BackgroundNotch signalling regulates cell fate in most tissues, promoting precursor cell proliferation in some, but differentiation in others. Accordingly, downregulation or overactivity variously contributes to cancer development. So far, little is known about Notch pathway activity and function in the normal urothelium and in urothelial carcinoma (UC). We have therefore investigated expression of Notch pathway components in UC tissues and cell lines and studied the function of one receptor, NOTCH1, in detail.MethodsExpression of canonical Notch pathway components were studied in UC and normal bladder tissues by immunohistochemistry and quantitative RT-PCR and in UC cell lines and normal cultured urothelial cells by qRT-PCR, immunocytochemistry and Western blotting. Pathway activity was measured by reporter gene assays. Its influence on cell proliferation was investigated by γ-secretase inhibition. Effects of NOTCH1 restoration were followed by measuring cell cycle distribution, proliferation, clonogenicity and nuclear morphology.ResultsNOTCH1 and its ligand, DLL1, were expressed at plasma membranes and in the cytoplasm of cells in the upper normal urothelium layer, but became downregulated in UC tissues, especially in high-stage tumours. In addition, the proteins were often delocalized intracellularly. According differences were observed in UC cell lines compared to normal urothelial cells. Canonical Notch pathway activity in reporter assays was repressed in UC cell lines compared to normal cells and a mammary carcinoma cell line, but was induced by transfected NOTCH1. Inhibitors of Notch signalling acting at the γ-secretase step did not affect UC cell proliferation at concentrations efficacious against a cell line with known Notch activity. Surprisingly, overexpression of NOTCH1 into UC cell lines did not significantly affect short-term cell proliferation, but induced nuclear abnormalities and diminished clonogenicity.ConclusionOur data indicate that canonical Notch signalling is suppressed in urothelial carcinoma mainly through downregulation of NOTCH1. These findings can be explained by proposing that canonical Notch signalling may promote differentiation in the urothelium, like in many squamous epithelia, and its suppression may therefore be advantageous for tumour progression. As an important corollary, inhibition of canonical Notch signalling is unlikely to be efficacious and might be counter-productive in the treatment of urothelial carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-628) contains supplementary material, which is available to authorized users.

Urothelial carcinoma (UC), the most common cancer of the urinary bladder causes severe morbidity and mortality, e.g. about 40.000 deaths in the EU annually, and incurs considerable costs for the health system due to the need for prolonged treatments and long-term monitoring. Extensive aberrant  DNA methylation is described to prevail in urothelial carcinoma and is thought to contribute to genetic instability, altered gene expression and tumor progression. However, it is unknown how this epigenetic alteration arises during carcinogenesis. Intact methyl group metabolism is required to ensure maintenance of cell-type specific methylomes and thereby genetic integrity and proper cellular function. Here, using two independent techniques for detecting DNA methylation, we observed DNA hypermethylation of the 5′-regulatory regions of the key methyl group metabolism genes ODC1, AHCY and MTHFR in early urothelial carcinoma. These hypermethylation events are associated with genome-wide DNA hypomethylation which is commonly associated with genetic instability. We therefore infer that hypermethylation of methyl group metabolism genes acts in a feed-forward cycle to promote additional DNA methylation changes and suggest a new hypothesis on the molecular etiology of urothelial carcinoma.

To obtain T2Ã values in histologically evaluated healthy ovine intervertebral discs of the cervical and lumbar spine. Intervertebral discs of nine sheep and nine lambs underwent histological assessment with the modified Boos score for grading of disc degeneration. Discs with a score <10 points (maximum ¼ 40 points) underwent T2Ã mapping (n ¼ 64). Mid-sagittal T2 Ã values were obtained in five regions: Anterior annulus fibrosus, anterior nucleus pulposus, central nucleus pulposus, posterior nucleus pulposus, and posterior annulus fibrosus. We noted a zonal T2Ã distribution with high values in the central nucleus and low T2 Ã values in the anterior and posterior annulus fibrosus. The T2 Ã values were higher in lamb than in sheep IVDs for both cervical and lumbar spine (p < 0.001). The T2 Ã values were also higher in the cervical than in the lumbar spine (p ¼ 0.029 for sheep and p < 0.001 for lamb IVDs). The T2Ã values obtained in these ovine intervertebral discs can serve as baseline values for future T2 Ã measurements both in health and disease. ß

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