Studies on ribonucleoside-diphosphate reductase in permeable animal cells. II. Catalytic and regulatory properties of the enzyme in mouse L cells

Ribonucleoside-diphosphate reductase (EC 1.17.4.1) was studied in mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (R. Kucera and H. Paulus, 1982, Arch. Biochem. Biophys. 214, 102–113). The reduction of CDP was almost completely dependent on added ATP or adenyl-5′-yl imidodiphosphate, and that of GDP on dTTP. The pattern of inhibition by deoxyribonucleoside triphosphates was similar to that observed by others in cell-free preparations except for a somewhat higher sensitivity to inhibition. The substrate saturation curves for CDP and GDP were hyperbolic with apparent K_m values of 0.05 and 0.24 mm, respectively. The maximum velocities for CDP and GDP reduction were close to the in vivo rate of DNA synthesis. Ribonucleotide reductase activity was not affected by the addition of ferric salts but was inhibited by the chelators bathophenanthroline sulfonate and thenoyltrifluoroacetone and also by hydroxyurea. EDTA caused a reversible stimulation of GDP reduction and an irreversible inhibition of CDP reduction; the latter could be partially reactivated by the addition of magnesium salts. Ribonucleotide reductase activity was inhibited by arsenite but only slightly stimulated by NADPH or dithiols; however, if the cells were first treated with 2,6-dichlorophenolindophenol, an almost complete dependence on NADPH was observed which could also be met by dithiothreitol or dihydrolipoic acid but not by reduced glutathione. This suggests that ribonucleotide reductase in dextran sulfate-treated L cells is relatively tightly coupled to an endogenous hydrogen donor system.     

Cell cycle regulation of ribonucleoside diphosphate reductase activity in permeable mouse L cells and in extracts

Ribonucleoside diphosphate reductase (EC1.17.4.1) was previously characterized in exponentially growing mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (Kucera and Paulus, 1982b). This characterization has now been extended to cells in specific phases of the cell cycle and in transition between cell cycle phases, with activity studied both in situ (permeabilized cells) and in cell extracts. Cells at various stages in the cell cycle were obtained by unit-gravity sedimentation employing a commercially available reorienting chamber device, by G1 arrest induced by isoleucine limitation, and by metaphase arrest induced by Colcemid. G1 cells from both cycling and noncycling populations had negligible levels of ribonucleotide reductase activity as measured by CDP reduction both in situ and in extracts. When G1 arrested cells were allowed to progress to S phase, ribonucleotide reductase activity increased in parallel with [3H]thymidine incorporation into DNA. Ribonucleotide reductase activity in extracts increased at a somewhat greater rate than in situ activity. S phase ribonucleotide reductase activity measured in situ resembled the previously characterized activity in exponentially growing cells with respect to an absolute dependence on ATP or its analogs as positive allosteric effector, sensitivity to the negative allosteric effector dATP, and low susceptibility to stimulation by NADPH, dithiothreitol, and FeCl3. Disruption of permeabilized cells caused reductase activity to become highly dependent on the presence of both dithiothreitol and FeCl3. As synchronized cultures progressed from S into G2/M phase, no significant change in ribonucleotide reductase activity was seen. On the other hand, when cells that had been arrested in metaphase by Colcemid were allowed to resume cell cycle traversal by removing the drug, in situ ribonucleotide reductase activity decreased by 75% within 2.5 h. This decrease seemed to be a late mitotic event, since it was not correlated with the percentage of cells entering G1 phase. The cause of a subsequent slight increase of in situ ribonucleotide reductase activity is not clear. Parallel measurements of ribonucleotide reductase activity in cell extracts indicated also an initial decline accompanied by increasing dependence on added dithiols and FeCl3, followed by complete activity loss. Our results suggest a cell cycle pattern of ribonucleotide reductase activity that involves negligible levels in G1 phase, a progressive increase of activity upon entry into S phase paralleling overall DNA synthesis, continued retention of significant ribonucleotide reductase activity well into the metaphase period of mitosis, and a very rapid decline in activity during the later phases of mitosis. The periods of increase and decrease of ribonucleotide reductase activity were accompanied by modulation of the properties of the enzyme as indicated by differential changes in enzyme activity measured in situ and in extracts.     

Changes in colonic mucosal permeability in mouse colitis induced with dextran sulfate sodium.

