Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells.

Molecular mechanisms involved in the cytotoxicity induced by coumarins from Calophyllum brasiliense in K562 leukaemia cells.

OBJECTIVES: The aim of this study was to determine the cellular and molecular mechanisms of cell death induced by mammea A/BA and A/BB (3 : 1) on K562 cells. METHODS: These compounds were isolated from Calophyllum brasiliense and its cytotoxicity was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell death was evaluated by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunocytofluorescence of active caspase-3. Genotoxicity was tested using comet assay. Lastly, a chemoinformatic analysis was performed with Osiris-Molinspiration software. KEY FINDINGS: The mixture of mammea A/BA and A/BB (3 : 1) showed cytotoxic activity against K562 cells (IC50 = 43.5 µm). TUNEL positive cells and active caspase-3 were detected after treatment. Genotoxicity of mammea A/BA and A/BB on K562 was detected since first hour of treatment. Additionally, mammea A/BA and A/BB were found to be in compliance with Lipinski 'rule of 5' suggesting that they possess strong potential of druglikeness. CONCLUSIONS: The overall results confirm and extend the knowledge about coumarins as an important resource of antitumor drugs, and indicate that these compounds could be used in further preclinical studies against leukaemia.

Differential DNA methylation pattern in the A and B promoters of the progesterone receptor is associated with differential mRNA expression in the female rat hypothalamus during proestrus.

In rodents, the display of reproductive behavior occurs during the proestrus-estrus transition of the estrus cycle. This behavior is regulated by estradiol and progesterone mainly via their intracellular receptors. Two isoforms of the progesterone receptor have been described (A and B), and they have different promoters for their regulation. It has been demonstrated that the mRNA for both isoforms changes during the proestrus-estrus transition. It has been recently established that DNA methylation can be transient and cyclical in gene promoters, however, these changes have only been reported in vitro but not in physiological models. The aim of this study was to analyze the pattern of DNA methylation in the PR (A and B) promoter regions during the proestrus-estrus transition in the rat hypothalamus and its correlation with the regulation of mRNA expression. The results demonstrated a differential mRNA expression of the progesterone receptor (A and B) isoforms. The expression of total PR did not change significantly during the proestrus day, while the expression of isoform B increased significantly at 17:00 h, followed by a significant decrease at 21:00 h of the proestrus day. Interestingly, we also found that the isoform A promoter was mainly unmethylated at all studied time points. In contrast, the isoform B promoter showed a transient methylation increase during the evening of proestrus. The overall results indicate that there is a switch of progesterone receptor isoforms expression during the evening of proestrus that is related to the differential gene methylation patterns of their promoter regions, mainly for the isoform B promoter.