ABSTRACT
Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at gamma-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after gamma-ray doses of 0.5-2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.
Subject(s)
DNA Repair , DNA/radiation effects , Oxygen/metabolism , Radiation, Ionizing , Blotting, Western , Cell Line, Tumor , DNA Damage , Dose-Response Relationship, Radiation , Gamma Rays , Humans , Oligonucleotides/chemistry , RNA/chemistry , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription, Genetic , Up-RegulationABSTRACT
We have investigated the biochemical basis of the mevalonate dependence of DNA replication. Stimulating quiescent rat hepatoma cells to proliferate in the presence of compactin, an inhibitor of mevalonate synthesis, prevented DNA replication in as many as 80% of these cells. The percentage of cells that failed to replicate DNA increased with the increased duration of quiescence. Aphidicolin-sensitive DNA polymerase and ornithine decarboxylase activities were selectively decreased in compactin-treated cells, whereas RNA and protein synthesis, the level of dihydrofolate reductase and aphidicolin-resistant DNA polymerase activity were unaffected. Adding putrescine, the product of ornithine decarboxylase and the precursor of other polyamines, did not restore DNA replication. Our results demonstrate that the decreased activities of at least two DNA-replication enzymes are among the proximal causes of the failure of mevalonate-deprived cells to synthesize DNA. More importantly, our data indicate that a mevalonate-dependent factor(s) is progressively depleted during quiescence, and that inability to resynthesize this factor(s) may be the ultimate cause of the failure of resting cells to replicate DNA when stimulated to proliferate in the absence of mevalonate.
Subject(s)
DNA Replication/physiology , DNA/biosynthesis , Liver Neoplasms, Experimental/pathology , Mevalonic Acid/metabolism , Amino Acids/deficiency , Animals , Cell Nucleus/metabolism , DNA-Directed DNA Polymerase/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Liver Neoplasms, Experimental/enzymology , Lovastatin/analogs & derivatives , Lovastatin/pharmacology , Ornithine Decarboxylase/metabolism , Protein Biosynthesis , RNA/biosynthesis , Rats , Resting Phase, Cell Cycle/physiology , Tetrahydrofolate Dehydrogenase/metabolism , Time Factors , Tumor Cells, CulturedABSTRACT
It is well established that either exogenous or endogenous cholesterol is required for both cell growth and proliferation. This laboratory has recently discovered that, in baby hamster kidney-21 cells, independent of its role as a cholesterol precursor, mevalonic acid plays an essential role in S phase DNA replication. It was later shown that isopentenyl adenine, a known product of mevalonate in prokaryotes and lower eukaryotes, is 100 to 200 times more effective than mevalonate in restoring DNA replication in cells in which mevalonic acid synthesis is blocked with the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor, compactin. The present study was designed to determine the relationship in the cell cycle between the known requirement for cholesterol and the newly discovered effect of mevalonic acid and isopentenyl adenine on S phase DNA synthesis. Employing cells arrested by serum depletion, it was shown that the cholesterol requirement is limited to the early and mid-G1 phases, whereas the isopentenyl effect is required at the late G1-S interphase of the cell cycle. The evidence supporting these conclusions involves: first, in serum-arrested cells blocked early in G1 by compactin, only the combination of cholesterol added in early G1 and either mevalonate or isopentenyl adenine in late G1 permitted progression through the G1 and S phase DNA synthesis. Neither isopentenyl adenine added early in G1 nor cholesterol in late G1 was capable of restoring DNA synthesis in this system. Second, in accord with the above formulation, inhibition of cholesterol synthesis with the oxidosqualene cyclase inhibitor, dl-4,4,10 beta-trimethyl-trans-decal-3 beta-ol, affected only the early G1 phase of the cell cycle, but had no late G1 effect on DNA replication.