Effect of hammerhead ribozyme against human thymidylate synthase on the cytotoxicity of thymidylate synthase inhibitors.

One of the resistance mechanisms to folate-based thymidylate synthase (TS) inhibitors is the increase in TS activity in tumor cells. Human B lymphoblastoid cell line (W1L2) was made resistant to a lipophilic non-polyglutamatable TS inhibitor (ZM249148), and the subline (W1L2:R179) showed a 20-fold increase in TS enzyme activity with concomitant overexpression of TS mRNA. To overcome the resistance, we designed a ribozyme that can cleave the CUC sequences in a triple tandemly repeated sequence of TS mRNA. Expression of this ribozyme in W1L2:R179 cells transfected with Epstein Barr virus-based expression vector resulted in sensitization to TS inhibitors concomitantly with a decrease of TS expression. The ribozyme expressed in transfectants was shown to be functional in cleaving artificial TS RNA in vitro.

N10‐propargyl‐5,8‐dideazafolic acid

dihydrofolate reductase

dimethyl sulfoxide

fetal bovine serum

folylpolyglutamate synthetase

glyceraldehyde‐3‐phosphate dehydrogenase

drug concentration producing 50% inhibition of cell growth

3‐[4,5‐dmiethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide

methotrexate; PCR, polymerase chain reaction

trimetrexate

thymidylate synthase