Modulation of regulatory proteins of G1/S by variations in the levels of H2O2 in tumor cells.

IBAÑEZ IL; POLICASTRO LL; TROPPER I; MOLINARI B; DURAN HA

Montevideo, Uruguay

Congreso; V Meeting of Society for Free Radical Biology and Medicine-South American Group, V International Conference on Peroxynitrite and Reactive Nitrogen Species.; 2007

High levels of reactive oxygen species (ROS) induce cytotoxic effects, whereas at physiological concentrations, ROS are involved in the regulation of cellular functions, such as proliferation and apoptosis. In a previous report we demonstrated an endogenous imbalance of antioxidant enzymes coupled to a rise in H2O2 levels in cancer cells. Moreover, scavenging H2O2 with exogenous catalase (CAT) or by transfection with cDNA of CAT inhibited cell proliferation.The aim of the present study was to evaluate the mechanisms by which the modulation of the levels of ROS regulates cell proliferation in tumor cells, by analyzing cell cycle and regulatory proteins of G1/S in different types of tumor cell lines.The PIG1 (melanocytes), A375 (melanoma), PAJU (neuroblastoma), and LoVo (colon carcinoma) human cell lines and the CH72-T4 (spindle-cell carcinoma) murine cell line were used. Cell cycle analysis was performed by flow cytometry. Cyclins D1 and E, CDK4, CDK2 and p27 were determined by western blot. The levels of reactive oxygen species (ROS) were measured by flow cytometry using the DCFH assay and proliferation was evaluated by the MTT assay.The comparison between normal melanocytes (PIG1) and melanoma (A375) showed higher levels of endogenous ROS (p<0.05) and increased constitutive expression of cyclin D1 in malignant cells.Scavenging of H2O2 resulted in a significant (p<0.05) cell cycle arrest at G1 phase associated  with the inhibition of cell proliferation in all the cell lines studied. Regarding the regulatory proteins of G1/S, the diminution of H2O2 levels induced an increase in p27 and a decrease in cyclin D1.In conclusion, the regulation of cell proliferation by H2O2 is related with the modulation of specific regulatory proteins of early to mid G1 (cyclin D1) and transition G1/S (p27). We suggest that this regulation is mediated by the control of MAPKs by H2O2.