Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells

Abstract

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.

Figure 1

Antisense TS ODN 83 inhibits HeLa cell proliferation. (A) Cells were transfected with 0, 25, 50 or 100 nM antisense TS ODN 83 or scrambled control ODN 32 in the presence of 2 μg ml⁻¹ LFA, and counted 4 days later as described in Materials and methods. The mean number of cells in three independent growth chambers±s.e. is plotted. Where error bars are not apparent, they are smaller than the symbol. Asterisks (^*) indicate data significantly different from that obtained from cells treated with LFA alone, or scrambled control ODN 32 plus LFA (P⩽0.02, Student t-test). (B) Cells were transfected as for A, except that 4 μg ml⁻¹ LFA transfection reagent was used. (C) Cells were transfected with 50 nM antisense TS ODN 83 or 50 nM scrambled control ODN 32 in the presence of 3 μg ml⁻¹ LFA. They were counted 1,2,5 and 6 days later. Control cells were plated without exposure to LFA or DNA. As in A and B, data points indicate the mean cell number in three independent growth chambers±s.e. Error bars are smaller than the symbols in every case.

Figure 2

Antisense TS ODN 83 suppresses HeLa cell growth after transfection, followed by recovery to control proliferation rate after 48 h. HeLa cells were transfected with 50 nM antisense TS ODN 83 or 50 nM scrambled control ODN 32 as described in the legend to Figure 1C. Values derived from cells transfected with ODN 32 were normalized to 100%. Each bar indicates the difference from that value induced by treatment with ODN 83 (mean±s.d. of four independent experiments). Asterisks (^*) indicate data significantly different from cells treated with scrambled control ODN 32 (P⩽0.02, Student t-test).

Figure 3

Treatment of HeLa cells with ODN 83 leads to decreased TS mRNA levels. HeLa cells were transfected with ODN 83 or scrambled control ODN 32, or treated with LFA alone (LF) as described in Material and methods. Cells were harvested at 1, 2, and 4 days post-transfection and total cellular RNA isolated, reverse-transcribed, and TS and GAPDH cDNA amplified by 24 PCR cycles, in the same reaction vessel. TS (208 b.p.) and GAPDH (752 b.p.) RT–PCR products were confirmed by Southern blotting and hybridization to specific radioactively-labelled probes.

Figure 4

TS protein levels (inferred by measurements of 5-FdUMP binding) are diminished by antisense TS ODN 83 but not scrambled control ODN 32. 5-FdUMP binding was measured in cells transfected with ODN 83 (hatched bars) or ODN 32 (open bars) at different times following transfection. (A) Results are plotted as a per cent of 5-FdUMP binding in cells transfected with control ODN 32±s.e. (n=5). The values for ODN 32 (n=5) were normalized to 100% and are shown without error bars. (B) Results are plotted as pmol 5-FdUMP bound per mg total protein (×10⁻³) to reveal that transfection with control ODN 32 had no significant effect on TS protein levels. Error bars indicate s.e.mean calculated according to a Student t-test, and indicate error due to differences in experimental conditions in four measurements taken on different days, as well as differences due to transfection with different ODNs. The asterisks indicate significant differences (P⩽0.02) according to a paired Student t-test, which controls for differences in experimental conditions.

Figure 5

Antisense TS ODN 83 sensitizes HeLa cells to the toxic effects of 5-FU, 5-FUdR, and Tomudex, but not cisplatin or chlorambucil. HeLa cells were transfected with ODN 83 or control ODN 32, and then continuously exposed to various concentrations of 5-FU (A), 5-FUdR (B), Tomudex (C), cisplatin (D) or chlorambucil (E) for 4 days, beginning 24 h after transfection, as described in Materials and methods. Data points indicate the number of cells (mean±s.e. of four independent cultures) at the 4 day time point, as a percentage of the number of cells in cultures treated with ODN 83 or 32 (as appropriate) but unexposed to drug. Where error bars are not apparent, they are obscured by the symbol. Asterisks (^*) indicate significant differences (P⩽0.02, Student t-test).

Figure 6

Overall effect of antisense TS ODN 83 on cell proliferation in the absence or presence of 5-FU, 5-FUdR, and Tomudex. As described in the legend to Figure 5, HeLa cells were transfected with ODN 83 or control ODN 32, and then continuously exposed to various concentrations of 5-FU (A), 5-FUdR (B), or Tomudex (C) for 4 days, beginning 24 h after transfection. Data points indicate the number of cells (mean±s.e. of four independent cultures) at the 4 day time point, as a percentage of the number of cells in cultures left untreated with DNA, LFA or toxic drug. Where error bars are not apparent, they are obscured by the symbol. Asterisks (^*) indicate significant differences (P⩽0.02, Student t-test).