Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Breuer, M.
Expression of AKT/PKB, PTEN and CLOCK in Merkel cell carcinoma cell lines
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2018. pp.
[OPEN ACCESS]
FullText
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Sattler Wolfgang
- Altmetrics:
- Abstract:
- Loss or alterations of the prominent tumor suppressor Phosphatase and Tensin homolog deleted on chromosome Ten (PTEN) is clearly implicated with the pathogenesis of a variety of cancers. This association has not yet been demonstrated for the tumorigenesis of Merkel cell carcinoma (MCC); a highly aggressive and rare cutaneous neuroendocrine carcinoma with poor therapeutic options. Nevertheless, the discovery of the Merkel cell polyomavirus (MCPyV) was an enormous step towards revealing underlying mechanisms of its pathogenesis. Whether MCPyV alters the coupling of the molecular circadian cycle to the cell cycle in order to favor viral transcription in a circadian/cellular cycle fixed manner is a hypothesis worth to be explored in respect to a potential chronotherapeutic approach. The aim of this thesis is to clarify whether MCPyV-positive MCC determines expression levels of PTEN as well as activation/phosphorylation of AKT in comparison to MCPyV-negative MCC cell lines. For western blotting, total cell lysates of four MCPyV-negative cell lines, as well as nine MCC cell lines which harbor viral DNA, were investigated by employing primary antibodies targeted against PTEN, pAKTS473, AKT and ß-Tubulin as a loading control. Revealing that all cell lines tested express PTEN but without significant differences regarding MCPyV status. pAKT S473 levels are highly variable amongst the cell lines and by far expressed at a lesser extent. Additionally, to characterize the subcellular localization of PTEN and to evaluate Clock’s colocalization, nuclear and cytosolic as well as total cell lysates of MCPyV-positive WaGa cells of either heat-shocked or untreated/desynchronized samples were used to be probed with primary antibodies specific for Clock and PTEN; as well as ß-Actin and ß-Tubulin as cytosolic controls and Lamin B1 as nuclear loading control. PTEN and Clock were detected solely in the nucleus. Despite the clear evidence of PTEN activation concerning total cell lysates of all thirteen MCC cell lines as well as for the experimental design nuclear and cytosolic extraction regarding WaGa cells; this established experimental set-up could provide a solid and reliable basis for further experiments to elucidate mechanisms relating to the circadian timing system and modulation of cell growth potentially exploited by MCPyV.