Kumar Pabbisetty Sudheer, Shiras Anjali, Das Gowry, Jagtap Jayashree C, Prasad Vandna, Shastry Padma
National Centre for Cell Science, NCCS, Ganeshkhind, Pune, India.
Mol Cancer. 2007 Jun 12;6:42. doi: 10.1186/1476-4598-6-42.
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.
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.
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.
肿瘤坏死因子-α（TNF-α）对肿瘤命运的影响存在争议，一些研究报道了TNF-α的凋亡或坏死作用，而另一些研究则表明内源性TNF-α促进肿瘤的生长和发展。了解TNF-α介导的生长停滞机制对于阐明组织相关巨噬细胞在肿瘤抵抗中的作用至关重要。本研究旨在探讨细胞周期蛋白依赖性激酶抑制剂（CDKI）——p21cip/waf1和p27kip1在TNF-α介导的反应中与p53以及核因子-κB（NF-κB）和蛋白激酶B（Akt）信号通路激活的关系。本研究以人胶质瘤细胞系-LN-18和LN-229细胞为研究对象，采用单层培养和多细胞球体（MCS）作为体外模型。
TNF-α诱导LN-18细胞增殖抑制，并增强p21cip/waf1和p27kip1的表达。p21在暴露于TNF-α时被诱导，仅定位于细胞核，作为细胞周期抑制剂发挥作用，但不作为抗凋亡蛋白。相反，p27组成性表达，主要定位于细胞质，不参与增殖停滞。我们使用IkappaBalpha突变型LN-18细胞和磷脂酰肌醇3激酶/蛋白激酶B抑制剂-LY294002的数据表明，p21的表达受NF-κB调节。LN-229细胞（p53阳性）中IkappaBalpha功能的丧失不影响TNF-α诱导的磷酸化p53（Ser-20 p53）的积累，表明p53不在NF-κB的下游。球体形成增强了p27的表达，与单层培养相比，TNF-α诱导的p21在MCS中显著增加。
本研究明确了CDKIs-p21cip/waf1和p27kip1在TNF-α刺激的LN-18胶质瘤细胞反应中的功能作用。我们的研究结果表明，TNF-α诱导的p21可能独立地受NF-κB或p53调节。p21作为细胞增殖的抑制剂，在使细胞对TNF-α介导的细胞毒性产生抗性方面没有直接作用。
