Abstract
BackgroundPreviously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance.MethodsSensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR.ResultsHuman ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes encoding glutathione-metabolizing enzymes in 293/B5-11 cells was similar to that in 293/mock cells. The cellular content of Glu, a precursor of glutathione, in 293/B5-11 and 293/mb5-8 cells was higher than that in 293/mock cells.ConclusionsABCB5/Abcb5-transfected cells showed resistance to BSO, which is not a substrate of ABCB5. Our results suggest that ABCB5/Abcb5 upregulates cellular glutathione levels to protect cells from various poisons.
Highlights
We have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein
buthionine sulfoximine (BSO) is an inhibitor of glutamylcysteine ligase (GCL), a rate-limiting enzyme of glutathione synthesis. 293/B5-11 and 293/B5-126 cells, which expressed high amounts of ABCB5, showed 6.5- and 4.6-fold higher resistance to BSO than 293/mock cells, respectively, whereas 293/B5-104 and 293/B5-118 cells, which expressed low amounts of ABCB5, showed only 1.4- and 1.7-fold higher resistance to BSO than 293/ mock cells, respectively (Fig. 1a and b)
We examined the sensitivity of the ABCB5- and Abcb5-transfected cells to methionine sulfoximine (MSO), another GCL inhibitor with a similar structure to BSO
Summary
We have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCC1 is expressed in the gastrointestinal tract, liver, kidney and capillary endothelial cells, and mediates the efflux of a variety of organic anions, including glutathione and glucuronide conjugates, as well as unconjugated organic anions such as reduced glutathione (GSH) and folate derivatives. These findings suggest that ABC transporters have two major roles: (1) transporting natural substances and xenobiotics across the lipid bilayer membrane, and (2) protecting important organs and cells such as the brain, testis, and hematopoietic and tissue stem cells from toxic substances
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