Differentiation Phenotypes Of K562 Cells Induced By Exogenous Wnt5a

ObjectiveWnt5a,a member of the Wnt family,plays essential organizing roles in the development.Wnt5a has been recently found to be correlated with tumorigenesis,but what is the relations and the mechanism are still not clear, especially concerning leukemia.This study was aimed to observe Wnt5a expression in samples of myeloid leukemia patients and myeloid leukemia cell lines,and to attain the relation of Wnt5a expression with leukemiagenesis. Furthermore,myeloid leukemia cell lines K562 were treated by exogenous Wnt5a and the differentiation of the cells was studied.This study established the foundation for further experimental studies on the function and mechanism of wnt5a in leukemia,and for wnt5a being regarded as a tumor marker and a gene therapy target for leukemia.Methods1.Peripheral blood and bone marrow samples collected from 6 patients with chronic myeloid leukemia and 5 with acute myeloid leukemia,as well as leukemia cell lines Jurkat,K562 and HL-60 were analyzed for the expression of Wnt5a by RT-PCR.2.Recombinant adenovirus vectors AdWnt5a(with GFP labeling) and AdGFP(as control ) respectively infected HEK293 cells for amplifying these virus.The wnt5a and GFP condition mediums(CM) were separately prepared by collecting the supernatants 48 h after recombinant adenoviral vector AdWnt5a and AdGFP infecting CHO cells.The GFP expression was observed by fluorescence microscope in CHO cells 16 h after infection.Wnt5a protein expression was studied by Western blot analysis after the CM was frozen and dried. 3.K562 cells were treated with CM of Wnt5a and the control respectively for 1-5 d.Cell proliferation was evaluated by direct cell counting after trypan blue staining.The total cell counting was carried out and a growth curve was plotted.4.Flow cytometry was used to observe the growth cycle of K562 cells 1,3 and 5 d after the treatment of Wnt5a CM and control CM.5.The morphology and ultrastructure of K562 cells were observe by light microscope and transmission electron microscope after they were treated with Wnt5a.6.After k562 cells were treated with Wnt5a and control CM for 1-7 d, cytochemical staining of POX,PAS andα-NAE were made respectively to identify positive cells.7.Treated with Wnt5a and control CM for 1-7 d,K562 cells were observed by immunocytochemical methods for the expressions of monocytic differentiation antigen CD14 and CD68,and granulocytic differentiation antigen CD13.Results1.Wnt5a was not expressed in all of 6 cases of chronic myeloid leukemia, and K562 and HL-60 cells,and had no or weakly expression in 4 eases of acute myeloid leukemia.However,Wnt5a was found to be strongly expressed in Jurkat cells and one case of acute myeloid leukemia that was in complete remission.2.Viral particles of AdWnt5a and AdGFP at high titer were obtained after HEK293 cells infection.Wnt5a and control CM were collected after AdWnt5a and AdGFP infected the CHO cells with high intensity under a fluorescence microscope.Meanwhile,Wnt5a protein was expressed in Wnt5a the CM by Western blot assay.3.The growth of K562 cells was inhibited after treated by Wnt5a CM,but their vitality was not affected.4.The cell number at G₂ phase were 3.22 times more in K562 cells treated by Wnt5a CM than in those by control CM. 5.The morphological and ultrastructure studies showed that differentiated K562 cells tended to be more mature in treated group than in control group.In brief,K562 cells treated by Wnt5a CM demonstrated smaller volume,decreased nuclei/cytoplasm ratio,condensed chromatin,reduced and disappear of nucleolus,and even some cells had typical features of mononuclear cells. Ultrastructually,the cells had more heterochromatin,reduced or smaller nucleolus,more cytoplasm,decreased nuclei/cytoplasm ratio,and increased cell organs.6.Both POX and PAS positive cells covered significantly more in treated group than in control group.Theα-NAE staining also was positive,but its positive intensity could be inhibited up to 70%by NaF in treated group.7.The expressions of monocytic differentiation antigen of CD14,CD68 were significantly increased in treated group,and the expressions of granuloeytic differentiation antigen CD13 was increased too,but no significant difference was found on statistics.Conclusion1.RT-PCR detected that 90%Wnt5a was not expressed or down-regulated expressed in 11 cases acute and chronic myeloid leukemia,and neither was in K562,HL-60 cells.However,it was highly expressed in one case of acute myeloid leukemia with complete remission.These results indicate that loss of Wnt5a expression may be related with leukemiagenesis.2.The growth of K562 cells was inhibited after treated by Wnt5a condition medium.The cell cycle in treated group compared with control group was arrested in G₂ phase,because the cell could not make normal mitosis and thus suppress the cell proliferatio.3.Morphological changes of K562 cells showed that the cells displayed differentiation phenotypes and typical monocytic form in individual cells.POX, PAS,andα-NAE staining + NaF,proved that Wnt5a could induce K562 cell to differentiate into granulocytic and monocytic lineage.Immunocytochemical studies on CD14 and CD68 further confirmed exogenous Wnt5a may induce the K562 cell to differentiate into monocytic lineage. Conclusively,the loss of Wnt5a expression may be related with leukemiagenesis.Wnt5a possibly played the role of tumour suppressor gene. This study provide the foundation for further experimental studies on the gene diagnosis and therapy.