Antisense-induced down-regulation of thymidylate synthase and enhanced cytotoxicity of 5-FUdR in 5-FUdR-resistant HeLa cells

Abstract

1. Thymidylate synthase (TS) is a target for several anticancer drugs. We previously showed that an antisense oligodeoxynucleotide (ODN) directed against TS mRNA down-regulated TS protein and enhanced cytotoxicity of TS-targeting drugs [including 5-fluorodeoxyuridine (5-FUdR)] in HeLa cells. Patient tumours with increased TS expression are resistant to TS-targeting drugs. It was hypothesized that TS mRNA and consequently TS protein could be down-regulated in 5-FUdR-resistant cells that overexpress TS, sensitizing them to 5-FUdR cytotoxicity. In this study we assessed the capacity of an anti-TS antisense ODN to circumvent resistance dependent on TS overexpression. 2. Variant HeLa clones exhibiting 2 - 20 fold resistance to 5-FUdR were selected by exposing cultured cells to drug. Clones FUdR-5a, -25b, and -50a expressed TS protein levels 10 fold, 10 fold, and 17 fold higher (respectively) than parental cells. Cells were treated with antisense ODN 83 (a 2'-methoxy-ethoxylated, phosphorothioated 20-mer, complementary to a portion of the 3'-untranslated region of TS mRNA), or ODN 32 (a control ODN with the same base composition as ODN 83, but in randomized order). Twenty-four and 48 h following transfection (50-100 nM ODN, plus polycationic liposome), TS mRNA levels (by RT-PCR) and protein levels (by radiolabelled 5-FUdR-monophosphate binding) were decreased by at least 60% in ODN 83-treated cells compared with control ODN 32-treated cells. ODN 83 enhanced the cytotoxicity of 5-FUdR by up to 85% in both parental and 5-FUdR-resistant cell lines. 3. Antisense ODN can be used to down-regulate TS and attenuate drug resistance in TS-overexpressing cells.

Figure 1

Reduction of cellular levels of TS mRNA in HeLa cells and FUdR-resistant variants following treatment with antisense ODN 83. Following administration of ODNs to cultures, cells were harvested on days 1 and 2. Relative TS mRNA was visualized by RT – PCR, using GAPDH as a control for RNA integrity. This image is typical and representative of assays of cells harvested from three separate experiments.

Figure 2

Reduction in thymidylate synthase mRNA and protein levels by treatment of HeLa cells and FUdR-resistant variants with antisense ODN 83. Following a 4 h transfection of cells with ODN at the concentration indicated, one volume of medium was added, and cells were further incubated in ODN at 50% of the original concentration. Cells were harvested 1 and 2 days following initiation of exposure to ODN. (A) relative TS mRNA was assayed by RT – PCR, and compared with the level of the housekeeping gene product GAPDH. Data are summarized from the analysis of two separate determinations for day 1 and 3 for day 2, as represented by Figure 1. (B) TS protein level was measured by [³H]-FdUMP-binding. The TS content in the ODN 32-treated parental HeLa line was 0.983±0.132 pmol mg⁻¹ (n=5) on Day 1, and 0.792±0.232 pmol mg⁻¹ (n=5) on Day 2. All values presented are significantly different from the respective ODN 32-treated control (P<0.01).

Figure 3

Enhancement of FUdR cytotoxicity in HeLa cells and FUdR-resistant variants by treatment with antisense ODN 83. Cells were treated with ODNs for 4 h, followed by administration of FUdR without changing the medium. Cell number was determined after 4 days, and proliferation calculated as a percentage of that of control ODN-treated cells. (A) HeLa; (B) FUdR-5a; (C) FUdR-25b; (D) FUdR-50a. Error bars are smaller than the symbols.

Figure 4

Enhancement of FUdR cytotoxicity in HeLa cells and in FUdR-resistant variants by antisense ODN 83. Cells were treated with antisense ODN 83 or scrambled control ODN 32 for 4 h, followed immediately by coincubation with FUdR for 4 days. Proliferation was determined by increase in cell number. IC₅₀ values were interpolated from plotted data, from a series of experiments represented by Figure 3. The value presented is the IC₅₀ of the ODN 83-treated cells as a percentage of that of the control ODN 32-treated cells, calculated within individual experiments. All values for relative resistance are significantly different from that of control ODN-treated cells, P<0.001. ^aThis value is reproduced from Table 1 (see table for s.d. and n values for these averages). Resistance index was calculated by dividing the IC₅₀ of the FUdR-resistant line by that of the parent HeLa cell line, within each individual experiment. ^bRelative proliferation is the relative increase in cell number, as a percentage of the control ODN-treated cells, of the cultures treated with antisense ODN 83 alone.