Transient transfection of a synthetic hammerhead ribozyme targeted againsthuman MGMT gene to cells in culture potentiates the genotoxicity of the alkylation damage induced by mitozolomide
L. Citti et al., Transient transfection of a synthetic hammerhead ribozyme targeted againsthuman MGMT gene to cells in culture potentiates the genotoxicity of the alkylation damage induced by mitozolomide, ANTISENSE N, 9(2), 1999, pp. 125-133
Unmodified and chemically modified forms of a synthetic hammerhead ribozyme
with the mRNA of methylguanine-DNA methyltransferase (MGMT) gene as substr
ate were characterized for their in vitro and in vivo activities. The unmod
ified ribozyme efficiently cleaved in vitro a short synthetic substrate, an
d it was rapidly degraded in fetal bovine serum (FBS), The introduction of
phosphorothioates and the substitution of uridine with thymidine at probabl
e nuclease-sensitive sites slightly increased the nuclease resistance of th
e 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 ex
changes was higher in ribozyme-transfected than in untransfected cells, ind
icating that the synthetic ribozymes potentiated the genotoxicity of mitozo
lomide, Moreover, the concomitant occurrence of messenger RNA reduction in
ribozyme-transfected cells indicated that the inhibition of MGMT resynthesi
s was the basis of the enhanced genotoxicity.