Effect of 2'-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells.
Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2'-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5' upstream cis-acting regulatory element (nucleotides 80-109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of beta-actin, alpha-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.
Schmitz
J C
JC
Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA Connecticut Healthcare System, New Haven, CT 06520, USA.
Yu
D
D
Agrawal
S
S
Chu
E
E
D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D020320:Oligoribonucleotides, Antisense / Q000494:pharmacology*; D011799:Quinazolines / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D020320:Oligoribonucleotides, Antisense; D011799:Quinazolines; D013876:Thiophenes; D013940:Thymidylate Synthase; C068874:raltitrexed