DIFFERENTIATION-INDUCED ENHANCEMENT OF THE ABILITY OF CULTURED HUMAN KERATINOCYTES TO SUPPRESS OXIDATIVE STRESS

Human keratinocytes in culture were harvested at different stages of d ifferentiation. Both the level of antioxidants and the response of cel ls to oxidative stress were measured as a function of growth and diffe rentiation. As the keratinocyte cultures became confluent and began to differentiate, the cellular levels of glutathione, glutathione peroxi dase, glutathione S transferase, and glucose-6-phosphate dehydrogenase increased. This higher level of antioxidants was maintained until the cells began to lose viability, Further, as the keratinocyte cultures began to differentiate, they became more resistant to the toxic effect of cumene hydroperoxide in terms of both of the rate of loss of cell mass and total glutathione and of the rate of decline in the activity of oxidation-sensitive enzymes, To determine how tightly the observed effects are linked to the calcium-dependent aspects of differentiation and to rule out effects related to time in culture, the cells were sw itched from 1.2 mM Ca++ to 0.03 mM Ca++ to suppress Ca++-dependent dif ferentiation. After 4 d, these cells were then treated with 0.5 mM cum ene hydroperoxide. The switch to 0.03 mM Ca++ blocked the normal incre ases in both glutathione peroxidase and glucose-6-phosphate dehydrogen ase activities. Further, cells in 0.03 mM Ca++ had reduced resistance to cumene hydroperozide relative to cells cultured for the same length of time in 1.2 mM Ca++. This indicates that there is a differentiatio n-associated, Ca++-specific increase in both the level of antioxidants and in tolerance to organic hydropepoxides.