Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Two Tales of Antioxidant Enzymes on β Cells and Diabetes

Pancreatic islets contain low activities of catalase, selenium-dependent glutathione peroxidase 1 (GPX1), and Cu,Zn-superoxide dismutase 1 (SOD1). Thus, enhancing expression of these enzymes in islets has been unquestionably favored. However, such an attempt has produced variable metabolic outcomes. While β cell-specific overexpression of Sod1 enhanced mouse resistance to streptozotocin-induced diabetes, the same manipulation of catalase aggravated onset of type 1 diabetes in nonobese diabetic mice. Global overexpression of Gpx1 in mice induced type 2 diabetes-like phenotypes. Although knockouts of Gpx1 and Sod1 each alone or together decreased pancreatic β cell mass and plasma insulin concentrations, these knockouts improved body insulin sensitivity to different extents. Pancreatic duodenal homeobox 1, forkhead box A2, and uncoupling protein 2 are three key regulators of β cell mass, insulin synthesis, and glucose-stimulated insulin secretion. Phenotypes resulted from altering GPX1 and/or SOD1 were partly mediated through these factors, along with protein kinase B and c-jun terminal kinase. A shifted reactive oxygen species inhibition of protein tyrosine phosphatases in insulin signaling might be attributed to altered insulin sensitivity. Overall, metabolic roles of antioxidant enzymes in β cells and diabetes depend on body oxidative status and target functions. Revealing regulatory mechanisms for this type of dual role will help prevent potential pro-diabetic risk of antioxidant over-supplementation to humans. Antioxid. Redox Signal. 14, 489–503