Mechanistic studies on the induction of fluoropyrimidine resistance by E. colidUTPase expression, wild typep53 induction or thymidylate synthase overexpression in human cells.

Abstract

One limitation of fluoropyrimidine chemotherapy is the sensitivity of normal host tissues to these drugs. The development of techniques required for the selective induction of genes in vivo has led to the consideration of gene therapy-directed host protection strategies designed to enhance chemotherapy by allowing the administration of previously intolerable doses of drug. This dissertation presents an evaluation of the relative abilities of three genes: E. coli dUTPase (dutE), wt-p53 and thymidylate synthase (TS) to confer resistance in TS inhibitors in vitro. Previous studies indicated dUTPase has a role in determining the sensitivity of a cell to TS inhibition. We tested the hypothesis that induction of dutE expression could protect human cells from TS inhibitors. Although dutE expression reduced drug-induced DNA damage 5 fold in HT29 cells, protection from cytotoxicity was only 2 fold, and dutE did not protect HuTu80 cells from the cytotoxic effects of 5-fluorodeoxyuridine (FdUrd). These findings indicate dutE would not be a good gene for induction of fluoropyrimidine resistance in vivo. We also compared the relative abilities of two forms of TS, human (hTS) and E. coli (eTS), to protect human cells from FdUrd. E. coli TS was expressed at slightly higher levels (2-4 fold) than hTS in human cells, and FdUrd sensitivity in eTS-expressing cells was correspondingly lower. Cells expressing eTS were much more resistant to FdUrd when leucovorin (LV) was also present. While LV potentiated FdUrd-induced cytotoxicity in hTS-expressing cells about 10 fold, it had no effect in eTS-expressing cells. Cells expressing eTS were also relatively more resistant to AG337-induced cytotoxicity. These results suggest that eTS would be the gene of choice for a liver-directed host protection with either of these drugs. Finally, we examined the ability of TS-overexpressing cells to protect cells with normal TS content from the cytotoxic effects of TS inhibitors. Cells expressing eTS were twice as effective as hTS-overexpressing cells at protecting neighboring lacZ cells from FdUrd and AG337-induced cytotoxicity, further suggesting that eTS may be more effective than hTS at conferring fluoropyrimidine resistance in vivo.