WT1 Gene And Its Isoforms In Different Cell Subsets Of Normal Human And Acute Myelogenous Leukemia Bone Marrow

The Wilms' tumor gene(WT1)is a transcription factor involved in tumorigenesis especially leukemogenesis.However,the role of WT1 expression in nonmalignant hematopoietic cells remains unclear.Furthermore,due to alternative splicing at two sites:the 17 amino acids of exon 5(+17AA)and the 3 amino acids(+KTS)between exons 9 and 10,WT1 gene has four main isoforms(17AA+/KTS+,17AA+/KTS-,17AA-/KTS+,17AA-/KTS-, abbreviation:+/+,+/-,4+,4-).The isoforms probably existed in haematopoietic cells,which makes the research more complex.Many studies showed that the four isoforms of WT1 may be involved in transcriptional and post-transcriptional regulation,cell proliferation,differentiation and apoptosis.Many studies showed that WT1 was strongly expressed in almost all leukemia samples, regardless of disease types,as was proposed that WT1 was a key molecule in leukemogenesis. Still,it remains unknown whether WT1 gene exerts an oncogenic function in leukemogenesis or it is a necessary gene for differentiation.Early studies showed that WT1 was only expressed in normal bone marrow CD34⁺ cells,but disappeared in mature cells.However,Ellisen et al showed that the overexpression of total WT1 turned up once more during the course of hematopoietic cells differentiation.The real expression of WT1 is as yet unclear especially the ratio of the four splice variants in different cell stages.Acute myelogenous leukemia(AML)cell are organized in a hierarchical fashion,with only the most primitive rare population(leukemia stem cell,LSC)of AML cells capable of maintaining the leukemic clone.A broad range of studies has indicated that AML results from mutations at the level of the stem cells of AML cells.The changes of cellular and molecular features in these malignant stem cells determine the features of leukemia clone and give rise to different subtypes of AML.LSCs share some similar characteristics with normal hematopoietic stem cells(HSC)including the ability to self-renew,and also have the potential of limited differentiation.Except CD34⁺ and CD38^- the same as HSC,LSCs,also have some features that are not found in normal HSC.LSCs have unique phenotype such as CD90^-,CD117 and CD123⁺.Due to CD123⁺ existing in LSC uniquely,it can be a marker of LSC.Gilliland had put forward a two-hit modal of leukemogenesis that activating of protein kinase and function altering of transcription factor are the basic mechanism in leukemogenesis. Recent years,lots of studies showed the wild-type WT1 gene overexpressed in almost all kinds of leukemia,but the expression of WT1 gene and its isoforms in LSC has not yet been reported.In the present study,To elucidate the expression and its isoforms of WT1 gene in different cell subsets of healthy bone marrow and different differentiation stage of leukemia cell line HL-60,fluorescence RT-PCR detection system was established to measure the expressions of full-length WT1,WT1(+17AA)and WT1(+KTS)in CD34⁺CD38(stem cell), CD34⁺CD38⁺(progenitor cell),CD15⁺CD11b⁺(granulocyte),CD33⁺CD14⁺(monocyte), CD20⁺CD5^-(B-lymphocyte)and CD20^-CD5⁺(T-lymphocyte)subsets sorted by fluorescence-activated cell sorting(FACS)from 18 bone marrow samples and different cell stage of HL-60 differentiation induced by ATRA.usingβ-actin as a internal standard.The relative ratios of the four splice variants WT1(+/+),WT1(+/-),WT1(-/+),WT1(-/-)were calculated.Through discussing the relationship between WT1 gene and hematogenic cell differentiation,we try to elucidate the function of WT1 gene during cell proliferation and differentiation.Next,CD34⁺CD38^-CD123⁺ and