Role of Ribonucleotide Reductase and Deoxynucleotide Pools in the Oxygen‐Dependent Control of DNA Replication in Ehrlich Ascites Cells

Brischwein, Klaus; Engelcke, Michael; Riedinger, Hans-Jörg; Probst, Hans

doi:10.1111/j.1432-1033.1997.00286.x

Cited by 24 publications

(24 citation statements)

References 42 publications

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“…It seems, from the present data, that the pRB status survey the physiological growth conditions of the cell, and that it may, thus, function as a stress indicator in the cell. This is, however, a slowlyacting regulation and even in cells with functional pRB the immediate arrest in S-phase after the onset of moderate hypoxia is believed to be a consequence of blocking de novo synthesis of pyrimidine deoxynucleotides due to inhibition of the two oxygendependent enzymes, dihydroorotate dehydrogenase and ribonucleotide reductase (Probst et al, 1989;Loffler, 1992;Amellem et al, 1994;Brischwein et al, 1997). An additional explanation proposed by Probst et al (1988) suggests that hypoxia inhibits the initiation of new replicones, whereas DNA chain elongation and termination proceed during hypoxia.…”

Section: Discussion Cell Cycle Regulation During Moderate Hypoxia In mentioning

confidence: 99%

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein

Åmellem

Sandvik²,

Stokke³

et al. 1998

Br J Cancer

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Summary We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G, phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21 WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p1 6INK4a was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclindependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p1 6INK4a would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions.

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Section: Discussion Cell Cycle Regulation During Moderate Hypoxia In mentioning

confidence: 99%

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein

Åmellem

Sandvik²,

Stokke³

et al. 1998

Br J Cancer

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“…4 We are not the first to have observed a specific decline in the dCTP pool in hypoxic mammalian cells. Probst et al (5) observed a rapid inhibition of DNA replication during transient hypoxia in Ehrlich ascites cells, and Brischwein et al (6) observed a specific decline in the dCTP pool under these conditions and also a decline in the ribonucleotide reductase free radical content of the cells. Similar results have been noted for cultured human cell lines (19).…”

Section: Effects Of Hypoxia Upon Dntp Pools-mentioning

confidence: 99%

“…O 2 is required for formation of the oxygen-bridged iron center that participates in forming the catalytically essential tyrosine free radical for ribonucleotide reductase (7)(8)(9). For this reason, Brischwein et al (6) proposed that hypoxic conditions affect ribonucleotide reductase so as to cause a specific decline in the dCTP pool, either directly, by modifying the specificity of the enzyme for different rNDP substrates, or indirectly, by an unknown mechanism.…”

Section: Effects Of Hypoxia Upon Dntp Pools-mentioning

confidence: 99%

“…1 The type I RNR, which is found in all mammalian systems examined to date, requires O 2 for formation of a catalytically essential diferric iron center and tyrosine radical (7-9). Brischwein et al (6) proposed that hypoxic conditions somehow affect ribonucleotide reductase activities so that the dCTP pool is specifically lowered, either by a direct or indirect mechanism, and that the dCTP pool size, as proposed earlier (10), exerts a specific regulatory effect upon the DNA replication machinery.…”

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Ribonucleotide Reductase, a Possible Agent in Deoxyribonucleotide Pool Asymmetries Induced by Hypoxia

Chimploy

Tassotto

Mathews

2000

Journal of Biological Chemistry

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While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O 2 is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O 2 is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension.This investigation arose from our attempts to identify the biochemical basis for a natural asymmetry in the pool sizes of the four deoxyribonucleoside triphosphates (dNTPs) in mammalian cells (1). In nearly every cell line analyzed, dGTP represents just 5-10% of the sum of the four dNTP pools (dATP ϩ dCTP ϩ dGTP ϩ dTTP).A possible explanation for this asymmetry came from the discovery that the mutT gene product in Escherichia coli is a nucleoside triphosphatase, with particular activity against an oxidized guanine nucleotide, 8-oxo-dGTP (2), and that mammalian cells contain a similar enzyme (3). An 8-oxo-dGMP residue in DNA stimulates mutagenesis via a transversion pathway because of its ready tendency to base pair with dAMP (4). Therefore, the mutT gene product and its homolog are thought to minimize replication errors by eliminating from the cell an oxidized nucleotide, 8-oxo-dGTP, whose incorporation into DNA would otherwise be a strongly mutagenic event.We hypothesized that turnover of dGTP, via its oxidation and subsequent degradation, might contribute to its underrepresentation in the DNA precursor pool, and we asked whether culturing cells at low oxygen tension, where guanine nucleotide oxidation might be minimized, would cause dGTP to accumulate. However, as detailed below, we found that culturing V79 hamster lung cells in an artificial atmosphere containing 2% oxygen instead of at its ...

