title: Cytometric study of intracellular P-gp expression and reversal of drug resistance.
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.
authors.affiliation: Laboratoire d'Hématologie, Hôpital du Haut-Lévêque, Pessac, France.
authors.forename: G
authors.initials: G
authors.lastname: Labroille
authors.forename: F
authors.initials: F
authors.lastname: Belloc
authors.forename: C
authors.initials: C
authors.lastname: Bilhou-Nabera
authors.forename: S
authors.initials: S
authors.lastname: Bonnefille
authors.forename: E
authors.initials: E
authors.lastname: Bascans
authors.forename: M R
authors.initials: MR
authors.lastname: Boisseau
authors.forename: P
authors.initials: P
authors.lastname: Bernard
authors.forename: F
authors.initials: F
authors.lastname: Lacombe
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D020126:Brefeldin A; D002455:Cell Division / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D007938:Leukemia; D018942:Macrolides; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016315:Proto-Oncogene Proteins c-abl / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D017931:DNA Primers; D018942:Macrolides; D009838:Oligodeoxyribonucleotides; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D020126:Brefeldin A; D003513:Cycloheximide; D016315:Proto-Oncogene Proteins c-abl