PMC

Methylation status of TNFα gene promoter in ALK+ TCL cells. (A) Schematic map of the CpG sites in the TNFα gene promoter. Methylation status of TNFα gene promoter in ALK+ TCL and control ALK- TCL cell lines (B) and ALK+ TCL tissues (C) determined by pyrosequence analysis of the bisulfate-converted DNA.

Impact of DNA methylation on activity of TNFα gene promoter. (Left column) Patterns of DNA methylation introduced into the TNFα promoter (open ovals depict unmethylated and solid ovals methylated CpG sites). (Right column) The corresponding impact of the complete methylation or hemimethylation of proximal or distal TNFα promoter on the promoter's activity as detected by the Luciferase gene reporter assay. Activity of the unmethylated TNFα promoter examined in parallel, served as a reference.

Lack of TNFα expression in ALK+ TCL cells. (A) The depicted 6 ALK+ TCL cell lines as well as 2 ALK- TCL cell lines (2A and 2B) derived from the CD30+ cutaneous lymphoproliferative T-cell disorder were examined for expression of TNFα mRNA by standard RT-PCR (A) and TNFα protein by EIA (B). Mitogen(PHA)-stimulated normal PBMC served as a positive control in the protein detection assay.

Effect of DNA methyltransferase inhibitor 5-ADC on expression of TNFα gene in ALK+ TCL cells. (A) Changes in TNFα promoter methylation induced in SUDHL-1 cells by treatment with 5-ADC for 0, 24, and 72 h. (B) Kinetic of 5-ADC-induced changes in TNFα protein expression in SUDHL-1 (left panel) and Karpas 299 (right panel) detected by EIA. (C) Changes in TNFα mRNA expression in SUDHL-1 cells exposed to 5-ADC for the depicted periods of time. (Upper panel) Standard RT-PCR, (lower panels) quantitative RT-PCR with GAPDH expression serving as a positive control.

TNFα-induced apoptotic cell death of ALK+ TCL cells. (A) Impact of TNFα used at the depicted concentrations on growth of the listed ALK+ and ALK- TCL cell lines as determined in the MTT enzymatic conversion assay. (B) Effect of TNFα on caspase 8 and caspase 3 activation status in the depicted ALK+ and ALK- TCL cell lines. Expression of TNF-R1 mRNA (C) and protein (D) by the depicted 6 ALK+ and 2 ALK- TCL cell lines determined by standard RT-PCR and flow cytometry, respectively. In panel D, the orange color line reflects staining with an anti-TNF-R1 antibody, the violet color line with an isotype-matched control antibody.

Gene expression pattern of the TNFα apoptotic pathway in ALK+ TCL cells. (A) The presence (red bar) or absence (blue bar) of expression of the TNFα apoptotic pathway genes detected in the ALK+ TCL-derived SUDHL-1 cell line by using the genome-scale DNA oligonucleotide array. (B) Expression of the TNFα gene determined by the DNA oligonucleotide array in the SUDHL-1 cells cultured in the presence of medium or 5-ADC for 24, 48, or 72 h. The data are depicted as fold increase in the TNFα mRNA expression in the 5-ADC-treated cells as compared to the cells exposed to medium containing the drug vehicle. The experiments presented in both panels were performed as triplicate cultures.