Though clinical trials for medical applications of dimethyl sulfoxide (DMSO) reported toxicity in the 1960s, later, the FDA classified DMSO in the safest solvent category. DMSO became widely used in many biomedical fields and biological effects were overlooked. Meanwhile, biomedical science has evolved towards sensitive high-throughput techniques and new research areas, including epigenomics and microRNAs. Considering its wide use, especially for cryopreservation and in vitro assays, we evaluated biological effect of DMSO using these technological innovations. We exposed 3D cardiac and hepatic microtissues to medium with or without 0.1% DMSO and analyzed the transcriptome, proteome and DNA methylation profiles. In both tissue types, transcriptome analysis detected >2000 differentially expressed genes affecting similar biological processes, thereby indicating consistent cross-organ actions of DMSO. Furthermore, microRNA analysis revealed large-scale deregulations of cardiac microRNAs and smaller, though still massive, effects in hepatic microtissues. Genome-wide methylation patterns also revealed tissue-specificity. While hepatic microtissues demonstrated non-significant changes, findings from cardiac microtissues suggested disruption of DNA methylation mechanisms leading to genome-wide changes. The extreme changes in microRNAs and alterations in the epigenetic landscape indicate that DMSO is not inert. Its use should be reconsidered, especially for cryopreservation of embryos and oocytes, since it may impact embryonic development.

Key Words: cell adhesion Ⅲ endothelial cells Ⅲ angiogenesis Ⅲ biochemistry Ⅲ signaling C rosstalk between integrins and growth factor receptors plays an important role in vascular development and its maintenance. Several examples demonstrated that crosstalk between integrins and tyrosine kinase receptors is required for growth factor-induced biological processes to ensure cell growth, survival, and differentiation in normal and pathological processes. 1 The angiogenic processes require the coordination of signals from the extracellular environment to activates specific tyrosine kinase receptors and integrins. 1,2 Integrins associating with growth factor receptors regulate the capacity of the integrin/receptor complexes to propagate downstream signals. 3,4 Integrin-dependent activation of receptor tyrosine kinases is a general mechanism to enhance growth factor signals, the recruitment of transducing proteins to membrane cytoskeletal complexes as well as nuclear responses. This cooperation has been shown for several signaling pathways including insulin, epidermal growth factor (EGF), platelet-derived growth factor, fibroblast growth factor, and vascular endothelial growth factor (VEGF) signaling. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] Integrins are able to trigger ligand-independent EGF receptor autophosphorylation leading to activation of the downstream pathway. 16 Integrins, c-Src, p130Cas and EGF receptor associate in a macromolecular complex on the cell membrane and integrin-dependent adhesion induces phosphorylation of specific tyrosine residues of EGF receptor, distinct from those obtained by the soluble ligand EGF. 11,19 A large body of evidence demonstrates that the angiogenic process is initiated by mitogenic signals induced by growth factors such as VEGF and the interplay between the tyrosine 34 -36 VEGFR-3, in contrast to its highly related endothelial receptor VEGFR-2 (which, after VEGF stimulation, forms a complex with integrin ␤3 subunit 8,20,21,24,25,37 ), associates selectively with integrin ␤1. 10,12 Cell attachment to fibronectin or collagen induces the phosphorylation of VEGFR-3 in the absence of a ligand and significantly enhances the phosphorylation of the receptor induced by its ligand. 10,12 However, the mechanism of integrin activation of VEGFR-3 has not been clarified.Here, we demonstrate that collagen I-induced activation of VEGFR-3 is independent from the intrinsic catalytic activity of the receptor but it is a direct target of c-Src. c-Src phosphorylates VEGFR-3 at specific tyrosine residues with a pattern of phosphorylation that is distinct from the pattern induced by the ligand. Cell adhesion induces the phosphorylation of the tyrosine residues 1063 and 1337, which are known binding sites for CRKI/II and SHC. Pull-down assays demonstrated that integrin-mediated receptor phosphorylation induces the recruitment of CRKI/II and SHC second messengers to the receptor suggesting that the integrin/receptor complex can activate growth and survival signaling in the absence ...

Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.

No abstract