Differentiation of Intracellular p16INK4a Expression in the Circulating Human Mononuclear Isolated Cells after ADMA and H2O2 Exposure

Abstract

p16INK4a (CDKN2A) represent as primary cell-cycle regulation, which arranges the moment of continued or interrupt cell proliferation. Cells expressing p16INK4a accumulate in aging tissue and age-related accumulation DNA damage, yet their physiologic compensated effects in human peripheral blood mononuclear cells (PBMNCs) after different stress inducer are poorly understood. Blood samples were obtained from healthy and non-sedentary lifestyle volunteers. Human mononuclear cells (MNCs) were purified from peripheral blood with Ficoll-density gradient centrifugation subsequently seeded into a medium culture. 80% confluence cells were divided into untreated cells and four treated cells with Asymmetric dimethylarginine (ADMA) and H2O2 in different doses for 24 h. Quantification p16INK4a positive cells were analyzed by FACS. The difference of p16INK4a positive cells after ADMA treated cells, H2O2 treated cells and untreated cells were also analyzed with a statistical test. We found that ADMA and H2O2 treatment in human MNCs induce elevation of p16INK4a (p=0.001), continuous p16INK4a expression incline to increase attendant extension dose of cellular stress inducer (p=0.000). Expression of p16INK4a has been proven higher in ADMA treated cells (1.43% ± 0.21) than control cells (0.34% ± 0.125) after 24h, the number of p16INK4a positive cells tended to increase when the ADMA exposure dose is added at 500µM (2.76% ± 1.39) compare with H2O2 treated cells (1.22% ± 0.33). These findings showed that p16INK4a positive cells are a part of the cellular stress response that results in temporary adaptation to some stressors, and may promote inhibition of inappropriate cell division.