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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. Induction of p21 and p27 expression by TNF-α does not involve PI3K/Akt pathway. (A) LN-18 cells grown on coverslips, were sensitized with LY294002 (50 μM) for 2 hr and treated with TNF-α (10 ng/ml) for 3 hr and the expression of p21 or (B) p27 was studied by confocal laser scanning microscopy. Cells were stained with p21/p27 antibodies followed by secondary antibody labeled with Cy3. DAPI was used for nuclear staining (blue). Merged images depict nuclear localization for p21 and predominant cytoplasmic localization of p27. (Magnification 63×).
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Figure 8: A-B. Induction of p21 and p27 expression by TNF-α does not involve PI3K/Akt pathway. (A) LN-18 cells grown on coverslips, were sensitized with LY294002 (50 μM) for 2 hr and treated with TNF-α (10 ng/ml) for 3 hr and the expression of p21 or (B) p27 was studied by confocal laser scanning microscopy. Cells were stained with p21/p27 antibodies followed by secondary antibody labeled with Cy3. DAPI was used for nuclear staining (blue). Merged images depict nuclear localization for p21 and predominant cytoplasmic localization of p27. (Magnification 63×).

Mentions: The CDKIs, p21 and p27 are reported to be also regulated by PI3K/Akt pathway [35-38], we therefore examined the role of PI3K/Akt pathway by treating LN-18 cells sensitized with LY294002, a pharmacological inhibitor of PI3K/Akt pathway with TNF-α. The inhibitor had no effect on the fluorescence intensity or the percent positivity of p21 or p27 positive cells in TNF-α stimulated cells, suggesting that PI3K/Akt pathway was not involved in regulation of these CDKIs in LN-18 cells (Fig. 8A and 8B).


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. Induction of p21 and p27 expression by TNF-α does not involve PI3K/Akt pathway. (A) LN-18 cells grown on coverslips, were sensitized with LY294002 (50 μM) for 2 hr and treated with TNF-α (10 ng/ml) for 3 hr and the expression of p21 or (B) p27 was studied by confocal laser scanning microscopy. Cells were stained with p21/p27 antibodies followed by secondary antibody labeled with Cy3. DAPI was used for nuclear staining (blue). Merged images depict nuclear localization for p21 and predominant cytoplasmic localization of p27. (Magnification 63×).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: A-B. Induction of p21 and p27 expression by TNF-α does not involve PI3K/Akt pathway. (A) LN-18 cells grown on coverslips, were sensitized with LY294002 (50 μM) for 2 hr and treated with TNF-α (10 ng/ml) for 3 hr and the expression of p21 or (B) p27 was studied by confocal laser scanning microscopy. Cells were stained with p21/p27 antibodies followed by secondary antibody labeled with Cy3. DAPI was used for nuclear staining (blue). Merged images depict nuclear localization for p21 and predominant cytoplasmic localization of p27. (Magnification 63×).
Mentions: The CDKIs, p21 and p27 are reported to be also regulated by PI3K/Akt pathway [35-38], we therefore examined the role of PI3K/Akt pathway by treating LN-18 cells sensitized with LY294002, a pharmacological inhibitor of PI3K/Akt pathway with TNF-α. The inhibitor had no effect on the fluorescence intensity or the percent positivity of p21 or p27 positive cells in TNF-α stimulated cells, suggesting that PI3K/Akt pathway was not involved in regulation of these CDKIs in LN-18 cells (Fig. 8A and 8B).

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