ALKBH3 promoter CpG island hypermethylation and transcriptional silencing in Hodgkin lymphoma cells. (A) Percentage of ALKBH3 methylation in the Sanger set of cancer cell lines according to tumor type. Number of cell lines studied for each tumor type is shown on top of each column. (B) ALKBH3 hypermethylation is associated with loss of the mRNA in cell lines from the Sanger panel (n = 957). (C) Percentage of ALKBH3 methylation in the Sanger set of cell lines derived from hematological malignancies according to subtype. Number of cell lines studied for each tumor type is shown on top of each column. (D) ALKBH3 methylation is associated with loss of the transcript in the Sanger set of cell lines derived from hematological malignancies (n = 162). (E) Bisulfite genomic sequencing of the ALKBH3 promoter CpG island in Hodgkin lymphoma cell lines and naive B cells from healthy donors. CpGs are represented as short vertical lines; the transcription start site (TSS) is represented as a black arrow. Single clones are shown for each sample. Presence of an methylated or unmethylated cytosine is indicated by a black or white square, respectively. (F) DNA-methylation profile of the ALKBH3 promoter CpG island analyzed using the 450 000 DNA-methylation microarray. Single CpG-methylation levels (0-1) are shown. Red, methylated; green, unmethylated. Data from the 4 studied Hodgkin lymphoma cell lines. (G) Expression levels of ALKBH3 in Hodgkin lymphoma cell lines assessed by quantitative reverse transcription PCR (data shown represent mean plus or minus standard deviation [SD] of biological triplicates) (left) and western blot (right). (H) Expression of the ALKBH3 RNA transcript (data shown represent the mean plus or minus SD of biological triplicates) and protein was recovered in the ALKBH3-hypermethylated and -silenced KM-H2 and L540 cells upon use of the DNA-demethylating agent 5-aza-2′-deoxycytidine (AZA). *P < .05; **P < .01; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSCLC, non–small cell lung carcinoma; SCLC, small cell lung carcinoma.