Reversion of hydroxyurea resistance, decline in ribonucleotide reductase activity, and loss of M2 gene amplification

A key rate-limiting reaction in the synthesis of DNA is catalyzed by ribonucleotide reductase, the enzyme which reduces ribonucleotides to provide the deoxyribonucleotide precursors of DNA. The antitumor agent, hydroxyurea, is a specific inhibitor of this enzyme and has been used in the selection of drug resistant mammalian cell lines altered in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of mammalian cells with elevated ribonucleotide reductase activity has been used to isolate three stable subclones with varying sensitivities to hydroxyurea cytotoxicity and levels of ribonucleotide reductase activities. These subclones have been analyzed at the molecular level with cDNA probes encoding the two nonidentical subunits of ribonucleotide reductase (M1 and M2). Although no significant differences in M1 mRNA levels or gene copy numbers were detected between the three cell lines, a strong correlation between cellular resistance, enzyme activity, M2 mRNA and M2 gene copies was observed. This is the first demonstration that reversion of hydroxyurea resistance is directly linked to a decrease in M2 mRNA levels and M2 gene copy number, and strongly supports the concept that M2 gene amplification is an important mechanism for achieving resistance to this antitumor agent through elevations in ribonucleotide reductase.     

Altered ribonucleotide reductase activity in mammalian tissue culture cells resistant to hydroxyurea

Four Chinese hamster ovary cell lines and one mouse L cell line have been isolated which are resistant to the cytotoxic effects of hydroxyurea and guanazole. These five cell lines contain an altered ribonucleotide reductase activity as judged by a decreased sensitivity to the inhibitory action of both drugs. This is strong evidence that ribonucleotide reductase is one of the lethal sites of action for these two antitumour agents. The results are also consistent with the view that mammalian cell variants can arise from structural gene mutations.     

Deoxyadenosine toxicity and cell cycle arrest in hydroxyurea-resistant S49 T-lymphoma cells

Hydroxyurea-resistant S49 T-lymphoma cells have increased ribonucleotide reductase activity and deoxyribonucleoside triphosphate pools when compared with wild-type cultures. If ribonucleotide reductase inhibition is the mechanism by which deoxyadenosine is cytotoxic, then hydroxyurea (HU)-resistant S49 cells might be more resistant to deoxyadenosine toxicity when adenosine deaminase is inhibited than wild-type cells. Five S49 cell lines resistant to varying concentrations of HU were compared with wild-type cells by measuring CDP reductase activity, deoxyribonucleoside triphosphate pools, and deoxyadenosine toxicity. All five cell lines resistant to increasing concentrations of HU exhibited a twofold increase in resistance to deoxyadenosine toxicity when compared to wild type, and the resistance was proportional to the twofold increased pools of dNTPs in these cell lines but was less than the six- to eight fold increase in ribonucleotide reductase activity. In both wild-type and mutant cell lines, deoxyadenosine toxicity was accompanied by the accumulation of deoxyadenosine triphosphate and reduction of the other dNTPs; however, only dGTP greatly diminished. Exogenous addition of deoxycytidine decreased the dATP accumulation by about 20%, but also resulted in increases in the dCTP, dTTP, and dGTP pools. The S49 cells arrested in G1 phase when exposed to dAdo, although hydroxyurea-resistant cells required higher dAdo concentrations to elicit G1-phase arrest than wild-type cells. Deoxycytidine prevented dAdo-induced G1 arrest in all cell types. In summary, these data support the hypothesis that deoxyadenosine-induced dATP accumulation results in inhibition of ribonucleotide reductase and that this may be the mechanism for both cell cycle arrest and cytotoxicity in S49 T-lymphoma cells.     

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines.

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of ribonucleotide reductase activities in normal human fibroblast strains with their transformed counterparts

The objective of this investigation was to examine the relationship between levels of ribonucleotide reductase activity and transformation of two human cell strains. Enzyme activity levels were elevated by 3.2- to 3.5-fold in transformed cells compared directly to the normal human fibroblast strains from which they were derived. There did not appear to be a general correlation between elevated ribonucleotide reductase and increased proliferation abilities as has been previously observed with some rodent tumor cell lines. In keeping with the rise in reductase activity, human transformed cells were relatively more resistant to the cytotoxic effects of hydroxyurea, an antitumor agent whose site of action is ribonucleotide reductase. This indicates that an important point to be considered during drug therapy aimed at the reductase, is the greater sensitivity of normal compared to transformed cells due to differences in enzyme activity. The results of this investigation support the view that an increased ability to reduce ribonucleotides is an important step towards the development of a neoplastic program in human cells.     

