Browsing by Author "Räth, Sebastian"

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Open Access
Population and single-cell based quantitative analysis of protein kinase D-mediated regulation of the cell cycle
(2014) Räth, Sebastian; Pfizenmaier, Klaus (Prof. Dr.)
The cell cycle consists of G1, G2, S, and M phase and is a tightly regulated process with various checkpoints to control order and length of the separate phases. A multitude of signal molecules and pathways are involved in this process. In cancer, cell cycle control is often changed and understanding of these changes may result in new therapeutic targets in the treatment of patients. Additionally, cell cycle control is of special interest in stem cells as important decisions of cell fate – to proliferate or to differentiate - are part of cell cycle control. The success of adult stem cell therapeutic applications is thus dependent on in-depth understanding of this regulation. The Fluorescent ubiquitination-based cell cycle indicator (Fucci) is a sophisticated technology, which can easily determine G1 and/or S/G2/M phases of the cell cycle. The technology analyzes living cells in a spatio-temporal manner using fusion proteins consisting of two distinct cell cycle proteins fused to two fluorophores - a dual color scheme of orange and green. The aim of this thesis was to characterize the influence of Protein kinase D (PKD) using this technology in cells with adult stem cell characteristics and an established human cancer cell line. At first, a characterization of primary human mesenchymal stromal cells (MSC) derived from umbilical cord (UC) and bone marrow (BM) was performed. Furthermore, murine bone marrow stromal cells (mBMSCs) were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and host derived hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy. Consequently, multipotent mBMSC lines were established from transgenic Fucci mice. Single cell analysis of cell cycle progression was performed in these Fucci-mBMSCs and Fucci transgenic human HeLa cells. Specifically, the influence of protein kinase D (PKD) and the RAF/MEK/ERK pathway on progression through S/G2/M phase was investigated in detail. Inhibition of PKD but not of MEK resulted in a delay in progression through S/G2/M phase in HeLa cells and mBMSCs. Furthermore, MAPK pathway activation was quantitatively assessed during the synchronous progression of HeLa cells through S/G2/M and successfully used to develop a quantitative mathematical model describing this pathway. Taken together this study demonstrates the benefit of quantitative and single cell analysis in cells with stem cell characteristics and an established cell line to enlighten the role of PKD in cell cycle control and, on top of that, support the notion that PKD is a potential new target for cancer therapy.