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The impact of cell architecture on activation and output of the p53 stress response pathway


Most studies of cell signaling in vitro use cells grown as monolayers on rigid and non-physiological substrata. However, epithelial cells in vivo are attached on malleable extracellular matrix (ECM) and are organized into three dimensional (3D) structures with the aid of cell-cell and cell-ECM interactions. The rigidity of the ECM and the 3D cellular architecture have a profound influence on cell growth, survival and differentiation mediated through changes in cell signaling and gene transcription. P53 is a transcription factor capable of responding to a variety of stresses and signals. Furthermore, the level of P53 negative regulator Hdm2 is influenced by mitogenic signalings from the environment. Since the activity of P53 is influenced by the levels of its upstream regulators, changes in cell architecture may influence Hdm2 level and P53 activity. To study the modulation of P53 activity by different transgenes, an improved Cre-loxP recombination system was characterized. The new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA at a defined locus with incoming DNA. When used in combination with a stringent inducible system, the level of transgene expression can be controlled at physiologically relevant levels in an in vitro setting. The study of the influence of cell architecture and the impact of neighboring stromal cells on P53 activity and levels of its upstream regulators were described in subsequent chapters. We found that cells grown in 3D were more resistant to apoptosis induced by a combination of doxorubicin and Nutlin. The sensitivity to apoptosis correlated with the efficiency of Hdmx downregulation. Lowering Hdmx level by shRNA in 3D cells sensitizes these cells to apoptosis induced by doxorubicin and Nutlin. Hence, the cell architecture is one factor to consider in determining the efficacy of chemotherapeutic agents as it can impact on Hdmx level to reduce P53-dependent apoptosis

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