Normal human keratinocytes are stimulated to proliferate in serum-free medium containing subphysiological concentrations of calcium (0.09 mM, low calcium). In this study, we examined the effect of increased levels of extracellular calcium (2.0 mM, normal calcium) on UVB-induced apoptosis. Apoptosis was assessed by changes in cellular morphology, annexind V-FITC flow cytometry, and the formation of internucleosomal DNA ladders. High doses of UVB induced keratinocytes grown in low calcium medium to undergo apoptosis. In contrast, keratinocytes grown for 72 h in normal calcium medium were completely resistant to UVB-induced apoptosis. No apoptosis was observed even at UVB doses as high as 1200 J/m2. However, despite the lack of UVB-induced cell death, keratinocytes grown in normal calcium medium lost the ability to proliferate following high levels of UVB irradiation. High doses of UVB also increased the expression of the differentiation-specific proteins involucrin and cytokeratin 10 in a dose-dependent manner. In addition, growth in normal calcium medium lowered the UVB-induced stimulation of the p53 protein and altered the normal subcellular localization pattern of p53. UVB irradiation of human keratinocytes grown in normal calcium medium may be inducing further cell differentiation in the absence of overt cell death.
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