Expression of the tumor suppressor p16INK4a after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16INK4a on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of b1,4-galactosyltransferases (down-regulation of b1,4-galactosyltransferases-I ⁄ V and up-regulation of b1,4-galactosyltransferase-IV) as well as decreased a2,3-sialylation of O-glycans and a2,6-sialylation of N-glycans. The changes are compatible with increased b 1 -integrin maturation, subunit assembly and binding activity of the a 5 b 1 -integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16 INK4a-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16 INK4aAbbreviations

Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the ␣5␤1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G 1 cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the ␣5␤1 integrin.

The tumor suppressor p16INK4a has functions beyond cell‐cycle control via cyclin‐dependent kinases. A coordinated remodeling of N‐ and O‐glycosylation, and an increase in the presentation of the endogenous lectin galectin‐1 sensing these changes on the surface of p16INK4a‐expressing pancreatic carcinoma cells (Capan‐1), lead to potent pro‐anoikis signals. We show that the p16INK4a‐dependent impact on growth‐regulatory lectins is not limited to galectin‐1, but also concerns galectin‐3. By monitoring its expression in relation to p16INK4a status, as well as running anoikis assays with galectin‐3 and cell transfectants with up‐ or downregulated lectin expression, a negative correlation between anoikis and the presence of this lectin was established. Nuclear run‐off and northern blotting experiments revealed an effect of the presence of p16INK4a on steady‐state levels of galectin‐3‐specific mRNA that differed from decreasing the transcriptional rate. On the cell surface, galectin‐3 interferes with galectin‐1, which initiates signaling toward its pro‐anoikis activity via caspase‐8 activation. The detected opposite effects of p16INK4a at the levels of growth‐regulatory galectins‐1 and ‐3 shift the status markedly towards the galectin‐1‐dependent pro‐anoikis activity. A previously undescribed orchestrated fine‐tuning of this effector system by a tumor suppressor is discovered.

BackgroundAvailability of oxygen and nutrients in the coronary circulation is a crucial determinant of cardiac performance. Nutrient composition of coronary blood may significantly vary in specific physiological and pathological conditions, for example, administration of special diets, long-term starvation, physical exercise or diabetes. Quantitative analysis of cardiac metabolism from a systems biology perspective may help to a better understanding of the relationship between nutrient supply and efficiency of metabolic processes required for an adequate cardiac output.ResultsHere we present CardioNet, the first large-scale reconstruction of the metabolic network of the human cardiomyocyte comprising 1793 metabolic reactions, including 560 transport processes in six compartments. We use flux-balance analysis to demonstrate the capability of the network to accomplish a set of 368 metabolic functions required for maintaining the structural and functional integrity of the cell. Taking the maintenance of ATP, biosynthesis of ceramide, cardiolipin and further important phospholipids as examples, we analyse how a changed supply of glucose, lactate, fatty acids and ketone bodies may influence the efficiency of these essential processes.ConclusionsCardioNet is a functionally validated metabolic network of the human cardiomyocyte that enables theorectical studies of cellular metabolic processes crucial for the accomplishment of an adequate cardiac output.

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