New molecular targets in radiotherapy: DNA damage signalling and repair in targeted and non-targeted cells

Ionising radiation plays a key role in therapy due to its ability to directly induce DNA damage, in particular DNA double-strand breaks leading to cell death. Cells have multiple repair pathways which attempt to maintain genomic stability. DNA repair proteins have become key targets for therapy, using small molecule inhibitors, in combination with radiation and or chemotherapeutic agents as a means of enhancing cell killing. Significant advances in our understanding of the response of cells to radiation exposures has come from the observation of non-targeted effects where cells respond via mechanisms other than those which are a direct consequence of energy-dependent DNA damage. Typical of these is bystander signalling where cells respond to the fact that their neighbours have been irradiated. Bystander cells show a DNA damage response which is distinct from directly irradiated cells. In bystander cells, ATM- and Rad3-related (ATR) protein kinase-dependent signalling in response to stalled replication forks is an early event in the DNA damage response. The ATM protein kinase is activated downstream of ATR in bystander cells. This offers the potential for differential approaches for the modulation of bystander and direct effects with repair inhibitors which may impact on the response of tumours and on the protection of normal tissues during radiotherapy. (C) 2009 Elsevier B.V. All rights reserved.

VEGF-induced retinal angiogenic signalling is critically dependent on Ca2+ signalling via Ca2+/calmodulin-dependent protein kinase II

Purpose. The authors conducted an in vitro investigation of the role of Ca2+-dependent signaling in vascular endothelial growth factor (VEGF)-induced angiogenesis in the retina.

Methods. Bovine retinal endothelial cells (BRECs) were stimulated with VEGF in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; intracellular Ca2+ chelator), U73122 (phospholipase C (PLC) inhibitor), xestospongin C (Xe-C), and 2-aminoethoxydiphenyl borate (2APB) (inhibitors of inositol-1,4,5 triphosphate (IP3) signaling). Intracellular Ca2+ concentration ([Ca2+]i) was estimated using fura-2 Ca2+ microfluorometry, Akt phosphorylation quantified by Western blot analysis, and angiogenic responses assessed using cell migration, proliferation, tubulogenesis, and sprout formation assays. The effects of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 were also evaluated on VEGF-induced Akt signaling and angiogenic activity.

Results. Stimulation of BRECs with 25 ng/mL VEGF induced a biphasic increase in [Ca2+]i, with an initial transient peak followed by a sustained plateau phase. VEGF-induced [Ca2+]i increases were almost completely abolished by pretreating the cells with BAPTA-AM, U73122, Xe-C, or 2APB. These agents also inhibited VEGF-induced phosphorylation of Akt, cell migration, proliferation, tubulogenesis, and sprouting angiogenesis. KN93 was similarly effective at blocking the VEGF-induced activation of Akt and angiogenic responses.

Conclusions. VEGF increases [Ca2+]i in BRECs through activation of the PLC-IP3 signal transduction pathway. VEGF-induced phosphorylation of the proangiogenic protein Akt is critically dependent on this increase in [Ca2+]i and the subsequent activation of CaMKII. Pharmacologic inhibition of Ca2+-mediated signaling in retinal endothelial cells blocks VEGF-induced angiogenic responses. These results suggest that the PLC/IP3/Ca2+/CaMKII signaling pathway may be a rational target for the treatment of angiogenesis-related disorders of the eye.

Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells.

The Jagged/Notch pathway has been implicated in TGFß1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFß1-stimulated gene responses in human kidney epithelial cells in vitro. TGFß1 treatment of HK-2 and RPTEC cells for 24 h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of ?-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFß1-responsive genes associated with epithelial–mesenchymal transition such as E-cadherin and vimentin were also affected by ?-secretase inhibition, but other TGFß1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFß1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFß1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFß1, but did not affect a-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFß1-responsive gene subset. Increased Jagged1 expression upon TGFß1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFß1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFß1 and Notch cascades in kidney epithelia.


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Performance of calcium deficient hydroxyapatite-polyglycolic acid composites: An in vitro study

The strategic incorporation of bioresorbable polymeric additives to calcium-deficient hydroxyapatite cement may provide short-term structural reinforcement and modify the modulus to closer match bone. The longer-term resorption properties may also be improved, creating pathways for bone in-growth. The aim of this study was to investigate the resorption process of a calcium phosphate cement system containing either in polyglycolic acid tri-methylene carbonate particles or polyglycolic acid fibres. This was achieved by in vitro aging in physiological conditions (phosphate buffered solution at 37°C) over 12 weeks. The unreinforced CPC exhibited an increase in compressive strength at 12 weeks, however catastrophic failure was observed above a critical loading. The fracture behaviour of cement was improved by the incorporation of PGA fibres; the cement retained its cohesive structure after critical loading. Gravimetric analysis and scanning electron microscopy showed a large proportion of the fibres had resorbed after 12 weeks allowing for the increased cement porosity, which could facilitate cell infiltration and faster integration of natural bone. Incorporating the particulate additives in the cement did not provide any mechanism for mechanical property augmentation or did not demonstrate any appreciable level of resorption after 12 weeks.