Differential expression and role of p21cip/waf1 and p27kip1 in TNF-alpha-induced inhibition of proliferation in human glioma cells.

BACKGROUND

The role of TNF-alpha in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-alpha, others provide evidence that endogenous TNF-alpha promotes growth and development of tumors. Understanding the mechanism(s) of TNF-alpha 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)--21cip/waf1 and p27kip1 in TNF-alpha mediated responses in context with p53 and activation of NF-kappaB 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-alpha induced inhibition of proliferation and enhanced the expression of p21cip/waf1 and p27kip1 in LN-18 cells. p21 was induced on exposure to TNF-alpha, 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 IkappaBalpha mutant LN-18 cells and PI3K/Akt inhibitor-LY294002 revealed that the expression of p21 is regulated by NF-kappaB. Loss of IkappaBalpha function in LN-229 cells (p53 positive) did not influence TNF-alpha induced accumulation of pp53 (Ser-20 p53) suggesting that p53 was not down stream of NF-kappaB. Spheroidogenesis enhanced p27 expression and p21 induced by TNF-alpha was significantly increased in the MCS compared to monolayers.

CONCLUSION

This study demarcates the functional roles for CDKIs-p21cip/waf1 and p27kip1 during TNF-alpha stimulated responses in LN-18 glioma cells. Our findings provide evidence that TNF-alpha-induced p21 might be regulated by NF-kappaB 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-alpha mediated cytotoxicity.