Estrogen receptor beta growth-inhibitory effects are repressed through activation of MAPK and PI3K signalling in mammary epithelial and breast cancer cells.

Two thirds of breast cancers express estrogen receptors (ER). ER alpha (ERα) mediates breast cancer cell proliferation, and expression of ERα is the standard choice to indicate adjuvant endocrine therapy. ERbeta (ERß) inhibits growth in vitro; its effects in vivo have been incompletely investigated and its role in breast cancer and potential as alternative target in endocrine therapy needs further study. In this work, mammary epithelial (EpH4 and HC11) and breast cancer (MC4-L2) cells with endogenous ERα and ERß expression and T47-D human breast cancer cells with recombinant ERß (T47-DERß) were used to explore effects exerted in vitro and in vivo by the ERß agonists 2,3-bis (4-hydroxy-phenyl)-propionitrile (DPN) and 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY). In vivo, ERß agonists induced mammary gland hyperplasia and MC4-L2 tumour growth to a similar extent as the ERα agonist 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) or 17ß-estradiol (E2) and correlated with higher number of mitotic and lower number of apoptotic features. In vitro, in MC4-L2, EpH4 or HC11 cells incubated under basal conditions, ERß agonists induced apoptosis measured as upregulation of p53 and apoptosis-inducible factor protein levels and increased caspase 3 activity, whereas PPT and E2 stimulated proliferation. However, when extracellular signal-regulated kinase 1 and 2 (ERK ½) were activated by co-incubation with basement membrane extract or epidermal growth factor, induction of apoptosis by ERß agonists was repressed and DPN induced proliferation in a similar way as E2 or PPT. In a context of active ERK ½, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (AKT) signalling was necessary to allow proliferation stimulated by ER agonists. Inhibition of MEK ½ with UO126 completely restored ERß growth-inhibitory effects, whereas inhibition of PI3K by LY294002 inhibited ERß-induced proliferation. These results show that the cellular context modulates ERß growth-inhibitory effects and should be taken into consideration upon assessment of ERß as target for endocrine treatment.

Asynchronous DNA replication detected by fluorescence in situ hybridisation as a possible indicator of genetic damage in human lymphocytes.

The present study aimed to correlate the DNA replication timing of different genes with genetic damage and frequency of cancer. Using a fluorescence in situ hybridisation (FISH) approach, the replication timing of three loci, two human genes possessing transcriptional capability and involved in both the cellular response to genetic damage and cancer development (TP53 and RB1) and the non-coding locus D22S163, was evaluated. The data obtained show that normal human lymphocytes exposed in vitro to known DNA-damaging agents, e.g. H2O2, ionizing radiation and mitomycin C, exhibit an asynchronous replication of the genes TP53 and RB1. In vivo studies were performed in three different populations from Kazakhstan. In two of these populations that are living in polluted areas and have higher cancer mortalities than people living in a control area, a DNA replication behaviour similar to that observed in human lymphocytes exposed in vitro to known genotoxic agents was detected. The results obtained further indicate that DNA damage hampers replication and FISH represents a fast and accurate method of assessing asynchronous replication by providing an important tool to evaluate DNA damage at a populational level.