Cytometric study of intracellular P-gp expression and reversal of drug resistance.
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.
Labroille
G
G
Laboratoire d'Hématologie, Hôpital du Haut-Lévêque, Pessac, France.
Belloc
F
F
Bilhou-Nabera
C
C
Bonnefille
S
S
Bascans
E
E
Boisseau
M R
MR
Bernard
P
P
Lacombe
F
F
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
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