RESUMO
N-alkyl-nitrosoureas and alkyl-triazenes are alkylating antineoplastic drugs, the efficacy of which is strongly affected by the level of expression of the DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). In tumors, MGMT activity reduces the chemotherapeutic potential of alkylating drugs; therefore, efforts have been made to down-regulate the protein. A partial sensitization of Mex+ cells to alkylating drugs has been obtained using either free alkylated bases or oligonucleotides targeted against MGMT mRNA. In the present work, O6-methylguanine and a chemically modified ribozyme, without a cationic liposome as a carrier, were coadministered to CHO47 cells, which express a high level of human MGMT protein. The reduction of MGMT mRNA and protein enhanced the genotoxicity of the alkylating drug mitozolomide. Furthermore, the sensitivity of CHO47 cells is the same as that of CHO5 cells, which lack MGMT protein. These data indicate that a strategy in which both mRNA and protein are degradation targets can be successfully applied to down-regulate the MGMT gene.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Compostos de Mostarda Nitrogenada/farmacologia , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , RNA Catalítico/fisiologia , Animais , Sequência de Bases , Células CHO/enzimologia , Terapia Combinada , Cricetinae , Primers do DNA , Eletroforese em Gel de Ágar , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Humanos , Dados de Sequência Molecular , Estrutura Molecular , O(6)-Metilguanina-DNA Metiltransferase/genética , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Catalítico/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Troca de Cromátide Irmã/efeitos dos fármacos , TransfecçãoRESUMO
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.
Assuntos
Alquilantes/farmacologia , Mutagênicos/farmacologia , Compostos de Mostarda Nitrogenada/farmacologia , O(6)-Metilguanina-DNA Metiltransferase/efeitos dos fármacos , RNA Catalítico/farmacologia , Animais , Células CHO , Cricetinae , Regulação para Baixo , Sinergismo Farmacológico , Humanos , O(6)-Metilguanina-DNA Metiltransferase/biossíntese , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Troca de Cromátide Irmã/efeitos dos fármacos , Tionucleotídeos , TransfecçãoRESUMO
The cleavage activity of synthetic ribozymes needs to be characterized by reliable and rapid methods. A chromatographic method to simultaneously quantitate the amounts of substrate, cleavage fragments and ribozyme is described. The method allows the rapid normalization of analytical data because the sum of the 260-nm peak areas of remaining substrate and obtained fragments is essentially equal to the initial substrate peak area. Moreover, the simultaneous determination of the ribozyme content improves the accuracy of the evaluation of kinetic parameters compared with conventional densitometric methods. Finally, the characterization of two different hammerhead motifs indicated that the method is suitable for a rapid screening of synthetic ribozyme activity.