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 c ellular injury and the magnitude of the change in GADD 153 expression is pr oportional 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 int egrated into the genome of a clone of UMSCC 10b head and neck carcinoma cel ls. Activation of the exogenous GADD153 promoter was quantified using flow cytometric measurement of EGFP expression following drug exposure. The exog enous GADD153 promoter in this clone was activated by N-methl-N'-nitro-N-ni trosoguanidine (MNNG) in a concentration-dependent manner with kinetics tha t closely paralleled perturbation of cell cycle phase distribution. EGFP ex pression was strongly activated by a variety of genotoxic agents including DNA cross-linking and methylating agents, oxygen free radicals, DNA interca lator, UV and gamma -radiation and hypoxia. When grown as a xenograft in nu de mice, the stably transfected clone also demonstrated dose dependent EGFP expression when measured 4 days after cisplatin treatment. The reporter sy stem accurately categorized the relative potency of adducts produced by 6 r elated platinum-containing drugs. In conclusion, this reporter system can f acilitate in vitro and in vivo screening for agents capable of producing cy totoxicity via a wide variety of different mechanisms, and can be utilized to investigate the relative potency of structurally related DNA adducts. (C ) 2001 Wiley-Liss, Inc.