A 96-well plate cell-based assay to quantify undesired cytotoxic effects against quiescent non-dividing cells

1157 A major issue in the development of new chemotherapeutic agents is the occurrence of undesired toxicity in normal tissues. In fact, toxicity against normal non-dividing cells within the context of affected organs is the most common reason that compounds fail in the clinic. Here we describe the development of an assay to measure cytotoxicity against quiescent, or non-dividing, cells. IMR-90 human lung fibroblasts were plated at 1.0 x 104 cells/well in 96-well plates and grown in EMEM + 10% FBS. After 5 days in culture, media for the now confluent cells was replaced with EMEM + 0.1% FBS for three days to ensure total quiescence. Quiescence was verified using [3H]-thymidine incorporation with signal to noise ratio calculated by the ability of aphidicolin to further reduce signal. Test compounds were plated at final concentrations of 0.03nM to 100uM, and cell viability determined by measuring intracellular ATP after 1-3 days using a luciferase-based monitoring system (ATPLite, Perkin Elmer). Several clinical and laboratory compounds were evaluated in the quiescent IMR-90 cytotoxicity assay including the clinical drugs paclitaxel, oxaliplatin, 5-FU, doxorubicin, etoposide, SN-38 (active metabolite of CPT-11), gemcitabine and mitomycin C, as well as the laboratory tools actinomycin D and cycloheximide. Results indicate that clinical agents which act solely within proliferative phases of the cell cycle show good cytotoxicity windows between cytotoxic effects on quiescent fibroblasts and growth inhibitory effects against proliferating cancer cells. For example, the mitotic blocker paclitaxel inhibits cancer cell growth at low nM concentrations, yet is not cytotoxic to quiescent fibroblasts until approximately 10 μM even after 72 hours. Interestingly, agents such as doxorubicin, mitomycin C, and actinomycin D, which act via direct interactions with nucleic acids, show time-dependent increases in cytotoxicity against quiescent fibroblasts. Overall, results from a spectrum of drugs tested in this assay indicate that agents with mechanisms which are not exclusive to the proliferative phases of the cell cycle will show cytotoxicity towards quiescent fibroblasts. Most assays currently in use examine both cell proliferation and cytotoxicity in dividing cells, whether malignant or nonmalignant. However, assays which can quantify cytotoxic effects against truly non-proliferating cells have not been described. Development of the quiescent IMR-90 cytotoxicity assay to quantify cytotoxicity in non-dividing cells provides an in vitro tool to gauge the likelihood of undesirable cytotoxicity against non-dividing cells. These data may help guide the decision to subject anticancer leads to in vivo evaluation and thus accelerate progression of only the most promising drug candidates toward clinical testing.