Modifications in Status Methylation of Global DNA and p53 and p21 Gene Promoters during the Development of Experimental Pituitary Tumors.

SOSA LDV; DA SILVA R; SABATINO ME; GRONDONA E; TORRES A; LATINI A; DE PAUL AL

Praga

Congreso; XII International Congress of Cell Biology; 2016

Pituitary tumors are common and mostly benign neoplasia in thepopulation. In an experimental model of pituitary tumor wedemonstrated the activation of ATM signaling indicating thepossibility of DNA damage accompanied by the up-regulation thecell-cycle regulators p21. Considering that epigenetic changes ofgenes involved in regulating cell proliferation could considered aspotential initiators of tumorigenesis pituitary, in the present work weevaluated possible epigenetic modifications occurring along thedevelopment of experimental pituitary tumors. Male Wistar rats weresubcutaneously exposed to estradiol benzoate (10mg) for 10, 20, 40and 60 days (E10-60). Then, pituitaries were dissected and DNA, RNAand protein were extracted. Global DNA methylation was analyzed byMspI/HpaII endonucleases and gene expression of DNMTs (1, 3A and3B), TETs (1, 2, 3) and 8-oxiguanine DNA glycosylase 1 (OGG-1) bysemiquantitative RT-PCR. The promoter methylation for P53 and P21was carried out by methylation-sensitive restriction enzymes. Theprotein content and the Dmtn 1 subcellular localization wereanalyzed by epifluorescence microscopy and western blot. Increasedglobal DNA hypomethylation was observed from E20 to E60, incorrelation with decreased DNMT1 expression and lack of DNMT1nuclear localization. The mRNA expression of DNMT3A and DNMT3Band TETs family enzymes were not modified along the pituitary tumordevelopment. An increase OGG-1 gene expression was observedalong the estrogen treatment. Finally, a marked hypermethylation ofp53 and p21 promoters was detected at E20 and E60, but p21 genepromoter showed also a hypomethylated status at E40, datacorrelated with higher p21 nuclear protein levels detected in thiscondition previously. Our findings demonstrate the existence ofalterations in the maintenance methylation by the DNAmethyltransferase, DNMT1, DNA damage and that epigeneticmodifications could alter the p53 and p21 gene expression, thuscontributing to the control of pituitary tumoral growth.