The Contribution of Mitochondrial Ion Channels to Cancer Development and Progression

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Quantitation of cellular deoxynucleoside triphosphates

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP

p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage.

Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-(3)H]cytidine, [6-(3)H]deoxycytidine, and [C(3)H(3)]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication

Nucleotide excision repair efficiency in quiescent human fibroblasts is modulated by circadian clock

The efficiency of Nucleotide Excision Repair (NER)process is crucial for maintaining genomic integrity because in many organisms, including humans, it represents the only system able to repair a wide range of DNA damage. The aim of the work was to investigate whether the efficiency of the repair of photoproducts induced by UV-light is affected by the circadian phase at which irradiation occurred. NER activity has been analyzed in human quiescent fibroblasts (in the absence of the cell cycle effect), in which circadian rhythmicity has been synchronized with a pulse of dexamethasone. Our results demonstrate that both DNA damage induction and repair efficiency are strictly dependent on the phase of the circadian rhythm at which the cells are UV-exposed. Furthermore, the differences observed between fibroblasts irradiated at different circadian times (CTs) are abolished when the clock is obliterated. In addition, we observe that chromatin structure is regulated by circadian rhythmicity. Maximal chromatin relaxation occurred at the same CT when photoproduct formation and removal were highest. Our data suggest that the circadian clock regulates both the DNA sensitivity to UV damage and the efficiency of NER by controlling chromatin condensation mainly through histone acetylatio

Human mitochondrial 5'-deoxyribonucleotidase. Overproduction in cultured cells and functional aspects.

Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5′-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover

Nucleotide excision repair efficiency in quiescent human fibroblasts is modulated by circadian clock

The efficiency of Nucleotide Excision Repair (NER)process is crucial for maintaining genomic integrity because in many organisms, including humans, it represents the only system able to repair a wide range of DNA damage. The aim of the work was to investigate whether the efficiency of the repair of photoproducts induced by UV-light is affected by the circadian phase at which irradiation occurred. NER activity has been analyzed in human quiescent fibroblasts (in the absence of the cell cycle effect), in which circadian rhythmicity has been synchronized with a pulse of dexamethasone. Our results demonstrate that both DNA damage induction and repair efficiency are strictly dependent on the phase of the circadian rhythm at which the cells are UV-exposed. Furthermore, the differences observed between fibroblasts irradiated at different circadian times (CTs) are abolished when the clock is obliterated. In addition, we observe that chromatin structure is regulated by circadian rhythmicity. Maximal chromatin relaxation occurred at the same CT when photoproduct formation and removal were highest. Our data suggest that the circadian clock regulates both the DNA sensitivity to UV damage and the efficiency of NER by controlling chromatin condensation mainly through histone acetylation

Transient Exposure of Endothelial Cells to Doxorubicin Leads to Long-Lasting Vascular Endothelial Growth Factor Receptor 2 Downregulation

Doxorubicin (Dox) is an effective antineoplastic drug with serious cardiotoxic side effects that persist after drug withdrawal and can lead to heart failure. Dysregulation of vascular endothelium has been linked to the development of Dox-induced cardiotoxicity, but it is unclear whether and how transient exposure to Dox leads to long-term downregulation of Endothelial Vascular Endothelial Growth Factor Receptor type2 (VEGFR2), essential for endothelial cells function. Using an in vitro model devised to study the long-lasting effects of brief endothelial cells exposure to Dox, we show that Dox leads to sustained protein synthesis inhibition and VEGFR2 downregulation. Transient Dox treatment led to the development of long-term senescence associated with a reduction in VEGFR2 levels that persisted days after drug withdrawal. By analyzing VEGFR2 turnover, we ruled out that its downregulation was depended on Dox-induced autophagy. Conversely, Dox induced p53 expression, reduced mTOR-dependent translation, and inhibited global protein synthesis. Our data contribute to a mechanistic basis to the permanent damage caused to endothelial cells by short-term Dox treatment