2005 Abstracts: Robo 1 and Robo 4: Validation of Differentially Expressed Genes Associated with Angiogenesis in Colorectal Cancer
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Robo 1 and Robo 4: Validation of Differentially Expressed Genes Associated with Angiogenesis in Colorectal Cancer
Oliver Doebler, Departement of Surgery Charite-Berlin Campus Benjamin Franklin, Berlin, Berlin, Germany; Joern Groene, Heinz J. Buhr, Sarah Bhargava, Department of Surgery, Campus Benjamin Franklin, Charité Berlin, Berlin, Berlin, Germany
Introduction:
The family of the roundabout (Robo) proteins is related to the transmembrane receptors and plays a major role during the process of axonal guidance in neurogenesis. Furthermore, it was shown that Robo proteins are also associated with angiogenesis in cancer. We have been able to demonstrate in large scale gene expression studies (Affymetrix U133A), that Robo1 and Robo4 are up-regulated in tumor tissue vs. normal tissue. The aim of this study was to validate the differential expression by means of real-time fluorescence PCR and to analyze the in situ expression of Robo1 and Robo4 in tissue sections. Material and methods: Shock-frozen samples of normal and colorectal tumor tissues were obtained from 50 patients with informed consent. After RNA isolation of macrodissected tissues and reverse transcription, Robo1 and Robo4 were quantified using real-time PCR with specific hybridization probes. As a reference gene â-2-Mikroglobulin was established. The expression rates were evaluated with algorithms of the Relative Quantification Software. For in situ localization immunhistochemistry and in-situ hybridization was performed. Results: Real-time PCR analysis of Robo1 showed an over-expression in 76 % of the patients (38/50) in the tumor epithelium compared to normal epithelium. In 40 % of the patients (20/50) a fourfold over-expression of Robo1 could be proved. In 12 % of the tumors (6/50) a more than twelvefold over-expressioncould be detected. An up-regulation of Robo4 was measured in 74 % of the patient samples (37/50), whereas 44 % of the patients (22/50) have shown a twofold, and 16% (8/50) a fourfold over-expression in the tumor tissue. Immunohistochemical staining and in situ hybridization revealed an expression of Robo1 and Robo4 in the tumor vessels. Conclusion: The over-expression of Robo1 and Robo4 in tumor vs. normal tissue was confirmed by means of real-time PCR. The histological tissue analyses showed an expression of these receptors in the tumor vessels. Thus, the Robo proteins provide potential target structures for the anti-angiogenic therapy of colorectal carcinoma.
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