Insulin-like growth factor-binding protein-2 inhibits proliferation of human embryonic kidney fibroblasts and of IGF-responsive colon carcinoma cell lines

AbstractSo far, the physiological role of insulin-like growth factor binding protein-2 (IGFBP-2) has not been demonstrated directly. Therefore, we transfected 293 cells with an expression vector containing the CMV promoter and the complete cDNA of mouse IGFBP-2. Secretion of bioactive IGFBP-2 into conditioned medium was demonstrated by Western ligand and Western immunoblotting and quantified by specific RIA. For the analysis of cell proliferation three clones exhibiting either high or low/no IGFBP-2 expression were selected and compared to non-transfected parental 293 cells. IGFBP-2 secreting clones displayed reduced conversion of thiazolyl blue when compared to negative clones or non-transfected parental 293 cells (P<0.01). The lower growth activity measured in the IGFBP-2 secreting clones was compensated in great part by the administration of exogenous IGF-I or -II. Conditioned media of IGFBP-2 secreting clones inhibited growth of IGF-responsive colon tumor cell lines (LS513, HT-29) while those of negative clones did not. In addition, conditioned medium from a clone expressing high levels of IGFBP-2 inhibited anchorage-independent growth of LS513 and HT-29 cells. In contrast, growth of an IGF-unresponsive tumor cell line (Co-115) was not affected by the conditioned media. We hypothesize that IGFBP-2 might sequester the IGFs and thus prevent them from transferring their mitogenic signals

IGF-II transgenic mice display increased aberrant colon crypt multiplicity and tumor volume after 1,2-dimethylhydrazine treatment

In colorectal cancer insulin-like growth factor II (IGF-II) is frequently overexpressed. To evaluate, whether IGF-II affects different stages of tumorigenesis, we induced neoplastic alterations in the colon of wild-type and IGF-II transgenic mice using 1,2-dimethylhydrazine (DMH). Aberrant crypt foci (ACF) served as markers of early lesions in the colonic mucosa, whereas adenomas and carcinomas characterized the endpoints of tumor development. DMH-treatment led initially to significantly more ACF in IGF-II transgenic than in wild-type mice. This increase in ACF was especially prominent for those consisting of ≥three aberrant crypts (AC). Nevertheless, adenomas and adenocarcinomas of the colon, present after 34 weeks in both genetic groups, were not found at different frequency. Tumor volumes, however, were significantly higher in IGF-II transgenic mice and correlated with serum IGF-II levels. Immunohistochemical staining for markers of proliferation and apoptosis revealed increased cell proliferation rates in tumors of IGF-II transgenic mice without significant affection of apoptosis. Increased proliferation was accompanied by elevated localization of β-catenin in the cytosol and cell nuclei and reduced appearance at the inner plasma membrane. In conclusion, we provide evidence that IGF-II, via activation of the β-catenin signaling cascade, promotes growth of ACF and tumors without affecting tumor numbers

A novel de novo TBX5 mutation in a patient with Holt-Oram syndrome leading to a dramatically reduced biological function

BACKGROUND: The Holt-Oram syndrome (HOS) is an autosomal dominant disorder affecting 1/100.000 live births. It is defined by upper limb anomalies and congenital heart defects with variable severity. We describe a dramatic phenotype of a male, 15-month-old patient being investigated for strict diagnostic criteria of HOS. ----- METHODS AND RESULTS: Genetic analysis revealed a so far unpublished TBX5 mutation, which occurs de novo in the patient with healthy parents. TBX5 belongs to the large family of T-box transcription factors playing major roles in morphogenesis and cell-type specification. The mutation located in the DNA-binding domain at position 920 (C→A) leads to an amino acid change at position 85 (proline → threonine). Three-dimensional analysis of the protein structure predicted a cis to trans change in the respective peptide bond, thereby probably provoking major conformational and functional alterations of the protein. The p.Pro85Thr mutation showed a dramatically reduced activation (97%) of the NPPA promoter in luciferase assays and failed to induce NPPA expression in HEK 293 cells compared to wild-type TBX5 protein. The mutation did not interfere with the nuclear localization of the protein. ----- CONCLUSION: These results suggest that the dramatic functional alteration of the p.Pro85Thr mutation leads to the distinctive phenotype of the patient

