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Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using
J E Gervasoni1, S Z Fields, S Krishna
1Department of Medicine, Columbia University, New York, New York 10032.
Abstract:
Four well defined multidrug-resistant cell lines and their drug-sensitive counterparts were examined for intracellular distribution of daunorubicin (DNR) by laser-assisted confocal fluorescence microscopy: P-glycoprotein-negative HL-60/AR cells, and P-glycoprotein-positive P388/ADR, KBV-1, and MCF-7/ADR cells. Both drug sensitive cell lines (HL-60/S, P388/S, KB3-1, and MCF-7/S) and drug-resistant cell lines (HL-60/AR, P388/ADR, KBV-1, and MCF-7/ADR) exposed to DNR showed a similar rapid distribution of drug from the plasma membrane to the perinuclear region within the first 2 min. From 2-10 min, the drug sensitive HL-60/S, P388/S, and MCF-7/S cells redistributed drug to the nucleus and to the cytoplasm in a diffuse pattern. In contrast, drug-resistant HL-60/AR, P388/ADR, and MCF-7/ADR redistributed DNR from the perinuclear region into vesicles distinct from nuclear structures, thereby assuming a "punctate" pattern. This latter redistribution could be inhibited by glucose deprivation (indicating energy dependence), or by lowering the temperature of the medium below 18 degrees C. The differences in distribution between sensitive and resistant cells did not appear to be a function of intracellular DNR content, nor the result of drug cytotoxicity. Drug-sensitive KB3-1 and -resistant KBV-1 cells did not fully follow this pattern in that they demonstrated an intracellular DNR distribution intermediate between HL-60/S and HL-60/AR cells with both "punctate" and nuclear/cytoplasmic uptake sometimes in the same cell. These data indicate that the intracellular distribution of DNR is an important determinant of drug resistance regardless of the overexpression of P-glycoprotein. The intracellular movement of drug requires the presence of glucose and a temperature above 18 degrees C, implicating energy-dependent processes and vesicle fusion in the distribution process. This intracellular transport of DNR away from the nucleus in multidrug-resistant cells may protect putative cell targets such as DNA against drug toxicity.
Insights
Multidrug-resistant cells show altered daunorubicin (DNR) distribution, moving it to vesicles instead of the nucleus. This energy-dependent process, independent of P-glycoprotein, contributes to drug resistance.
Area of Science:
- Cell biology
- Pharmacology
- Cancer research
Background:
- Multidrug resistance (MDR) is a major challenge in cancer chemotherapy.
- P-glycoprotein (P-gp) is a known transporter involved in MDR.
- Understanding drug intracellular distribution is crucial for overcoming resistance.
Purpose of the Study:
- To investigate the intracellular distribution of daunorubicin (DNR) in multidrug-resistant and drug-sensitive cell lines.
- To determine if DNR distribution is linked to P-glycoprotein expression or other energy-dependent mechanisms.
- To explore the role of DNR intracellular localization in mediating drug resistance.
Main Methods:
- Utilized laser-assisted confocal fluorescence microscopy to visualize DNR.
- Examined four pairs of multidrug-resistant and drug-sensitive cell lines.
- Assessed the impact of glucose deprivation and temperature on DNR distribution.
Main Results:
- Both sensitive and resistant cells initially showed rapid DNR distribution to the perinuclear region.
- Sensitive cells redistributed DNR to the nucleus and cytoplasm.
- Resistant cells exhibited a punctate pattern, sequestering DNR into vesicles, an energy-dependent process.
- This punctate distribution was inhibited by glucose deprivation and low temperatures (<18°C).
- The KBV-1 cell line showed an intermediate distribution pattern.
Conclusions:
- Intracellular DNR distribution is a significant factor in multidrug resistance, irrespective of P-glycoprotein overexpression.
- Energy-dependent processes, including vesicle fusion, are critical for DNR intracellular transport in resistant cells.
- This altered intracellular transport may protect cellular targets like DNA from daunorubicin toxicity, contributing to drug resistance.