M2 subunit of ribonucleotide reductase is a target of cyclic AMP-dependent protein kinase

Cyclic AMP arrests T lymphocytes in the G1 phase of the cell cycle, and prolonged exposure results in cytolysis. Both of these effects require cyclic AMP-dependent protein kinase. We recently observed that some S49 mouse T lymphoma cell lines selected for hydroxyurea resistance were not arrested in G1 by cyclic AMP. Further analysis revealed that these cell lines were cyclic AMP-dependent protein kinase deficient, and conversely, other cyclic AMP-dependent protein kinase deficient cell lines not selected for hydroxyurea resistance were two- to threefold more hydroxyurea resistant. However, hydroxyurea is a specific inhibitor of ribonucleotide reductase and does not inhibit this kinase. We subsequently showed that cyclic AMP-dependent protein kinase will phosphorylate the M2 but not the M1 subunit of ribonucleotide reductase in vitro, and this phosphorylation will diminish CDP reductase activity. In vivo phosphorylation of M2 occurred under conditions similar to those that generate cell cycle arrest. We conclude that the M2 subunit of ribonucleotide reductase can be a target of cyclic AMP-dependent protein kinase. The phosphorylated enzyme has diminished activity, and this may play a role in cyclic AMP-induced lymphocyte cell cycle arrest.     

Relationships between reversion of hydroxyurea resistance in hamster cells and the co-amplification of ribonucleotide reductase M2 component, ornithine decarboxylase and P5-8 genes

Hydroxyurea is a specific inhibitor of ribonucleotide reductase, which is a rate-limiting enzyme activity in DNA synthesis. Cells selected for resistance to hydroxyurea contain alterations in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of hamster cells has been used to isolate a stable drug resistant cell line, and two stable revertant lines with different sensitivities to hydroxyurea cytotoxicity and different ribonucleotide reductase activity levels. We show for the first time that a decrease in hydroxyurea resistance is accompanied by a parallel decline in gene copies for the M2 component of ribonucleotide reductase, ornithine decarboxylase and a gene of unknown function called p5-8, indicating that the co-amplification of the three genes is associated with drug resistance, and supporting the concept that M2, ornithine decarboxylase and p5-8 are closely linked, and form part of a single amplicon in hamster cells.     

Involvement of ribonucleotide reductase in cellular differentiation

L₆ and L₈ rat myoblast cell lines have been selected for resistance to hydroxyurea, an antineoplastic agent whose intracellular target is the rate-limiting enzyme activity of DNA synthesis, ribonucleotide reductase. In contrast to the differentiation-competent parental lines from which they were selected, the drug-resistant lines exhibit a grossly altered or absent myogenic capacity. Independent selections have revealed a strong correlation between changes in ribonucleotide reductase, as determined by velocity levels and product pool analyses, and altered myogenic potential. These results provide the first indication that alterations in this key enzyme activity and its accompanying deoxyribonucleoside triphosphate pools can affect cellular differentiation.     

Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.     

Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12–0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair. © 1994 Wiley-Liss, Inc.     

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity.

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.     

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.     

Ribonucleotide reductase from wild type and hydroxyurea-resistant chinese hamster ovary cells

The kinetic properties of partially purified ribonucleotide reductase from Chinese hamster ovary cells have been investigated. Double reciprocal plots of velocity against substrate concentration were found to be linear for three the substrates tested, and yielded apparent Km values of 0.12 mM for CDP, 0.14 mM for ADP and 0.026 mM for GDP. Hydroxyurea, a potent inhibitor of ribonucleotide reduction, was tested against varying concentrations of ribonucleotide substrates and inhibited the enzyme activity in an uncompetitive fashion. Intercept replots were linear and exhibited Ki values for hydroxyurea of 0.08 mM for CDP reduction, 0.13 mM for ADP reduction and 0.07 mM for GDP reduction. Guanazole, another inhibitor of ribonucleotide reductase, interacted with the enzyme in a similar manner to hydroxyurea showing an uncompetitive pattern of inhibition with CDP reduction and yielding a Ki value of 0.57 mM. Partially purified ribonucleotide reductase from hydroxyurea-resistant cells was compared to enzyme activity from wild type cells. Significant differences were observed in the hydroxyurea Ki values with the three ribonucleotide substrates that were tested. Also, CDP reductase activity from the drug-resistant cells yielded a significantly higher Ki value for guanazole inhibition than the wild type activity. The properties of partially purified ribonucleotide reductase from a somatic cell hybrid constructed from wild type and hydroxyurea-resistant cells was also examined. The Ki value for hydroxyurea inhibition of CDP reductase was intermediate between the Ki values of the parental lines and indicated a codominant expression of hydroxyurea-resistance at the enzyme level. The most logical explanation for these results is that the mutant cells contain a structurally altered ribonucleotide reductase whose activity is less sensitive to inhibition by hydroxyurea or guanazole.     

Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea-resistant S49 T-lymphoma cells

We investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea-resistant (HYUR) murine S49 T-lymphoma cell lines. Cell lines 10- to 40-fold more hydroxyurea-resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5- to 8-fold) and increased dNTP pools (up to 5-fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase. These characteristics remained stable when the cells were grown in the absence of hydroxyurea for up to 2 years. In both wild type and hydroxyurea-resistant cell populations synchronized by elutriation, dCTP and dTTP pools increased in S phase, whereas dATP and dGTP pools generally remained the same or decreased, suggesting that allosteric effector mechanisms were operating to regulate pool sizes. Additionally, CDP reductase activity measured in permeabilized cells increased in S phase in both wild type and hydroxyurea-resistant cells, suggesting a nonallosteric mechanism of increased ribonucleotide reductase activity during periods of active DNA synthesis. While wild type S49 cells could be arrested in the G1 phase of the cell cycle by dibutyryl cyclic AMP, hydroxyurea-resistant cell lines could not be arrested in the G1 phase by exogenous cyclic AMP or agents that elevate the concentration of endogenous cyclic AMP. These data suggest that cyclic AMP-generated G1 arrest in S49 cells might be mediated by the M2 subunit of ribonucleotide reductase.     

Iron deprivation decreases ribonucleotide reductase activity and DNA synthesis

The effects of the iron-chelator, desferrioxamine, and monoclonal antibodies against transferrin receptors of DNA synthesis and ribonucleotide reductase activity were examined in human leukemia K562 cells. Treatment of the cells with desferrioxamine resulted in decreases of ribonucleotide reductase activity, DNA synthesis, and cell growth. Exposure of the cells to anti-transferrin receptor antibody, 42/6, which blocks iron supplement into cells caused decreases of ribonucleotide reductase activity and DNA synthesis, in a parallel fashion. Decreases of ribonucleotide reductase activity and DNA synthesis by 42/6 were restored by the addition of ferric nitriloacetate. These results indicate that ribonucleotide reductase activity is dependent on the iron-supply and also regulates cell proliferation.     

Inhibition of mammalian ribonucleotide reductase by cis-diamminedichloroplatinum(II).

Ribonucleotide reductase is a highly regulated, rate-limiting activity in the synthesis of DNA. A previous study has shown that the Escherichia coli enzyme is inhibited by the clinically important antitumor agent cis-diamminedichloroplatinum(II) (DDP), and this has led to the hypothesis that ribonucleotide reductase is an important site of action for this chemotherapeutic agent. This hypothesis has been directly tested in this investigation. We observed that DDP inhibits the mammalian ribonucleotide reductase, with 50% inhibition occurring at 0.3 mM. Unlike the E. coli enzyme where only one of the two protein components is targeted by DDP, we observed that both of the mammalian proteins (R1 and R2) were sites for the inhibitory activity of the drug. Colony-forming experiments, enzyme activity studies, and analyses of R1 and R2 message levels in mutant cell lines containing either high levels of ribonucleotide reductase activity or exhibiting resistance to the cytotoxic effects of DDP were used to further investigate the potential role of ribonucleotide reductase in DDP cytotoxic action and drug resistance. These studies did not support a hypothesis formulated in the earlier investigation that inhibition of ribonucleotide reductase is an important component of DDP cytotoxic activity or that it is a major participant in DDP resistance mechanisms. From a biological point of view, DDP is a very active drug, and in addition to its cytotoxic effects it is capable of inducing a variety of cellular changes. Whether or not the inhibition of mammalian ribonucleotide reductase activity that we have described in this study plays a role in mediating any of these other effects remains to be determined.     

Selection and characterization of mutant S49 T-lymphoma cell lines resistant to phosphonoformic acid: evidence for inhibition of ribonucleotide reductase

Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T-lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild-type S49 cells. However, one line (PFA 3-9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase alpha activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3-9 cells decreased to wild-type control levels, and the CDP reductase activity of wild-type cells was also greatly reduced when PFA (2-3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild-type or PFA 3-9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.     

Ribonucleotide reductase activity and deoxyribonucleoside triphosphate metabolism during the cell cycle of S49 wild-type and mutant mouse T-lymphoma cells

We investigated deoxyribonucleoside triphosphate metabolism in S49 mouse T-lymphoma cells synchronized in different phases of the cell cycle. S49 wild-type cultures enriched for G1 phase cells by exposure to dibutyryl cyclic AMP (Bt2cAMP) for 24 h had lower dCTP and dTTP pools but equivalent or increased pools of dATP and dGTP when compared with exponentially growing wild-type cells. Release from Bt2cAMP arrest resulted in a maximum enrichment of S phase occurring 24 h after removal of the Bt2cAMP, and was accompanied by an increase in dCTP and dTTP levels that persisted in colcemid-treated (G2/M phase enriched) cultures. Ribonucleotide reductase activity in permeabilized cells was low in G1 arrested cells, increased in S phase enriched cultures and further increased in G2/M enriched cultures. In cell lines heterozygous for mutations in the allosteric binding sites on the M1 subunit of ribonucleotide reductase, the deoxyribonucleotide pools in S phase enriched cultures were larger than in wild-type S49 cells, suggesting that feedback inhibition of ribonucleotide reductase is an important mechanism limiting the size of deoxyribonucleoside triphosphate pools. The M1 and M2 subunits of ribonucleotide reductase from wild-type S49 cells were identified on two-dimensional polyacrylamide gels, but showed no significant change in intensity during the cell cycle. These data are consistent with allosteric inhibition of ribonucleotide reductase during the G1 phase of the cycle and release of this inhibition during S phase. They suggest that the increase in ribonucleotide reductase activity observed in permeabilized S phase-enriched cultures may not be the result of increased synthesis of either the M1 or M2 subunit of the enzyme.     

Early induction of ribonucleotide reductase gene expression by transforming growth factor beta 1 in malignant H-ras transformed cell lines.

Previous investigations have indicated that the suppression of proliferation by transforming growth factor (TGF) beta 1 is often lost upon cellular transformation, and that proliferation of some tumors is stimulated by TGF-beta. The present study provides the first observation of a link between TGF-beta 1 regulation of this process and alterations in the expression of ribonucleotide reductase, a highly controlled rate-limiting step in DNA synthesis. A series of radiation and T24-H-ras-transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was evaluated for TGF-beta 1 induced alterations in ribonucleotide reductase M1 and M2 gene expression. Early increases in M1 and/or M2 message and protein levels were observed only in malignant cell lines. The TGF-beta 1 induced changes in M1 and/or M2 gene expression occurred prior to any detectable changes in the rates of DNA synthesis, supporting the novel concept that ribonucleotide reductase gene expression can be elevated by TGF-beta 1 without altering the proportion of cells in S phase. T24-H-ras-transformed 10T1/2 cells were transfected with a plasmid containing the coding region of TGF-beta 1 under the control of a zinc-sensitive metallothionein promoter. When these cells were cultured in the presence of zinc, a large induction of TGF-beta 1 message was observed within 1 h. Both M1 and M2 genes were also induced, with increased mRNA levels appearing 2 h after zinc treatment, or 1 h after TGF-beta 1 message levels were clearly elevated. In total, the data suggests a mechanism of autocrine stimulation of malignant cells by TGF-beta 1, in which early alterations in the regulation of ribonucleotide reductase may play an important role.