Small Interfering Double-Stranded RNAs as Therapeutic Molecules to Restore Chemosensitivity to Thymidylate Synthase Inhibitor Compounds

John C. Schmitz, Tian-min Chen, Edward Chu
2004 Cancer Research  
RNA interference is a post-transcriptional mechanism by which double-stranded RNA specifically silence expression of a corresponding gene. Small interfering double-stranded RNA (siRNA) of 21-23 nucleotides can induce the process of RNA interference. Studies from our laboratory have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this process gives rise to increased synthesis of TS and
more » ... hesis of TS and leads to the development of cellular drug resistance to TS-targeted compounds. As a strategy to inhibit TS expression at the mRNA level, siRNAs were designed to target nucleotides 1058 -1077 on human TS mRNA. Transfection of TS1058 siRNA into human colon cancer RKO cells resulted in a dose-dependent inhibition of TS expression with an IC 50 value of 10 pM but had no effect on the expression of ␣-tubulin or topoisomerase I. Inhibition of TS expression by TS1058 was maximal at 48 h and remained suppressed for up to 5 days. Pretreatment of RKO cells with TS1058 siRNA suppressed TS protein induction following exposure to raltitrexed. In addition, TS1058 restored chemosensitivity of the resistant RKO-HTStet cell line to various TS inhibitor compounds. On treatment with TS1058, IC 50 values for raltitrexed, 1843U89, and 5-fluoro-2-deoxyuridine decreased by ϳ15-16-fold. These studies suggest that TS-targeted siRNAs are effective inhibitors of TS expression and may have therapeutic potential by themselves or as chemosensitizers in combination with TS inhibitor compounds. MATERIALS AND METHODS siRNA Synthesis. siRNA duplexes were designed to target sequences on human TS mRNA corresponding to nt 1058 -1077 (5Ј-GGAUAUUGUCAGU-CUUUAGG-3Ј). The selected sequence was screened in a BLAST search against all of the known human genes to verify that only human TS mRNA was targeted. siRNA duplexes were obtained from Dharmacon (Lafayette, CO). Each RNA contained two additional 2Ј-deoxythymidine nts on the 3Ј end. In addition, a mismatch siRNA, TS1058M10, and two control siRNA duplexes, GL2 and SCRII, were obtained from Dharmacon. Cell Culture. The human colon cancer cell lines RKO and RKO-HTStet were maintained in 75-cm 2 tissue culture flasks (BD Bioscience, San Jose, CA)
doi:10.1158/0008-5472.can-03-1203 pmid:14973067 fatcat:bejbu62frzdjpnvo565axeqqxe