Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0027651 (tumor)
685,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonucleotide reductase provides the four deoxyribonucleotides required for the synthesis of DNA. In this study, we examined the hypothesis that alterations in the regulation of ribonucleotide reductase activity may be necessary to provide the deoxyribonucleotides required for DNA repair, following exposure of mammalian cells to DNA damaging agents such as the antitumor agent chlorambucil. We observed a marked transient increase in ribonucleotide reductase activity within 2 h of exposing BALB/c 3T3 mouse cells to DNA damaging concentrations of chlorambucil. Northern blot analysis showed that elevations in activity were accompanied by transient increases in the mRNA levels of both genes (R1 and R2) that code for ribonucleotide reductase. Western blot analysis indicated that only the protein for the limiting component for enzyme activity, R2, was significantly elevated in chlorambucil treated cultures. The chlorambucil effects upon activity and regulation of ribonucleotide reductase occurred without any detectable changes in the rate of DNA synthesis, as would be expected if the elevation in enzyme activity is required for DNA repair. The chlorambucil-induced elevations in R1 and R2 message levels were blocked by treatment of cells with actinomycin D or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating the importance of the reductase transcriptional process in responding to the action of chlorambucil and providing evidence for the involvement of a protein kinase C pathway in the regulation of mammalian ribonucleotide reductase. In addition to the chlorambucil-induced elevations in enzyme activity, message, and protein levels, the drug was also shown to be an inhibitor of ribonucleotide reductase activity in cell-free preparations. Separation of ribonucleotide components on an affinity column followed by selective exposure of the protein components to chlorambucil showed that both R1 and R2 proteins were targets for chlorambucil, in keeping with the known alkylating abilities of the drug. These observations provide the first direct demonstration of a link between the regulation of mammalian ribonucleotide reductase and the process of DNA repair and contribute to our understanding of the mode of action of a class of drugs represented by chlorambucil, in which chemotherapeutic activity has been attributed to DNA damaging effects.
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PMID:Alterations in the activity and regulation of mammalian ribonucleotide reductase by chlorambucil, a DNA damaging agent. 155 13

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.
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PMID:Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. 171 30

Fotemustine is a highly reactive chloroethyl-nitrosourea anti-tumor drug that is currently undergoing phase III clinical trials in stage IV metastatic malignant melanoma. The drug is a potent alkylating agent and rapidly chloroethylates the active sites of the important thioproteins thioredoxin reductase (TR), glutathione reductase (GR) and ribonucleotide reductase (RR). These enzymes control ribonucleotide reduction and, consequently, DNA synthesis in the S phase of the cell cycle. Side effects are minor due to the rapid metabolism of the drug. [14C]Fotemustine exhibited a half-life of 90 min in the vascular system after the administration of 100 mg/m2. Fotemustine was shown to yield the volatile degradation product acetylene (a) in distilled water (4.1%/h), (b) in melanoma cell culture medium (MCDB) supplemented with 10% fetal calf serum (33%/h) and (c) in fotemustine-sensitive human melanoma cells in culture medium (70.5%/h). Due to its rapid metabolism and its low toxicity, high concentrations of fotemustine (55 x 10(-3) M) were injected directly into cutaneous and subcutaneous melanoma metastases (n = 36) of seven patients, resulting in minor necrosis followed by total remission of the metastases. Untreated metastases adjacent to the treated tumors were not affected by fotemustine, confirming that rapid local metabolism of this drug occurs only in the vicinity of injected tumors without producing any systemic effects.
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PMID:Local treatment of cutaneous and subcutaneous metastatic malignant melanoma with fotemustine. 176 Aug 62

Activity of replicase complex enzymes involving thymidine kinase (TK), ribonucleotide reductase (RR), DNA-polymerases alpha and beta as well as DNA synthesis and single breaks in DNA were studied during growth of P388 ascites tumor. Under these conditions the rate of DNA synthesis was distinctly decreased via salvage pathway and de novo. Single breaks were not detected in the preexistent DNA within various periods after transplantation of P338 leukemic cells. Retardation of DNA synthesis during tumor growth correlated with a decrease in TK, RR and DNA-polymerase alpha activities, while DNA-polymerase beta activity was markedly increased. Growth of melanoma B16 was accompanied by a decrease in content of ATP, ADP, NAD, phosphocreatine and phosphosaccharides as well as by an increase in the level of inorganic phosphates.
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PMID:[Changes in the replication apparatus and phosphorus-containing metabolite pool in experimental tumors in animals during development]. 181 11

