title: Antisense-induced down-regulation of thymidylate synthase and enhanced cytotoxicity of 5-FUdR in 5-FUdR-resistant HeLa cells.
abstract: 1. 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. 2. 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 nM 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. 3. Antisense ODN can be used to down-regulate TS and attenuate drug resistance in TS-overexpressing cells.
authors.affiliation: Cancer Research Laboratories, London Regional Cancer Centre, London, Ontario, Canada.
authors.forename: P J
authors.initials: PJ
authors.lastname: Ferguson
authors.forename: J M
authors.initials: JM
authors.lastname: DeMoor
authors.forename: M D
authors.initials: MD
authors.lastname: Vincent
authors.forename: J
authors.initials: J
authors.lastname: Koropatnick
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects* / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D005467:Floxuridine / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D000964:Antimetabolites, Antineoplastic; D016373:DNA, Antisense; D012333:RNA, Messenger; D005467:Floxuridine; D013940:Thymidylate Synthase