells, and Southern blot analysis revealed that characteristic restriction sizes of cDNA were detected in the clones. RNA blot analysis using a cDNA for the transferase as a probe showed that the level of mRNA for the transferase in transfected cells was higher than that in control cells, and mRNA for the exogenous transferase was still detectable 2 days after the transfected cells were treated with interferon-gamma. This indicates that the exogenous transferase gene was expressed in transfected cells. RNA blot analysis with a cDNA for the Ia antigen and flow-cytometric analysis showed that the Ia antigen was induced much less in the transfected cells by interferon-gamma, in terms of the mRNA and the Ia antigen. The results suggest that down-regulation of the transferase is required for the induction of the Ia antigen in P388D1 cells by interferon-gamma.     

Sequential X-chromosome reactivation and inactivation in cell hybrids between murine embryonal carcinoma cells and female rat thymocytes

By means of a 5-bromodeoxyuridine (BrdU) incorporation and an acridine orange fluorescence staining method together with [3H]thymidine ([3H]TdR) autoradiography, we studied the chronology of X-chromosome replication in newly formed cell hybrids between the hypoxanthine phosphoribosyl transferase (HPRT)-deficient OTF9-63 murine embryonal carcinoma (EC) cell with 43 +/- chromosomes and the female rat thymocyte having 42 chromosomes. Most near-tetraploid hybrid cells retained all chromosomes from both parents including one mouse X (XM) and two rat X (XR) chromosomes throughout the period of this study. Data showing changes in the chronology of X-chromosome replication obtained here were indicative of reactivation of the inactive X chromosome from the rat thymocyte, and de novo X-inactivation of one or two chromosomes. The extinction of lymphocyte phenotypes from the hybrids and their subsequent differentiation to the cell type resembling endoderm found in the peri-implantation mouse embryo apparently occurred in parallel with the above changes. These hybrids also showed an interesting possibility of preferential reinactivation of the reactivated XR chromosome in the early stages after cell fusion.     

Endogenous non-cyclooxygenase metabolites of arachidonic acid modulate growth and mRNA levels of immediate-early response genes in rat mesangial cells

The role of endogenous arachidonic acid and its metabolites as mediators of cell growth was studied in rat mesangial cells. Inhibitors of the cytochrome P450 monooxygenase and lipoxygenase systems (nordihydroguaiaretic acid (NDGA), SK&F 525A, and ketoconazole) significantly reduced serum-stimulated cell growth as determined by cell counts and incorporation of [3H]thymidine. Inhibition of cyclooxygenase or lipoxygenases alone had no effect on cell growth. Stimulation with arginine vasopressin, epidermal growth factor, or phorbol myristate acetate increased [3H]thymidine incorporation and mRNA levels of the immediate-early response genes c-fos and Egr-1. These increases in [3H]thymidine incorporation and mRNA levels were reduced by NDGA and ketoconazole. NDGA, SK&F 525A, and ketoconazole had no effect on cellular ATP levels. Indomethacin had no effect upon cell growth. 14,15-Epoxyeicosatrienoic acid potentiated the effect of arginine vasopressin to enhance [3H]thymidine incorporation. Reverse-phase high pressure liquid chromatography analysis of lipid extracts from cells prelabeled with [3H]arachidonic acid resulted in the detection of a radioactive peak which eluted with lipoxygenase and monooxygenase products, with the same retention time as vicinal dihydroxyeicosatrienoic acids. This peak increased after stimulation with arginine vasopressin or epidermal growth factor and was reduced by preincubation with NDGA. Furthermore, analysis of unlabeled cell extracts by gas chromatography-mass spectrometry revealed the presence of a compound with epoxyeicosatrienoic acid-like characteristics. These results indicate that mesangial cells in culture likely produce products of the cytochrome P450 monooxygenase system that are important endogenous mediators of the growth response to mitogenic agents.     

Expression of ribosomal protein L4 (rpL4) during neurogenesis and 5-azacytidine (5AzC)-induced apoptotic process in the rat

5-Azacytidine (5AzC) induces neuronal apoptosis in rat and mouse fetuses. 5AzC also induces apoptosis in undifferentiated PC12 cells, and ribosomal protein L4 (rpL4) mRNA expression increases prior to apoptosis. To clarify the roles of rpL4 during neurogenesis, we first examined the distribution of rpL4 mRNA in the developing rat brain by in situ hybridization and RT-PCR, and compared the results to the distribution of TUNEL- or PCNA-positive cells. rpL4 mRNA expression was strong in the ventricular zone (VZ), subventricular zone (SVZ), cortical plate (CP), cerebral cortex, granule cell layer (GCL), pyramidal cell layer (Py) and external granular layer (EGL) during embryonic and early postnatal days, and it was remarkably weakened thereafter. A lot of PCNA-positive cells were observed in VZ, SVZ, and EGL during embryonic and early postnatal days, and such distribution of PCNA-positive cells was almost identical to rpL4 mRNA distribution. Only few TUNEL-positive cells were observed in VZ, SVZ, cerebral cortex, EGL, and hippocampus during embryonic and early postnatal days, and the regions with TUNEL-positive cells were not identical to rpL4 mRNA distribution. Next, the changes of rpL4 mRNA expression in the brain of 5AzC-treated rat fetuses were examined by in situ hybridization and RT-PCR. Apoptotic cells appeared at 9 to 24 hours after treatment (HAT). However, the rpL4 mRNA expression was unchanged during the apoptotic process. From the results, it is suggested that rpL4 would have certain roles in cell proliferation and differentiation during neurogenesis, but have no roles in 5AzC-induced apoptosis in the fetal brain.