Role of    DNA-methylation in the branching    pattern of e   strogen-transformed human   breast epithelial cells. Free

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The lost of the branching pattern of the ductal structures is an early event in breast cancer. In our in vitro in vivo model of the estrogen induced cell transformation, the E₂-transformed MCF-10F (trMCF) cells exhibit progressive loss of ductulogenesis, invasive (bsMCF) and tumorigenic (caMCF) phenotypes (Russo et al. 2006, FASEB J, 20:1622-34). Using this model, we have previously reported that 39 genes were found hypermethylated using Restriction Landmark Genomic Scanning (RLGS) technique. Seventeen of them were potentially involved in the branching process: HOXA9, HOXB5, EPB49, FGF14, SYNE1, FOXD1, BMP6, SPRY1, TM4SF9, SNIP, STXBP6, TLL1, CSS3, OTP, TBR1, NRG1 and ITGA11. The transformed cells trMCF showed low expression of NRG1, STXBP6, BMP6 and TM4SF9 compared to MCF-10F. The bsMCF shown low expression of NRG1, STXBP6, BMP6, SPRY1, CSS3 and SNIP although, they had an increase in TM4SF9 expression. In caMCF, no expression of NRG1 and CSS3 was detected and these cells had low expression of BMP6, SPRY1 and SNIP however they showed higher expressions of STXBP6 and TM4SF9. In the present work we are aiming to determine that the genes identified by RLGS were epigenetically silenced by studying their expression after treatment with the demethylating agent 5-aza-dC and the inhibitor of histone deacetylase TSA. The expression of SPRY1, NRG1, STXBP6, BMP6, CSS3, TM4SF9 and SNIP showed differences after 5-aza-dC and/or TSA treatments, indicating that they are epigenetically regulated. The expressions of SPRY1 and NRG1 were higher after 5-aza-dC but they did not show differences after treatment with TSA in MCF-10F, trMCF and bsMCF, an indication that DNA methylation but not histone modifications was important in the regulation of SPRY1 and NRG1 in MCF-10F and its derivative cells Treatment of MCF-10F, trMCF or bsMCF with TSA and /or 5-aza-dC increased the expression of STXBP6, BMP6, CSS3, TM4SF9 and SNIP indicating that both DNA methylation and histone modification play an important role in their regulation. Further relevance of these data is that the changes observed in the early phases of cell transformation and can be followed through multiple generations of cell populations. To test if the reversion of the methylation status was responsible for the loss of ductulogenesis induced by E2, one of the clones of the E2-70 nM (E2-70- clone 11) that formed spherical masses in collagen was treated with 2.5 µM 5-aza-dC during 5 days. The treatment with 5-aza-dC revert the formation of spherical masses forming instead duct-like structures Altogether our data indicate that the loss of ductulogenesis induced by estrogen can be epigenetically modulated. (This work was supported by grants DMAD17-00-1-0247 and DMAD17-03-1-0229)