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Brk tyrosine kinase signaling in the gastrointestinal tractHägebarth, Andrea 07 December 2005 (has links)
Die Tyrosin Kinase Brk stellt den Prototypen nicht N-terminal myristoylierter, Nicht-Rezeptor Tyrosin Kinasen dar. Die Expression dieser Kinase ist auf epitheliale Gewebe beschränkt und wird während der Entwicklung differentiell reguliert. In normalen Geweben ist die Brk Expression auf nichtproliferierende, terminal differenzierte Zellen beschränkt. Um die regulatorische Funktion von Brk im murinen Darmepithel zu untersuchen, wurde das brk Gen in der Maus inaktiviert. Brk knockout Mäuse zeigten keine offensichtlichen Defekte in ihrer Entwicklung jedoch eine erweiterte Proliferationszone in den Krypten des Darmepithels und verlängerte Villi. Die Inaktivierung von Brk führte zu einer erhöhten Akkummulation von nukleärem (-catenin sowie einer Hochregulierung des (-catenin Zielgens c-myc in den Krypten der knockout Mäuse. Zusätzlich zeigten Brk knockout Mäuse eine Aktivierung des Akt-Signaltransduktionswegs in ihrem Darmepithel. Im Gegensatz zu Wildtyp Mäusen waren Brk knockout Mäuse resistent gegenüber (-Strahlung, was die Anhäufung onkogener Mutationen und damit die Entwicklung von Krebs fördert. Eine Induktion der Expression des Brk-Proteins im Darmepithel behandelter Wildtyp Mäuse wurde festgestellt. Weiterhin traten bei Brk knockout Mäusen chronische Entzündungen des Darmepithels sowie eine erhöhte Sensibilität gegenüber dem Reizmittel DSS auf. Im Gegensatz dazu, zeigten Wildtyp Mäuse eine mit der Literatur Übereinstimmende Reaktion zu DSS verbunden mit einer Induktion der Brk Expression im Darmepithel. Zusammenfassend kann gesagt werden, dass die Brk Tyrosin Kinase eine entscheidende Rolle in der Aufrechterhaltung der Homöstase und Integrität des Darmepithels spielt. Insbesondere scheint Brk als wichtiger Faktor zur Bestimmung der Sensitivität epithelialer Zellen zu genotoxischem Stress zu fungieren. Entgegen der bisher vermuteten onkogenen Funktion in epithelialen Tumoren scheint Brk im normalen Darmepithel "Tumor Suppressor" Ähnliche Funtionen innezuhaben. / The Breast tumor kinase Brk is a prototypical non-myristoylated, non-receptor tyrosine kinase. Brk expression is epithelial-specific and ,in normal tissues, restricted to cells exiting the cell cycle and undergoing terminal differentiation. To determine the biological role of Brk in the gastrointestinal tract, we disrupted mouse brk by homologous recombination. Loss of Brk in the mouse resulted in increased intestinal epithelial cell turnover and the appearance of longer small intestinal villi. Brk deficient mice displayed enhanced accumulation of nuclear (-catenin and upregulation of the (-catenin target gene c-myc in the crypt compartment of small and large intestine. In addition, Brk deficient mice exhibited increased Akt kinase activity. Even though, there was no corresponding difference in base-line apoptosis in untreated wild-type and knockout animals. However, subjected to (-irradiation, Brk deficient animals were significantly impaired in the apoptotic response. Wild-type mice, however, exhibited normal levels of apoptosis following (-irradiation accompanied by a rapid induction of Brk expression in crypt cells. Furthermore, chronic inflammation was observed in Brk deficient mice, and they showed increased susceptibility to a colon injury model utilizing DSS. Interestingly, wild-type mice exhibited a significant upregulation of nuclear Brk protein throughout the intestinal epithelium in response to DSS. These recent findings suggest that Brk plays a crucial role in the maintenance of intestinal tissue homeostasis and integrity. In addition, Brk may function to protect the intestinal epithelium against DNA-replication-induced errors and hence the development of cancer. Contrary to reported oncogenic properties of Brk in other epithelial tissues, Brk appears to have tumor suppressor-like functions in the mouse gastrointestinal epithelium.
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Roles of EEF1A2 & PTK6 in breast cancerFida, Mariam January 2011 (has links)
Eukaryotic Translation Elongation Factor 1 Alpha (EEF1A) exists as two forms with different tissue specificities and encoded by separate loci: eEF1A1 on 6q13 and eEF1A2 on 20q13.3. eEF1A1 is ubiquitously expressed whereas eEF1A2 expression is normally limited to the heart, brain and skeletal muscles. eEF1A proteins are GTP-binding proteins that recruit an amino-acylated tRNA to the ribosome during the elongation phase of protein translation. eEF1A2 mRNA and protein are highly expressed in 50–60% of primary human breast tumors and metastases but not in normal breast epithelium. Since it is also overexpressed in 30% of primary human ovarian tumors, transforms rodent fibroblasts and increases their tumorigenicity in nude mice, eEF1A2 is considered to be a potential human oncogene. The mechanism of eEF1A2 expression is yet to be determined. Studies showed no gene mutation and no correlation between locus amplification or methylation and gene expression. Phosphate Tyrosine Kinase-6 (PTK6) is also located on 20q13.3. It is 48kb upstream of EEF1A2. PTK6 is a non-receptor tyrosine-kinase that is normally expressed in epithelial linings, prostate, skin and oral epithelium but it is not detected in the normal human mammary epithelium. PTK6 has been found to be expressed in many breast cancer cell lines and in approximately 60% of primary human breast tumors but it has not been detected in normal human breast tissue nor in fibroadenomas. Like other tyrosine kinases, PTK6 phosphorylates and activates downstream substrates that would be predicted to lead to increased transcriptional activity and therefore mediates proliferation of breast cancer cells. PTK6 is considered a prognostic marker of metastasis-free survival in breast cancer independent of the classical markers of tumor size, lymph node involvement and HER2 status. The aim of this project was to characterize for the first time the genomic region containing the two mentioned breast cancer oncogenes and understand their various roleswhether they act in tandem or independently in breast tumorigenesis. Immunohistochemistry was performed on tissue microarrays from 300 breast cancer patients to detect the expression levels of eEF1A2 and PTK6. Tumors that showed a high co-expression were analyzed for the genes’ copy number. An increased copy number of PTK6 was detected but not of eEF1A2 nor of adjacent genes on the 20q13.3 amplicon. To understand the effect of EEF1A2 expression on other genes, microarray analysis was performed on NIH-3T3 cells stably transfected with EEF1A2. Many upregulated genes were associated with different types of cancers. This was further confirmed by real-time PCR. However, when the NIH-3T3 cells were transiently transfected with EEF1A2, the genes that were upregulated in the microarray study showed no change in expression. In conclusion, EEF1A2 and PTK6 act independently and each acts through a different mechanism in breast tumorigenesis.
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