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Cytometric study of intracellular P-gp expression and reversal of drug resistance
G Labroille1, F Belloc, C Bilhou-Nabera
1Laboratoire d'Hématologie, Hôpital du Haut-Lévêque, Pessac, France.
Abstract:
Expression of the multidrug resistance (MDR) phenotype is responsible for chemotherapy failure in numerous cancers. This phenotype is generally due to the expression of the mdr1 gene-encoded P-gp. Modulation of P-gp activity by chemotherapy has limited possibilities because of toxicity and poor specificity. In contrast, specific transcription blockage of the mdr1 gene can be obtained by oligonucleotides forming a triple helix structure at the DNA level. We used here immunofluorescence and both flow cytometry and image analysis to evaluate surface and total P-gp content in K562 MDR cells. The mdr1 mRNA content was measured by RT-PCR. We confirm the capacity of a 27-mer oligodeoxynucleotide, targeted to an mdr1 DNA fragment, to cause a 10-fold decrease in mdr1 mRNA level. However, this specific genetic inhibition was functionally limited because cellular growth was not modified in a cytotoxic environment. We found that total P-gp content was reduced in resistant cells treated with the mdr1-targeted oligonucleotide, while it remained in high levels on the cell surface, suggesting the existence of a large cytoplasmic pool of P-gp (approximately 50% of the total cellular P-gp). Moreover, when cycloheximide was used for 72 h to suppress protein synthesis, surface P-gp expression showed no decrease, whereas total P-gp was considerably lowered. A rapid 35% decrease in surface P-gp level was reached when resistant cells were treated for 24 h with brefeldin A, an inhibitor of intracellular protein trafficking. Simultaneously, the total P-gp level remained stable, thus indicating a probable accumulation of cytoplasmic P-gp, in agreement with the interruption of protein migration. We propose that the cytoplasmic P-gp pool could be a storage pool consumed for maintaining a steady-state level of surface P-gp. Cytometry could be a useful tool to study such a mechanism of P-gp trafficking and cellular distribution, which could explain the difficulties encountered in achieving stable and rapid effects of MDR reversal with oligonucleotides.
Insights
Targeting the multidrug resistance (MDR) gene with oligonucleotides reduces P-gp mRNA and total P-gp. However, surface P-gp levels remain high, suggesting a cytoplasmic pool that maintains surface expression, limiting MDR reversal strategies.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Multidrug resistance (MDR) is a major cause of chemotherapy failure in cancer.
- The MDR phenotype is often mediated by P-glycoprotein (P-gp), encoded by the mdr1 gene.
- Targeting P-gp transcription offers a potential strategy to overcome MDR.
Purpose of the Study:
- To investigate the functional impact of specific mdr1 gene transcription blockage on P-gp expression and cellular behavior in MDR cancer cells.
- To characterize the distribution and trafficking of P-gp within MDR cells following genetic inhibition of mdr1.
Main Methods:
- K562 MDR cells were treated with a 27-mer oligodeoxynucleotide targeting the mdr1 gene.
- P-gp expression was evaluated using immunofluorescence, flow cytometry, and image analysis.
- mRNA levels were quantified by RT-PCR; protein synthesis and trafficking were assessed using cycloheximide and brefeldin A.
Main Results:
- Oligonucleotide treatment decreased mdr1 mRNA by 10-fold and total P-gp content but did not affect cellular growth.
- Surface P-gp levels remained high despite reduced total P-gp, indicating a significant cytoplasmic P-gp pool.
- Inhibition of protein trafficking with brefeldin A reduced surface P-gp without altering total P-gp, suggesting a dynamic cytoplasmic storage mechanism.
Conclusions:
- A substantial cytoplasmic pool of P-gp exists and appears to serve as a reservoir for maintaining surface P-gp levels.
- The intracellular trafficking and distribution of P-gp influence the efficacy of MDR reversal strategies.
- Cytometry is a valuable tool for studying P-gp trafficking and cellular distribution in the context of MDR.