In this study we examined changes in colonic mucosal permeability induced by dextran sulfate sodium (DSS) during the acute phase of mouse colitis. To induce colitis, the mice were given drinking water containing 5% (w/v) DSS (MW = 40,000) ad libitum. Colonic mucosal permeability was evaluated by the permeation of Evans blue (EB) from the lumen into the wall of the colon on 1, 2, 3 and 7 days postadministration of DSS. Mucosal changes were also histologically examined daily for 7 days postadministration. The permeation of EB increased significantly by days 3 and 7 postadministration. Histological analysis showed that crypt loss was the initial change, with no inflammatory process and the surface mucosal epithelial cells remained morphologically intact. These histological changes developed on 2 to 3 days postadministration. Erosion was first recognized at 5 days postadministration. These findings indicated that the increase in colonic mucosal permeability may have occurred in 3 days postadministration, and the increase in mucosal permeability occurred before the appearance of the inflammatory process. This suggests that an increase in colonic mucosal permeability, leading to the destruction of mucosal barrier function, may play an important role in the induction of DSS-induced murine colitis.     

Characterization of ribonucleotide reductase activity from mouse L cells

We describe some fundamental properties of the cytidine 5'-diphosphate (CDP) and guanosine 5'-diphosphate (GDP) reductase activity from mouse L cells. Both activities increased in a nonlinear fashion at low protein concentrations; this may be due to dissociation of two protein subunits of the enzyme at very low concentrations. CDP reductase activity was greatly stimulated in the presence of ATP and required magnesium and iron for maximum activity. GDP reductase required 2'-deoxythymidine 5'-triphosphate for maximum activity. Also apparent Km values of 0.14 mmol/l for CDP and 0.05 mmol/l for GDP were determined from double reciprocal plots of velocity against substrate concentrations. Activity in extracts of logarithmically growing mouse L cells was very high indicating that attempts to purify the enzyme from this source should be rewarding.     

Hydroxyurea-resistant mouse L cells with elevated levels of drug-resistant ribonucleotide reductase activity

We describe the isolation and partial characterization of a mouse L-cell line which is resistant to normally highly cytotoxic concentrations of hydroxyurea. A detailed analysis of the target enzyme ribonucleotide reductase in both wild-type and hydroxyurea-resistant enzyme preparations suggests that the drug-resistant cells form a ribonucleotide reductase enzyme which contains a structural alteration, rendering it less sensitive to inhibition by hydroxyurea. K₁ values for hydroxyurea inhibition of ribonucleotide reduction in enzyme preparations from hydroxyurea-resistant cells were significantly higher than corresponding values from preparations from wild-type cells. The K_m for CDP reduction in enzyme preparations of drug-resistant cells was approximately threefold higher than the corresponding parental wild-type value. In addition, in vivo enzyme assays detected a major difference between the temperature profiles of ribonucleotide reduction in nucleotide-permeable drug-resistant and wild-type cells. When levels of ribonucleotide reductase activity were measured in vivo, it was found that the drug-resistant cells contained approximately 3 times the wild-type level of CDP reductase activity and twice wild-type level of GDP reductase activity. This combination of enhanced enzyme levels plus an altered sensitivity to drug inhibition can easily account for the drug-resistance phenotype. The properties of these hydroxyurea-resistant cells indicate that they will be useful for genetic and biochemical studies.This work was supported by the N.S.E.R.C. of Canada and the Muscular Dystrophy Association of Canada through research funds (J. A. W.) and by the N.R.C. of Canada through a graduate scholarship (B. A. K.).     

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Dose-dependent promoting effect of dextran sodium sulfate on mouse colon carcinogenesis initiated with azoxymethane

We previously reported a powerful tumor-promoting ability of dextran sodium sulfate (DSS) in a novel mouse model for colitis-related colon carcinogenesis initiated with azoxymethane (AOM). To determine the dose-dependent influence of DSS in our animal model, male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight), followed by DSS at dose levels of 2, 1, 0.5, 0.25, and 0.1% (w/v) in drinking water for 1 week. All animals were sacrificed at week 14 and histological alterations in their colon and nitrotyrosine immunohistochemistry were examined to evaluate the nitrosative stress. In the mice which received AOM and 2% DSS, the incidences (multiplicity) of colonic tubular adenoma and adenocarcinoma were 75% (1.25+/-1.26/mouse) and 100% (2.75+/-2.22/mouse), respectively. Mice given AOM and 1% DSS had 80% incidence of adenoma (1.00+/-0.71/mouse) and 60% incidence of adenocarcinoma (1.40+/-2.07/mouse) in the colon. In a mouse treated with AOM and 0.5% DSS, only one colonic adenoma (20% incidence with 0.20+/-0.45 multiplicity) developed. Higher frequency of high-grade colonic dysplasia was noted in mice given AOM and 2% or 1% DSS when compared with mice treated with AOM and lower doses of DSS. Also, scoring of inflammation and nitrotyrosine immunoreactivity suggested that severe inflammation and nitrosation stress caused by high-doses (2% and 1%) of DSS contribute its tumor-promoting effects in mouse colon carcinogenesis initiated with a low dose of AOM. Thus, our findings indicate that a tumor-promoting effect of DSS was dose-dependent (1% or more) and the effect might occur under the condition of inflammation and nitrosation stress.     