Pulsatile ex vivo perfusion of human saphenous vein grafts under controlled pressure conditions increases MMP-2 expression

<p>Abstract</p> <p>Background</p> <p>The use of human saphenous vein grafts (HSVGs) as a bypass conduit is a standard procedure in the treatment of coronary artery disease while their early occlusion remains a major problem.</p> <p>Methods</p> <p>We have developed an <it>ex vivo </it>perfusion system, which uses standardized and strictly controlled hemodynamic parameters for the pulsatile and non-static perfusion of HSVGs to guarantee a reliable analysis of molecular parameters under different pressure conditions. Cell viability of HSVGs (n = 12) was determined by the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) into a purple formazan dye.</p> <p>Results</p> <p>Under physiological flow rates (10 mmHg) HSVGs remained viable for two weeks. Their exposure to arterial conditions (100 mmHg) was possible for one week without important reduction in viability. Baseline expression of matrix metalloproteinase-2 (MMP-2) after venous perfusion (2.2 ± 0.5, n = 5) was strongly up-regulated after exposure to arterial conditions for three days (19.8 ± 4.3) or five days (23.9 ± 6.1, p < 0.05). Zymographic analyses confirmed this increase on the protein level. Our results suggest that expression and activity of MMP-2 are strongly increased after exposure of HSVGs to arterial hemodynamic conditions compared to physiological conditions.</p> <p>Conclusion</p> <p>Therefore, our system might be helpful to more precisely understand the molecular mechanisms leading to an early failure of HSVGs.</p

Myeloid Zinc Finger 1 (Mzf1) Differentially Modulates Murine Cardiogenesis by Interacting with an Nkx2.5 Cardiac Enhancer

Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations

Region and cell-type resolved quantitative proteomic map of the human heart

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular level. Analysis of cardiac fibroblasts identifies cellular receptors as potential cell surface markers. Application of our heart map to atrial fibrillation reveals individually distinct mitochondrial dysfunctions. The heart map is available at maxqb. biochem. mpg. de as a resource for future analyses of normal heart function and disease

IGF-II transgenic mice display increased aberrant colon crypt multiplicity and tumor volume after 1,2-dimethylhydrazine treatment

<p>Abstract</p> <p>In colorectal cancer insulin-like growth factor II (IGF-II) is frequently overexpressed. To evaluate, whether IGF-II affects different stages of tumorigenesis, we induced neoplastic alterations in the colon of wild-type and IGF-II transgenic mice using 1,2-dimethylhydrazine (DMH). Aberrant crypt foci (ACF) served as markers of early lesions in the colonic mucosa, whereas adenomas and carcinomas characterized the endpoints of tumor development. DMH-treatment led initially to significantly more ACF in IGF-II transgenic than in wild-type mice. This increase in ACF was especially prominent for those consisting of &#8805;three aberrant crypts (AC). Nevertheless, adenomas and adenocarcinomas of the colon, present after 34 weeks in both genetic groups, were not found at different frequency. Tumor volumes, however, were significantly higher in IGF-II transgenic mice and correlated with serum IGF-II levels. Immunohistochemical staining for markers of proliferation and apoptosis revealed increased cell proliferation rates in tumors of IGF-II transgenic mice without significant affection of apoptosis. Increased proliferation was accompanied by elevated localization of &#946;-catenin in the cytosol and cell nuclei and reduced appearance at the inner plasma membrane. In conclusion, we provide evidence that IGF-II, via activation of the &#946;-catenin signaling cascade, promotes growth of ACF and tumors without affecting tumor numbers.</p