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“…The effect of hypoxia on DSBR is further complicated by the suggestion that acute and chronic hypoxia may lead to different tumor biology that might directly affect tumor therapy [8]. In addition, studies on the effect of hypoxia on cell proliferation and cell cycle progression using mammalian cells in culture have indicated that oxygen availability reversibly arrests DNA replication [9] by directly regulating Ribonucleotide reductase (RNR) function [10] resulting in cell cycle arrest either at metaphase or just before S phase [11].…”

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Homologous Recombination is Activated at Early Time Points Following Exposure to Cobalt Chloride Induced Hypoxic Conditions in Saccharomyces cerevisiae

2011

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DNA repair functions are essential for the maintenance of genetic integrity and are regulated in response to both environmental and chemical stressors in mammalian and yeast cells in culture. The inhibitory effect of limited O 2 availability on DNA repair functions in general and on homologous recombination (HR) in particular, correlates with increased chromosomal abnormalities in hypoxic cancer cells. Given the above, we have investigated the effects of CoCl 2 ,--a hypoxia mimetic agent on HR and genetic aberrations in Saccharomyces cerevisiae. Our studies demonstrate that both acute and chronic exposure to CoCl 2 activated HR and increased genetic aberrations in S. cerevisiae D7 cells. At early time points following addition of CoCl 2 to the growth media, cells were briefly arrested in the G1-S boundary concomitant with a transient increase in Rad52-GFP foci formation and induction of low levels of DNA damage. The mode of action of CoCl 2 is thus similar to that of DNA synthesis inhibitors, the later are known to induce HR and cause G1-S arrest. We propose that the activation of HR in the presence of the hypoxia mimetic agent may be attributed to the replication stress and/or DNA damage induced by the stressor.Keywords Saccharomyces cerevisiae Á Hypoxia mimetic Á Homologous recombination Á DNA damage Á G1-S arrest Intracellular DNA double-strand breaks (DSB) generated by exposure to ionizing radiation, chemotherapeutic drugs and reactive oxygen species, as well as during V(D)J recombination and immunoglobulin class switching are repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Failure to repair DSBs results in various chromosomal abnormalities that may ultimately lead to neoplastic transformation [1]. In S. cerevisiae, HR is the major pathway for DNA double strand break repair (DSBR) and is carried out by members of the RAD52 epistasis group that include RAD52, RAD50, RAD51, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2 and requires extensive DNA sequence homology between the DNA molecules involved in recombination. HR is initiated by DSBs followed by 5 0 to 3 0 resection of the ends. The 3 0 ends thus formed are recombinogenic that bind Rad51 in an ATP dependent reaction and invades homologous template to initiate DNA replication by strand displacement. The inhibitory effect of RPA on Rad51 binding to DNA is overcome by Rad52. Broken ends are then joined by ligation leading to the formation of two Holliday junctions that are subsequently resolved to yield either crossover or non-crossover products [2]. DSBR by NHEJ requires little or no sequence homology and is carried out by the MRX complex along with Yku80, Yku70, DNA Pol4 and DNA ligase IV [3]. While DSB repair by HR is error-proof, that by NHEJ is an error-prone process.DNA replication and repair functions are regulated in response to environmental stress conditions in S. cerevisiae. Hypoxic stress results in transient down-regulation in the expression of MCB and SCB regulated genes required

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Role of Ribonucleotide Reductase and Deoxynucleotide Pools in the Oxygen‐Dependent Control of DNA Replication in Ehrlich Ascites Cells

Cited by 24 publications

References 42 publications

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein

Ribonucleotide Reductase, a Possible Agent in Deoxyribonucleotide Pool Asymmetries Induced by Hypoxia

Homologous Recombination is Activated at Early Time Points Following Exposure to Cobalt Chloride Induced Hypoxic Conditions in Saccharomyces cerevisiae

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