Quantification of tumor cell injury in vitro and in vivo using expression of green fluorescent protein under the control of the GADD153 promoter.
The GADD153 gene is strongly transcriptionally activated by many types of cellular injury and the magnitude of the change in GADD153 expression is proportional to the extent of damage. We developed a novel reporter system in which a chimeric gene containing the GADD153 promoter linked to the coding region of an enhanced green fluorescent protein (EGFP) gene was stably integrated into the genome of a clone of UMSCC10b head and neck carcinoma cells. Activation of the exogenous GADD153 promoter was quantified using flow cytometric measurement of EGFP expression following drug exposure. The exogenous GADD153 promoter in this clone was activated by N-methl-N'-nitro-N-nitrosoguanidine (MNNG) in a concentration-dependent manner with kinetics that closely paralleled perturbation of cell cycle phase distribution. EGFP expression was strongly activated by a variety of genotoxic agents including DNA cross-linking and methylating agents, oxygen free radicals, DNA intercalator, UV and gamma-radiation and hypoxia. When grown as a xenograft in nude mice, the stably transfected clone also demonstrated dose-dependent EGFP expression when measured 4 days after cisplatin treatment. The reporter system accurately categorized the relative potency of adducts produced by 6 related platinum-containing drugs. In conclusion, this reporter system can facilitate in vitro and in vivo screening for agents capable of producing cytotoxicity via a wide variety of different mechanisms, and can be utilized to investigate the relative potency of structurally related DNA adducts.
Department of Medicine and the Cancer Center, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0058, USA.
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D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics*; D002453:Cell Cycle; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis; D019175:DNA Methylation; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005720:Gamma Rays; D017930:Genes, Reporter; D005822:Genetic Vectors; D016678:Genome; D049452:Green Fluorescent Proteins; D006258:Head and Neck Neoplasms / Q000235:genetics; D006801:Humans; D000860:Hypoxia; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D000970:Antineoplastic Agents; D022762:CCAAT-Enhancer-Binding Proteins; D003432:Cross-Linking Reagents; C497249:DDIT3 protein, human; D018736:DNA Adducts; C497250:Ddit3 protein, mouse; D008164:Luminescent Proteins; D014157:Transcription Factors; D008769:Methylnitronitrosoguanidine; D051742:Transcription Factor CHOP; D049452:Green Fluorescent Proteins; D004247:DNA; D002945:Cisplatin