OBJECTIVETo explore expression ratio alteration between WT1 gene and its isomers during differentiation of leukemia cell line HL-60 induced by all-trans retinoic acid (ATRA) and the relationship existed between them.

METHODSThe degree of cellular maturation was verified by NBT reduction test and immunophenotyping. Expression of unspliced WT1, WT1 (17AA+) and WT1 (KTS+) were determined by real-time fluorescence quantitative RT-PCR during the induced differentiation of HL-60 cells. The relative ratio of four splicing variants WT1 (+/+), WT1 (+/-), WT1 (-/+), WT1 (-/-) were analyzed.

RESULTSDuring the induced differentiation of HL-60 cells, NBT reduction rate and CD11b positive rate increased significantly (P < 0.05 and P < 0.001, respectively). The expression of WT1 gene decreased from (4.17 +/- 2.21) x 10(-3) at 0 hour to (7.53 +/- 2.30) x 10(-4) at the 96th hour. The ratio of 17AA+decreased from 0.60 +/- 0.05 at 0 hour to 0.42 +/- 0.08 at the 96th hour. The ratio of KTS+ decreased from 0.53 +/- 0.08 at 0 hour to 0.41 +/- 0.04 at the 96th hour. The ratio of WT1 (+/+) decreased gradually from 0.32 +/- 0.06 at 0 hour to 0.17 +/- 0.03 at the 96th hour. Change of ratios of other two isomers not significant.

CONCLUSIONSDuring the induced differentiation of HL-60 cells, WT1 gene expression decreases gradually. The phenotype of the majority of uninduced HL-60 cells is WT1 (+/+), in contrast to WT1 (-/-) phenotype after the induction of cell differentiation, indicating that WT1 gene may participate in the regulation of hematopoietic cell differentiation through modulation of the expression ratios of its four spliced variants.