(Authors): Department of Hematology/Oncology and Center for Cancer and Transplantation Biology, Children's Research Institute, Children's National Medical Center
P-gp and MRP-expressing cell lines and peripheral blood cells were used in evaluating accumulation and retention of rhodamine 123 (rho123) and calcein AM to define functional assays for drug resistance that are relevant in clinical samples. Accumulation was measured by 1 hour dye uptake. Retention was measured by loading the cells with dye +/- verapamil to overcome poor accumulation and allowing efflux for 2 hours. Using the P-gp+ CEM VCR30, the MRP+ UMCC-1/VP, and the WT CEM and WT UMCC-1 cell lines, we showed that rho123 is a substrate for P-gp but not for MRP. In the P-gp cell line, rho123 accumulation was enhanced and efflux was blocked by verapamil (VRP) and cyclosporin A (CSA) but not probenecid. MK571 caused higher levels of rho123 accumulation in these cells but had no effect on retention. Impaired calcein AM accumulation by P-gp cells was enhanced by VRP, CSA, slightly enhanced by MK571, but unaffected by probenecid. Calcein AM retention was normal in P-gp cells. The MRP cell line exhibited normal rho123 accumulation and retention, but these cells showed diminished accumulation of calcein AM which was reversed by VRP, CSA, probenecid and MK571. Low levels of calcein AM retention after 2 hrs were not reversed by VRP or CSA, partially reversed by MK571, and reversed by probenecid. P-gp function was detected in peripheral CD34 cells, NK cells, CD4 and CD8 memory and naive cells and in CD38+ and CD38- B cells. Functional MRP was not detected in peripheral blood cells using calcein AM accumulation and retention.
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