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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

, : 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. Japanese Journal Of Cancer Research. 86(6): 607-615

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 SARED670 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 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 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 (AML) 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.


PMID: 7622426

DOI: 10.1111/j.1349-7006.1995.tb02441.x

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