Genes associated with an anti-proliferative function are silenced by DNA methylation in tamoxifen-resistant cells.

<p>A. MeDIP coupled PCR to determine the methylation status of RASAL1, DUSP7, ATP2B4 and GDF15 in MCF-7, TAM-R and TAM-Wd cells, relative the expression of PEG3 (an imprinted gene). Data is expressed as 2^∧−ΔΔct (n = 3). GAPDH is included as a negative control to demonstrate the successful enrichment of methylated material. The data demonstrate a greater enrichment of methylated DNA in resistant vs tamoxifen-sensitive MCF-7 cells for all four genes (*p<0.05). B. RT-qPCR evaluation of GDF15 expression in MCF-7, TAM-R and TAM-Wd cells +E2 (1×10⁻⁹ M), +5-Aza (1×10⁻⁶ M), 5-Aza + E2 or 5-Aza + E2+ TAM (1×10⁻⁷ M) for 48 hrs. Data shown are normalised to GAPDH and presented relative to the expression calculated for vehicle-treated cells. In the absence of 5-Aza, GDF15 expression is only significantly increased by E2 challenge in MCF-7 cells (*p<0.001). The lower panel shows a western blot of mature, processed GDF15 protein (18 KDa) and β-actin (loading control) (representative of 3 independent experiments). C. Clonal bisulphite sequencing analysis for the GDF15 promoter region in TAM-Wd cells ±5-Aza (1×10⁻⁶ M) for 48 hrs. Colours and symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040466#pone-0040466-g003" target="_blank">Figure 3</a>.