It is known that one of main reasons of refractory and relapsed acute myeloid leukemia (AML) is multidrug resistant (MDR). Despite it was showed that 14-3-3ζ was over- expressed in HL-60/VCR MDR cells than in HL-60 sensitive AML cells by examining the difference of gene expression profile with Affymetrix GeneChip Human Genome U133 set A. Yet, the understanding on role of 14-3-3ζ in the survival of AML cells remains poor.
Semi-quantitative RT-PCR method was used to examine the expression of mdr1 mRNA in AML cell lines to validate the results of microarray. Western blot was performed to investigate Pgp#x2610;A14-3-3ζ#x2610;ABCL-2#x2610;AMCL-1 proteins expression. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscope. 14-3-3ζ knockdown cells were obstained by transduction with lentivirus-mediated shRNA to silence 14-3-3ζ in AML cell lines. MTT and cell count method were used to analyze the changes of growth of AML cells.
mdr1 mRNA and Pgp was not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells(P<0.01). Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than in HL-60 cells, especially 14-3-3ζ(P<0.01). The higher increased expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than in HL-60 cells. These results were same as gene chip. It was also noticed that14-3-3ζ was located in the cytoplasma and nuclear of AML cell lines, especially over-expressed in HL-60/VCR cells(P<0.05). Furthermore, suppression of14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with BCL-2 and MCL-1 decreased protein expression, especially in HL-60/VCR cells(P<0.01).
It was implied that14-3-3ζ played an important role in AML MDR and was associated with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.