Wednesday, June 8, 2011

Functional Characterization of Multi-Drug Resistance Transporters in Living Cells

Multi-drug resistance (MDR) relates to resistance of tumor cells to a broad range of chemotherapy drugs with different structures and cellular targets. This phenomenon is a well-known problem in oncology and thus requires careful consideration in cancer treatment. One of the underlying molecular rationales for MDR is the up-regulation of a family of transmembrane ATP binding cassette (ABC) transporter proteins that are present in practically all living organisms. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. We have used a novel fluorescent probe from Enzo Life Sciences that serves as a substrate for all three ABC transporter types to characterize MDR in microplate-based assays using several different mesothelioma tumor cell lines. The probe exhibits fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multi-drug resistance activity factor (MAF).


Figure 1. eFluxx-ID® Green signal from CHO-M1, HepG2, LP9 and HT 1080 cell lines.
Figure 1. eFluxx-ID® Green signal from CHO-M1, HepG2, LP9 and HT 1080 cell lines.
We observed that different cell lines exhibited expression of one or more of the transporter proteins tested. Many of these cell lines have been in culture for decades under various physiologic and chemical stresses, which would provide a selective advantage to cells that can rapidly remove large amounts of a xenobiotic. Interestingly, cell lines recently isolated from mesothelioma tumors also demonstrate expression of the transporter proteins. These cell lines have not had the lengthy stay in culture, yet exhibit the same phenomenon. Despite being from the same tumor type the different cell lines have very different transporter phenotypes, suggesting that chemical resistance need not always follow the same path with tumor cells.


Figure 2. Characterization of different mesothelial and mesothelioma cell lines.
Figure 2. Characterization of different mesothelial and mesothelioma cell lines.
While these assays can be run manually, we used the EL406 Washer Dispenser from BioTek to perform the fluid handling tasks necessary to wash the cells and add reagent. The EL406 Washer Dispenser combines several existing fluid handling technologies into one instrument.

By, BioTek Instruments, Paul Held PhD, Laboratory Manager

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