WNT5A is epigenetically silenced in hematologic malignancies and inhibits leukemic cell growth. (A) Expression levels of WNT5A mRNA in 5-aza-2′-deoxycytidine (Aza)–treated and untreated nasal NK/T-cell lymphoma (NK-YS and SNK-6) and NK leukemia (YT) cell lines. The data were obtained with microarray (Human Genome U133 Plus 2.0 Microarray, Affymetrix, Santa Clara, CA) expression analysis by GeneSpring version 7.3.1 software (Agilent, Palo Alto, CA). The degree of WNT5A up-regulation after Aza and TSA treatment is also shown. Red color represents high expression whereas green color represents low expression. (B) The CpG island (CGI) of the WNT5A promoter includes the core promoter, exon 1, and part of intron 1. The transcription start site is indicated by a curved arrow. The MSP and BGS regions analyzed in the CGI are indicated. (C) Semiquantitative RT-PCR and MSP analyses of WNT5A in normal cells and tissues and tumor cell lines. WNT5A expression in cell lines after treatment with 5 μM Aza⁷  is shown on the right. GAPDH was used as a control. The WNT5A expression levels among different PBMC samples could be variable. M indicates methylated; and U, unmethylated. (D) High-resolution methylation analysis of the WNT5A promoter in 2 leukemia cell lines by BGS, showing the methylation status of every CpG site in the studied region. Each row in the grid represents an individual allele of the WNT5A promoter. ■ and □ represent methylated or unmethylated CpG sites, respectively. (E) Representative MSP results of primary BL, nasal NK/T-cell lymphoma, normal PBMCs, and LN tissues. RT-PCR results of several primary BL and 1 nasal NK/T-cell lymphoma are also shown. (F) Expression levels of WNT5A in silenced K562 cells before and after transfection were determined by RT-PCR. (G) Growth curves of K562 cells after transfection with pcDNA3.1-WNT5A or control vector. At indicated time points after transfection, cell numbers were counted and plotted. Mean values plus or minus SD of triplicate experiments are shown (*P < .05). (H) Quantitative analysis of colony numbers after transfection with pcDNA3.1-WNT5A and G418 selection in K562. The number of G418-resistant colonies in the control vector–transfected cell line was set to 100%. Mean values plus or minus SD of 3 separate experiments are shown.