Unmethylated DNA in mouse L cells. I. DNA fibre autoradiography.

Enzymatic methylation of DNA in mouse L cells has been studied using DNA fibre autoradiography to analyse the distribution of 5-methylcytosine in chromosomal DNA. The autoradiographic pattern of DNA labelled in the 5-methylcytosine is in several respects similar to the pattern of DNA replication. Two mean features are apparent: (1) the silver grains appear in well defined sections, and (2) the labelled sections are arranged in tandem along each DNA double helix. After a short pulse of radioactivity in the rate of growth of labeled sections in the pattern of DNA replication and the enzymatic methylation of DNA are identical. Unlike the replication pattern, DNA labeled during the S phase with L-[Me-3H] methionine is not completely labeled. There are distinct, 8-20 mum intervals in the autoradiographic pattern of this DNA. The length of these intervals may correspond to unmethylated sections of chromosomal DNA of about 23 to 58 kilo base pairs. These unmethylated sections of chromosomal DNA represent about 10% of the total genome.     

Stabilization of basic fibroblast growth factor with dextran sulfate.

Dextran sulfate protected bFGF from heat and acid inactivation and from proteolytic degradation. The protective effect was stronger than that of heparin which is known as a stabilizer of bFGF. Dextran sulfate and bFGF formed a high molecular weight complex via ionic interaction when mixed together in aqueous solution. The complex was dissociated when the ionic strength was increased and the protective effect was completely abolished. Successive digestion of bFGF with Staphylococcus aureus V8 protease and pepsin followed by affinity chromatography on an immobilized dextran sulfate column and reversed-phase high performance liquid chromatography yielded three positively charged fragment peptides, Tyr24-Phe30, Tyr106-Trp114 and Tyr124-Leu138. These results suggest that dextran sulfate stabilizes bFGF by binding close to the putative heparin binding sites of the bFGF molecule.     

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.     

Plant originated glycoprotein has anti-oxidative and anti-inflammatory effects on dextran sulfate sodium-induced colitis in mouse

Summary We investigated the preventive effect of glycoprotein (27 kDa) isolated from Gardenia jasminoides Ellis (GJE) fruits on colitis in dextran sulfate sodium (DSS, 3%)-induced A/J mice which were administrated orally for 7 days. Anti-inflammatory activity of GJE glycoprotein was assessed by neutrophil infiltration and colonic lipid peroxidation, and determined by myeloperoxidase (MPO) activity and levels of thiobarbituric acid reactive substances (TBARS), respectively in DSS treatment system. The activities of antioxidative enzymes [catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)], activation of inflammation related mediators (iNOS, COX-2, and NF- B), and production of nitric oxide (NO) and reactive oxygen species (ROS) were also measured. The results of this study showed that GJE glycoprotein (g/ml) has a scavenging property to inhibit the intracellular ROS production in RAW 264.7 cells and that GJE glycoprotein (80 mg/kg BW) significantly suppressed the increase in the MPO activity, TBARS level, and NO production, inflammation related mediators [iNOS, COX-2, and NF-B (p50)] activity in DSS-induced mice. Interestingly, the activities of CAT, SOD, and GPx were gradually augmented after a supplement of GJE glycoprotein. Therefore, we suggest that GJE glycoprotein is preventive and therapeutic agent for the ulcerative colitis.     

Gene amplification in methotrexate-resistant mouse cells. I. DNA rearrangement accompanies dihydrofolate reductase gene amplification in a T-cell lymphoma

Mouse EL4 lymphoma cells have been selected in vitro for resistance to methotrexate. Four independently derived resistant cell lines are described. Each has amplified dihydrofolate reductase (DHFR) genes, and overproduces DHFR RNA and DHFR protein. In three of the four cell lines DNA rearrangement has occurred near the ends of the DHFR gene. The rearrangement is different in each case, but always involves only a proportion of the DHFR genes.