Altered DNA Methylation in Leukocytes with Trisomy 21
Figure 5
Effects of the demethylating drug 5aza-dC on expression of TMEM131 short isoform, TCF7, and NPDC1 mRNAs in Jurkat cells and normal human T-cells expanded with mitogens.
A, Increased expression of TMEM131 short isoform mRNA in Jurkat cells and PBMC exposed to 5aza-dC at the indicated concentrations for 3 days. The short-term cultures of PBMC were grown in the presence of IL-15 to induce proliferation of cytotoxic T-cells and NK cells. B, Decreased expression of TCF7 mRNA in Jurkat cells and PBMC exposed to 5aza-dC at the indicated concentrations for 3 days. The short term cultures of PBMC were grown in the presence of PHA to induce a polyclonal proliferation of T-cells. PHA rather than IL-15 stimulation was utilized for assessing TCF7 expression and response to 5aza-dC because the baseline expression of TCF7 mRNA is high after PHA stimulation but very low after stimulation with IL-15. C, Increased expression of NPDC1 mRNA in Jurkat cells and PBMC exposed to 5aza-dC at the indicated concentrations for 3 days. The short term cultures of PBMC were grown in the presence of PHA to induce a polyclonal proliferation of T-cells. In each experiment a 25 – 40 percent reduction in DNA methylation of the index regions of interest after exposure to the highest dose of 5aza-dC was confirmed by MS-Pyroseq or bisulfite sequencing (data not shown). Cell viability was preserved in each experiment, but net cell proliferation was reduced by approximately 50% at the highest doses of 5aza-dC.