Abrogation of the radiation-induced G2 checkpoint by the staurosporine derivative UCN-01 is associated with radiosensitisation in a subset of colorectal tumour cell lines

Ionising radiation is commonly used in the treatment of colorectal cancer. Tumour cells with mutant p53 undergo cell cycle arrest at G2/M after ionising radiation and evidence suggests that abrogation of this G2 arrest can lead to a premature, aberrant mitosis, thus enhancing ionising radiation-induced cell killing. The G2 checkpoint inhibitor UCN-01 was thus investigated to determine whether it would abrogate the G2 checkpoint induced by 5 Gy ionising radiation in a range of colorectal tumour cell lines. Data presented show that, at doses that are alone non-toxic to the cells, UCN-01 inhibits the ionising radiation-induced G2 checkpoint in five colorectal tumour cell lines with mutant p53. The ability of UCN-01 to sensitise cells to ionising radiation-induced growth inhibition and apoptosis was also investigated and UCN-01 was found to radiosensitise two out of five cell lines. These results were confirmed by long-term colony forming efficiency studies. These results demonstrate that abrogation of the ionising radiation-induced G2 checkpoint is not necessarily associated with sensitisation to ionising radiation, however, some colorectal tumour cell lines can be radiosensitised by UCN-01. Although the mechanism of radiosensitisation is not clear, this may still be an important treatment strategy

Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana

Amidase 1 (AMI1) from Arabidopsis thaliana converts indole-3-acetamide (IAM), into indole-3-acetic acid (IAA). AMI1 is part of a small isogene family comprising seven members in A. thaliana encoding proteins which share a conserved glycine- and serine-rich amidase-signature. One member of this family has been characterized as an N-acylethanolamine-cleaving fatty acid amidohydrolase (FAAH) and two other members are part of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) or mitochondria (Tom complex) and presumably lack enzymatic activity. Among the hitherto characterized proteins of this family, AMI1 is the only member with indole-3-acetamide hydrolase activity, and IAM is the preferred substrate while N-acylethanolamines and oleamide are not hydrolyzed significantly, thus suggesting a role of AMI1 in auxin biosynthesis. Whereas the enzymatic function of AMI1 has been determined in vitro, the subcellular localization of the enzyme remained unclear. By using different GFP-fusion constructs and an A. thaliana transient expression system, we show a cytoplasmic localization of AMI1. In addition, RT-PCR and anti-amidase antisera were used to examine tissue specific expression of AMI1 at the transcriptional and translational level, respectively. AMI1-expression is strongest in places of highest IAA content in the plant. Thus, it is concluded that AMI1 may be involved in de novo IAA synthesis in A. thaliana

Pharmacologic Inhibition of the TGF-β Type I Receptor Kinase Has Anabolic and Anti-Catabolic Effects on Bone

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-β has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-β signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-β signaling on bone remain unclear. To examine the role of TGF-β in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-β type I receptor (TβRI) kinase on bone mass, architecture and material properties. Inhibition of TβRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TβRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TβRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TβRI inhibitors may be effective in treating conditions of skeletal fragility