Altered Drug Membrane Permeability in a Multidrug-Resistant Leishmania tropica Line

doi:10.1016/S0006-2952(97)00385-7

Biochemical Pharmacology

Volume 55, Issue 2, 15 January 1998, Pages 131-139

https://doi.org/10.1016/S0006-2952(97)00385-7 Get rights and content

Abstract

We selected a Leishmania tropica cell line resistant to daunomycin (DNM) that presents a multidrug-resistant (MDR) phenotype characterized by overexpression of a P-glycoprotein of 150 kDa. The resistant line overexpressed an MDR-like gene, called ltrmdr1, located in an extrachromosomal circular DNA. DNM uptake experiments using laser flow cytometry showed a significant reduction in drug accumulation in the resistant parasites. The initial stages of the interaction of DNM with membranes from wild-type and DNM-resistant parasites were defined by a rapid kinetic stopped-flow procedure which can be described by two kinetic components. On the basis of a previous similar kinetic study with tumor cells, we ascribed the fast component to rapid interaction of DNM with membrane surface components and the slow component to passive diffusion of the drug across the membranes. The results reported here indicate that entrance of DNM into wild-type parasites was facilitated in respect to the resistant ones. We propose that resistance to DNM in L. tropica is a multifactorial event involving at least two complementary mechanisms: an altered drug membrane permeability and the overexpression of a protein related to P-glycoprotein that regulates drug efflux.

Section snippets

Drugs and Chemicals

DNM and doxorubicin hydrochloride were purchased from Farmitalia Carlo Erba. Vinblastine was obtained from Lilly S.A. Puromycin was from Sigma Chemical Co. Glutathione Sepharose 4B, Protein A Sepharose CL-4B and CNBr-activated Sepharose 4B were purchased from Pharmacia LKB Biotechnology. All other reagents were of standard laboratory grade.

Parasite Culture and Selection of DNM Resistant Line

L. tropica LRC-L39 was obtained from Dr. L. F. Schnur (Kuvin Center for the Study of Infectious and Tropical Diseases, Jerusalem, Israel). The WT line used

Characterization of the MDR Phenotype in a L. tropica Line

The L. tropica DNM-R150 line was generated in vitro using a stepwise selection process initiated with a concentration of 25 μM DNM. The time required to induce resistance at the maximum concentration of 150 μM DNM was approximately 5 months. The resistance index at the maximum DNM concentration was 62.3-fold (Table 1). In Rv₁, Rv₃ and Rv₆ parasites, the ic₅₀ values for DNM reverted to those for WT, ranging from 10 μM in the Rv₁ (Table 1) to 6 μM in the Rv₃ and 3 μM in the Rv₆ line. These

Discussion

A main objective in this work has been to study whether modifications other than overexpression of a Pgp can contribute to the resistant phenotype in an L. tropica line resistant to DNM. The line shows an MDR-like phenotype similar to that described in mammalian cells. It is characterized by the amplification of the ltrmdr1 gene as an extrachromosomal element (DN circle) and the overexpression of a Pgp of 150 kDa. The resistance of parasites to DNM is unstable without drug pressure, as

Acknowledgements

The authors thank Dr. Ulisses Gazos (Federal University of Rio de Janeiro, Brazil) for the generous gift of the pLa06 probe and Dr. Antonio González (Instituto de Parasitologı́a y Biomedicina “Lopez-Neyra”, CSIC, Granada, Spain) for the β-tubulin gene from T. cruzi. We also thank Dr. Gregory Schneider (School of Medicine, St. Louis University, MO, USA) for revision and critical reading of the manuscript and Pharmacia Farmitalia (Barcelona, Spain) for the daunomycin used in this study. This work

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Research PapersAltered Drug Membrane Permeability in a Multidrug-Resistant Leishmania tropica Line

Abstract

Section snippets

Drugs and Chemicals

Parasite Culture and Selection of DNM Resistant Line

Characterization of the MDR Phenotype in a L. tropica Line

Discussion

Acknowledgements

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J Biol Chem

FEBS Lett

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J Biol Chem

Parasitol Today

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J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

Cell

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Cell biological mechanisms of multidrug resistance in tumors

Proc Natl Acad Sci USA

The biochemistry of P-glycoprotein-mediated multidrug resistance

Annu Rev Biochem

Research Papers
Altered Drug Membrane Permeability in a Multidrug-Resistant Leishmania tropica Line