Effect of 2'-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells

Abstract

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.

Figure 1

Effect of ratio of Eufectin to HYB0432 on TS expression. RKO cells were incubated with different Eufectin:HYB0432 combinations. After 6 h, complexes were removed and fresh RPMI medium was added. After an additional 24 h, cells were harvested and processed for western blot analysis as described in Materials and Methods. Lane 1 contains cell extracts from untreated RKO cells. Lanes 2–6 contain 190 nM HYB0432 complexed with Eufectin in weight ratios of 1:1 (lane 2), 1:3 (lane 3), 1:5 (lane 4), 1:7 (lane 5) and 1:9 (lane 6). Lane 7 contains cell extracts treated with Eufectin alone (10 µg/ml).

Figure 2

Western blot analysis of RKO cells after treatment with HYB0432. Cells were incubated in the absence (lane 1) or presence (lanes 2–8) of Eufectin–ORN complexes (5:1 ratio; µg:µg) for 6 h, grown for an additional 24 h and then harvested and processed for western blot analysis as described in Materials and Methods. Extracts in lanes 2–5 were treated with complexes containing 63, 126, 190 and 250 nM HYB0432, respectively. Cells were treated with various control ORNs: lane 6, sense ORN HYB0848 (250 nM); lane 7, mismatch ORN HYB0503 (250 nM). Lane 8 contains cell extracts treated with Eufectin alone (13 µg/ml).

Figure 3

Duration of effect of HYB0432 on expression of TS protein in RKO cells. After a 6 h transfection with HYB0432–Eufectin complexes (190 nM), cells were harvested prior to transfection (lane 1) and at the following times: 0 (after transfection, lane 2), 6 (lane 3), 12 (lane 4), 24 (lane 5), 36 (lane 6), 48 (lane 7) and 72 h (lane 8). Cell extracts were processed for western blot analysis as described in Materials and Methods.

Figure 4

Effect of antisense treatment on expression of TS mRNA and TS protein. RKO cells were treated for 6 h in the absence (lane 1) or presence of oligo–Eufectin complexes (190 nM), grown for an additional 24 h, harvested and processed for northern (A) and western (B) blot analysis as described in Materials and Methods. Lanes 2–5 contain cell extracts treated with Eufectin alone (lane 2) or complexed with either HYB0432 (lane 3), a TS antisense 30mer PS-DNA oligo (lane 4) or TS sense 30mer RNA HYB0848 (lane 5).

Figure 5

Effect of antisense treatment on the half-life of TS protein in RKO cells. Cells were incubated with Eufectin alone (squares) or Eufectin complexed with HYB0432 (190 nM) (circles) for 6 h, followed by a 2 h incubation with ³⁵S-labeled methionine-containing medium. After removal of the radiolabel, cells were harvested at the indicated times and processed as described in Materials and Methods. The amount of radiolabeled TS protein (treated with Eufectin alone) at 0 h equals 100%. Points represent the mean ± SD from three separate experiments.

Figure 6

Effect of 18 nt HYB0504 on expression of TS protein in RKO cells. Cells were incubated in the absence (lane 1) or presence of Eufectin–ORN complexes (9:1 ratio, µg:µg) for 3 h, grown for an additional 24 h and then harvested and processed for western blot analysis as described in Materials and Methods. Lanes 2–5 were treated with complexes containing 53, 106, 159 and 212 nM HYB0504, respectively.

Figure 7

Effect of HYB0504 on expression of TS protein in TDX-resistant, RKO-TDX cells. Cells were treated for 12 h with increasing concentrations of HYB0504–Eufectin complexes: untreated (lane 2), 53 nM (lane 3), 105 nM (lane 4), 158 nM (lane 5), 315 nM (lane 6). Lanes 7 and 8 contain extracts from cells treated with 315 nM TSAS18-MM, a 5-base mismatch 18mer RNA and 210 nM TS18, a sense 18mer RNA, respectively. Lane 9 contains cell extracts treated with Eufectin alone (18 µg/ml). Lane 1 contains protein extract from the parent RKO cell line. After 24 h, cells were harvested and processed for western blot analysis as described in Materials and Methods.