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.

DNA repair induced by various mutagens in rat hepatocyte primary cultures measured in the presence of hydroxyurea, guanazole or aphidicolin.

Guanazole and aphidicolin were chosen as candidates in the search for a selective, non-genotoxic inhibitor of DNA replication which could be used instead of hydroxyurea to measure DNA repair synthesis in rat hepatocyte primary cultures by liquid scintillation counting. The genotoxicity of these 3 chemicals was studied using the Salmonella/liver homogenate assay and the autoradiographic UDS test in hepatocytes. Hydroxyurea was positive in both of these assays. Guanazole and aphidicolin did not induce DNA repair in hepatocytes. Aphidicolin was not mutagenic for Salmonella typhimurium, whereas guanazole increased the revertant numbers of strain TA102 slightly. The incorporation of [3H]thymidine was measured by liquid scintillation to determine DNA repair induced by 2-acetylaminofluorene (2-AAF), aflatoxin B1, benzo[a]pyrene, cyclophosphamide, H2O2, 6-hydroxydopamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 4-nitroquinoline-N-oxide and UV irradiation in the presence of either 10 mM hydroxyurea, 15 mM guanazole or 0.015 mM aphidicolin. Aphidicolin had an inhibitory effect on DNA repair. Except for the 3 chemicals mentioned below, the sensitivity of the DNA repair measurement was the same, no matter whether hydroxyurea or guanazole was used to inhibit replicative DNA synthesis. In the presence of hydroxyurea, DNA repair synthesis was found at lower concentrations in the case of aflatoxin B1, due to differences in the solvent control values, and in the case of H2O2, possibly due to a synergistic effect between hydroxyurea and H2O2. Guanazole allowed the detection of DNA repair induced by MNNG at lower concentrations, probably because of an antagonistic effect between hydroxyurea and MNNG. Based on these results, it was concluded that guanazole, but not aphidicolin, could be used instead of hydroxyurea to measure DNA repair synthesis by liquid scintillation in rat hepatocyte primary cultures. Although guanazole does not completely fulfill the criteria for an ideal DNA replication inhibitor, it has the advantage of being less genotoxic than hydroxyurea, and also appears to have a smaller potential to falsify the results by interacting with the test compounds.

Drug-induced loss of unstably amplified genes.

Methotrexate-resistant R500 cells slowly lose amplified dihydrofolate reductase (dhrf) genes with biphasic kinetics when grown in the absence of methotrexate. Both phases of gene loss were markedly accelerated by subcytotoxic drug treatments. R500 cells were passed in low concentrations of cytotoxic drugs (inhibitors of ribonucleotide reductase, type I and type II topoisomerases, and polyamine synthesis). At each passage, relative dhfr gene copy number was determined by slot blot analysis. All of these drugs were able to induce rapid loss of dhfr gene dosage in the R500 cell population. The ability of these treatments to cause the rapid emergence of a cell population with substantially reduced dhfr gene dosage indicates that either the amplified genes or those cells with the highest levels of gene amplification are selectively targeted by low-level cytotoxic stress. The complex kinetics of amplified gene loss are suggestive of differential targeting of resistant cell subpopulations.

Herpes simplex type 1 ribonucleotide reductase. Mechanism studies with inhibitors.

Several known inhibitors of mammalian ribonucleotide reductase were studied for their interactions with herpes simplex virus type 1 (HSV-1) ribonucleotide reductase. MAIQ (4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone) produced apparent inactivation of HSV-1 ribonucleotide reductase. Only catalytically cycling, not resting, enzyme could be inactivated. Double reciprocal replots of the rates of inactivation versus the concentration of MAIQ indicated that a reversible complex with the enzyme was formed prior to inactivation. In the presence of 10 microM CDP, the maximum rate of inactivation was 20 per h (t1/2 = 3 min). The half-maximum rate was achieved at about 15 microM MAIQ. INOX (periodate-oxidized inosine) also appeared to inactivate HSV-1 ribonucleotide reductase. In contrast to MAIQ, it readily inactivated resting as well as cycling enzyme. CDP retarded the rates of inactivation by INOX. An initial reversible complex between INOX and enzyme was not detectable under the conditions used. IMPY (2,3-dihydro-1H-pyrazolo(2,3-a)imidazole) and guanazole (3,5-diamino-1,2,4-triazole) produced reversible inhibition. Although the data with both inhibitors were most consistent with the noncompetitive inhibition model (versus CDP), the data with guanazole were also marginally consistent with the uncompetitive model.

Studies on the differential mechanisms of inhibition of ribonucleotide reductase by specific inhibitors of the non-heme iron subunit.

