RESUMO
One of the advantages of viral-directed enzyme prodrug therapy (VDEPT) is its potential for tumor-specific cytotoxicity. However, the viruses used to deliver cDNAs encoding prodrug-activating enzymes transduce normal cells as well as tumor cells, and several approaches to achieve tumor-specific expression of the delivered cDNAs are being investigated. One such approach is to regulate transcription of the prodrug-activating enzyme with a promoter that is preferentially activated by tumor cells. Published data suggest that the most promising transcription factor/promoter/enhancer combinations are those activated by a tumor-specific transcription factor to retain tumor cell specificity but that are equal in strength to nonspecific viral promoters in their ability to up-regulate target cDNAs. This report identifies MYC-responsive, modified ornithine decarboxylase (ODC) promoter/enhancer sequences that up-regulate target protein expression in tumor cells overexpressing either N-MYC or c-MYC protein. The most efficient of the four constructs assessed contained six additional CACGTG MYC binding sites 5' to the endogenous ODC promoter (R6ODC). Reporter assays with this chimeric promoter/enhancer regulating expression of chloramphenicol acetyltransferase demonstrated 50-250-fold more activity in MYC-expressing cells compared with similar assays with promoterless plasmids. The R6ODC regulatory sequence was approximately equivalent to the CMV promoter in inducing expression of the neomycin resistance gene in c-MYC-expressing SW480 and HT-29 colon carcinoma cells and in N-MYC-expressing NB-1691 neuroblastoma cells. The modified ODC promoter may, therefore, be useful in achieving tissue-specific expression of target proteins in tumor cells that overexpress c- or N-MYC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Ornitina Descarboxilase/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/biossíntese , Animais , Antineoplásicos Fitogênicos/farmacocinética , Biotransformação , Camptotecina/farmacocinética , Carboxilesterase , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/genética , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Genes Reporter , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Immunoblotting , Irinotecano , Proteína MyoD/biossíntese , Proteína MyoD/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Coelhos , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Transfecção , Transgenes , Células Tumorais CultivadasRESUMO
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.