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

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Abstract
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 MRK16 (b‐MRK16) and SA‐RED670 resulted in higher sensitivity as compared with standard methods such as the use of streptavidin‐phycoerythrin (SA‐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 gene. P‐gp positively 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, hut not in NOMO‐1 and HL60 cells. In K562 cells, mdr‐1 was found after 40 cycles of PCR, but not 25 cycles. These data are well correlated with those from the flow cytometry. We then studied the P‐gp expression on normal peripheral blood cells and acute leukemia cells. P‐gp was little expressed on peripheral lymphocytes, monocytes and granulocytes. It was also little expressed on blast cells from 5 patients with acute promyelocytic leukemia (APL) at diagnosis, ranging from 0.2 to 10.6% (4.6±3.9%). Ten other acute myeloid leukemia (AMD and 5 acute lymphocytic leukemia (ALL) expressed P‐gp at diagnosis, ranging 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 were found in APL, CD34‐positive and relapse and refractory cases (P=0.0006, 0.0007 and 0.0088, respectively). These results indicate that this flow cytometric analysis is useful for the evaluation of clinical MDR status and can identify a group of patients with resistant leukemia.