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 de novo synthesis of thymidine, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide down-regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyrimidine 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-theon yl-L-glutamic acid). 2. 2'-Methoxyethoxylated, phosphorothioated 20-mer oligodeoxynucleotides (ODNs), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomized order, against which all the biological effects were compared. Following a 6-h transfection of HeLa cells using polycationic liposome at 3 microg ml(-1), ODN 83 (50 nM), complementary to a region in the 3'-untranslated region 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 to radiolabelled 5-fluorodeoxyuridine monophosphate. In addition to inhibiting proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxicity of Tomudex or 5-FU, added 1 day following transfection, by 50 - 60%. ODN 83 also enhanced sensitivity to 5-FUdR by 70%, but did not affect the toxicity 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 drugs.
Ferguson
P J
PJ
Department of Oncology, University of Western Ontario, and London Regional Cancer Centre, London, Ontario, Canada.
Collins
O
O
Dean
N M
NM
DeMoor
J
J
Li
C S
CS
Vincent
M D
MD
Koropatnick
J
J
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002452:Cell Count / Q000187:drug effects; D015536:Down-Regulation; D005260:Female; D005467:Floxuridine / Q000494:pharmacology*; D005472:Fluorouracil / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D011799:Quinazolines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000964:Antimetabolites, Antineoplastic; D016376:Oligonucleotides, Antisense; D011799:Quinazolines; D012333:RNA, Messenger; D013876:Thiophenes; D005467:Floxuridine; D013940:Thymidylate Synthase; C068874:raltitrexed; D005472:Fluorouracil