CD34⁺CD38^-CD123^- leukemic cell populations from 47 AML samples were FACS-sorted.The expressions of full-length WT1,WT1(+17AA) and WT1(+KTS)were detected to compare with HSC.In this study,experiments were performed to try revealing the real function of WT1 gene in leukemia.Partâ… The Expression WT1 Gene and Its lsoforms in Different Cell Subsets of Normal Human Bone Marrow1.standard curves were set upFour standard curves were formed by positive standard and the coefficient of correlation was all above 0.996.Fluorescence RT-PCR detection system was established to measure the expressions of full-length WT1,WT1(+17AA)and WT1(+KTS)in CD34⁺CD38^- CD123-(stem cell),CD34⁺CD38⁺(progenitor cell),CD15⁺CD11b⁺(granulocyte),CD33⁺CD14⁺(monocyte), CD20⁺CD5^-(B-lymphocyte)and CD20^-CD5⁺(T-lymphocyte)subsets from 18 bone marrow samples.The selected splice of WT1(17AA)and WT1(KTS)was independent,so the relative ratio of the four splice variants WT1(+/+),WT1(+/-),WT1(-/+)and WT1(-/-)can be calculated by multiply with the ratio of 17AA+,17AA-,KTS+ and KTS-.2.The expression of WT1 gene in different cell subsets of normal human bone marrowCompared with the positive control K562 cell line(4.67×10^-3±2.95),results showed that WT1 expressed lowly in CD34⁺CD38^-CD123^-,CD34⁺CD38⁺,CD15⁺CD11b⁺ and CD33⁺CD14⁺ but not in CD20⁺CD5 and CD20-CD5⁺ subsets.The highest was in CD34⁺CD38^- CD123^-(7.92×10^-4±1.07)but decreased gradually in CD15⁺CD11b⁺(9.68×10^-5±3.24)and CD33⁺CD14⁺ (7.08×10^-5±2.57)subsets.3.The expression and ratios of +17AA,+KTS isoforms in different cell subsets of normal human bone marrowThe expression level of +17AA,+KTS isoforms had the same tendency with full-length WT1,that the highest was in CD34⁺CD38^-CD123^- and the lowest in CD33⁺CD14⁺ subsets.WT1(+17AA)and WT1(+KTS)isoforms were predominant in CD34⁺CD38^- and CD34⁺CD38⁺ primitive subsets,while in CD15⁺CD11b⁺ and CD33⁺CD14⁺ the dominant isoforms were WT1(-17AA)and WT1(-KTS),WT1(+17AA)and WT1(+KTS)isoforms decreased obviously,especially the WT1(+17AA).4.The expression and ratios of four isoforms in different cell subsets of normal human bone marrowWT1(+/+)isoform was predominant in CD34⁺CD38^- CD123^-and CD34⁺CD38⁺ primitive subsets and K562 cell line and the ratios were 0.40±0.09,0.37±0.03 and 0.32±0.05,respectively. There were significant differences between different groups(P＜0.05).WT1(-/-)isoform was main in CD15⁺CD11b⁺ and CD33⁺CD14⁺ cell subsets.Partâ…¡The Expression of WT1 Gene and its Isoforms During the Differentiation of Leukemia Cell Line HL-601.Granulocytic differentiation of HL-60 by ATRAThe optimal density(1μmol/L)of ATRA-induced HL-60 granulocytic differentiation was determined by cell proliferation curve and NBT reduction rate.Cell differentiation was judged by morphology,NBT reduction rate and the CD11b positive rate.Results showed that the NBT reduction rate and the CD11b positive rate both increased gradually during the differentiation of HL-60 cell.At the same time,the morphology of granulocytic nuclear can be seen.Consistent with the previous data,ATRA can induce granulocytic differentiation of HL-60.2.The expression of WT1 gene during the differentiation of leukemia cell line HL-60During the course of differentiation in HL-60 cell,WT1 gene expressions were downregulated gradually.0 hour was 4.67×10^-3±1.11×10^-3,96 hour was 7.53×10^-4±2.30×10^-4.Western blot showed that WT1 protein decreased gradually with the differentiation of HL-60.3.The expressions and ratios of +17AA,+KTS isoforms during the differentiation of leukemia cell line HL-60The expression level of +17AA,+KTS isoforms had the same tendency with full-length WT1.With the differentiation of HL-60 cell,the