Cytometric study of intracellular P-gp expression and reversal of drug resistance

Cytometry. 1998 Jun 1;32(2):86-94.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis*
  • Anti-Bacterial Agents / pharmacology
  • Base Sequence
  • Brefeldin A
  • Cell Division / drug effects
  • Cycloheximide / pharmacology
  • Cyclopentanes / pharmacology
  • Cytoplasm / metabolism
  • DNA Primers
  • Drug Resistance, Multiple*
  • Flow Cytometry / methods
  • Fluorescent Antibody Technique
  • Gene Expression Regulation, Neoplastic / drug effects*
  • HL-60 Cells
  • Humans
  • Kinetics
  • Leukemia
  • Macrolides
  • Oligodeoxyribonucleotides / pharmacology
  • Polymerase Chain Reaction
  • Protein Synthesis Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-abl / biosynthesis
  • RNA, Messenger / metabolism
  • Transfection
  • Tumor Cells, Cultured


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Anti-Bacterial Agents
  • Cyclopentanes
  • DNA Primers
  • Macrolides
  • Oligodeoxyribonucleotides
  • Protein Synthesis Inhibitors
  • RNA, Messenger
  • Brefeldin A
  • Cycloheximide
  • Proto-Oncogene Proteins c-abl