Inhibition of thymidylate synthase activity by antisense oligodeoxynucleotide and possible role in thymineless treatment

Thymidylate synthase (TS) is an important target for chemotherapeutic treat ment of cancer. However, efficacy of TS-targeted anticancer drugs is limite d by the development of drug resistance as a result of TS gene amplificatio n. In this work, a phosphorothioated antisense oligonucleotide (ODN), desig nated ATS-2, was used to suppress cellular synthesis of TS. ATS-2 at 0.2 mu M concentration was mixed with lipofectin in a charge ratio of 1:1 and was used to treat the human embryonic kidney (HEK) cell line. A reduction of TS mRNA and protein was achieved. Furthermore, a dose-dependent reduction of cumulative viable cells of up to 98% was observed. Flow cytometer analysis of cell cycle progression indicates that ATS-2-treated cells were arrested and went into apoptosis at the S phase, possibly because of thymidine short age, suggesting that ATS-2 is specifically effective for dividing cells. Wh en used in combination with the anticancer drug FdUrd, ATS-2 exerted a addi tive inhibitory effect on cellular proliferation. To elucidate the possible role of cellular thymidine kinase (TdR kinase) in ATS-2 treatment, a secon d cell line, HeLa, was used. Both HEK and HeLa have similar rates of cell d ivision and ODN uptake. In contrast to HEK, which was shown to have very lo w levels of TdR kinase activity in [H-3]thymidine incorporation experiments , [H-3]thymidine incorporation in HeLa. was 15-fold greater than that of HE K. We found that HeLa cells were sensitive to FdUrd but were rather resista nt to ATS-2. On the contrary, HEK cells were sensitive to ATS-2 but insensi tive to FdUrd. Effects of ATS-2 and FdUrd are, therefore, complementary in thymineless treatment too.