Insights into ectopic estrogen receptor expression, nucleocytoplasmic distribution and interaction with chromatin obtained with new antibodies to estrogen receptors α and ß.

Recent reports have indicated that in cells ectopically expressing only ERα or the full-length hormone-binding isoform of ERß (ERß1), the receptors interact with chromatin with different efficacies and that antibodies capable of probing such interactions by chromatin immunoprecipitation (ChIP) are scarce. We therefore produced nine subtype and isoform-specific antibodies to ERα or ERß and validated their performance in receptor probing in cell lines and tissue biopsies by various immunochemical methods, including ChIP. We also produced clones of HEK-293 cells stably transfected with an estrogen response element (ERE)-dependent luciferase reporter and ERα or ERß1, in order to comparatively study their interaction with reporter ERE. We show that ERα was located in the nucleus and ERß1 in the cytoplasm as well as the nucleus of the stably transfected cells, while both receptors were found predominantly in the nucleus in transiently transfected cells and in all estrogen target tissues examined using the same antibodies. The cells displayed wild-type transcriptional activity and canonical regulation of ERE-dependent luciferase expression by estrogen agonists and antagonists. However, unlike ERα, ERß1 recruitment to the reporter ERE could be probed only by sequential ChIP with antibodies to receptor N- and C-terminus. These data suggest that in HEK-293 cells stably expressing ERα or ERß1, ER subtype-specific constraints apply to ERß1 nuclear entry; and that in cells displaying cytoplasmic as well as nuclear localization of ERß1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells.

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H2O2, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H2O2 stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 microM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination.