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.     

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 novel transforming growth factor-beta 1 responsive cytoplasmic trans-acting factor binds selectively to the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability.

Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-beta 1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3'-untranslated region of R2 mRNA and did not bind to the 5'UTR, the coding region of the R2 message or to the 3'UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-beta 1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3'-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-beta 1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-beta 1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-beta 1 mediated stabilization of R2 message involves a specific interaction of a p75-trans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.     

Enzymatically active mammalian ribonucleotide reductase exists primarily as an alpha6beta2 octamer

Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an alpha(2)beta(2) complex consisting of R1 (alpha) and R2 (beta) proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (alpha(6)beta(2)). The newly discovered alpha(6)beta(2) complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.     

Ribonucleotide reductases: the evolution of allosteric regulation.

Ribonucleotide reductases catalyze in all living organisms the production of the deoxyribonucleotides required for DNA replication and repair. Their appearance during evolution was a prerequisite for the transition from the "RNA world," where RNA sufficed for both catalysis and information transfer, to today's situation where life depends on the interplay among DNA, RNA, and protein. Three classes of ribonucleotide reductases exist today, widely differing in their primary and quaternary structures but all with a highly similar allosteric regulation of their substrate specificity. Here, I discuss the diversities between the three classes, describe their allosteric regulation, and discuss the evidence for their evolution. The appearance of oxygen on earth provided the likely driving force for enzyme diversification. From today's characteristics of the three classes, including their allosteric regulation, I propose that the anaerobic class III reductases with their iron-sulfur cluster and the requirement for S-adenosylmethionine for the generation of a glycyl protein free radical are the closest relatives to an ancestor ribonucleotide reductase.     

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.     

Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.     

Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells

Recently, a homologue of the small subunit of mammalian ribonucleotide reductase (RNR) was discovered, called p53R2. Unlike the well characterized S phase-specific RNR R2 protein, the new form was induced in response to DNA damage by the p53 protein. Because the R2 protein is specifically degraded in late mitosis and absent in G0/G1 cells, the induction of the p53R2 protein may explain how resting cells can obtain deoxyribonucleotides for DNA repair. However, no direct demonstration of RNR activity of the p53R2 protein was presented and furthermore, no corresponding RNR large subunit was identified. In this study we show that recombinant, highly purified human and mouse p53R2 proteins contain an iron-tyrosyl free radical center, and both proteins form an active RNR complex with the human and mouse R1 proteins. UV irradiation of serum-starved, G0/G1-enriched mouse fibroblasts, stably transformed with an R1 promoter-luciferase reporter gene construct, caused a 3-fold increase in luciferase activity 24 h after irradiation, paralleled by an increase in the levels of R1 protein. Taken together, our data indicate that the R1 protein can function as the normal partner of the p53R2 protein and that an R1-p53R2 complex can supply resting cells with deoxyribonucleotides for DNA repair.     

An active ribonucleotide reductase from Arabidopsis thaliana cloning, expression and characterization of the large subunit.

In all living organisms, deoxyribonucleotides, the DNA precursors, are produced by reduction of the corresponding ribonucleotides catalyzed by ribonucleotide reductase. In mammals as in Escherichia coli, the enzyme consists of two proteins. Protein R1 is the proper reductase as it contains, in the substrate binding site, the reducing active cysteine pair. Protein R2 provides a catalytically essential organic radical. Here we report the cloning, expression, purification and characterization of protein R1 from Arabidopsis thaliana. Expression in E. coli was made possible by coexpression of tRNAArg4 which is required for the utilization of AGA and AGG as codons for arginines. Protein R1 shows extensive similarities with protein R1 from mammals: (a) it shows 69% amino-acid sequence identity to human and mouse R1 protein; (b) it is active during CDP reduction by dithiothreitol, in the presence of protein R2 [Sauge-Merle, S., Laulhère, J.-P., Coves, J., Ménage, S., Le Pape, L. & Fontecave, M. (1997) J. Biol. Inorg. Chem. 2, 586-594]; (c) activity is stimulated by thioredoxin and ATP and is inhibited by dATP, showing that as in the mammalian enzyme, the plant ribonucleotide reductase seems to be allosterically regulated by positive (ATP) and negative (dATP) effectors.     

