Use of the ornithine decarboxylase promoter to achieve N-MYC-mediated overexpression of a rabbit carboxylesterase to sensitize neuroblastoma cells toCPT-11

Overexpression of specific transcription factors by tumor cells can be expl oited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor-selective toxicity. A majority of advance d-stage neuroblastomas overexpress the transcription factor N-MYC, and this overexpression is associated with poor prognosis. This study describes reg ulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promo ter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT -11. Chloramphenicol acetyltransferase reporter assays and CE activity assa ys in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4 NB-1 691) and rhabdomyosarcoma cell lines (JR1neo20, jR1Nmyc6, JR1Nmyc9) support this approach as a potential method for sensitizing tumor cells to CPT-11. Clonogenic assays with IMR32 human neuroblastoma cells which express N-MYC and that had been stably transfected with a plasmid containing an ODC prom oter/CE cassette corroborated results of enzyme activity assays. Specifical ly, IMR32.ODC.CE cells expressed approximately eightfold more CE activity t han IMR32.CMV.neo cells; and 5 muM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 muM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on aden oviral delivery of an ODC promoter/CE cDNA cassette for potential virus-dir ected enzyme prodrug therapy applications.