Differential expression and role of p21(cip/waf1 )and p27(kip1 )in TNF-α-induced inhibition of proliferation in human glioma cells

BACKGROUND: The role of TNF-α in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-α, others provide evidence that endogenous TNF-α promotes growth and development of tumors. Understanding the mechanism(s) of TNF-α mediated growth arrest will be important in unraveling the contribution of tissue associated macrophages in tumor resistance. The aim of this study was to investigate the role of Cyclin Dependent Kinase Inhibitors (CDKI) – p21(cip/waf1 )and p27(kip1 )in TNF-α mediated responses in context with p53 and activation of NF-κB and Akt pathways. The study was done with human glioma cell lines -LN-18 and LN-229 cells, using monolayer cultures and Multicellular Spheroids (MCS) as in vitro models. RESULTS: TNF-α induced inhibition of proliferation and enhanced the expression of p21(cip/waf1 )and p27(kip1 )in LN-18 cells. p21 was induced on exposure to TNF-α, localized exclusively in the nucleus and functioned as an inhibitor of cell cycle but not as an antiapoptotic protein. In contrast, p27 was constitutively expressed, localized predominantly in the cytoplasm and was not involved in arrest of proliferation. Our data using IκBα mutant LN-18 cells and PI3K/Akt inhibitor-LY294002 revealed that the expression of p21 is regulated by NF-κB. Loss of IκBα function in LN-229 cells (p53 positive) did not influence TNF-α induced accumulation of pp53 (Ser-20 p53) suggesting that p53 was not down stream of NF-κB. Spheroidogenesis enhanced p27 expression and p21 induced by TNF-α was significantly increased in the MCS compared to monolayers. CONCLUSION: This study demarcates the functional roles for CDKIs-p21(cip/waf1 )and p27(kip1 )during TNF-α stimulated responses in LN-18 glioma cells. Our findings provide evidence that TNF-α-induced p21 might be regulated by NF-κB or p53 independently. p21 functions as an inhibitor of cell proliferation and does not have a direct role in rendering the cells resistant to TNF-α mediated cytotoxicity.