Transforming growth factor-beta 1 mediated alterations in ribonucleotide reductase gene expression in BALB/c 3T3 fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) stimulated DNA synthesis (3-fold) in BALBc/3T3 fibroblasts following 24 hours of growth factor exposure. Since ribonucleotide reductase is important for the coordination of DNA synthesis and cell proliferation, we investigated the hypothesis that cells like BALB/c 3T3, which are TGF-beta 1 responsive, would exhibit modifications in expression of the gene for ribonucleotide reductase following growth factor treatment. We observed 2.6, 4.1, and 4.8-fold increases in ribonucleotide reductase activity following TGF-beta 1 exposure for 6, 12, and 24 hours, respectively. Increased ribonucleotide reductase R2 gene expression (3, 3.7, and 4.5-fold) and R1 gene expression (2,2.5, and 2.6-fold) were observed following 6, 12, and 24 hours of TGF-beta 1 treatment, respectively. Western blots indicated 2.2, 3.1, and 4.1-fold increases in protein R2 levels at 6, 12, and 24 hours exposure to TGF-beta 1, whereas 2.6 and 3.3-fold elevations in R1 protein levels were observed at 12 and 24 hours post-TGF-beta 1 exposure. These TGF-beta 1 mediated modifications in ribonucleotide reductase gene expression occurred, in part, prior to any detectable changes in the rate of DNA synthesis, demonstrating alterations in the normal regulation of ribonucleotide reductase. Furthermore, these alterations could be markedly reduced by prolonged pretreatment with 12-O-tetradecanoylphorbol-13-acetate (R2 gene expression increased by only 1.3, 1.5 and 2.3-fold after 6, 12, and 24 hours of TGF-beta 1 treatment, respectively), suggesting a role for a protein kinase C pathway in the TGF-beta 1 regulated changes in ribonucleotide reductase gene expression. These results indicate for the first time that TGF-beta 1 can regulate the expression of the two genes for ribonucleotide reductase in BALB/c 3T3 fibroblasts, and suggest that regulation of these genes plays an important role in critical events involved in growth factor modulation of normal and transformed cell proliferation.     

Relationships between sensitivity to hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIO) and ribonucleotide reductase RNR2 mRNA levels in strains of Saccharomyces cerevisiae

Ribonucleotide reductase is responsible for providing the deoxyribonucleotide precursors for DNA synthesis. In most species the enzyme consists of a large and a small subunit, both of which are required for activity. In mammalian cells, the small subunit is the site of action of several antitumor agents, including hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ). The mRNA levels for the small subunit of ribonucleotide reductase (RNR2) and sensitivity to hydroxyurea and MAIQ were determined in four strains of the yeast, Saccharomyces cerevisiae. Two strains exhibited significantly different sensitivities to both hydroxyurea and MAIQ, which closely correlated with differences in the levels of RNR2 mRNA. These results are consistent with recent observations with mammalian cells in culture, and indicate that a common mechanism of resistance to hydroxyurea and related drugs occurs through the elevation in ribonucleotide reductase message levels. A transplason mutagenized strain with marked structural modifications in RNR2 DNA and mRNA showed an extreme hypersensitivity to hydroxyurea but not to MAIQ, providing evidence that the two drugs do not inhibit the RNR2 subunit by the same mechanism. In addition, a yeast strain isolated for low but reproducible resistance to MAIQ exhibited a sensitivity to hydroxyurea similar to the parental wild-type strain, supporting the idea that the two drugs inhibit the activity of RNR2 by unique mechanisms. These yeast strains provide a useful approach for further studies into the regulation of eucaryotic ribonucleotide reduction and drug resistance mechanism involving a key rate-limiting step in DNA synthesis.     

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.     

Cloning and characterization of ribonucleotide reductase from Chlamydia trachomatis

In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.     

The prototypic class Ia ribonucleotide reductase from Escherichia coli: still surprising after all these years

RNRs (ribonucleotide reductases) are key players in nucleic acid metabolism, converting ribonucleotides into deoxyribonucleotides. As such, they maintain the intracellular balance of deoxyribonucleotides to ensure the fidelity of DNA replication and repair. The best-studied RNR is the class Ia enzyme from Escherichia coli, which employs two subunits to catalyse its radical-based reaction: β2 houses the diferric-tyrosyl radical cofactor, and α2 contains the active site. Recent applications of biophysical methods to the study of this RNR have revealed the importance of oligomeric state to overall enzyme activity and suggest that unprecedented subunit configurations are in play. Although it has been five decades since the isolation of nucleotide reductase activity in extracts of E. coli, this prototypical RNR continues to surprise us after all these years.     

Direct photoaffinity labeling of the catalytic site of mouse ribonucleotide reductase by CDP

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.     

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.