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

1 Thymidylate synthase (TS) is a target for several anticancer drugs. We pr eviously showed that an antisense oligodeoxynucleotide (ODN) directed again st TS mRNA down-regulated TS protein and enhanced cytotoxicity of TS-target ing drugs [including 5-fluorodeoxyuridine (5-FUdR)] in HeLa cells. Patient tumours with increased TS expression are resistant to TS-targeting drugs. I t was hypothesized that TS mRNA and consequently TS protein could be down-r egulated 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. 2 Variant HeLa clones exhibiting 2-20 fold resistance to 5-FUdR were select ed by exposing cultured cells to drug. Clones FUdR-5a, -25b, and -50a expre ssed 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 b ase composition as ODN 83, but in randomized order). Twenty-four and 48 h f ollowing transfection (50-100 nm ODN, plus polycationic liposome), TS mRNA levels (by RT-PCR) and protein levels (by radiolabelled 5-FUdR-monophosphat e 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-FU dR by up to 85% in both parental and 5-FUdR-resistant cell lines. 3 Antisense ODN can be used to down-regulate TS and attenuate drug resistan ce in TS-overexpressing cells.