The data presented here show that while the non-heme iron subunit of ribonucleotide reductase is inhibited by IMPY, hydroxyurea and MAIQ, the mechanism of inhibition by hydroxyurea and IMPY is distinct from that for MAIQ. This difference in mechanisms is expressed not only in effects of iron-chelating agents on enzyme activity and of L1210 cell growth in culture, but also in differences in responses to catalase and peroxidase. Further, these data suggest that the inhibition of reductase activity by IMPY and IMPY/iron-chelator occurs through different pathways. The same conclusion can be drawn for the inhibition of reductase by hydroxyurea and hydroxyurea/iron-chelator. It is clear that additional studies will be required to understand the exact mechanism by which hydroxyurea or IMPY and the thiosemicarbazones inhibit the non-heme iron component of ribonucleotide reductase. It will also be necessary to better define the pathways of inhibition of reductase activity by IMPY and the IMPY/iron-chelator combination (or hydroxyurea and hydroxyurea/iron-chelator combination). From these studies may come information which will allow these antitumor agents to have greater utilization in the clinical management of neoplastic diseases.

A series of N-carbamoyloxyurea resistant cell lines with alterations in ribonucleotide reductase: lack of coordination in pyrimidine and purine reductase activity.

N-Carbamoyloxyurea is cytotoxic for cells in culture and, like hydroxyurea and guanazole, the drug is an effective inhibitor of mammalian ribonucleotide reductase and thus DNA synthesis. In addition to ribonucleotide reductase, N-carbamoyloxyurea has a second site of action which also appears to be in the pathway of DNA synthesis. A series of drug-resistant cell lines, which contain alterations in ribonucleotide reduction, have been sequentially selected in the presence of increasing concentrations of N-carbamoyloxyurea. CDP and ADP reductase activities in these drug-resistant lines have been investigated and two types of alterations have been identified: elevated levels of enzyme activity with wild-type sensitivity to drug and altered levels of reductase with reduced drug sensitivity, probably owing to structural modification of the enzyme. Furthermore, N-carbamoyloxyurea resistant lines contain another alteration as well, presumably at a second site of drug action. They are also cross-resistant to hydroxyurea and guanazole, and studies on enzyme activity levels support our previous findings with cells selected for resistance to hydroxyurea, which showed changes in CDP reductase activity are not always coordinated with changes in ADP reductase. Although several possibilities exist, these observations are most easily explained by the existence of independent enzyme substrate binding subunits which are regulated by different mechanisms. Moreover, increases in cellular resistance were accompanied by significant increases in CDP but not ADP reductase, suggesting that an ability to maintain an adequate level of CDP reductase activity is especially important to achieve resistance to DNA synthesis inhibitors like N-carbamoyloxyurea, hydroxyurea, and guanazole.

N-carbamoyloxyurea-resistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity.

We describe the isolation and characterization of a Chinese hamster ovary cell line selected for resistance to N-carbamoyloxyurea. Using the mammalian cell permeabilization assay developed in our laboratory, a detailed analysis of the target enzyme, ribonucleotide reductase (EC 1.17.4.1), was carried out. Both drug-resistant and parental wild-type cells required the same optimum conditions for enzyme activity. The Ki values for N-carbamoyloxyurea inhibition of CDP reduction were 2.0 mM for NCR-30A cells and 2.3 mM for wild-type cells, while the Ki value for ADP reduction was 2.3 mM for both cell lines. Although the Ki values remained essentially unchanged, the Vmax values for NCR-30A cells were 1.01 nmoles dCDP formed/5 X 10(6) cells/hour and 1.83 nmoles dADP/5 X 10(6) cells/hour, while those for the wild-type cells were 0.49 nmoles dCDP produced/5 X 10(6) cells/hour and 1.00 nmoles dADP/5 X 10(6) cells/hour. This approximate twofold increase in reductase activity as least partially accounts for a 2.6-fold increase in D10 value for cellular resistance to N-carbamoyloxyurea exhibited by NCR-30A cells. The NCR-30A cell line was also cross-resistant to the antitumor agents, hydroxyurea and guanazole. No differences in Ki values for inhibition of CDP and ADP reduction by these two drugs were detected and cellular resistance could be entirely accounted for by the elevation in activity of the reductase in the NCR-30A cell line. The properties of N-carbamoyloxyurea-resistance cells indicate they should be useful for further investigations into the regulation of mammalian enzyme activity.

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.

A simple method for long-term drug infusion in mice: evaluation of guanazole as a model (38488).

A simplified technique for iv infusion in unrestricted DBA/2-J inbred mice has been described. The method, which involves direct cannulation of the tail vein with polyethylene tubing, is suitable for routine use. Guanazole, an antileukemic agent with a short plasma half-life, was evaluated as a model compound. After administration for 47 hr at the rate of 0.3 ml/hr, guanazole (30 mg/ml) caused a marked inhibition of incorporation of 14 C-uridine, administered 15 min before sacrifice, primarily into DNA of spleen, thymus and bone marrow in decreasing order. Inhibition of incorporation into RNA was less marked but followed a similar pattern. The effects on the incorporation of uridine in nucleic acids of kidney, heart and brain were minimal. Increased incorporations into RNA and DNA occurred in liver. The data for the hemopoietic and lymphoid organs, namely spleen, thymus and marrow, are consistent with the reported immunosuppressive and mylelosuppressive effects of the drug and also with the inhibition of ribonucleoside diphosphate reducaste by guanazole.