MDR—ABC transporter activity in normal population and in rheumatoid arthritis patients. A predictive tool of biological therapeutic response.

In the past 10 years, a paradigm shift has taken place in understanding the clinical significance of mechanism of action of multidrug resistance (MDR) transporters and their function as potential biomarkers. Earlier, it was assumed that the mechanism behind impaired therapeutic efficacy of certain drugs and multidrug resistance - ATP-binding cassette (MDR—ABC) transporter activity is solely associated with the drug efflux function. In addition to drug transport more and more evidence emerged that the transporters have an important role as regulators of immune response and inflammation representing the most important efflux mechanism for several inflammatory signaling molecules, such as platelet-activating factor (PAF), phingosine-1-phosphate (S1P), and eicosanoids (prostanoids and leukotrienes) which are among the mediators of chronic inflammation. MDR—ABC transporters may also modulate cellular redox homeostasis. In addition, several studies have showed the clinical significance of MDR MDR—ABC transporters as prognostic and/or predictive marker in immunosuppressive therapies for active rheumatoid arthritis (RA) using methotrexate, other synthetic disease-modifying anti-rheumatic drugs (sDMARDs) or biological DMARDs. According to the new concept MDR—ABC transporters are biomarkers. Their role in the immune processes and MDR is a rapidly developing field and it will be likely evaluated as part of a complex panel of biomarkers for prognostic scoring, for monitoring disease activity or to predict the responsiveness to certain medications (e.g. immunosuppressive treatments or chemotherapy in malignancies). However, translation of MDR—ABC transporter activity into clinical decisions and treatment regimen requires well defined normal reference and pathological activity values. This thesis explores how the new scientific findings might establish MDR—ABC transporters as predictive biomarkers in rheumatoid arthritis (RA). Focusing on the importance of the multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) transporters in rheumatoid arthritis

mRNA levels of related Abcb genes change opposite to each other upon histone deacetylase inhibition in drug-resistant rat hepatoma cells.

The multidrug-resistant phenotype of tumor cells is acquired via an increased capability of drug efflux by ABC transporters and causes serious problems in cancer treatment. With the aim to uncover whether changes induced by epigenetic mechanisms in the expression level of drug transporter genes correlates with changes in the drug resistance phenotypes of resistant cells, we studied the expression of drug transporters in rat hepatoma cell lines. We found that of the three major rat ABC transporter genes Abcb1a, Abcb1b and Abcc1 the activity of only Abcb1b increased significantly in colchicine-selected, drug-resistant cells. Increased transporter expression in drug-resistant cells results primarily from transcriptional activation. A change in histone modification at the regulatory regions of the chromosomally adjacent Abcb1a and Abcb1b genes differentially affects the levels of corresponding mRNAs. Transcriptional up- and down-regulation accompany an increase in acetylation levels of histone H3 lysine 9 at the promoter regions of Abcb1b and Abcb1a, respectively. Drug efflux activity, however, does not follow tightly the transcriptional activity of drug transporter genes in hepatoma cells. Our results point out the need for careful analysis of cause-and-effect relationships between changes in histone modification, drug transporter expression and drug resistance phenotypes

Determination of reference values of MDR-ABC transporter activities in CD3+ lymphocytes of healthy volunteers using a flow cytometry based method

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Determination of reference values of MDR-ABC transporter activities in CD3+ lymphocytes of healthy volunteers using a flow cytometry based method

BACKGROUND: MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined. METHODS: The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells. RESULTS: MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = -0.27 and P = 0.0001, r = -0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age. CONCLUSIONS: The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools

<i>Abcb1a</i> and <i>Abcb1b</i> mRNA and protein levels in drug-sensitive and resistant rat hepatoma cells.

<p>Relative mRNA levels of <i>Abcb1a</i> (A) and <i>Abcb1b</i> (B) in hepatoma cells were determined by quantitative real-time PCR. RNA samples were isolated from drug-sensitive parental (D12) and drug-resistant (col500 and col1000) cells, which were cultured for 3 days in the absence of colchicine. (*p<0.05, Mann Whitney test) The protein levels of ABCB1a (C) and ABCB1b (D) were determined by Western blot. Protein samples obtained from drug-sensitive and drug-resistant cells were separated on 10% SDS-polyacrylamide gels and transferred to immobilon membranes. Western blots were developed using ABCB1a and ABCB1b pan-specific primary antibodies. The loading control was developed with anti-tubulin Ab.</p

Activity of drug transporters in drug-sensitive and drug-resistant cell lines.

<p>MAF values reflecting ABCB1 (A), ABCC1 (B), ABCB1a (C) and ABCB1b (D) transporter activities were determined as described in the Materials and Methods and are shown as the percent of the total drug transporter activity of the indicated cell lines. (*p<0.05, Mann Whitney test).</p