Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicityand antitumor drug potential of platinum complexes

Background: The need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds. Combinatorial chemistry could aid in th e search for cisplatin analogs if fast, high-throughput assays were availab le. Our goal was to develop rapid cell-based assays suitable for high-throu ghput screening that accurately predict the cytotoxicity of platinum comple xes. We examined the effects of platinum complexes and other agents on repo rter-gene expression in cancer cells. Results: HeLa Tet-On cells with inducible enhanced green fluorescent protei n (EGFP) were prepared. Cisplatin and other cis-disubstituted platinum comp lexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity. By contrast, trans-[Pt(NH3)(2)Cl-2], oth er trans-platinum complexes, methyl methanesulfonate or heat shock stimulat ed EGFP expression. Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription. In another r eporter-gene assay in Jurkat cells, cisplatin, but not trans [Pt(NH3)(2)Cl- 2] or K-2[PtCl4], inhibited beta-lactamase expression, as measured by hydro lysis of the fluorescent substrate CCF2. Conclusions: The EGFP results indicate that cytotoxic stress enhances trans cription from the inducible promoter, whereas compounds able to form the 1, 2-intrastrand platinum-DNA cross-links repress transcription, Both fluoresc ence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery.