The goal of this project is enhancing the therapeutic index of the chloroethylnitrosourea (CENU) class of anti-cancer drugs. In order to inhibit the repair of the cytotoxic O6-akylguanine DNA adduct produced by these agents, this project targets the major repair enzyme for this lesion, methylguanine DNA methyltransferase (MGMT). The strategy will be to produce genetic suppressor elements (GSEs) from the MGMT cDNA. Briefly, the cDNA will be fragmented by DNase I digestion and the size- fractionated (100-200 base pairs), blunt-ended fragments cloned into the eukaryotic expression vector pcDNA3.1. These fragments of the MGMT cDNA will contain linkers providing a translational start site and stop codons in all three reading frames. Transcription from the antisense strand of the MGMT cDNA element will produce short antisense RNAs in the cells able to hybridize to the full length MGMT mRNA and bring about translational arrest of the MGMT protein and possibly the degradation of the mRNA itself. Protein synthesis from sense-oriented GSEs should produce polypeptides capable of interfering with normal MGMT enzyme activity through domain interactions possibly by perturbing its localization in the nucleus or by interfering with the active site of the enzyme among various possibilities. After transfection and selection for stable transformants, 1000-2000 clones will be subjected to selection with 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU) at a sublethal dose. From a master plate, cells determined to be hypersensitive to BCNU selection will be grown out and their in vitro MGMT activity determined. DNA sequencing of the GSEs after PCR amplification should reveal whether the elements work through an antisense or dominant negative peptide modality although this will be rigorously confirmed with several in vitro and in vivo assays. GSEs derived from this selection strategy should be exceptional candidates for gene therapy waiting only for specific delivery mechanisms in order to maximize tumor cell/normal cell kill ratios.
| Zhang, Qiwei; Ohannesian, David W; Erickson, Leonard C (2004) Hammerhead ribozyme-mediated sensitization of human tumor cells after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea. J Pharmacol Exp Ther 309:506-14 |
| Zhang, Q; Ohannesian, D W; Kreklau, E L et al. (2001) Modulation of 1,3-bis-(2-chloroethyl)-1-nitrosourea resistance in human tumor cells using hammerhead ribozymes designed to degrade O6-methylguanine DNA methyltransferase mRNA. J Pharmacol Exp Ther 298:141-7 |
