Growth factors transmit biological signals for the stimulation of cell growth and their stimulation may be involved in turnourigenesis. It is therefore of great importance to understand growth factor receptor reactions in response to stimuli such as calcium depletion or ultraviolet radiation, which normal human cells are invariably exposed to during their growth cycle. We have studied human skin cells i.e. fibroblasts, lceratinocytes and melanocytes and their growth factor receptor expression on the surface of cells, reactions in the plane of the cell membrane, intracellular trafficlcing, and gene expression after exposure to their ligand, serum, UVB radiation and calcium depletion. We have used Fluorescence recovery after photo bleaching (FRAP) to assess receptor characteristics in cell membranes, confocal laser scanning microscopy to visualize receptor internalization, Ratio imaging for calcium studies, Northern blot for detection of the gene for the epidermal growth factor receptor (EGF-R) and flow cytometry for cell surface receptor determination. Platelet-derived growth factor receptors (PDGF-R) and EGF-R were studied since they affect cell proliferation and viability.
Our results showed that the addition of PDGF increased receptor mobility characteristics in normal fibroblasts, also "starvation" of cells increased their receptor mobility. We could show that changes in both intra-and extracellular free Ca2+ influence the mobility characteristics of PDGF-ß2 receptors. We have demonstrated that the three celltypes display different basal EGF-R mobility characteristics. After UVB irradiation mobility characteristics increased in all cell types but with differences in diffusion coefficients or mobile fractions. Addition of antioxidant enzymes, catalase and superoxide dismutase, prior to UVB irradiated cells abolished the UY-induced receptor mobility changes. We have shown that a single physiologic dose ofUVB radiation alters the intracellular EGF-R distribution and intracellular transport in melanocytes. It also significantly alters the melanocyte phenotype. We were able to detect a constitutive EGF-R gene expression and showed that UVB-radiation induces a time-dependent induction in EGF-R mRNA in melanocytes. Human melanocytes express EGF-R on their cell surface and UVR induces time dependent changes in the number of receptors but the number of receptors does not correlate with the level of UV-induced EGF-R gene expression.
It is concluded that UV-radiation, growth factors and calcium, ubiquitous constituents of every day life, all having tremendous effects in vivo, also affect human cells in vitro in parameters studied that are of importance for proliferation, survival and tumourigenesis.
Linköping: Linköpings universitet , 2001. , 88 p.
2001-05-18, Administrationshusets Aula, Universitetssjukhuset, Linköping, 09:30 (Swedish)