Inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation.

We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-alpha, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis.

Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis.

Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, required for DNA synthesis. Ribonucleotide reductase consists of two dissimilar protein components often called M1 and M2, which are independently regulated during cell proliferation. The M1 component contains multiple effector binding sites and is responsible for the complex allosteric regulation of the enzyme, whereas the M2 protein contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction. Since the reaction is rate limiting for DNA synthesis, ribonucleotide reductase plays an important role in regulating cell division, and hence, cell proliferation. There are many inhibitors of ribonucleotide reductase and perhaps the most valuable one from a cell biology, biochemistry, and clinical point of view is the hydroxamic acid, hydroxyurea. This drug has also been very useful as a selective agent for isolating a variety of mammalian mutant cell lines altered in ribonucleotide reductase gene expression. Regulatory, structural, and biological characteristics of ribonucleotide reductase are reviewed, including evidence that ribonucleotide reductase, particularly the M2 protein, has an important early role to play in tumor promotion. In addition, modifications in the expressions of genes altered in hydroxyurea-resistant mutants and cultured in the absence or presence of hydroxyurea are discussed, with emphasis on changes in M2 protein, M1 protein, and the iron-storage protein ferritin.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Molecular mechanisms responsible for the drug-induced posttranscriptional modulation of ribonucleotide reductase levels in a hydroxyurea-resistant mouse L cell line.

Ribonucleotide reductase, which catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, is the rate-limiting enzyme in DNA synthesis. The enzyme consists of two nonidentical subunits called M1 and M2, both of which are required for activity. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. Previously, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyurea. This mutant cell line contains elevated quantities of both proteins M1 and M2 as a result of corresponding increases in the levels of mRNAs for both subunits. Interestingly, both M1 and M2 protein levels were further elevated when mutant cells were cultured in the presence of hydroxyurea, and this elevation was not accompanied by increases in their corresponding mRNAs. These results indicated that hydroxyurea can modulate ribonucleotide reductase expression posttranscriptionally. In this report, we show that the level of both subunits of ribonucleotide reductase responds to hydroxyurea in a drug concentration dependent manner. Furthermore, results from kinetic studies indicate that protein M2 levels rise much more rapidly than protein M1. Pulse-chase experiments indicated that the half-lives of both the M1 and M2 polypeptides are increased by approximately 2-fold when the mutant cells are cultured in the presence of hydroxyurea. We also present evidence indicating that exposure of these cells to hydroxyurea leads to a relatively slow but specific increase in the rate of biosynthesis of both proteins M1 and M2, as assayed by pulse labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanisms of drug resistance involving ribonucleotide reductase: hydroxyurea resistance in a series of clonally related mouse cell lines selected in the presence of increasing drug concentrations.

Mammalian ribonucleotide reductase is a highly regulated, rate-limiting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins often called M1 and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucleotide reductase by interacting with the M2 component specifically at a unique tyrosyl free radical. To obtain further information about drug resistance mechanisms, we have used M1 and M2 complementary DNAs and monoclonal antibodies to investigate the properties of a series of clonally related drug-resistant mouse cell lines, selected by a step-wise procedure for increasing levels of resistance to the cytotoxic effects of hydroxyurea. Several interesting mechanisms have been identified. Each successive drug selection step leading to the isolation of highly resistant cells was accompanied by stable elevations in cellular resistance and ribonucleotide reductase activities. The changes that occurred at each step involved the M2 component. A very early event, occurring at the first step in the selection process, was the amplification of the M2 gene accompanied by an increase in M2 messenger RNA. Although cellular resistance and M2 protein levels increased significantly during drug selection, only a modest change in M2 gene copy number was observed after the initial selection step. Analysis of wild type, moderately resistant, and highly resistant cells indicated that, in addition to M2 gene amplification, posttranscriptional modification also occurred during drug selection. This second mechanism was not due to alterations in protein M2 half-life, but involved an increase in translational efficiency. By increasing the rate of M2 synthesis, without altering degradation rates, resistant cells were able to accumulate high levels of this key regulatory protein. Cells selected for the ability to proliferate in concentrations of drug as high as 4 mM exhibited changes that involved M2, without detectable changes to M1. These results provide further evidence that M1 and M2 levels are controlled by different mechanisms in mammalian cells. Eventually, however, cells required an elevation in the M1 protein, as well as the M2 protein, to survive in a hydroxyurea concentration of 5 mM. These results illustrate the complexity of the drug-resistant phenotype and provide further information about the molecular processes that lead to the development of cells resistant to low, intermediate, and high concentrations of hydroxyurea.

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.

Expression of H-ras correlates with metastatic potential: evidence for direct regulation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells.

Using three independent approaches, we studied the effects of H-ras on metastasis formation. Analysis of five in vitro-ras-transfected 10T1/2 clones with either flat or refractile morphologies revealed a relationship between metastatic potential, H-ras expression, and anchorage-independent growth. Four metastatic variants derived from a poorly metastatic, low-H-ras-expressing line all expressed high levels of H-ras RNA and grew efficiently in soft agar. Activation of H-ras expression in the metastatic tumors had occurred through amplification and rearrangement of H-ras sequences. In addition, preinduction of p21 synthesis in NIH 3T3 line 433, which contains v-H-ras under transcriptional control of the glucocorticoid-sensitive mouse mammary tumor virus long terminal repeat, significantly increased metastatic efficiency. Glucocorticoid treatment of normal or pEJ-transformed NIH 3T3 cells did not affect metastatic potential. These data reveal a direct relationship between ras expression and metastasis formation and suggest that metastatic and transformed phenotypes may be coregulated in ras-transformed 10T1/2 and NIH 3T3 cells.

Hydroxyurea-induced conversion of mammalian ribonucleotide reductase to a form hypersensitive to bleomycin.

Bleomycin is a commonly used chemotherapeutic agent known to cause extensive DNA damage. In this paper we show that bleomycin inhibits ribonucleotide reductase activity in mouse L-cells. The effectiveness of the drug is a result of its metal-chelating properties which enable it to inactivate the iron containing M2 subunit of the enzyme. A hydroxyurea-resistant mouse L-cell line was used to show that the degree of inhibition caused by bleomycin can be greatly enhanced if ribonucleotide reductase has been previously exposed in vivo or in vitro to agents, such as hydroxyurea, which destroy the tyrosine free radical of subunit M2. The increased effectiveness of bleomycin appears to result from a decrease in the stability of the iron center of protein M2 following exposure to hydroxyurea. These findings have important implications in terms of the use of bleomycin as an anticancer agent, especially in combination chemotherapy where it can be used with other drugs that act at ribonucleotide reductase.

Ribonucleotide reductase: an intracellular target for the male antifertility agent, gossypol.

Gossypol is a yellow phenolic compound which reversibly inhibits spermatogenesis making it one of the few effective male antifertility drugs. The cytotoxic effects of gossypol have been associated with its ability to irreversibly inhibit DNA synthesis by a previously unknown mechanism. The results of this study indicate that gossypol is a potent inhibitor of ribonucleotide reductase the rate limiting enzyme activity in DNA synthesis. Furthermore, in agreement with these enzyme studies, DNA synthesis in a hydroxyurea resistant cell line with high levels of ribonucleotide reductase activity showed increased resistance to gossypol when compared to wild type cells with normal levels of reductase activity. Ribonucleotide reductase is the first specific site of action documented for gossypol which can explain its recently described antiproliferative, cell cycle and toxic effects.