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.     

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.     

Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure.

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates but not deoxyribonucleotide triphosphates. Ribonucleotide reductase is the only enzyme known to catalyze the direct conversion of a ribonucleotide to a deoxyribonucleotide. Hydroxyurea inhibits ribonucleotide reductase by inactivating the tyrosine free radical present in the small subunit of the enzyme. In this report, we show that Chlamydia trachomatis growth is inhibited by hydroxyurea in both wild-type mouse L cells and hydroxyurea-resistant mouse L cells. Hydroxyurea was used as a selective agent in culture to isolate, by a stepwise procedure, a series of C. trachomatis isolates with increasing levels of resistance to the cytotoxic effects of the drug. One of the drug-resistant C. trachomatis isolates (L2HR-10.0) was studied in more detail. L2HR-10.0 retained its drug resistance phenotype even after passage in the absence of hydroxyurea for 10 growth cycles. In addition, L2HR-10.0 was cross resistant to guanazole, another inhibitor of ribonucleotide reductase. Results obtained from hydroxyurea inhibition studies using various host cell-parasite combinations indicated that inhibition of host cell and C. trachomatis DNA synthesis by hydroxyurea can occur but need not occur simultaneously. Crude extract prepared from highly purified C. trachomatis reticulate bodies was capable of reducing CDP to dCDP. The CDP reductase activity was not inhibited by monoclonal antibodies to the large and small subunits of mammalian ribonucleotide reductase, suggesting that the activity is chlamydia specific. The CDP reductase activity was inhibited by hydroxyurea. Crude extract prepared from drug-resistant L2HR-10.0 reticulate bodies contained an elevation in ribonucleotide reductase activity. In total, our results indicate that C. trachomatis obtains the precursors for DNA synthesis as ribonucleotides with subsequent conversion to deoxyribonucleotides catalyzed by a chlamydia-specific ribonucleotide reductase.     

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)     

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.     

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.     

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.).     

Transient elevation of ribonucleotide reductase activity, M2 mRNA and M2 protein in BALB/c 3T3 fibroblasts in the presence of 12-O-tetradecanoylphorbol-13-acetate

A rapid elevation of ribonucleotide reductase activity was observed with BALB/c 3T3 fibroblasts within 1/2 to 1 hour treatment with 0.1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA). This increase in activity was transient, and returned to about normal levels within 24 to 48 hours. Northern analysis of the two components of ribonucleotide reductase showed a slight transient elevation of M1 mRNA and a marked transient elevation of M2 mRNA after 1/2 hour TPA treatment. As a positive control, ornithine decarboxylase message levels were also observed to be transiently elevated following identical treatment with TPA. Western blot analysis with M1 and M2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the transient elevation of the M2 but not the M1 protein during treatment with 0.1 microM TPA. This first demonstration that the tumor promotor, TPA, can cause rapid and transient alterations in ribonucleotide reductase suggests that the enzyme, particularly the M2 component, may play an important role in the critical events involved in the process of tumor promotion.     

Ribonucleotide reductase, a possible agent in deoxyribonucleotide pool asymmetries induced by hypoxia

While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O(2) is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O(2) is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension.     

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.     

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.     

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.     

Manganese-dependent ribonucleotide reductase ofPropionibacterium freudenreichii subsp.shermanii: Partial purification, characterization, and role in DNA biosynthesis

LikeLactobacillus leichmanii, Rhizobium meliloti, andEuglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids,P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with partially purified ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein components, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficientP. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.     

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.     

Deoxyribonucleotide biosynthesis in yeast: assay and properties of ribonucleotide reductase in permeabilized Saccharomyces cerevisiae cells

Yeast cells permeabilized by freeze-thaw cycles in a sorbitol-containing medium provide an experimentally favorable system for the study of ribonucleotide reduction in a small number of cells or in mutant strains. Ribonucleotide reductase activities determined in such cells are about twice those found in cell extracts but properties of the enzyme, except pH optimum, are closely comparable in both assay procedures. In contrast with other organisms, the activities measured in permeabilized cells from both diploid or haploid strains exceed the demand for deoxyribonucleotide formation during replication of the yeast genome. The method has been applied to yeast cultures growing in the presence of the ribonucleotide reductase inhibitor hydroxyurea and a twofold increase of enzyme activity has been established in such cells. On the other hand, analysis of a series of hus mutants, selected for hydroxyurea sensitivity in the laboratory of Singer and Johnston did not reveal obvious alterations of the enzyme vs the parental strains, suggesting that the hus phenotype may be due to lesions other than in ribonucleotide reductase.     

Manganese-dependent ribonucleotide reductase from Propionibacterium freudenreichii subsp. Shermanii: partial purification, characteristics and role in DNA biosynthesis

Like Lactobacillus leichmanii, Rhizobium meliloti, and Euglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids, P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with crude ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein subunits, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficient P. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.