Transient Transfection of a Synthetic Hammerhead Ribozyme Targeted Against Human MGMT Gene to Cells in Culture Potentiates the Genotoxicity of the Alkylation Damage Induced by Mitozolomide

Unmodified and chemically modified forms of a synthetic hammerhead ribozyme with the mRNA of methylguanine-DNA methyltransferase (MGMT) gene as substrate were characterized for their in vitro and in vivo activities. The unmodified ribozyme efficiently cleaved in vitro a short synthetic substrate, and it was rapidly degraded in fetal bovine serum (FBS). The introduction of phosphorothioates and the substitution of uridine with thymidine at probable nuclease-sensitive sites slightly increased the nuclease resistance of the ribozyme. Conversely, pyrimidine nucleoside substitution with 2′NH2 and 2′F nucleosides strongly enhanced nuclease resistance. The in vivo activity was determined by measuring the genotoxicity induced by the alkylating drug mitozolomide, the damage of which is repaired by MGMT enzyme. CHO/47 cells, temporarily depleted of the MGMT protein, were first transfected with the various synthetic ribozymes and subsequently treated with mitozolomide. At equivalent concentration of the drug, the induction of sister chromatid exchanges was higher in ribozyme-transfected than in untransfected cells, indicating that the synthetic ribozymes potentiated the genotoxicity of mitozolomide. Moreover, the concomitant occurrence of messenger RNA reduction in ribozyme-transfected cells indicated that the inhibition of MGMT resynthesis was the basis of the enhanced genotoxicity.