Hydrogen Peroxide Modulation Leads To Post-translational Modifications Of The G1/S Cell Cycle Inhibitor p27 KIP1

BRACALENTE C; IBAÑEZ I; NOTCOVICH C; POLICASTRO L; MOLINARI B; DURAN H

Congreso; VII Meeting of South American Group of the SFRBM; 2011

CDK inhibitor p27KIP1 (p27) is a key protein in the decision between proliferation and cell cycle exit. This protein staysin the nucleus in quiescent cells, but it is exported to the cytoplasm in response to proliferating signals, where it can bedegraded or stabilized to be part of other processes, such as cell growth and migration (1). The actions of p27 arecontrolled by its concentration, subcellular localization and phosphorylation. H2O2 is involved in the activation of mitogenicpathways that might modulate p27 (2) and its phosphorylation (3-4).Thus we evaluated the effffects of H2O2 levels on the regulation of p27 in difffferent human cell lines (melanocytes andmelanoma, colorectal carcinoma and neuroblastoma cells).We observed a high percentage of p27 positive nuclei by immunocytoflfluorescence in cells overexpressing or treated withexogenous catalase as compared to controls, which showed a lower signal and cytoplasmic p27. Then we studied the levelsof phosphorylated p27 (p27p) at serine 10 (S10) and at threonine 198 (T198) because p27p at those sites allows its nuclearexportation, leading to accumulation and stabilization of p27pT198 in the cytoplasm (5-6). We demonstrated by westernblot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in tumor cells, associated with nuclearlocalization of p27. Normal cells also exhibited lower levels of p27p at those sites than their tumor counterpart, whichshowed p27 cytoplasmic localization. We also showed that the addition of H2O2 (0.1 μM) to G1-arrested cells by serumstarvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization ofp27.In conclusion, we showed herein another possible mechanism by which cancer cells taking advantage of their intrinsic prooxidantstate would favor cell proliferation, altering p27 subcellular localization through modulation of p27phosphorylations.