Multidrug resistance modulators PSC 833 and CsA show differential capacity to induce apoptosis in lymphoid leukemia cell lines independently of their MDR phenotype.
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 0.1 microg ml(-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 (1 microg ml(-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 (1 microg ml(-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 (0.1 microg ml(-1)) or by direct impact on the cells through induction of apoptosis (1 microg ml(-1)), in the latter case through a mechanism that might act independent of the Bcl-2 family proteins.
Lopes
Eloisi C
EC
Department of Immunology, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA) IDEHU-CONICET, Junin 956 4 piso, 1113, Buenos Aires, Argentina. meclopes@hotmail.com
Garcia
Mariana
M
Benavides
Fernando
F
Shen
Jianjun
J
Conti
Claudio J
CJ
Alvarez
Elida
E
Hajos
Silvia E
SE
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle; D016572:Cyclosporine / Q000494:pharmacology*; D003524:Cyclosporins / Q000494:pharmacology*; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005091:Exons / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic; D019450:Genes, MDR; D019254:Genes, bcl-2; D016158:Genes, p53; D015458:Leukemia, T-Cell / Q000235:genetics / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D010641:Phenotype; D018807:Polymorphism, Single-Stranded Conformational; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology; D014750:Vincristine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; C494827:Bax protein, mouse; C494812:Bcl2l1 protein, mouse; D003524:Cyclosporins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D014750:Vincristine; D004317:Doxorubicin; D016572:Cyclosporine; C095122:multidrug resistance protein 3; C070272:valspodar