P-glycoprotein expression and function in circulating blood cells from normal volunteers.
In contrast to its clearly defined role as a multidrug efflux pump in neoplastic cells, the physiologic function of P-glycoprotein (P-gly) in normal cells is unclear. Recent reports identifying P-gly in normal blood and bone marrow suggest that hematopoietic development or function may be dependent on P-gly. To understand the normal function of P-gly in the blood, its level of expression and function must first be quantitated relative to a known standard. In this study, P-gly, MDR1 gene expression, and P-gly function were quantitated in normal leukocytes. P-gly and MDR1 expression were analyzed in individual leukocyte lineages (T-helper, T-suppressor, monocyte, granulocyte, B-lymphocyte, NK cell) from normal volunteers. P-gly on the cell surface was detected by fluorescent double-labeling for lineage (CD4, CD8, CD14, CD15, CD19, CD56, respectively) and P-gly (MRK16) with analysis by flow cytometry and in some cases immunoblot analysis. MDR1 mRNA analysis on purified lineages was performed using quantitative reverse transcription-polymerase chain reaction. P-gly function was determined for each lineage using dual-labeling for lineage and P-gly substrate (rhodamine 123). The P-gly expressing human myeloma cell line, 8226/Dox6, was used as a reference of comparison for levels of P-gly, MDR1 mRNA, and function. CD56+ cells expressed the highest levels of MDR1 mRNA followed by CD8+ > CD4+ approximately equal to CD15+ > CD19+ > CD14+, with percentage values relative to Dox6 of 49%, 17%, 8%, 8%, 4%, and 2%, respectively. The assays for P-gly immunofluorescence and function correlated well with mRNA analysis except for CD15+ cells (granulocytes), which showed a moderate MDR1 mRNA level with a lack of both function and surface P-gly staining. Granulocyte membranes did show P-gly on immunoblot analysis when probed with either C219 or JSB1. We conclude that (1) P-gly and the MDR1 mRNA are expressed in normal leukocytes, (2) this P-gly expression is lineage specific with relatively high levels among CD56+ cells, and (3) the expression of P-gly in granulocytes is not associated with transport of the P-gly substrate, rhodamine 123, out of the cell.
Klimecki
W T
WT
Arizona Cancer Center, University of Arizona, Tucson 85724.
Futscher
B W
BW
Grogan
T M
TM
Dalton
W S
WS
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD / Q000032:analysis; D001402:B-Lymphocytes / Q000378:metabolism; D002352:Carrier Proteins / Q000097:blood* / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007694:Killer Cells, Natural / Q000378:metabolism; D007962:Leukocytes / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000097:blood* / Q000235:genetics; D009000:Monocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000097:blood; D012333:RNA, Messenger / Q000097:blood; D006377:T-Lymphocytes, Helper-Inducer / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D012313:RNA