Figure 4.

Long-term treatment with cytokines causes DNA de-methylation together with aberrant gene expression. Chondrocytes were cultured with 5-aza-dC (A,B); or IL-1β or TNF-α/OSM (C,D,E,F). Relative mRNA expression of IL1B was compared with the % DNA methylation at −299bp in the IL1B promoter.

(A,B) Culture per se induced some expression and a 20% loss in methylation, 5-aza-dC increased expression 5-fold compared to control culture (A) and caused a further loss of DNA methylation (B). Means and SDs of six samples; * = P<0.05. (C) Relative expression of IL1B in chondrocytes from six individual patients, labelled with the patients' numbers and treated as indicated in the key. No IL1B was detectable in non-cultured chondrocytes. Culture itself induced low expression (set to =1), but IL-1β, and TNF-α/OSM increased expression considerably. (D) The % DNA methylation in the same samples as above. Before culture ~60% of cells were methylated. Culture alone could reduce DNA methylation, which was significant in patient 406 and 421. However, cytokine treatment caused greater loss of DNA methylation, particularly the combined treatment. (E,F) Means and S.D. of the samples shown in C and D. The differences between all groups are significant with P<0.05.