RT Journal Article
SR Electronic
T1 Hammerhead Ribozyme-Mediated Sensitization of Human Tumor Cells after Treatment with 1,3-Bis(2-chloroethyl)-1-nitrosourea
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 506
OP 514
DO 10.1124/jpet.103.061507
VO 309
IS 2
A1 Zhang, Qiwei
A1 Ohannesian, David W.
A1 Erickson, Leonard C.
YR 2004
UL http://jpet.aspetjournals.org/content/309/2/506.abstract
AB O6-Methylguanine DNA methyltransferase (MGMT) protects tumor cells from the cytotoxic effects of DNA-alkylating agents such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). To improve the therapeutic index of BCNU, biochemical strategies to inhibit MGMT temporarily by systemic administration of small molecules, such as O6-benzylguanine, have been developed and are showing promise in clinical trials. In this study, an alternative molecular strategy for modulating BCNU resistance was explored using hammerhead ribozymes (Rz) designed to degrade the long-lived MGMT mRNA. We had previously identified several ribozymes capable of decreasing MGMT levels in HeLa cells. Using colony formation assays, the BCNU-induced cell kill was shown to be increased by 1 to 3 logs in the HeLa/Rz clones compared with wild-type HeLa cells at a BCNU dose of 100 μM. In the current study, 10 randomly selected clones of Rz161, 212, and a reconstructed Rz178/212 were assayed for MGMT activity, MGMT mRNA, and sensitivity to BCNU. The 30 clones exhibited almost identical results in the three assays, i.e., nearly undetectable MGMT activity, greatly diminished MGMT mRNA, and comparable sensitivity to BCNU using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) viability assay. The effects of catalytically inactive ribozymes carrying a single point mutation were compared with their active counterparts in vitro and in stably transfected clones to determine whether antisense inhibition was a contributor to the inhibition of MGMT activity we observed. Collectively, these results suggest that the hammerhead ribozymes characterized in this study will be excellent candidates for future gene therapy approaches targeting MGMT. The American Society for Pharmacology and Experimental Therapeutics