Phosphorylation of histone H3 by protein kinase C signaling plays a critical role in the regulation of the developmentally important TBX2 gene.

The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.

Live Imaging of Neural Crest and Pigment Cells and Transient Transgenic Manipulation of Gene Activity.

Neural crest cells are a highly multipotent and migratory cell type that are important for adult pigment pattern formation, cellular homeostasis, and regeneration. The optical transparency and accessibility of fish embryos makes them particularly well-suited to high-resolution analysis of neural crest development. However, the dispersive nature of these cells adds to the challenge of their study. We describe key protocols for the analysis of neural crest development in zebrafish and medaka, including live imaging of neural crest cells and differentiating pigment cells and transient transgenesis assays that can be used to manipulate neural crest development.

The palladacycle complex AJ-5 induces apoptotic cell death while reducing autophagic flux in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Recently, a novel binuclear palladacycle, AJ-5, was shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. This study investigates the anti-cancer activity of AJ-5 in alveolar and embryonal RMS. IC50 values of ≤ 0.2 µM were determined for AJ-5 and it displayed a favourable selectivity index of >2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate, respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptosis and necrosis. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1 and a significant reduction in autophagosome flux J. Finally, pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. Together these findings suggest that AJ-5 may be an effective chemotherapeutic with a desirable mechanism of action for treating drug-resistant and advanced sarcomas.

The ulnar-mammary syndrome gene, Tbx3, is a direct target of the retinoic acid signaling pathway, which regulates its expression during mouse limb development.

TBX3, a member of the T-box transcription factor gene family, is a transcriptional repressor that is required for the development of the heart, limbs, and mammary glands. Mutations in TBX3 that result in reduced functional protein lead to ulnar-mammary syndrome, a developmental disorder characterized by limb, mammary gland, tooth, and genital abnormalities. Increased levels of TBX3 have been shown to contribute to the oncogenic process, and TBX3 is overexpressed in several cancers, including breast cancer, liver cancer, and melanoma. Despite its important role in development and postnatal life, little is known about the signaling pathways that modulate TBX3 expression. Here we show, using in vitro and in vivo assays, that retinoic acid (RA) activates endogenous TBX3 expression, which is mediated by an RA-receptor complex directly binding and activating the TBX3 promoter, and we provide evidence that this regulation may be functionally relevant in mouse embryonic limb development. Our data identify TBX3 as a direct target of the RA signaling pathway and extend our understanding of the role and regulation of TBX3 in limb development.