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Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells.

Ajay AK, Meena AS, Bhat MK - Cell Biosci (2012)

Bottom Line: However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells.Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53.E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Cell Science, NCCS Complex, Pune University Campus, Ganeshkhind, Pune - 411007, India. manojkbhat@nccs.res.in.

ABSTRACT

Background: In HPV infected cells p53 function is abrogated by E6 and even ectopically expressed p53 is unable to perform tumor suppressor functions. In addition to facilitating its degradation, E6 may also inhibit p53 transactivity, though the mechanisms are still poorly understood. It has been reported that inhibition of p300, an acetyltransferase responsible for p53 acetylation is inactivated by E6. Activation of overexpressed p53 to cause cell growth inhibition is facilitated by its phosphorylation. Previously, we reported that non-genotoxically overexpressed p53 in HeLa cells needs to be phosphorylated to perform its cell growth inhibitory functions. Since over expressed p53 by itself was not activated, we hypothesized an inhibitory role for E6.

Results: Majority of reports proposes E6 mediated degradation of p53 as a possible reason for its inactivation. However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells. Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53. However, inhibition of proteasomal degradation prevents the degradation of endogenous p53. These findings suggest that overexpressed p53 and endogenous p53 are differentially subjected to proteasomal degradation and the reasons for this discrepancy remain unclear. Our studies demonstrate that p53 over expression has no effect on anchorage independent cell-growth and E6 ifies its cell growth inhibitory effect. E6 overexpression abrogates OA induced p53 occupancy on the p21 promoter and cell death as well. E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

Conclusion: We report for the first time that E6 de-activates p53 by inhibiting its phosphorylation. This prevents p53 binding to p21 promoter and thereby restraining its cell-growth inhibitory functions. Our study provides new evidence indicating that viral protein E6 inhibits p53 transactivity by mechanism independent of degradation pathway.

No MeSH data available.


Related in: MedlinePlus

Overexpressed p53 is stable. (A and B) p53 was overexpressed with 1000 ng/ml of Dox for 48 h or not overexpressed and western blotswere performed after indicated time of Chx treatment in HTet23p53 and HTet26p53 cells. (C) Western blot for p53 was performed with or without addition of 1000 ng/ml Dox for 48 h followed by Chx treatment for indicated time points in HTet43GFP cells.(D) Graphical representation of percentage of p53 protein remaining after indicated time points in HTet23p53 and HTet26p53 cells by densitometric analysis following normalization with β-Actin. Protein percentage for 0 h Chx was taken as 100.
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Figure 4: Overexpressed p53 is stable. (A and B) p53 was overexpressed with 1000 ng/ml of Dox for 48 h or not overexpressed and western blotswere performed after indicated time of Chx treatment in HTet23p53 and HTet26p53 cells. (C) Western blot for p53 was performed with or without addition of 1000 ng/ml Dox for 48 h followed by Chx treatment for indicated time points in HTet43GFP cells.(D) Graphical representation of percentage of p53 protein remaining after indicated time points in HTet23p53 and HTet26p53 cells by densitometric analysis following normalization with β-Actin. Protein percentage for 0 h Chx was taken as 100.

Mentions: To perform tumor suppressor functions p53 stability is essential. Therefore to ascertain that overexpressed p53 is stable and it is not degraded by E6, cycloheximide (Chx) chase experiment was performed. Cells were treated with Chx for different time points to inhibit protein synthesis and then western blotted to detect p53. In the presence of Chx over expressed p53 was present in significant amount in HTet23p53 and HTet26p53 cells (Figure 4A and 4B) even after 6 h. Under these experimental conditions endogenous p53 in same cells (Figure 4A and 4B) or in HTet43GFP cells (Figure 4C) decreased to undetectable levels just within 1 h. The half-life of overexpressed p53 was calculated to be 6 h as compared to that of less than 1 h for endogenous p53 (Figure 4D).


Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells.

