Inhibition of DNA polymerase-alpha by ara-CMP in the presence of a regulatory protein extracted from human promyelocytic leukemic cells (HL-60).
Cytotoxicity of arabinofuranosylcytosine (ara-C) has been related in vitro to the inhibition of the DNA polymerase activities by arabinosylcytosine triphosphate (ara-CTP) and the incorporation of ara-C into the DNA where, acting as a chain terminator, it slows the chain elongation. Induced in vitro cellular resistance to ara-C was shown to be secondary to altered deoxycytidine (dCyd) kinase activity, dCyd deaminase activity, or deoxynucleotides triphosphates (dNTP) pools. Recent studies reported no differences of ara-C metabolism in cells obtained from leukemic patients at diagnosis and at relapse after ara-C therapy, suggesting that unknown cellular biochemical determinants may be involved in acquisition of ara-C resistance. Using dialysed crude extracts of leukemic cells obtained from patients at diagnosis, we observed variable inhibition of their DNA polymerase activities by arabinosylcytosine monophosphate (ara-CMP) at 2 mmol/L (0% to 50% inhibition). In similar conditions, ara-CMP reduced the polymerase activities of human thymus extract by 35% and 55% in extract of HL-60 cells (cultured human promyelocytic cells). The ara-CMP factor responsible for inhibition of DNA polymerase activity was nondialysable, heat labile, proteinase K sensitive, and has an estimated molecular mass of 30 kilodalton by gel filtration. After partial purification, this protein had no DNA polymerase RNA polymerase activities. In presence of the regulator and ara-CMP at 2 mmol/L, we observed no inhibition of the HL-60 3'----5' and 5'----3' exonucleases activities, suggesting the regulator interaction being mainly with the DNA polymerases in presence of ara-CMP. The relevance of the presence or absence of this protein regarding the cell sensitivity to ara-C is under investigation.
Leclerc
J M
JM
Grisé-Miron
L
L
Labonté
L
L
D000208:Acute Disease; D001088:Arabinonucleotides / Q000494:pharmacology*; D002460:Cell Line; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors*; D004268:DNA-Binding Proteins / Q000502:physiology*; D003843:Deoxycytidine Monophosphate / Q000378:metabolism; D006801:Humans; D007938:Leukemia / Q000503:physiopathology*; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology; D008970:Molecular Weight; D014542:Uridine Monophosphate / Q000378:metabolism
D001088:Arabinonucleotides; D003597:Cytosine Nucleotides; D004268:DNA-Binding Proteins; D003843:Deoxycytidine Monophosphate; C024888:1-beta-D-arabinofuranosylcytosine 5'-monophosphate; D014542:Uridine Monophosphate; D004257:DNA Polymerase II; D003568:Cytidine Monophosphate