Poly(ADP-ribose) synthesis in blocked and damaged cells and its relation to carcinogenesis.
There is compelling evidence showing that repair of DNA damage depends on the synthesis of poly(ADP-ribose) molecules at specific sites on histones and other proteins in nuclei of injured cells. In the present study, we have studied the effect of long-term exposure of mouse cells to nicotinamide and various cell cycle blockers on the ability of the cells to increase their levels of poly(ADP-ribose) in response to DNA methylation damage with dimethylsulfate (DMS). Of the cell cycle blockers used, hydroxyurea (HU) and cytosine arabinofuranoside (ara-C) inhibit DNA elongation at the replication fork and cause the appearance of small DNA molecules, whereas butyrate and colcemid block cells without interfering with DNA synthesis. In addition, cells were treated with nicotinamide, an inhibitor of poly(ADP-ribose) polymerase but also a precursor in NAD+ biosynthesis. Long-term exposure of cultured cells to these agents was followed with a short-term damage with DMS. The size-class distribution and concentration of poly(ADP-ribose) molecules were determined using high resolution polyacrylamide gel electrophoresis and were found to contain 1 to more than 30 ADP-ribosyl groups. On the contrary, histones from blocked cell nuclei were found to be mono- and oligo(ADP-ribosyl)ated. Thus, the large molecules of poly(ADP-ribose) were associated with nonhistone proteins and most likely with the enzyme pADP-R polymerase. DMS damage caused an increase in the levels of poly(ADP-ribose) that was synthesized in lysates from cells treated with any one drug alone. On the other hand, a dramatic decrease in total protein poly(ADP-ribosyl)ation resulted from DMS damage of cells treated with nicotinamide + HU or nicotinamide + ara-C. This decrease was not observed in combinations of nicotinamide with cell-cycle blockers that do not directly interfere with DNA synthesis (butyrate and colcemid). We suggest that nicotinamide + ara-C or nicotinamide + HU might be used as effective antineoplastic treatments by virtue of their ability simultaneously to damage DNA and reduce poly(ADP-ribosyl)ation and presumably DNA repair. A model for the facilitation of DNA repair by poly(ADP-ribosyl)ation of histones and of poly(ADP-ribose) polymerase is presented.
Boulikas
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Linus Pauling Institute of Science and Medicine, Palo Alto, California 94306.
D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002453:Cell Cycle / Q000187:drug effects; D003561:Cytarabine / Q000494:pharmacology*; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology*; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology*; D008745:Methylation; D051379:Mice; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D009536:Niacinamide / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis* / Q000302:isolation & purification; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured
D002087:Butyrates; D004273:DNA, Neoplasm; D006657:Histones; D010761:Phosphorus Radioisotopes; D013463:Sulfuric Acid Esters; D003561:Cytarabine; D009243:NAD; D014316:Tritium; D020148:Butyric Acid; D009536:Niacinamide; D011064:Poly Adenosine Diphosphate Ribose; C007482:dimethyl sulfate; D013936:Thymidine; D006918:Hydroxyurea