Ajay AK, Meena AS, Bhat MK - Cell Biosci (2012)

Overexpressed p53 is stable. (A and B) p53 was overexpressed with 1000 ng/ml of Dox for 48 h or not overexpressed and western blotswere performed after indicated time of Chx treatment in HTet23p53 and HTet26p53 cells. (C) Western blot for p53 was performed with or without addition of 1000 ng/ml Dox for 48 h followed by Chx treatment for indicated time points in HTet43GFP cells.(D) Graphical representation of percentage of p53 protein remaining after indicated time points in HTet23p53 and HTet26p53 cells by densitometric analysis following normalization with β-Actin. Protein percentage for 0 h Chx was taken as 100.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285035&req=5

Figure 4: Overexpressed p53 is stable. (A and B) p53 was overexpressed with 1000 ng/ml of Dox for 48 h or not overexpressed and western blotswere performed after indicated time of Chx treatment in HTet23p53 and HTet26p53 cells. (C) Western blot for p53 was performed with or without addition of 1000 ng/ml Dox for 48 h followed by Chx treatment for indicated time points in HTet43GFP cells.(D) Graphical representation of percentage of p53 protein remaining after indicated time points in HTet23p53 and HTet26p53 cells by densitometric analysis following normalization with β-Actin. Protein percentage for 0 h Chx was taken as 100.
Mentions: To perform tumor suppressor functions p53 stability is essential. Therefore to ascertain that overexpressed p53 is stable and it is not degraded by E6, cycloheximide (Chx) chase experiment was performed. Cells were treated with Chx for different time points to inhibit protein synthesis and then western blotted to detect p53. In the presence of Chx over expressed p53 was present in significant amount in HTet23p53 and HTet26p53 cells (Figure 4A and 4B) even after 6 h. Under these experimental conditions endogenous p53 in same cells (Figure 4A and 4B) or in HTet43GFP cells (Figure 4C) decreased to undetectable levels just within 1 h. The half-life of overexpressed p53 was calculated to be 6 h as compared to that of less than 1 h for endogenous p53 (Figure 4D).

Bottom Line: However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells.Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53.E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Centre for Cell Science, NCCS Complex, Pune University Campus, Ganeshkhind, Pune - 411007, India. manojkbhat@nccs.res.in.

ABSTRACT

Background: In HPV infected cells p53 function is abrogated by E6 and even ectopically expressed p53 is unable to perform tumor suppressor functions. In addition to facilitating its degradation, E6 may also inhibit p53 transactivity, though the mechanisms are still poorly understood. It has been reported that inhibition of p300, an acetyltransferase responsible for p53 acetylation is inactivated by E6. Activation of overexpressed p53 to cause cell growth inhibition is facilitated by its phosphorylation. Previously, we reported that non-genotoxically overexpressed p53 in HeLa cells needs to be phosphorylated to perform its cell growth inhibitory functions. Since over expressed p53 by itself was not activated, we hypothesized an inhibitory role for E6.

Results: Majority of reports proposes E6 mediated degradation of p53 as a possible reason for its inactivation. However, results presented here for the first time demonstrate that overexpressed p53 is not directly associated with E6 and therefore free, yet it is not functionally active in HPV positive cells. Also, the stability of overexpressed p53 does not seem to be an issue because inhibition of proteasomal degradation did not increase the half-life of overexpressed p53, which is more than endogenous p53. However, inhibition of proteasomal degradation prevents the degradation of endogenous p53. These findings suggest that overexpressed p53 and endogenous p53 are differentially subjected to proteasomal degradation and the reasons for this discrepancy remain unclear. Our studies demonstrate that p53 over expression has no effect on anchorage independent cell-growth and E6 ifies its cell growth inhibitory effect. E6 overexpression abrogates OA induced p53 occupancy on the p21 promoter and cell death as well. E6 did not decrease p53 protein but phospho-p53 level was significantly reduced.

Conclusion: We report for the first time that E6 de-activates p53 by inhibiting its phosphorylation. This prevents p53 binding to p21 promoter and thereby restraining its cell-growth inhibitory functions. Our study provides new evidence indicating that viral protein E6 inhibits p53 transactivity by mechanism independent of degradation pathway.

No MeSH data available.


Related in: MedlinePlus