Hydrogen peroxide modulation leads to post-translational modifications of the G1/S cell cycle inhibitor p27KIP1

BRACALENTE, CANDELARIA; IBAÑEZ, IRENE L.; NOTCOVICH, CINTIA; POLICASTRO, LUCÍA; MOLINARI, BEATRIZ L.; DURÁN HEBE

San Pablo

Congreso; Free Radicals Brazil 2011-VII South American Meeting of the SFRBM; 2011

South American Meeting of the SFRBM

The CDK inhibitor p27KIP1 (p27) is a key protein in the decision between proliferation and cell cycle exit. This protein stays in the nucleus in quiescent cells, but it is exported to the cytoplasm in response to proliferating signals, where it can be degraded or stabilized to be part of other processes, such as cell growth and migration. The actions of p27 are controlled by its concentration, subcellular localization and phosphorylation. H2O2 are involved in the activation of mitogenic pathways that might modulate p27 phosphorylation. Thus we evaluated the effects of H2O2 levels on the regulation of p27 in different human cell lines (melanocytes and melanoma, colorectal carcinoma and neuroblastoma cells). We observed a high percentage of p27 positive nuclei by immunocytofluorescence in cells overexpressing or treated with exogenous catalase as compared to controls, which showed a lower signal and cytoplasmic p27. Then we studied the levels of p27 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because p27p at those sites allows its nuclear exportation, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in tumor cells, associated with nuclear p27. Normal cells also exhibited lower levels of p27p at those sites than their tumor counterpart, which showed p27 cytoplasmic localization. We also showed that the addition of H2O2 (0.1 µM) to G1-arrested cells by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27. In conclusion, we showed herein another possible mechanism by which cancer cells taking advantage of their intrinsic pro-oxidant state would favor cell proliferation, altering p27 subcellular localization through modulation of p27 phosphorylations. Bracalente C and Ibañez IL contributed equally to this work. Comisión Nacional de Energía Atómica, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina; Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, Argentina; Agencia Nacional de Promoción Científica y Tecnológica, Argentina. Premio: Young Investigator Award 2011