Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency.
Thymidylate synthase (TS) is an important target of several chemotherapeutic agents. During TS inhibition, dTTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). BER has been hypothesized to play a role in the response to thymidylate deprivation, despite a lack of direct evidence. We previously found that beta-pol null murine fibroblasts were approximately six-fold more resistant than wild-type cells to raltitrexed, a folate-based inhibitor specific for TS. In this study, a number of endpoints were determined to understand the influence of BER and beta-pol during raltitrexed treatment. Raltitrexed induced apoptosis in wild-type cells to a greater extent than in beta-pol null cells. A PARP inhibitor decreased the sensitivity to raltitrexed, although the extent was not different between wild-type and beta-pol null cells. No evidence was seen for extensive strand break formation that preceded apoptosis, although raltitrexed induced more sister chromatid exchanges in wild-type cells. Increased levels of uracil in DNA were detected following treatment in wild-type and beta-pol null cells. However, uracil levels were only approximately two-fold higher in DNA from treated cells compared to untreated. Uracil DNA glycosylase activity was slightly higher in beta-pol null cells, although not sufficiently different to explain the difference in sensitivity to raltitrexed. Taken together, the data suggest that the sensitivity of the wild-type cells to raltitrexed is not associated with activation of PARP-1 dependent BER, extensive uracil incorporation into DNA and persistent strand breaks, but rather with changes suggestive of DNA recombination.
Li
Li
L
Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA.
Connor
Ellen E
EE
Berger
Sondra H
SH
Wyatt
Michael D
MD
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects*; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000473:pathology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D051379:Mice; D011064:Poly Adenosine Diphosphate Ribose / Q000037:antagonists & inhibitors / Q000494:pharmacology; D011799:Quinazolines / Q000037:antagonists & inhibitors / Q000633:toxicity; D012854:Sister Chromatid Exchange / Q000187:drug effects* / Q000502:physiology; D013876:Thiophenes / Q000037:antagonists & inhibitors / Q000633:toxicity; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000187:drug effects; D013997:Time Factors; D014498:Uracil / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
D011799:Quinazolines; D013876:Thiophenes; D011064:Poly Adenosine Diphosphate Ribose; D014498:Uracil; D013940:Thymidylate Synthase; D019951:DNA Polymerase beta; D051981:Uracil-DNA Glycosidase; C068874:raltitrexed