Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells

1 Thymidylate synthase (TS), the key enzyme in ne novo synthesis of thymidi ne, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide downregulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyr imidine analogues 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), and the folate analogue Tomudex (ICI D1694; N-(5-[N-(3,4-dihydro-2-methyl-4 -oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theonyl-L-glutamic acid). 2 2'-Methoxyethoxylated, phosphorothioated 20-mer oligodeoxynucleotides (OD Ns), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomi zed order, against which all the biological effects were compared. Followin g a 6-h transfection of HeLa cells using polycationic liposome at 3 mu g ml (-1), ODN 83 (50 nM), complementary to a region in the 3'-untranslated regi on of the TS mRNA, decreased TS mRNA levels by approximately 70% within 24 h. ODN 83 also decreased TS enzyme activity, as measured by binding of TS t o radiolabelled 5-fluorodeoxyuridine monophosphate. In addition to inhibiti ng proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxici ty of Tomudex or 5-FU, added 1 day following transfection, by 50-60%. ODN 8 3 also enhanced sensitivity to 5-FUdR by 70%, but did not affect the toxici ty of cisplatin, chlorambucil, melphalan, doxorubicin, ionizing radiation, paclitaxel, or irinotecan. 3 These data indicate that antisense ODN down-regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS-targeted dru gs.