Differential macrophage-mediated cytotoxicity to P388 leukemia cells and its drug-resistant cells examined by a new MTT assay

Abstract

After activation by interferon-γ (INF-γ) and lipopolysaccharide(LPS), mouse peritoneal macrophages were cocultured with P388 parental cell line(P388/PRT) and its adriamycin(ADM)-, cisplatin(CDDP)-, cyclophosphamide(CPM)-, and mitomycin-C(MMC)-resistant cell lines for one day at effector:target ratios(E:T) of 10:1, 5:1, and 2:1. The direct 3-(4,5-dimethylthiazol-2-yl)2,5diphenyl tetrazolium bromide (MTT) cleavage assay and a new indirect MTT assay as well as clonogenic assay were used to quantitate activated macrophage-mediated cytotoxicity to these nonadherent leukemia targets. The results revealed that all the P388 cell lines can be suppressed efficiently by activated macrophages, but P388 CPM- and MMC-resistant cell lines (P388/CPM, P388/MMC) were more susceptible than P388/PRT while P388 ADM- and CDDP-resistant cell lines (P388/ADM, P388/CDDP) shared equal level of survival rates with P388/PRT. This study also showed that both non-activated and activated macrophages can produce formazan in a high level, which can interfere with the final results of direct MTT assay. The new indirect MTT assay can avoid such interference by separating the effectors from the targets before performing the MTT assay and reflects the real viability of the targets so the indirect MTT assay developed in this study could be a better way to examine cytostatic and cytotoxic effect of activated macrophages an non-adherent tumor cells in vitro.