Induction of multidrug resistance in MOLT-4 cells by anticancer agents is closely related to increased expression of functional P-glycoprotein and MDR1 mRNA

Abstract.

Purpose: The aim of this study was to investigate the multidrug resistance (MDR) pattern, MDR gene and P-glycoprotein (P-gp) expression, and P-gp function in drug-induced human T-lymphoblastoid leukemia MOLT-4 sublines. Methods: The MDR sublines were developed by exposing the parental MOLT-4 cells to stepwise increasing concentrations of anticancer drugs daunorubicin (DNR), vinblastine (VBL) and doxorubicin (DOX). Degrees of resistance were assessed in terms of IC₅₀ values in an MTT assay and the P-gp function was evaluated in terms of rhodamine 123 (Rh123) accumulation and efflux. The percentage of cells undergoing apoptosis was determined by flow cytometry after staining with annexin V-FITC and propidium iodide. The levels of P-gp and MDR mRNA expression were estimated using the PE-conjugated anti-P-gp monoclonal antibody 17F9 and quantitative real-time reverse transcription-polymerase chain reaction. Results: Three MOLT-4 sublines were established and revealed a 2- to 115-fold resistance to the anticancer reagents DNR, VBL and DOX as compared to the parental cell line. The highest MDR was expressed in MOLT-4/DNR cells, which was overcome by the P-gp modulator, cyclosporin A (CsA). The resistant sublines showed a decreased accumulation and an increased efflux of Rh123 in proportion to the degree of resistance, and these were completely reversed in the presence of 8 µM CsA. The decreased apoptotic response in these cell lines was clearly associated with the degree of drug resistance. P-gp antigen and MDR1 mRNA were highly expressed in both the MOLT-4/DNR and MOLT-4/DOX sublines. Less-resistant MOLT-4/VBL cells expressed lower levels of MDR1 mRNA and P-gp, even though the cell line was established by exposing the parental MOLT-4 cells to VBL for longer (5 months) than to the other two reagents (3 months). Conclusions: MOLT-4 cells were able to acquire a high level of drug resistance by culturing the cells in the presence of certain anticancer drugs, and acquisition of the resistance was relatively reagent-specific. The degrees of resistance to the anticancer drugs were well correlated with the expressions of MDR1 mRNA and functional P-gp, and were also associated with a decreased response to apoptosis.