Matrix metalloproteinase (MMP)-mediated degradation of the extracellular matrix is a major factor for tumor development and expansion. This study analysed MMP-10 protein expression and activity in human lung tumors of various grade, stage, and type to address the relationship between MMP-10 and tumor characteristics and to evaluate MMP-10 as a therapeutic target in non small cell lung carcinoma (NSCLC). Unlike the majority of MMPs, MMP-10 was located in the tumor mass as opposed to tumor stroma. MMP-10 protein was observed at low levels in normal human lung tissues and at significantly higher levels in all types of NSCLC. No correlation was observed between MMP-10 protein expression and tumor type, stage, or lymph node invasion. To discriminate between active and inactive forms of MMP-10 in samples of human NSCLC, we have developed an ex vivo fluorescent assay. Measurable MMP-10 activity was detected in 42 of 50 specimens of lung cancer and only 2 of 10 specimens of histologically normal lung tissue. No relationship was observed between MMP-10 activity levels and clinicopathologic characteristics. Our results suggest that MMP-10 is expressed and active at high levels in human NSCLC compared to normal lung tissues, and, as such, is a potential target for the development of novel therapeutics for lung cancer treatment.

The aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC=55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6days. The data support the development of hydrogels specific for epithelial formation.

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 microM) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

Recent casework in Belgium involving the search for human remains buried with lime, demonstrated the need for more detailed understanding of the effect of different types of lime on cadaver decomposition and its micro-environment. Six pigs (Sus scrofa) were used as body analogues in field experiments. They were buried without lime, with hydrated lime (Ca(OH)(2)) and with quicklime (CaO) in shallow graves in sandy loam soil in Belgium and recovered after 6 months of burial. Observations from these field recoveries informed additional laboratory experiments that were undertaken at the University of Bradford, UK. The combined results of these studies demonstrate that despite conflicting evidence in the literature, hydrated lime and quicklime both delay the decay of the carcass during the first 6 months. This study has implications for the investigation of clandestine burials and for a better understanding of archaeological plaster burials. Knowledge of the effects of lime on decomposition processes also has bearing on practices involving burial of animal carcasses and potentially the management of mass graves and mass disasters by humanitarian organisations and DVI teams.

We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor alpha-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in gamma-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers.

PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.

Recently published research indicates that soluble oligomers of beta-amyloid (Abeta) may be the key neurotoxic species associated with the progression of Alzheimer's disease (AD) and that the process of Abeta aggregation may drive this event. Furthermore, soluble oligomers of Abeta and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Abeta on neuronal plasma membranes where exogenously applied Abeta in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL-induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Abeta accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. In addition, our data from fyn(-/-) transgenic mice show that accumulation of Abeta on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Abeta and subsequent cell death. These results identify fyn-dependent Abeta redistribution and accumulation in lipid rafts as being key to ADDL-induced cell death and defines a mechanism by which oligomers of Abeta and tau accumulate in lipid rafts.

Bone morphogenetic proteins (BMPs) play essential roles in the control of skin development, postnatal tissue remodelling and tumorigenesis. To explore whether some of the effects of BMP signalling are mediated by microRNAs, we performed genome-wide microRNA (miRNA) screening in primary mouse keratinocytes after BMP4 treatment. Microarray analysis revealed substantial BMP4-dependent changes in the expression of distinct miRNAs, including miR-21. Real-time PCR confirmed that BMP4 dramatically inhibits miR-21 expression in the keratinocytes. Consistently, significantly increased levels of miR-21 were observed in transgenic mice overexpressing the BMP antagonist noggin under control of the K14 promoter (K14-noggin). By in situ hybridization, miR-21 expression was observed in the epidermis and hair follicle epithelium in normal mouse skin. In K14-noggin skin, miR-21 was prominently expressed in the epidermis, as well as in the peripheral portion of trichofolliculoma-like hair follicle-derived tumours that contain proliferating and poorly differentiated cells. By transfecting keratinocytes with a miR-21 mimic, we identified the existence of two groups of the BMP target genes, which are differentially regulated by miR-21. These included selected BMP-dependent tumour-suppressor genes (Pten, Pdcd4, Timp3 and Tpm1) negatively regulated by miR-21, as well as miR-21-independent Id1, Id2, Id3 and Msx2 that predominantly mediate the effects of BMPs on cell differentiation. In primary keratinocytes and HaCaT cells, miR-21 prevented the inhibitory effects of BMP4 on cell proliferation and migration. Thus, our study establishes a novel mechanism for the regulation of BMP-induced effects in the skin and suggests miRNAs are important modulators of the effects of growth factor signalling pathways on skin development and tumorigenesis.

Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown whether phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. We found that protein kinase C-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both of these events are necessary for the internalization of GluK2-containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation led to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Our results suggest a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.

AIMS/HYPOTHESIS: We previously demonstrated that animals fed a high-fat (HF) diet for 10 weeks developed insulin resistance and behavioural inflexibility. We hypothesised that intervention with metformin would diminish the HF-feeding-evoked cognitive deficit by improving insulin sensitivity. METHODS: Rats were trained in an operant-based matching and non-matching to position task (MTP/NMTP). Animals received an HF (45% of kJ as lard; n = 24), standard chow (SC; n = 16), HF + metformin (144 mg/kg in diet; n = 20) or SC + metformin (144 mg/kg in diet; n = 16) diet for 10 weeks before retesting. Body weight and plasma glucose, insulin and leptin were measured. Protein lysates from various brain areas were analysed for alterations in intracellular signalling or production of synaptic proteins. RESULTS: HF-fed animals developed insulin resistance and an impairment in switching task contingency from matching to non-matching paradigm. Metformin attenuated the insulin resistance and weight gain associated with HF feeding, but had no effect on performance in either MTP or NMTP tasks. No major alteration in proteins associated with insulin signalling or synaptic function was detected in response to HF diet in the hypothalamus, hippocampus, striatum or cortex. CONCLUSIONS/INTERPRETATION: Metformin prevented the metabolic but not cognitive alterations associated with HF feeding. The HF diet protocol did not change basal insulin signalling in the brain, suggesting that the brain did not develop insulin resistance. These findings indicate that HF diet has deleterious effects on neuronal function over and above those related to insulin resistance and suggest that weight loss may not be sufficient to reverse some damaging effects of poor diet.