Modulation of 1,3-Bis-(2-chloroethyl)-1-nitrosourea Resistance in Human Tumor Cells Using Hammerhead Ribozymes Designed to DegradeO⁶-Methylguanine DNA Methyltransferase mRNA

Abstract

O⁶-Methylguanine DNA Methyltransferase (MGMT) protects tumor cells from the cytotoxic effects of the DNA alkylating agent 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). To improve the therapeutic index of BCNU, biochemical strategies to deplete MGMT activity have been developed. In the present study, a molecular strategy for modulating BCNU resistance was explored using hammerhead ribozymes (Rz) designed to degrade the long-lived MGMT mRNA. The ribozymes were designed against eight GUC sites within the MGMT mRNA. cDNAs of these ribozymes were cloned into an expression vector and then all eight vectors were pooled and stably transfected into HeLa cells. Several HeLa/Rz clones sensitive to a sublethal dose of BCNU were identified using a short-term cell proliferation assay. The ribozyme inserts were amplified from genomic DNA by polymerase chain reaction and sequenced in the BCNU-sensitive clones. The ribozyme inserts Rz161, 178, and 212, targeted against nucleotide 161, 178, and 212, respectively, in the MGMT mRNA, were found to be present in these clones. MGMT activity, Western, and Northern blot analyses revealed that two of the HeLa/Rz clones contained very low levels of MGMT activity, protein, and mRNA. Investigation of CpG methylation within the MGMT promoter indicated that the lack of MGMT expression in these HeLa/Rz clones was not likely due to methylation silencing of the MGMT gene. By colony formation, the cell killing induced by 100 μM BCNU was increased by 2 to 3 logs in the HeLa/Rz clones compared with wild-type HeLa cells.