Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency |
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Authors: | Li Li Connor Ellen E Berger Sondra H Wyatt Michael D |
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Affiliation: | Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA. |
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Abstract: | 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. |
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Keywords: | AP site, apurinic/apyrimidinic (abasic) site 3-AB, 3-aminobenzamide BER, base excision repair 5-FU, 5-fluorouracil FdUrd, 5-fluoro-2′-deoxyuridine MEFs, murine embryonic fibroblasts PARP, poly-ADP ribose polymerase PFGE, pulsed-field gel electrophoresis β-pol, DNA polymerase β RTX, raltitrexed (Tomudex) SCE, sister chromatid exchange TS, thymidylate synthase UDG, uracil DNA glycosylase |
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