Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells

Abstract

Purpose : We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. Methods : Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. Results : Following a 24-h treatment with 100 μ M CB3717 or 500 n M methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80 C and 80 K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. Conclusions : We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines.