The ability of hypoxia to modify the gene expression of thymidylate synthase in tumour cells in vivo

Abstract

Purpose: Hypoxic cells in tumours are resistant to 5-fluorouracil (5-FU). This in vivo study investigated the ability of hypoxia to regulate the gene expression of thymidylate synthase (TS), the target enzyme of 5-FU. Materials and methods: C3H mammary carcinomas, grown in the feet of female CDF1 mice, were used for all experiments. Mice were placed in a 10% oxygen environment for various time periods and the tumour oxygen status was determined with an Eppendorf oxygen electrode. The animals were then injected with BrdU (100mg/kg, i.p.). Tumours were excised and immediately frozen (-80 C) until isolation of total RNA. The mRNA was reversibly transcribed to complementary DNA and the resulting cDNA amplified in a multiplex PCR reaction, with beta-actin as the internal reference gene. Results: One hour of low oxygen breathing made tumours significantly more hypoxic. This increase was maintained for a maximum incubation period of 48h. In the same tumours, no change in TS gene expression was seen with up to 3h of low oxygen breathing. At longer times it decreased, reaching significance at 12-24h and remaining at this lower level for up to 48h. BrdU labelling was significantly reduced after breathing low O2 for 24h (p=0.001). Conclusion: Hypoxia-induced down-regulation of TS gene expression was observed. This would be expected to make hypoxic tumour cells more sensitive to 5-FU. Other mechanisms must be responsible for the previously reported resistance to this drug.