Localization of the multi-drug resistance gene product (LeMDR1) in Leishmania enriettii

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Localization of the multi-drug resistance gene product (LeMDR1) in Leishmania enriettii

 

Author: Tsang, Ming-san
Title: Localization of the multi-drug resistance gene product (LeMDR1) in Leishmania enriettii
Degree: M.Phil.
Year: 2004
Subject: Hong Kong Polytechnic University -- Dissertations
Multidrug resistance
Leishmania
Leishmaniasis
Department: Dept. of Applied Biology and Chemical Technology
Pages: xiii, 167 leaves : ill. (some col.) ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1749081
URI: http://theses.lib.polyu.edu.hk/handle/200/218
Abstract: Introduction Multi-drug resistance (MDR) in infectious diseases is one of the major problems all over the world. This problem is also emerging in Leishmania spp., a major parasitic disease. In previous studies, stepwise selected vinblastine resistant Leishmania enriettii cell line (with LeMDR1 amplification) and the "double-knockout" LeMDR1 mutant were developed. LeMDR1 was found to be correlated with vinblastine resistance. It was found that 65% of LeMDR1 was localized to mitochondria under electron microscopy observation. In this project, this panel of LeMDR1 mutant cell lines would be used to elucidate the LeMDR1 resistance mechanism by studying the fluorescent drugs accumulation and the LeMDR1 localization. Fluorescent Drugs Accumulation Accumulation profile of fluorescent mitochondria drugs rhodamine 123 (R123) and multidrug resistance (mdr) substrate calcein AM were studied in wild type and mutant cell lines. The results indicated that R123 accumulation was directly proportional to LeMDR1 copy number, whereas calcein AM was inversely proportional to the copy number. DiOC6(3) accumulation studies in these cell lines were similar, thereby eliminating the differences of membrane potential in these cell lines. When compared with cancer cell model, the R123 accumulation trend was opposite, indicating that LeMDR1 is not using the same efflux model as mdr model in cancer MDR. MDR1 is located on plasma membrane whereby LeMDR1 is not. We therefore hypothesize that the intracellularly located LeMIDR1 is sequestrating drugs into organelle. Studies on Influx and Efflux of Fluorescent Drugs In cancer MDR model, MDR1 was overexpressed on plasma membrane and it can pump R123 out of the cell by elevated efflux. LeMDR1 is not located on plasma membrane. In order to investigate differences between MDR1 and LeMDR1 models, the influx and efflux rates of fluorescent drugs were studied. In R123 studies, it was found that drug efflux rates were similar in all cell lines, but higher influx rates was detected in LeMDR1 overexpressed cell line. This is different from the MDR cancer model where MDR1 is responsible for elevated efflux. This result highly suggests that LeMDR1 and MDR1 work differently. Interestingly, influx rate of calcein AM was elevated in knockout mutant but the drug efflux rates were also similar in all cell lines. This result indicated that drug accumulation was affected probably by changing influx, not by efflux. Subcellular Fractionation In order to study the localization of LeMDR1, cells were analyzed by fractionation and western blotting. Antibodies against different organelles were used tp study the localization of LeMDR1. Results showed that LeMDR1 fraction was co-localized with DAPI (DNA) staining and cytochrome b antibodies (mitochondria). Although only 2 fold purification of mitochondria was obtained after fractionation, this result was the same as other group's studies. Mitochondria, therefore, is probably the localization of LeMDR1. Summary From the drug accumulation and influx/efflux studies, it was found that LeMDR1 is a different drug transport system compared to traditional mdr1 cancer cell model. LeMDR1 probably affects influx and not efflux. This is due to its intracellular localization. Fractionation studies also indicated that LeMDR1 was found in mitochondria. This data further strengthens our hypothesis that LeMDR1 is sequestrating drug into intracellular components, possibly mitochondria.

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