Induction of multidrug resistance in MOLT-4 cells by anticancer agents is closely related to increased expression of functional P-glycoprotein and MDR1 mRNA.
OBJECTIVE
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 IC(50) 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 microM 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.
Liu
Zhen-Li
ZL
Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
Onda
Kenji
K
Tanaka
Sachiko
S
Toma
Tsugutoshi
T
Hirano
Toshihiko
T
Oka
Kitaro
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003630:Daunorubicin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D007202:Indicators and Reagents; D012333:RNA, Messenger; D011061:Poly A; D014747:Vinblastine; D004317:Doxorubicin; D003630:Daunorubicin