Modifications in the status of global DNA methylation and p21 and p53 gene expression during the development of experimental pituitary tumors

RODRIGO AUGUSTO DA SILVA; TORRES ALICIA; SABATINO MARÍA EUGENIA; LATINI ALEXANDRA; SOSA LILIANA DEL VALLE; GRONDONA EZEQUIEL; DE PAUL ANA L

Praga

Congreso; 12th International Congress of Cell Biology; 2016

Czech Society for Cell Biology

Pituitary tumors are common and mostly benign neoplasia in the population. In an experimental model of pituitary tumor we demonstrated the emergence of cellular senescence, interpreted as a spontaneous initial barrier in tumorigenesis, by the activation of ATM signaling indicating the possibility of DNA damage accompanied by the up-regulation the cell-cycle regulators p21. Considering that epigenetic changes of genes involved in regulating cell proliferation could considered as potential initiators of tumorigenesis pituitary, in the present work we evaluated possible epigenetic modifications occurring along the development of experimental pituitary tumors. Male Wistar rats were subcutaneously exposed to estradiol benzoate (10mg) for 10, 20, 40 and 60 days (E10-60). Then, pituitaries were dissected and DNA, RNA and protein were extracted. Global DNA methylation was analyzed by MspI/HpaII endonucleases and gene expression of DNMTs (1, 3A and 3B), TETs (1, 2, 3) and 8-oxiguanine DNA glycosylase 1 (OGG-1) by semiquantitative RT-PCR. The promoter methylation for P53 and P21 was carried out by methylation-sensitive restriction enzymes. The protein content and the Dmtn 1 subcellular localization was analyzed by epifluorescence microscopy and western blot. Increased global DNA hypomethylation was observed from E20 to E60, in correlation with decreased DNMT1 expression and lack of DNMT1 nuclear localization. The mRNA expression of DNMT3A and DNMT3B and TETs family enzymes were not modified along the pituitary tumor development. An increase OGG-1 gene expression was observed along the estrogen treatment. Finally, a marked hypermethylation of p53 and p21 promoters was detected at E20 and E60, but p21 gene promoter showed also a hypomethylated status at E40, data correlated with higher p21 nuclear protein levels detected in this condition previously. Our findings demonstrate the existence of alterations in the maintenance methylation by the DNA methyltransferase, DNMT1, DNA damage and that epigenetic modifications could alter the p53 and p21 gene expression, thus contributing to the control of pituitary tumoral growth.