Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and 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 the search for cisplatin analogs if fast, high-throughput assays were available. Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes. We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells.


RESULTS
HeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared. Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity. By contrast, trans-[Pt(NH(3))(2)Cl(2)], other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression. Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription. In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-[Pt(NH(3))(2)Cl(2)] or K(2)[PtCl(4)], inhibited beta-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2.


CONCLUSIONS
The EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription. Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery.
Sandman
K E
KE
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Marla
S S
SS
Zlokarnik
G
G
Lippard
S J
SJ
D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D003001:Cloning, Molecular / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D005453:Fluorescence; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000096:biosynthesis; D009944:Organoplatinum Compounds / Q000138:chemical synthesis / Q000494:pharmacology*; D011237:Predictive Value of Tests; D014158:Transcription, Genetic / Q000187:drug effects; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000096:biosynthesis
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009944:Organoplatinum Compounds; D065093:beta-Lactamase Inhibitors; D049452:Green Fluorescent Proteins; D001618:beta-Lactamases; D002945:Cisplatin