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Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications
Published on: April 14, 2015
Use of fluorescent dyes as molecular probes for the study of multidrug resistance
1Interfaculty Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Abstract:
Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.
Insights
Multidrug-resistant cells show reduced staining with fluorescent dyes due to active dye efflux. Inhibitors of multidrug resistance restore normal staining, suggesting dye efflux as a key resistance mechanism.
Area of Science:
- Cell Biology
- Molecular Pharmacology
Background:
- Multidrug resistance (MDR) is a major challenge in cancer therapy.
- MDR involves the reduced intracellular accumulation of various drugs.
- The underlying mechanisms of MDR are complex and not fully understood.
Purpose of the Study:
- To investigate the relationship between cellular dye uptake and multidrug resistance.
- To explore the potential of fluorescent dye staining as a method for detecting MDR cells.
- To elucidate the role of active efflux in reduced dye accumulation in MDR cells.
Main Methods:
- Fluorescence microscopy was used to assess the intracellular staining of various cell lines.
- Cell lines included drug-sensitive (DM-15), multidrug-resistant (selected by colchicine, actinomycin D, etc.), and revertant cells.
- The effect of various MDR inhibitors (e.g., calcium channel blockers, reserpine) on dye staining was evaluated.
Main Results:
- Multidrug-resistant cells exhibited significantly reduced staining with 18 different fluorescent dyes compared to sensitive cells.
- This reduced staining was observed across multiple MDR cell lines from hamster and mouse origins.
- Inhibitors of multidrug resistance restored normal dye staining in resistant cells, and dye efflux was identified as an active, inhibitor-sensitive process.
Conclusions:
- Reduced fluorescent dye staining is a characteristic feature of multidrug-resistant cells.
- Active, non-specific efflux of substances is a fundamental mechanism underlying multidrug resistance.
- Fluorescent dye staining offers a simple and effective method for detecting and studying multidrug resistance.
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