다약물 내성 동계 생쥐 백혈병 아세포주의 선별 및 성격규명

Abstract

Background and purpose. The development of multidrug resistance (MDR) by tumor cells is a major obstacle to successful cancer chemotherapy. MDR cells can be sensitized to anticancer drugs when treated concomitantly with chemosensitizers. However, it is difficult to examine in vivo chemosensitizing effect using immune compromised animals. In this study, an attempt was made to select murine leukemic MDR sublines from P388 cells by chronic exposure to gradually increasing concentrations of daunorubicin and to characterize the mechanisms of their resistance to this drug for the determination of in vivo efficacy in a syngeneic mouse. Materials and Methods. Cytotoxicity and gene expression were determined by the MTT assay and reverse transcription-polymerase chain reaction (RT-PCR) assay or Western blot analysis. Functional drug accumulation was examined using flow cytometry. Daunorubicin-resistant subline P388/D100 cells have been selected. Results. The P388/D100 cells showed not only 31.3-fold to daunoubicin, as compared with each parental cell but also cross resistance to doxorubicin and vincristine but not paclitaxel and docetaxel. Drug sensitivity and intracellular drug accumulation of P388/D100 cells was increased by P-glycoprotein inhibitors, verapamil or PSC833. RT-PCR and Western blot analyses revealed that P388/D100 cells overexpressed P-glycoprotein. The RT-PCR and sequencing analysis indicates that there were mutation sites including C1017G (339 His-Glu), G1018C (340 Val-Leu), T2040A (680 Ala-Ala) and A2076C (692 Ala-Ala) in open reading frame of mdr1-a. Conclusion. lt is thought that P388/D100 cells can be used for the determination of in vitro and in vivo chemosensitizing efficacy for syngeneic mice. Sensitivity of P388/D100 cells to taxenes may be due to mutation of mdr1-a gene.