Multidrug resistance modulators PSC 833 and CsA show differential capacity to induce apoptosis in lymphoid leukemia cell lines independently of their MDR phenotype

Abstract 

Among the mechanisms that induce multidrug resistance (MDR), one of those most frequent is over-expression of a phosphoglycoprotein (Pgp) encoded in the mouse by the mdr-1 and mdr-3 genes. We have demonstrated that cyclosporin-A (CsA) as well as its analogue PSC 833 were able to revert the MDR phenotype in murine cell lines resistant to vincristine (LBR-V160) or doxorubicin (LBR-D160). The aim of this work was to evaluate the ability of PSC 833 and CsA to modulate mdr-1, mdr-3 and mrp-1 genes as well as to induce apoptosis analyzing the mechanism involved in the above tumor cell lines. By semi-quantitative RT-PCR, we demonstrated that mdr-3 was over-expressed in both resistant lines while mdr-1 was over-expressed only in LBR-V160; in contrast, mrp-1 expression was not evidenced in any of the cell lines. After treatment with 0.1μgml⁻¹ of either PSC 833 or CsA, LBR-V160 showed no changes in mdr-1 but decreased mdr-3 expression, while LBR-D160 failed to display any modification in the expression of these genes. Apoptosis was evidenced by fluorescence microscopy, S minuscule accumulation and agarose gel electrophoresis. Our results demonstrated that CsA (1μgml⁻¹) was able to induce apoptosis in all cell lines: 18.31% (±4.46) for LBR-, 25.96% (±5.24) for LBR-V160 and 27.36% (±4.12) for LBR-D160, while PSC 833 (1μgml⁻¹) only induced apoptosis 21.51% (±5.73) in LBR-V160 cell line. The expression of Bcl-2 family proteins (Bcl-2, Bax and Bcl-x_L) was analyzed by flow cytometry showing high expression of the three proteins which was not significantly modified after treatment with either PSC 833 or CsA on the sensitive as well as on the resistant cell lines. Single stranded conformation polymorphisms analysis of p53 (Trp53) gene in the cell lines showed no mutation in exons 5–8 of the tumor suppressor gene. We conclude that depending on the concentration used, PSC 833 and CsA may act either by modulating the mdr-3 gene (0.1μgml⁻¹) or by direct impact on the cells through induction of apoptosis (1μgml⁻¹), in the latter case through a mechanism that might act independent of the Bcl-2 family proteins.

Abbreviations:  ABCB1, ATP-binding cassette transporter, CsA, cyclosporin-A, DOX, doxorubicin, FACS, fluorescence activated cell sorter, FCS, fetal calf serum, LB, parental leukemic-T cells, LBR-, sensitive cell line, LBR-D160, doxorubicin resistant cell line, LBR-V160, vincristine resistant cell line, LRP, lung resistance protein, MDR, multidrug resistance, MRP, multidrug resistance protein, Pgp, phosphoglycoprotein, RF, resistance factor, RT-PCR, reverse transcriptase polymerase chain reaction, SSCP, single stranded conformation polymorphisms, VCR, vincristine

Keywords:  Multidrug resistance, Leukemia, Apoptosis, PSC 833, Cyclosporin-A, p53, mdr gene