1Christian Maercker, 2Amir Abdollahi, 1Christiane Rutenberg, 1Heidrun Ridinger, 3Ondrey Paces, 4James Wang, 3Wilhelm Ansorge, 1Bernhard Korn, 2Peter E. Huber
1RZPD German Resource Center for Genome Research, Heidelberg, Germany, email@example.com, 2German Cancer Research Center (DKFZ), Heidelberg, 3European Molecular Biology Laboratory (EMBL), Heidelberg, 4Hypromatrix, Worcester, USA
Angiogenesis is an important prerequisite for growth and survival of tumors. During this process, hypoxia inducible factor (HIF) stimulates vascular endothelial growth factor (VEGF) transcription. By it's binding to VEGF receptor, VEGF is stimulator of Ras proteins and other central signaling cascades, which lead to cell differentiation and proliferation. We want to identify players involved in promoting angiogenesis after oxygen depletion (hypoxia) and also in the opposite process after treatment with endostatin, a promising inhibitor of angiogenesis in tumors. On the transcriptional level, we used the Human Unigene RZPD-2 75k cDNA array to identify interesting new markers in HUVEC cells. One example is the Notch4 receptor, which decreases angiogenesis(Abdollahi et al., Mol. Cell., in press). After this we carried out profiling experiments on antibody arrays to confirm the RNA results and also to get more direct access to the cellular proteome. For pilot experiments, 35 rabbit and mouse monoclonal antibodies against well known human proteins involved in cell signaling and angiogenesis were spotted on a modified glass surface. For the overlay assay, protein lysates were labeled with NHS ester-linked Cy3 or Cy5, respectively, and incubated with the test array as well as with the 512 Ab array from BD. We found out that the results concerning cell cycle promoting factors and many other proteins matched very well with the transcription data, which was a strong argument for specific binding of the proteins to the respective antibodies. Moreover, other interesting candidates became visible, which are hardly detectable by RNA profiling experiments. One reason might be the short half-life of the RNAs encoding certain proteins, as, for example, signaling molecules of the phosphatidyl-inositol pathway. Together with phosphorylation studies on antibody arrays which we have started recently, we are confirmed that these techniques allow us to get a deeper insight into the molecular processes involved in angiogenesis.
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