Use of the Ornithine Decarboxylase Promoter to Achieve N-MYC-Mediated Overexpression of a Rabbit Carboxylesterase to Sensitize Neuroblastoma Cells to CPT-11
Authors: Cynthia A. Pawlik, Rekha V. Iyengar, Erik J. Krull, Stephen E. Mason, Ruchi Khanna, Linda C. Harris, Philip M. Potter, Mary K. Danks, Sylvie M. Guichard
Journal: Molecular Therapy (2000)

Overexpression of specific transcription factors by tumor cells can be exploited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor-selective toxicity. A majority of advanced-stage neuroblastomas overexpress the transcription factor N-MYC, and this overexpression is associated with poor prognosis. This study describes regulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promoter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT-11. Chloramphenicol acetyltransferase reporter assays and CE activity assays in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4, NB-1691) 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 promoter/CE cassette corroborated results of enzyme activity assays. Specifically, IMR32.ODC.CE cells expressed approximately eightfold more CE activity than IMR32.CMV.neo cells; and 5 μM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 μM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on adenoviral delivery of an ODC promoter/CE cDNA cassette for potential virus-directed enzyme prodrug therapy applications. Overexpression of specific transcription factors by tumor cells can be exploited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor-selective toxicity. A majority of advanced-stage neuroblastomas overexpress the transcription factor N-MYC, and this overexpression is associated with poor prognosis. This study describes regulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promoter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT-11. Chloramphenicol acetyltransferase reporter assays and CE activity assays in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4, NB-1691) 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 promoter/CE cassette corroborated results of enzyme activity assays. Specifically, IMR32.ODC.CE cells expressed approximately eightfold more CE activity than IMR32.CMV.neo cells; and 5 μM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 μM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on adenoviral delivery of an ODC promoter/CE cDNA cassette for potential virus-directed enzyme prodrug therapy applications.