expressions and ratios of +17AA,+KTS isoform decreased gradually.4.The expressions and ratios of the four isoforms during the differentiation of leukemia cell line HL-60 During the differentiation of HL-60 cell,the ratio of WT1(+/+)decreased gradually,0 hour 0.32±0.06,and 96 hour 0.17±0.03 respectively.However,the ratio of WT1(-/-)increased, 0 hour 0.19±0.04 and 96 hour 0.34±0.05.The other two isoforms ratios did not change significantly.Partâ…¢The Expression WT1 Gene and Its Isoforms in Different leukemic cell populations of AML1.The expression of WT1 gene in CD34+CD38-CD123+ and CD34+CD38-CD123leukemic cell populations of AMLThe average levels of WT1 expression in FACS-sorted CD34⁺CD38^-CD123⁺ and CD34⁺CD38^-CD123^- leukemic cell populations of 47 AML samples were 3.39×10^-2±3.38 and 5.25×10^-3±5.62,respectively.The WT1 expression levels in HSC(CD34⁺CD38^-CD123^-)were 7.94×10^-4±1.35 and at least 10 times lower than those in CD34⁺CD38^-CD123^- leukemic cell populations.Significant differences in the WT1 expression level were observed among the three different cell subsets(P＜0.05).2.The expression and ratios of +17AA,+KTS isoforms in CD34+CD38-CD123+ and CD34+CD38-CD123-leukemic cell populations of AMLThe expression levels of +17AA,+KTS isoforms had the same tendency with full-length WT1.Both isoforms expressed highest in CD34⁺CD38^-CD123⁺ primitive cell populations,while the lowest in HSC.+17AA isoforms was predominant in all the three cell populations and no significant differences were observed between the groups.The ratio of +KTS isoforms was the highest in HSC(0.57±0.04),while the lowest in CD34⁺CD38^-CD123^- leukemic cell populations(0.50±0.12)and there was great difference between the two groups(P＜0.05).But no significant differences were observed between CD34⁺CD38^-CD123⁺ leukemic cell populations and the other two(P＞0.05).3.The ratios of the four isoforms in CD34+CD38-CD123+ and CD34+CD38-CD123leukemic cell populations of AMLNo significant differences in the four isoforms ratio can be seen among CD34⁺CD38^-CD123⁺,CD34⁺CD38^-CD123^- leukemic cell populations and HSC(CD34⁺CD38^-CD123^-) (P＞0.05).4.The correlation between the prognosis and WT1 expression level,the proportion of CD34+CD38-CD123+ leukemic cell populations in primitive cellsNo association was found between WT1 expression level of CD34⁺CD38^-CD123⁺ leukemic cell populations and age,sex,FAB type,the amount of primitive cell.CR rate was 40.9%in WT1 expression level≥0.06 group,while 57.8%in＜0.06 group and there was no significant difference between the two groups(P>0.05).The proportion of CD34⁺CD38^-CD123⁺ leukemic cell populations in primitive cells is 0.03%～4.5%,median:0.1%.When the boundary is 0.1%,the survival time was 68.2%in <0.1%group and 26.3%in≥0.1%group and there was significant difference between the two groups(P<0.05).Long-term follow-up in 41 AML sample showed that≥0.1%group (n=19)was worse overall survival than<0.1%(n=22)group.Conclusion1.The WT1 expression in normal bone marrow cell subsets and HL-60 cell line decreased gradually with cell differentiation.2.WT1(+17AA),WT1(+KTS)and WT1(+/+)isoforms were predominant in hematopoetic progenitor cells,while in the mature cell the dominant isoforms were WT1(-17AA),WT1(-KTS) and WT1(-/-).Hematopoietic cells may adjust the ratios of WT1 isoforms to inhibit or promote cell differentiation.3.The high expression of WT1 gene in CD34⁺CD38^-CD123⁺ leukemic cell populations may be a phenomena that as a transcription factor WT1 gene is involved in leukemogenesis early.4.CD34⁺CD38^-CD123⁺leukemic cells may represent the rare cell populations LSC and its proportion in primitive cell of AML can affect prognosis directly.