Co-expression of several molecular mechanisms of multidrug resistance and their significance for paclitaxel cytotoxicity in human AML HL-60 cells

Abstract

Overexpression of P-glycoprotein (PGP), MRP or LRP has been characterized as the 'proximal', while overexpression of the anti-apoptosis Bcl-2 or Bcl-xL relative to the pro-apoptosis Bax protein has been recognized as the 'distal' mechanism of multidrug resistance in human AML cells. In the present studies, we examined whether these mechanisms can co-exist in human AML HL-60 cells. We also determined how these mechanisms would affect the accumulation and cytotoxicity of a PGP substrate, such as Taxol (paclitaxel). For this, immunoblot analyses were performed to determine the expression of PGP, MRP, Myc, Bcl-2, Bcl-xL and Bax on either the multidrug-resistant HL-60 sublines created under the selection pressure of doxorubicin (HL-60/AR), paclitaxel (HL-60/TAX1000) or vincristine (HL-60/VCR), or sublines created by transfection and overexpression of the bcl-2 (HL-60/Bcl-2) or bcl-xL gene (HL-60/Bcl-xL). As compared to the control HL-60, HL-60/AR cells possess high MRP while HL-60/TAX1000 and HL-60/VCR cells express high levels of the mdr-1 encoded PGP. In addition, these multidrug-resistant cells possess 1.5- to 2.5-fold higher Bcl-2, while their Bax and Myc levels are similar to those in the control HL-60 cells. HL-60/TAX1000 and HL-60/VCR cells also express three- and 2.5-fold higher Bcl-xL levels. PGP, but not MRP, overexpression significantly impaired paclitaxel accumulation and paclitaxel-induced apoptosis, as well as reduced its cytotoxic effects as determined by the MTT assay. In contrast, enforced and much higher expression of Bcl-2 in HL-60/Bcl-2 (five-fold) or Bcl-xL in HL-60/Bcl-xL cells (10-fold) significantly reduced paclitaxel-induced apoptosis and the loss of cell viability, without affecting its intracellular accumulation. These results confirm the possibility of co-expression of multiple mechanisms of multidrug resistance in human leukemic cells which had been selected by exposure to a single drug. The results also indicate that MRP overexpression does not confer resistance against paclitaxel. In addition, these findings suggest that, for Bcl-2 and Bcl-xL, enforced overexpression to high levels is necessary to induce paclitaxel resistance in HL-60 cells.