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

Kumar PS, Shiras A, Das G, Jagtap JC, Prasad V, Shastry P - Mol. Cancer (2007)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Cell Science, NCCS, Ganeshkhind, Pune, India. sudheercool@hotmail.com

ABSTRACT

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.

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A-B. Effect of TNF-α on expression of pp53 and p65 in LN-229 cells (p53 positive). A) LN-229 cells were exposed to TNF-α for 6 hr and stained with antibodies to phosphorylated Ser-20 p53 and p21. B) LN-229 cells were transfected with IκBα dominant negative construct and the expression of nuclear p65 was determined after exposure to TNF-α for 6 hr.
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Figure 4: A-B. Effect of TNF-α on expression of pp53 and p65 in LN-229 cells (p53 positive). A) LN-229 cells were exposed to TNF-α for 6 hr and stained with antibodies to phosphorylated Ser-20 p53 and p21. B) LN-229 cells were transfected with IκBα dominant negative construct and the expression of nuclear p65 was determined after exposure to TNF-α for 6 hr.

Mentions: p21 is regulated mainly via tumor suppressor protein-p53. Recently, p52/p100 NF-κB was described to regulate p53 function and hence influence the p53-regulated decision-making following DNA damage, a process involving p21 [32]. As LN-18 cells express mutant p53 protein, to examine if NF-κB influenced the p21 expression directly or whether it was via p53, experiments were performed using LN-229, a human glioma cell line expressing wild-type p53 [33]. Cells were exposed to TNF-α for 6 hr and analyzed for the expression of phosphorylated ser20-p53 (pp53) and p21. Stimulation with TNF-α led to enhanced accumulation of phosphorylated p53 in the nucleus, interestingly, the untreated control cells also displayed detectable levels of pp53. In contrast, the expression of nuclear p21 decreased in TNF-α treated cells compared to controls (Fig. 4A). To delineate the pathways regulating p21, the effect of TNF-α on pp53 and p21 was studied in LN-229 cells transfected with IκBα dominant negative construct. The loss of IκBα activity in the transfected cells was evident by the inhibition of p65 translocation to nucleus on stimulation with TNF-α compared with untransfected LN-229 cells (Fig. 4B). To address the question whether loss of NF-κB activation in these cells might influence the levels of pp53 and p21, transfected cells exposed to TNF-α for 6 hr were stained with antibodies to Ser20-p53 and p21. As shown in figure 5, there was no significant difference in the accumulation of pp53 or nuclear p21 between the transfected and untransfected cells. These findings led us to conclude that p53 might not function downstream of NF-κB and secondly, TNF-α-induced p21 can be regulated by p53 and NF-κB independently.


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

Kumar PS, Shiras A, Das G, Jagtap JC, Prasad V, Shastry P - Mol. Cancer (2007)

A-B. Effect of TNF-α on expression of pp53 and p65 in LN-229 cells (p53 positive). A) LN-229 cells were exposed to TNF-α for 6 hr and stained with antibodies to phosphorylated Ser-20 p53 and p21. B) LN-229 cells were transfected with IκBα dominant negative construct and the expression of nuclear p65 was determined after exposure to TNF-α for 6 hr.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904457&req=5

Figure 4: A-B. Effect of TNF-α on expression of pp53 and p65 in LN-229 cells (p53 positive). A) LN-229 cells were exposed to TNF-α for 6 hr and stained with antibodies to phosphorylated Ser-20 p53 and p21. B) LN-229 cells were transfected with IκBα dominant negative construct and the expression of nuclear p65 was determined after exposure to TNF-α for 6 hr.
Mentions: p21 is regulated mainly via tumor suppressor protein-p53. Recently, p52/p100 NF-κB was described to regulate p53 function and hence influence the p53-regulated decision-making following DNA damage, a process involving p21 [32]. As LN-18 cells express mutant p53 protein, to examine if NF-κB influenced the p21 expression directly or whether it was via p53, experiments were performed using LN-229, a human glioma cell line expressing wild-type p53 [33]. Cells were exposed to TNF-α for 6 hr and analyzed for the expression of phosphorylated ser20-p53 (pp53) and p21. Stimulation with TNF-α led to enhanced accumulation of phosphorylated p53 in the nucleus, interestingly, the untreated control cells also displayed detectable levels of pp53. In contrast, the expression of nuclear p21 decreased in TNF-α treated cells compared to controls (Fig. 4A). To delineate the pathways regulating p21, the effect of TNF-α on pp53 and p21 was studied in LN-229 cells transfected with IκBα dominant negative construct. The loss of IκBα activity in the transfected cells was evident by the inhibition of p65 translocation to nucleus on stimulation with TNF-α compared with untransfected LN-229 cells (Fig. 4B). To address the question whether loss of NF-κB activation in these cells might influence the levels of pp53 and p21, transfected cells exposed to TNF-α for 6 hr were stained with antibodies to Ser20-p53 and p21. As shown in figure 5, there was no significant difference in the accumulation of pp53 or nuclear p21 between the transfected and untransfected cells. These findings led us to conclude that p53 might not function downstream of NF-κB and secondly, TNF-α-induced p21 can be regulated by p53 and NF-κB independently.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Cell Science, NCCS, Ganeshkhind, Pune, India. sudheercool@hotmail.com

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus