Modulation of 1,3-bis-(2-chloroethyl)-1-nitrosourea resistance in human tumor cells using hammerhead ribozymes designed to degrade O-6-methylguanine DNA methyltransferase mRNA

O-6-Methylguanine DNA Methyltransferase (MGMT) protects tumor cells from th e cytotoxic effects of the DNA alkylating agent 1,3-bis-(2-chloroethyl)-1-n itrosourea (BCNU). To improve the therapeutic index of BCNU, biochemical st rategies to deplete MGMT activity have been developed, In the present study , a molecular strategy for modulating BCNU resistance was explored using ha mmerhead ribozymes (Rz) designed to degrade the long-lived MGMT mRNA. The r ibozymes 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/R z clones sensitive to a sublethal dose of BCNU were identified using a shor t-term cell proliferation assay. The ribozyme inserts were amplified from g enomic DNA by polymerase chain reaction and sequenced in the BCNU-sensitive clones. The ribozyme inserts Rz161, 178, and 212, targeted against nucleot ide 161, 178, and 212, respectively, in the MGMT mRNA, were found to be pre sent in these clones. MGMT activity, Western, and Northern blot analyses re vealed that two of the HeLa/Rz clones contained very low levels of MGMT act ivity, 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 fo rmation, the cell killing induced by 100 muM BCNU was increased by 2 to 3 l ogs in the HeLa/Rz clones compared with wild-type HeLa cells.