MECHANISM AND PHARMACOLOGICAL SPECIFICITY OF DUTPASE-MEDIATED PROTECTION FROM DNA-DAMAGE AND CYTOTOXICITY IN HUMAN TUMOR-CELLS

Purpose: We have reported previously that the expression of E. coli dU TPase (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 t hymidylate synthase (TS) inhibitors. In addition, we developed two HuT u80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytot oxicity. 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 te sted for drug-induced DNA damage with either alkaline elution or pulse d field gel electrophoresis and drug-induced loss of clonogenicity. Re sults: Following a 24-h treatment with 100 mu M CB3717 or 500 nM metho trexate (MTX), dutE7 cells were significantly less sensitive to drug-i nduced loss of clonogenicity than con3 cells. DutE7 cells were also re sistant to CB3717-induced DNA fragmentation at 24 h. However, followin g a 48-h treatment with CB3717 or MTX there was no difference in survi val between con3 and dutE7 cells, even though DNA damage was still gre atly attenuated in the dutE7 cell line. In addition, expression of dut E in two HuTu80 clones, 80 C and 80 K, did not protect these cells fro m FdUrd-induced DNA damage or cytotoxicity. Conclusions: We conclude t hat the role of uracil misincorporation and subsequent DNA damage in c ytotoxicity 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 i nability of dutE to protect HuTu80 cells from FdUrd further suggests t hat the significance of uracil misincorporation resulting from TS inhi bition varies among cell lines.