OBJECTIVETo explore the changes in expression of WT1 gene and ration of its isomers during phorbol ester (TPA) induced differentiation of leukemia cell line K562 by fluorescence quantitative RT-PCR and analysis the relationship between different isomers and hematogenic cell differentiation.

METHODSThe degree of cellular maturation were verified by NBT reduction test and immunophenotyping. Expression of WT1 gene was determined by fluorescence quantitative RT-PCR during differentiation of K562 cell line. The relative ratio of the four splicing variants WT1 ( + / + ), WT1 ( + / - ), WT1 ( - / + ), WT1 ( - / - ) were calculated.

RESULTSDuring the differentiation of K562 cell, the NBT reduction rate and the CD9 positive rate both increased significantly (P < 0. 05). The expression of WT1 gene decreased immediately to (1.67 +/- 0.45) x 10(-3) from (4.67 +/- 1.11) x 10(-3), and then increased again to (4.64 +/- 1.53) x 10(-3) at 96 hours. The ratio of WT1 ( + / + ) was decreased gradually, from 0 hour (39.65 +/- 19.46)% to 96 hour (15.25 +/- 7.27)%. While the ratio of WT1( - / - ) was increased, from 0 hour (15.38 +/- 11.34)%, to 96 hour (37.60 +/- 11.90)%. The other two isomers ratios did not change significantly.

CONCLUSIONDuring the TPA induced differentiation of K562 cell, there are two high expression levels of WT1 gene. Before differentiation, the majority is WT1 ( + / + ), and after differentiation, is WT1 ( - / - ). It indicates that WT1 gene may activate or inhibit cell differentiation by regulating the ratio of its four splicing variants.