Alternative splicing is a key regulatory mechanism for cellular metabolism controlling cell proliferation and angiogenesis, both of which are crucial processes for tumorigenesis under hypoxia. Human cells express two tissue factor (TF) isoforms, alternatively spliced TF (asTF) and 'full length' TF (flTF). flTF is the major source of thrombogenicity whereas, the function of soluble asTF, particularly in cancer, is widely unknown. In the present study, we examined the impact of alternative splicing on the pro-angiogenic potential and the TF expression pattern of A549 cells under hypoxia. We focused our efforts toward alternative splicing factors, such as Clk1, and pro-angiogenic proliferation-regulating factors, such as Cyr61. We further examined the influence of asTF overexpression on the expression of MCP-1, Cyr61 and VEGF, as well as on cell number and pro-angiogenic properties of A549 cells. Notably, we found hypoxia to induce the expression of alternative splicing factors (Clk1 and Clk4) as well as proliferation- and angiogenesis-promoting factors (Cyr61 and flTF). asTF overexpression in A549 cells also increased both cell number and tube formation. These effects were mediated by the induction of Cyr61, MCP-1 and VEGF, as well as by integrin α(v)β(3). Taken together, our results suggest that the pro-angiogenic potential of A549 lung cancer cells is modulated under hypoxic conditions via modulation of TF isoform expression which in turn is controlled by alternative splicing.

Purpose: Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells.Experimental Design: We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n ‫؍‬ 18), peripheral blood samples from healthy donors (n ‫؍‬ 21), and patients with cutaneous (n ‫؍‬ 122) and uveal (n ‫؍‬ 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples.Results: Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/l porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples).Conclusions: Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.

Tissue factor was down-regulated in the myocardium of DCM patients. The reduction in TF expression and change in localization may influence cell-to-cell contact stability and contractility, thereby contributing to cardiac dysfunction in DCM.

Non-small cell lung cancer (NSCLC) comprises of 75% of all lung cancers. Human full length tissue factor (flHTF), the physiological initiator of blood coagulation, is aberrantly expressed in certain solid tumors. FlHTF and its soluble isoform, alternatively spliced human tissue factor (asHTF), have been shown to contribute to thrombogenicity of the blood of healthy individuals. The aim of this study was to quantify flHTF and asHTF on mRNA and protein levels (using immunohistochemistry, immunoblotting, and ELISA) on a panel of human NSCLC tissue and plasma specimens. The tissue factor (TF) expression of 21 pulmonary adenomatous (AC) and 12 normal healthy tissues was assessed by real-time qRT-PCR. The TF protein concentration was quantified by ELISA in a subset of 11 AC and 9 normal tissue specimens as well as in the plasma of 13 lung cancer patients and 15 healthy controls. We found a significant increase in the ratio of flHTF/HGAPDH mRNA in AC (0.24±0.06 vs. 0.07±0.01; p=0.02 vs. controls) and in asHTF/HGAPDH mRNA (0.027±0.01 vs. 0.004±0.001; p=0.03 AC vs. controls). AsHTF mRNA expression was significantly lower in patients with stage IA disease compared to patients with higher grade stages, pointing to TF as being a marker of malignancy and metastases. TF protein of lung tumors was significantly increased in AC (p=0.004 vs. controls). TF in plasma was up-regulated in lung cancer patients (334.9±95.4 vs. 124.1±14.8 pg/ml; p=0.02 vs. controls). Immunohistochemical and immunoblotting data are in line with the increased TF expression, showing elevated blood thrombogenicity of NSCLC patients. The upregulation of flHTF and, especially, asHTF in AC suggests not only a raised risk of thrombosis, but also of tumor progression, thereby, indicating a poor prognosis in these patients.

Antioxidative treatment inhibited apoptosis and shedding of microparticles, thereby reducing thrombogenicity. Thus, antioxidants may help to prevent late thrombosis after antiproliferative treatment when used in combination with anticoagulants.

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 2 ABSTRACTThe renin-angiotensin system (RAS) plays a crucial role in cardiovascular and neuronal (patho-)physiology. The angiotensin AT2 receptor (AT2R) seems to counteract the proinflammatory, prohypertrophic and profibrotic actions of the AT1receptor. Recently, we identified a novel protein, termed "AT2R binding protein" (ATBP/ ATIP) which seems essential for AT2R mediated growth inhibition.Poly(ADP-ribose) polymerase-1 (PARP-1) can act as a nuclear integrator of angiotensin II-mediated cell signalling, and has been implicated in the pathogenesis of cardiovascular and neuronal disease.In this study, promoters of human AT2R and ATIP1 were cloned and two transcriptional start sites in the ATIP1 promoter were identified whereas only one was detected in the AT2R promoter. Promoter assays indicated that the exon 1-intron 1 region of AT2R is necessary and sufficient for AT2R promoter activity.Inverse cloning experiments indicated that this regulatory region is a promoter but not an enhancer element implicating (a) further start site(s) in this region.

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