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

1992 ◽  
Vol 70 (12) ◽  
pp. 1332-1338 ◽  
Author(s):  
Christine S. M. Chiu ◽  
Arthur K. Chan ◽  
Jim A. Wright
Keyword(s):  
Drug Resistance ◽  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Mutant Cell ◽  
Antitumor Agent ◽  
Resistance Mechanisms ◽  
Point Of View ◽  
Cytotoxic Action ◽  
Cytotoxic Effects ◽  
Ribonucleotide Reductase Activity

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.Key words: cisplatin, ribonucleotide reductase, drug resistance, hydroxyurea.

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells

1983 ◽  
Vol 3 (8) ◽  
pp. 741-748 ◽  
Author(s):  
Jim A. Wright ◽  
Joseph G. Cory
Keyword(s):  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Antitumor Agent ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Wild Type ◽  
Type Analysis ◽  
Similar Mode ◽  
Ribonucleotide Reductase Activity ◽  
Effector Binding

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NcR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NcR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heine-ironcontaining (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NcR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.

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

1990 ◽  
Vol 68 (12) ◽  
pp. 1364-1371 ◽  
Author(s):  
Jim A. Wright ◽  
Arthur K. Chan ◽  
Bob K. Choy ◽  
Robert A. R. Hurta ◽  
Grant A. McClarty ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Drug Resistance ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Cell Biology ◽  
De Novo ◽  
Mutant Cell ◽  
Resistance Mechanisms ◽  
M2 Protein ◽  
Iron Storage

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. Several regulatory models are presented, including a model showing the relationships between M2 protein levels, deoxyribonucleotide pools, and DNA synthesis, and a model demonstrating a linkage between M2 and ferritin proteins in regulating DNA synthesis in normal and hydroxyurea-resistant mammalian cells.Key words: DNA synthesis, cell proliferation, ribonucleotide reductase, drug resistance.

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity

1990 ◽  
Vol 10 (11) ◽  
pp. 5688-5699
Author(s):  
B E Wojcik ◽  
J J Dermody ◽  
H L Ozer ◽  
B Mun ◽  
C K Mathews
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster Ovary ◽  
Reductase Activity ◽  
Chinese Hamster ◽  
Temperature Shift ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Dntp Pool ◽  
Ribonucleotide Reductase Activity

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

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