Antisense‐induced down‐regulation of thymidylate synthase and enhanced cytotoxicity of 5‐FUdR in 5‐FUdR‐resistant HeLa cells

Thymidylate synthase (TS) is a target for several anticancer drugs. We previously showed that an antisense oligodeoxynucleotide (ODN) directed against TS mRNA down‐regulated TS protein and enhanced cytotoxicity of TS‐targeting drugs [including 5‐fluorodeoxyuridine (5‐FUdR)] in HeLa cells. Patient tumours with increased TS expression are resistant to TS‐targeting drugs. It was hypothesized that TS mRNA and consequently TS protein could be down‐regulated in 5‐FUdR‐resistant cells that overexpress TS, sensitizing them to 5‐FUdR cytotoxicity. In this study we assessed the capacity of an anti‐TS antisense ODN to circumvent resistance dependent on TS overexpression. Variant HeLa clones exhibiting 2 – 20 fold resistance to 5‐FUdR were selected by exposing cultured cells to drug. Clones FUdR‐5a, −25b, and −50a expressed TS protein levels 10 fold, 10 fold, and 17 fold higher (respectively) than parental cells. Cells were treated with antisense ODN 83 (a 2′‐methoxy‐ethoxylated, phosphorothioated 20‐mer, complementary to a portion of the 3′‐untranslated region of TS mRNA), or ODN 32 (a control ODN with the same base composition as ODN 83, but in randomized order). Twenty‐four and 48 h following transfection (50 – 100 n M ODN, plus polycationic liposome), TS mRNA levels (by RT – PCR) and protein levels (by radiolabelled 5‐FUdR‐monophosphate binding) were decreased by at least 60% in ODN 83‐treated cells compared with control ODN 32‐treated cells. ODN 83 enhanced the cytotoxicity of 5‐FUdR by up to 85% in both parental and 5‐FUdR‐resistant cell lines. Antisense ODN can be used to down‐regulate TS and attenuate drug resistance in TS‐overexpressing cells.British Journal of Pharmacology (2001) 134 , 1437–1446; doi: 10.1038/sj.bjp.0704394