Use of the ornithine decarboxylase promoter to achieve N-MYC-mediated overexpression of a rabbit carboxylesterase to sensitize neuroblastoma cells to CPT-11.
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 microM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 microM 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.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
C A
CA
Pawlik
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Iyengar
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EJ
Krull
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Mason
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Khanna
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Harris
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Potter
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Danks
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Guichard
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D006801:Humans; D000077146:Irinotecan; D009447:Neuroblastoma / Q000188:drug therapy* / Q000201:enzymology; D009955:Ornithine Decarboxylase / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D011817:Rabbits; D012208:Rhabdomyosarcoma / Q000188:drug therapy / Q000201:enzymology; D014162:Transfection; D014407:Tumor Cells, Cultured
D016271:Proto-Oncogene Proteins c-myc; D000077146:Irinotecan; D015500:Chloramphenicol O-Acetyltransferase; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D009955:Ornithine Decarboxylase; D002166:Camptothecin