The characteristic EPR doublet of tyrosine radicals of the growth-regulating enzyme ribonucleotide reductase was detected in human melanoma tissue grown in nude mice. This was possible through the use of an amelanotic melanoma that does not exhibit disturbing EPR signals from melanin. The content of tyrosine radicals is higher in young tumor tissues than in older ones. The clinically applied antimelanotic drug, 4-hydroxyanisole, inhibits ribonucleotide reductase in Ehrlich ascites tumor cells as demonstrated by a pronounced quenching of tyrosine radicals (IC50 = 5 microM). In amelanotic melanoma tissue tyrosine radicals of the enzyme are also quenched by 4-hydroxyanisole in concentrations down to 50 microM. Thus, the inactivation of ribonucleotide reductase, which provides deoxyribonucleotides for DNA synthesis, may be a hitherto unexpected mechanism for the antitumor action of 4-hydroxyanisole.
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PMID:Ribonucleotide reductase in melanoma tissue. EPR detection in human amelanotic melanoma and quenching of the tyrosine radical by 4-hydroxyanisole. 184 62

Tumor growth is primarily dependent on the fraction size of growing cells, their growth and proliferative rate, and the fractional cell death. In the present study, we focused specifically on the proliferative characteristics of squamous cell carcinomas of the head and neck region by using monoclonal antibodies and immunohistochemical methods. We studied two normally occurring antigens representative of cell proliferation: (1) ribonucleotide reductase, which is an independent cytoplasmatic enzyme and is intimately integrated in DNA synthesis, and (2) Ki-67, which is a nuclear antigen being expressed only in replicative cells. We also used intravenously injected bromodeoxyuridine (BRDU) for specific detection of tumor cells in the S phase of the cell cycle. In addition, in vivo injections of BRDU were also given to tumor-bearing mice to illustrate tumor cell kinetics by means of flow cytometry. The main observation was morphologic heterogeneity, with a high frequency of proliferative cell clusters in the cancer specimens interspersed among quiescent cells, which was demonstrated by the three monoclonal antibodies independent of each other. The experimental studies clearly visualized the transfer of BRDU throughout the tumor cell cycle. We conclude that immunohistochemical analysis provides valuable qualitative information on the proliferative pattern of tumor growth and, together with dynamic flow cytometry, may improve the clinical basis for individualized management of the cancer patient.
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PMID:Proliferative pattern of head and neck cancer. 195 1

Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase). 14C chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding 14C chloroethyl-thioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated beta-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with NADPH as electron donor and DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intracellularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase or glutathione reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.
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PMID:Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine. 206 1

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)
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PMID:Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis. 208 32

Treatment of Ehrlich ascites tumor cells with 2-difluoromethylornithine (F2MeOrn), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, resulted in depleted putrescine and spermidine content, and reduced growth rate. We have previously shown that adenine ribonucleotide levels are substantially increased in these polyamine-depleted cells. The present paper addresses the question whether the elevated ATP pool is accompanied by a concomitant increase in the dATP pool. If this is the case, the observed growth inhibition could be explained by the well-known dATP-mediated feedback inhibition of ribonucleotide reductase. We found that dNTP pools were not unbalanced and that dNTP synthesis was not arrested in polyamine-depleted cells. Moreover, the dNTP content and the activity of ribonucleotide reductase (CDP reduction) and thymidylate synthase, remained elevated despite the fact that the cells were inhibited in their growth by F2MeOrn treatment. Incorporation of a radiolabeled precursor into DNA was initially lower in F2MeOrn-treated. cells than in control cells. However, while incorporation of a radiolabeled precursor into DNA decreased markedly in plateau-phase control cells, it remained at a higher level in cells inhibited in growth by polyamine depletion. This discrepancy may be explained by the fact that polyamine-depleted cells accumulated in the S phase, and that they had an increased content of acid-soluble radiolabeled DNA precursor. Our data indicate that polyamine depletion adversely affects the DNA synthetic machinery by reducing the rate of elongation.
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PMID:Implications for a reduced DNA-elongation rate in polyamine-depleted cells. 211 38

The mechanism of action of a synthetic compound--2,2'-bipyridyl-6-carbothioamide--was investigated by developing tumor lines resistant to it (P388-R1.5 and P388-R4). P388-R4 is resistant to inhibitors of ribonucleotide reductase (RR) while no resistance was observed to antitumor drugs having other targets (except to bleomycin). The resistance to inhibitors of the RR M2 subunit is higher than that to compounds active on the RR M1 subunit. Moreover, murine chromosome 12, in which the M2 structural gene was recently localized, was trisomic in the resistant lines. We conclude that it is possible to consider BPYTA a new inhibitor of the RR M2 subunit.
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PMID:Isolation of two cellular lines resistant to ribonucleotide reductase inhibitors to investigate the inhibitory activity of 2,2'-bipyridyl-6-carbothioamide. 213 Oct 50


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