title: Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines.
abstract: BACKGROUND
  WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the "canonical" WNT/β-catenin signaling pathway, and the "non-canonical" pathways including WNT/Ca²⁺ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood.The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60.
  
  
  METHODS
  We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR.
  
  
  RESULTS
  WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB cells corroborated this observation. Interestingly, restoration of WNT4 expression in BJAB cells increased the accumulation of cells in G1 phase, and did not induce activation of canonical WNT/β-catenin target genes.
  
  
  CONCLUSIONS
  Our findings suggest that the WNT4 ligand plays a role in regulating the cell growth of leukemia-derived cells by arresting cells in the G1 cell cycle phase in an FZD6-independent manner, possibly through antagonizing the canonical WNT/β-catenin signaling pathway.
authors.affiliation: División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS), Sierra Mojada No, 800, Col, Independencia, 44340 Guadalajara, Jalisco, Mexico. adry.aguilar.lemarroy@gmail.com.
authors.forename: Beatriz
authors.initials: B
authors.lastname: García-Castro
authors.forename: Monserrat
authors.initials: M
authors.lastname: Alvarez-Zavala
authors.forename: Alma R
authors.initials: AR
authors.lastname: Riveros-Magaña
authors.forename: Pablo C
authors.initials: PC
authors.lastname: Ortíz-Lazareno
authors.forename: Sarah
authors.initials: S
authors.lastname: Ratkovich-González
authors.forename: Georgina
authors.initials: G
authors.lastname: Hernández-Flores
authors.forename: Alejandro
authors.initials: A
authors.lastname: Bravo-Cuellar
authors.forename: Luis F
authors.initials: LF
authors.lastname: Jave-Suarez
authors.forename: Adriana
authors.initials: A
authors.lastname: Aguilar-Lemarroy
mesh_terms: D017209:Apoptosis; D001402:B-Lymphocytes / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D051157:Frizzled Receptors / Q000378:metabolism; D059585:G1 Phase Cell Cycle Checkpoints; D015870:Gene Expression; D006801:Humans; D007938:Leukemia; D013601:T-Lymphocytes / Q000378:metabolism; D060449:Wnt Signaling Pathway; D060528:Wnt4 Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C484687:FZD6 protein, human; D051157:Frizzled Receptors; C555581:WNT4 protein, human; D060528:Wnt4 Protein