NEW FLOW CYTOMETRIC METHOD FOR DETECTION OF MINIMALLY EXPRESSED MULTIDRUG-RESISTANCE P-GLYCOPROTEIN ON NORMAL AND ACUTE-LEUKEMIA CELLS USING BIOTINYLATED MRK16 AND STREPTAVIDIN-RED670 CONJUGATE

To evaluate the expression of multidrug resistance (MDR) on normal and leukemia cells, we examined P-glycoprotein (P-gp) by a newly devised flow cytometric method, utilizing a biotinylated monoclonal antibody ( mAb) against P-gp (MRK16), a streptavidin-RED670 conjugate (SA-RED670) and appropriate emission filters. The combination of biotinylated MRK 16 (b-MRK16) and SA-RED670 resulted in higher sensitivity as compared with standard methods such as the use of streptavidin-phycoerythrin (S A-PE) conjugate. The sensitivity was examined in K562, K562/ADR, NOMO- 1, NOMO-1/ADR and HL60 cells, and compared with the data obtained from reverse transcription polymerase chain reaction (RT-PCR) of mdr-1 gen e. P-gp positivity on flow cytometry was 10.4%, 99.9%, 1.4%, 90.4% and 0%, respectively. Mdr-1 mRNA was well expressed in K562/ADR and NOMO- 1/ADR cells, but not in NOMO-1 and HL60 cells. In K562 cells, mdr-1 wa s found after 40 cycles of PCR, but not 25 cycles. These data are web correlated with those from the flow cytometry. We then studied the P-g p expression on normal peripheral blood cells and acute leukemia cells . P-gp was little expressed on peripheral lymphocytes, monocytes and g ranulocytes. It was also little expressed on blast cells from 5 patien ts with acute promyelocytic leukemia (APL) at diagnosis, ranging from 0.2 to 10.6% (4.6+/-3.9%). Ten other acute myeloid leukemia (AML) and 5 acute lymphocytic leukemia (ALL) expressed P-gp at diagnosis, rangin g from 8.5% to 34.5% (16.9+/-11.8%) and from 2.3% to 45.6% (24.0+/-17. 8%), respectively. All 9 relapsed or refractory cases expressed P-gp, ranging from 21.1% to 99.8% (52.2+/-29.9%). Significant differences we re found in APL, CD34-positive and relapse and refractory cases (P=0.0 006, 0.0007 and 0.0088, respectively). These results indicate that thi s flow cytometric analysis is useful for the evaluation of clinical MD R status and can identify a group of patients with resistant leukemia.