Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by a flow cytometric assay.
BACKGROUND
Chemoresistance in some hematologic malignancies has been associated with overexpression of P-glycoprotein, which is encoded by the MDR1 gene (also known as PGY1). However, inconsistencies in data on frequency and clinical relevance of multidrug resistance in B-cell chronic lymphocytic leukemia (B-CLL) may reflect a need for improved techniques to detect this overexpression.


OBJECTIVE
Our purpose was to measure P-glycoprotein activity in peripheral blood cells of B-CLL patients and to analyze possible clinical correlations (disease duration, prior treatment, Rai disease stage, lymphocyte counts, and disease progression).


METHODS
P-glycoprotein activity was assayed in peripheral blood cells of 42 consecutive B-CLL patients (22 treated and 20 untreated). We used dual fluorescence in a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123, which is transported from the cell by the P-glyprotein pump. Leukemia cells were costained with monoclonal antibody Leu12/CD19, and rhodamine 123 efflux was measured. Expression of MDR1 and MDR3 (also known as PGY3) messenger RNA (mRNA) was quantitatively evaluated by polymerase chain reaction (PCR) in 26 cases.


RESULTS
Marked rhodamine 123 efflux was observed in 34 (81%) of the 42 cases and was abolished in the presence of multidrug resistance inhibitors. Rhodamine 123 efflux was not associated with Rai stage, lymphocyte counts, duration of disease, or disease progression. Although rhodamine 123-negative cases were about equally distributed among untreated and previously treated patients, the percentage of cells with rhodamine 123 efflux was significantly lower for untreated patients than for those treated with chemotherapy regimens including at least one multidrug resistance-associated drug. MDR1 mRNA was detected in 25 of 26 cases and MDR3 mRNA in all 26. MDR1 mRNA expression was significantly correlated with rhodamine 123 efflux, whereas MDR3 mRNA expression was not significantly correlated; MDR1 and MDR3 mRNA expression was not significantly associated with Rai stage, prior treatment, or disease progresssion.


CONCLUSIONS
These findings suggest that P-glycoprotein overexpression in B-CLL is intrinsic rather than acquired and that P-glycoprotein activity is enhanced after exposure to multidrug resistance-associated drugs. This enhanced activity does not seem to be associated with more aggressive disease. Our results also indicate that an assay of P-glycoprotein function combined with PCR is suitable for clinical multidrug resistance screening.


CONCLUSIONS
Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.
Ludescher
C
C
Department of Internal Medicine, University of Innsbruck, Austria.
Hilbe
W
W
Eisterer
W
W
Preuss
E
E
Huber
C
C
Gotwald
M
M
Hofmann
J
J
Thaler
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D002352:Carrier Proteins / Q000097:blood*; D004351:Drug Resistance; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood* / Q000235:genetics; D008297:Male; D008562:Membrane Glycoproteins / Q000097:blood*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000097:blood*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000097:blood; D012334:RNA, Neoplasm / Q000097:blood; D020112:Rhodamine 123; D012235:Rhodamines
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012235:Rhodamines; D020112:Rhodamine 123