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Cellular Phenotype-Dependent and -Independent Effects of Vitamin C on the Renewal and Gene Expression of Mouse Embryonic Fibroblasts

2012-03-01 21:49:10

PLoS One; 2012 Mar; 7(3):e32957


Shiu-Ming Kuo, Lana R. Burl, Zihua Hu



Introduction



Vitamin C was first characterized as a coenzyme and a water-soluble antioxidant. Additional biological activities of vitamin C have been described subsequently. In primary cell culture, its ability to promote growth was reported to be due to an increased cell division and cell differentiation. A related property of vitamin C to delay senescence and enhance primary fibroblast reprogramming to pluripotency was further demonstrated. These in vitro observations made at physiologically relevant concentrations of vitamin C (10-5 M) are consistent with the results of in vivo studies. Patients with premature aging disorder, Werner Syndrome, display accelerated senescence in their fibroblasts and develop rare forms of cancer. In a mouse model of Werner Syndrome, vitamin C was found to promote normal growth and delay senescence. In this paper, gene expression profiling and immunofluorescence imaging were conducted to facilitate the understanding of molecular mechanisms mediating vitamin C-dependent growth promotion.



Little is known about the effect of vitamin C, at physiologically relevant conditions, on the growth of immortalized cells. Treating cancer cells with pharmacological levels of vitamin C (10-4 M and above) is known to lead to cell cycle arrest and apoptosis, as well as gene expression changes. While these observations were used to support the chemotherapeutic potential of vitamin C, a recent meta-analysis failed to conclude on the efficacy of vitamin C. Mutant mice that could not synthesize vitamin C endogenously, a trait similar to humans, had more aggressive growth of implanted tumor after vitamin C supplementation. In vitro studies using pharmacological doses of vitamin C were further complicated by the known free radical-generating interaction between vitamin C and culture medium components. In this paper, vitamin C dose-dependent effects were analyzed in immortalized cells using redox-active and -inactive vitamin C. Gene expression profiles of primary and immortalized cells were also compared after the same vitamin C treatment.



Mouse embryonic fibroblasts (MEF) were used as the cell model in this study. The vitamin C-synthesizing ability of mouse is limited to hepatocytes so MEF mimic human cells in their exogenous vitamin C requirement. In addition, MEF can undergo spontaneous immortalization, thus allowing the comparison of a single cell type at two phenotypes: primary MEF with limited mitotic life span; and immortalized MEF. Immortalization is considered a link between normal and tumorigenic cells. The relevancy of immortalized MEF to certain human cancer was previously discussed. Fibroblasts also contribute to the cancer stem cell niche and thus the response of fibroblasts to vitamin C could have additional implications. Besides the wildtype MEF, MEF from embryos that do not express functional sodium-dependent vitamin C transporter 2 (SVCT2-/-) were also used in the study to distinguish the effects of intracellular and extracellular vitamin C.



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