| Abstract |
Human Cytomegalovirus (HCMV) is a beta-herpes virus, and possesses a linear, double-stranded DNA genome. It is a pathogen with worldwide distribution and high clinical importance, causing severe implications in individuals with suppressed or underdeveloped immune systems. Also, HCMV can favor, through its oncomodulatory role, the progression of malignancy in many types of tumors, including glioblastoma, which is the most common malignant central nervous system (CNS) type of tumor. RhoA GTPase, which belongs to the family of Rho GTPases, is a regulator of the actin cytoskeleton, playing crucial role in processes like cell polarization, cell-cell adhesion and cell migration. Dysregulation of RhoA function has been found in a variety of cancers, including glioblastoma, in which RhoA is involved in cellular processes, such as cell proliferation and invasion. Besides, it has been shown that HCMV interacts with RhoA signaling pathways, favoring the progression of its infection in fibroblasts. The epigenetic factor, EZH2, is a histone methyltransferase, and it has been indicated to be involved in some important cellular processes, such as inhibition of cellular senescence and regulation of cell cycle. Except of its normal functions, EZH2 can play a crucial role in cancer progression, as it has been found that is generally up-regulated in many types of tumors, such as glioblastoma, having oncogenic role in them. Furthermore, it has been revealed that productive infection of fibroblasts with HCMV leads to increased EZH2 protein expression both in initial and intermediate stages of the viral life cycle, thus promoting the progression of viral infection. Given the above, in the present thesis, it was studied the effect of HCMV productive infection of U373MG glioblastoma (GBM) cells on the protein expression of RhoA and EZH2. The effect of viral infection on U373MG morphology was, also, examined. It was therefore found that, the protein expression of EZH2 is increased at all studied time points [6 hpi (hours post infection), 12 hpi, 24 hpi, 48 hpi, 72 hpi] during infection, compared to non-infected cells. RhoA expression levels change dynamically during temporal progression of HCMV infection. While its expression is elevated at 12 and 24 hpi, it significantly decreases at the subsequent time point of 48 hpi, maintaining its expression at the same level at 72 hpi. Regarding the effect of HCMV infection on GBM cells morphology, it was revealed that U373MG had stopped growing as monolayer culture at 48 hpi, since cellular “spheres” had started to form at the same time point, losing concurrently their ability to adhere to the flask surface. This phenotype was maintained and at 72 hpi, with formed spheres being
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larger in size and having an increased tendency to detach from the surface of the flask.
Taking into consideration the property of glioblastoma stem-cells (GSCs) to form spheres, and the fact that HCMV may drives development or maintenance of GSC phenotype, the “choice” of U373MG to form spheres upon HCMV infection is maybe one more indication of the ability of HCMV to induce this phenotype, thereby favoring malignant progression of glioblastoma. Surely, a more thorough investigation is needed, in order to detect more data, which would support more clearly the acquisition of GSC properties by U373MG upon HCMV infection.
The fact that expression levels of ΕΖΗ2 and RhoA altered during the course of infection of U373MG, indicates a possible involvement of these factors in the HCMV life cycle in glioblastoma. The increase in EZH2 expression, caused after infection of U373MG with HCMV, is consistent with the findings in the case of fibroblasts, thus EZH2 may has similar role in glioblastoma cells. The significant reduction in RhoA expression from 24 hpi to 48hpi may be linked with the property of U373MG to form spheres at the same time point, since the acquisition of this morphology presupposes the remodeling of actin cytoskeleton, which is one of the main functions of RhoA. The aforementioned observations are preliminary data, which can be useful for future research efforts, in order to clarify, first of all, the exact role of EZH2 and RhoA in HCMV life cycle in glioblastoma cells, and secondly, the probable manner of their involvement in the property of glioblastoma cells to form spheres, and generally to acquire GSC phenotype upon HCMV infection. Finally, the finding of more data, which connect HCMV infection with the malignant progression of glioblastoma, such as HCMV association with GSC phenotype, could offer more information in regard with therapeutic targeting of HCMV (+) glioblastoma tumors either using anti-viral therapy or targeting cellular factors which interact with this virus, such as RhoA and EZH2.
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Ο ρόλος της RhoA GTPάσης και του επιγενετικού παράγοντα ΕΖΗ2 σε καρκινικά κύτταρα γλοιοβλαστώματος μετά από μόλυνση από τον Ανθρώπινο Κυτταρομεγαλοϊό (HCMV)
