Increased drug resistance following retroviral gene transfer of a chimeric P-enolipyruvate carboxykinase (GTIP)-bacterial O6 alkylguanine-DNA alkyltransferase gene into NRK cells

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Abstract
Transfection of the Escherichia coli ada gene coding for O6 alkyltransferase results In expression of ada in mammalian cells and transmission of nitrosourea resistance to cells lacking alkyltransferase activity. We have used a replication-incompetent retrovirus to transfer into mammalian cells a chinieric gene consisting of 548 bp of the promoter-regulatory region of the gene for P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) linked to ada. The PEPCK promoter was used because it is inducible and highly expressed in liver and kidney cells both in vitro and in vivo. After retrovirus infection of the rat kidney cell line, NRK, intact proviral DNA was integrated into the genome of cloned cells. Individual NRK clones produced up to 20(1 units/mg protein of bacterial alkyltransferase activity compared to 65 units/mg protein of mammalian aikyl ransferase in the parent cell line. Transcription of ada mRNA originating from the PEPCK promoter was Induced with Bt2cAMP or dexamethasone and the combination caused a 4-fold Increase in ada mRNA while total alkyltransferase activity was induced up to 2-fold. NRK clones expressing ada had up to 2.0-fold increased resistance to 1,3-bis(2- chloro ethyl)-1-nitrosourea. Thus, retroviral gene transfer of the PEPCKada chimeric gene allows efficient and inducible expression of ada with a resulting increase in alkyltransferase activity and nitrosourea drug resistance. This retrovirus may be used to study the role of alkyltransferase in repair of mutagenic DNA lesions in mammalian cells in vivo.