Co-expression of several molecular mechanisms of multidrug resistance and their significance for paclitaxel cytotoxicity in human AML HL-60 cells.
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.
Huang
Y
Y
Division of Hematology/Oncology, Department of Medicine and Winship Cancer Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
Ibrado
A M
AM
Reed
J C
JC
Bullock
G
G
Ray
S
S
Tang
C
C
Bhalla
K
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects; D053938:DNA Fragmentation; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D016259:Genes, myc; D016158:Genes, p53; D018922:HL-60 Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D017239:Paclitaxel / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D014750:Vincristine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D004317:Doxorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D017239:Paclitaxel; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D014750:Vincristine; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C494826:BAX protein, human; C494811:BCL2L1 protein, human