Interaction of Human Genes WT1 and CML28 in Leukemic Cells

Abstract

The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms’ tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.

Keywords

Wilms’ tumor 1 suppressor gene / CML28 / K562 / siRNA

References

[1] BucklerA.J., PelletierJ., HaberD.A., et al.. Isolation, characterization, and expression of the murine Wilms’ tumor gene (WT1) during kidney development. Mol Cell Biol, 1991, 11(3): 1707-1712

[2] RauscherF.J.3rd, MorrisJ.F., TournayO.E., et al.. Binding of the Wilms’ tumor locus zinc finger protein to the EGR-1 consensus sequence. Science, 1990, 250(4985): 1259-1262

[3] DeyB.R., SukhatmeV.P., RobertsA.B., et al.. Repression of the transforming growth factor-beta 1 gene by the Wilms’ tumor suppressor WT1 gene product. Mol Endocrinol, 1994, 8(5): 595-602

[4] LoebD.M.. WT1 influences apoptosis through transcriptional regulation of Bcl-2 family members. Cell Cycle, 2006, 5(12): 1249-1253

[5] OhS., SongY., YimJ., et al.. The Wilms’ tumor 1 tumor suppressor gene represses transcription of the human telomerase reverse transcriptase gene. J Biol Chem, 1999, 274(52): 37 473-37 478

[6] CilloniD., GottardiE., De MicheliD., et al.. Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients. Leukemia, 2002, 16(10): 2115-2121

[7] InoueK., SugiyamaH., OgawaH., et al.. WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia. Blood, 1994, 84(9): 3071-3079

[8] BriegerJ., WeidmannE., MaurerU., et al.. The Wilms’ tumor gene is frequently expressed in acute myeloblastic leukemias and may provide a marker for residual blast cells detectable by PCR. Ann Oncol, 1995, 6(8): 811-816

[9] BergmannL., MiethingC., MaurerU., et al.. High levels of Wilms’ tumor gene (wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome. Blood, 1997, 90(3): 1217-1225

[10] BarraganE., CerveraJ., BoluferP., et al.. Prognostic implications of Wilms’ tumor gene (WT1) expression in patients with de novo acute myeloid leukemia. Haematologica, 2004, 89(8): 926-933

[11] ChiusaL., Francia di CelleP., CampisiP., et al.. Prognostic value of quantitative analysis of WT1 gene transcripts in adult acute lymphoblastic leukemia. Haematologica, 2006, 91(2): 270-271

[12] HuffV., SaundersG.F.. Wilms tumor genes. Biochim Biophys Acta, 1993, 1155(3): 295-306

[13] CallK.M., GlaserT., ItoC.Y., et al.. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell, 1990, 60(3): 509-520

[14] YamagamiT., SugiyamaH., InoueK., et al.. Growth inhibition of human leukemic cells by WT1 (Wilms’ tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis. Blood, 1996, 87(7): 2878-2884

[15] YangX.F., WuC.J., ChenL., et al.. CML28 is a broadly immunogenic antigen, which is overexpressed in tumor cells. Cancer Res, 2002, 62(19): 5517-5522

[16] GuoX., MaJ., SunJ., et al.. The zinc-finger antiviral protein recruits the RNA processing exosome to degrade the target mRNA. Proc Natl Acad Sci USA, 2007, 104(1): 151-156

[17] YangX.F., WuC.J., McLaughlinS., et al.. CML66, a broadly immunogenic tumor antigen, elicits a humoral immune response associated with remission of chronic myelogenous leukemia. Proc Natl Acad Sci USA, 2001, 98(13): 7492-7497

[18] WuC.J., BiernackiM., KutokJ.L., et al.. Graft-versus-leukemia target antigens in chronic myelogenous leukemia are expressed on myeloid progenitor cells. Clin Cancer Res, 2005, 11(12): 4504-4511

[19] ZhouH., ZhangD., WangY., et al.. Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector. Biochem Biophys Res Commun, 2006, 347(1): 200-207

[20] ZhangD.H., ZhouH.S., WangY.Y., et al.. Construction and expression of dendritic cell nucleic acid vaccine containing CML28 gene in human dendritic cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi (Chinese), 2005, 13(4): 631-636

[21] ZhangD.H., DaiM., ZhouH.S., et al.. Monitoring CML28 mRNA levels in patients before and after HSCT by real-time quantitative RT-PCR. Zhongguo Shi Yan Xue Ye Xue Za Zhi (Chinese), 2005, 13(5): 843-847

[22] BaiX.L., MaoX., ZhangB., et al.. The role of CML28 in regulation of proliferation and apoptosis of K562 cell. Med J Chin PAPF, 2012, 23(8): 678-681

[23] JacobsohnD.A., TseW.T., ChaleffS., et al.. High WT1 gene expression before haematopoietic stem cell transplant in children with acute myeloid leukaemia predicts poor event-free survival. Br J Haematol, 2009, 146(6): 669-674

[24] GlienkeW., MauteL., KoehlU., et al.. Effective treatment of leukemic cell lines with wt1 siRNA. Leukemia, 2007, 21(10): 2164-2170

[25] GaoF., MaitiS., AlamN., et al.. The Wilms tumor gene, Wt1, is required for Sox9 expression and maintenance of tubular architecture in the developing testis. Proc Natl Acad Sci USA, 2006, 103(32): 11 987-11 992

[26] Martínez-EstradaO.M., LetticeL.A., EssafiA., et al.. Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin. Nature Genet, 2010, 42(1): 89-93

[27] ChenJ., SantillanD.A., KoonceM., et al.. Loss of MLL PHD finger 3 is necessary for MLL-ENL-induced hematopoietic stem cell immortalization. Cancer Res, 2008, 68(15): 6199-6207

[28] RokudaiS., AikawaY., TagataY., et al.. Monocytic leukemia zinc finger (MOZ) interacts with p53 to induce p21 expression and cell-cycle arrest. J Biol Chem, 2009, 284(1): 237-244

[29] MorrisonA.A., VineyR.L., LadomeryM.R.. The post-trans-criptional roles of WT1, a multifunctional zinc-finger protein. Biochim Biophys Acta, 2008, 1785(1): 55-62

[30] BevilacquaA., CerianiM.C., CapaccioliS., et al.. Post-transcriptional regulation of gene expression by degradation of messenger RNAs. J Cell Physiol, 2003, 195(3): 356-372