A rapid assay for cdp reductase activity in mammalian cell extracts.
TL;DR: An assay for CDP reductase activity is described that utilizes standard methods of borate chromatography, which has the advantages of being more rapid and sensitive than previously described assay procedures, but the disadvantage of being useful only for reduction of cytidine nucleotides.
About: This article is published in Analytical Biochemistry.The article was published on 1970-03-01. It has received 236 citations till now. The article focuses on the topics: CDP reductase activity.
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TL;DR: No gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells, suggesting that cytostasis by activated Macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.
Abstract: Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega-methyl-L-arginine, or N omega-amino-L-arginine. L-Arginine, but not D-arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.
446 citations
Cites methods from "A rapid assay for cdp reductase act..."
...After the preincubation described above, RR activity was assayed by the conversion of radioactive CDP to dCDP, measured as described, with minor modifications (27-29)....
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...Dowex 1-borate was prepared as described with minor modifications (28)....
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TL;DR: The findings demonstrate the superiority of Triapine over hydroxyurea as an anticancer agent and suggest that prevention byTriapine of repair of DNA lesions created by agents that damage DNA may result in efficacious drug combinations for the treatment of cancer.
340 citations
TL;DR: In this article, the authors identify an essential function of the conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 in intracellular iron trafficking and sensing.
259 citations
TL;DR: Phytohaemagglutinin‐stimulated lymphocytes from patients with chronic iron deficiency showed lower levels of all four deoxyribonucleoside triphosphates than normal lymphocytes, suggested that this may be due to reduced ribonucleotide reductase activity of the iron‐deficient cells.
Abstract: Summary. Desferrioxamine (10-3m) caused a fall in the deoxyadenosine triphosphate level after 4 h incubation in normal phytohaemagglutinin-stimulated lymphocytes. There was a rise in the concentrations of the other three deoxyribonucleoside triphosphates (deoxythymidine-, deoxycytidine- and deoxyguanosine-triphosphate). The changes are similar to those caused by hydroxyurea, a known inhibitor of ribonucleotide reductase. Desferrioxamine (10-3m) was found to inhibit human lymphocyte ribonucleotide reductase to a mean of 11% of control activity after 45 min incubation. Both drugs, desferrioxamine and hydroxyurea, inhibited incorporation of [3H]thymidine DNA into lymphocytes in the presence or absence of deoxyuridine, and inhibited production of lymphocytic thymidine kinase, having opposite effects to methotrexate on both [3H]thymidine incorporation and thymidine kinase activity. Phytohaemagglutinin-stimulated lymphocytes from patients with chronic iron deficiency showed lower levels of all four deoxyribonucleoside triphosphates than normal lymphocytes. It is suggested that this may be due to reduced ribonucleotide reductase activity of the iron-deficient cells.
232 citations
Journal Article•
TL;DR: Contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.
Abstract: In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 microM dFdCyd, as compared with the KB-Gem clone IC50 of 32 microM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3-fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.
226 citations
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Journal Article•
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
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TL;DR: The results are interpreted in terms of metabolic control of the biosynthesis of deoxynucleotides by these nucleoside triphosphates serving as regulatory effectors.
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TL;DR: CDP and UDP inhibit the reduction of each other in a competitive manner, an indication that the same enzyme is involved in both reactions.
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