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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

p53 expression is regulated in a time dependent manner. (A, B and C) HTet23p53, HTet26p53 and HTet43GFP cells were treated with 1000 ng/ml Dox for indicated time points and western blots were performed for p53. (D) Fold induction was calculated by densitometric analysis of p53 in HTet23p53, HTet26p53 and HTet43GFP cells taking 0 h = 1 with normalization to β -Tubulin or GAPDH.
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Figure 2: p53 expression is regulated in a time dependent manner. (A, B and C) HTet23p53, HTet26p53 and HTet43GFP cells were treated with 1000 ng/ml Dox for indicated time points and western blots were performed for p53. (D) Fold induction was calculated by densitometric analysis of p53 in HTet23p53, HTet26p53 and HTet43GFP cells taking 0 h = 1 with normalization to β -Tubulin or GAPDH.

Mentions: To study the kinetics of p53 expression single dose (1000 ng/ml) of Dox was added for different time durations (1 h to 48 h) and western blot was performed. As shown in Figure 2, p53 expression was initiated within 1 h of Dox addition and it increased progressively up to 48 h of incubation in HTet23p53 (Figure 2A) and HTet26p53 cells (Figure 2B). No changes in basal p53 levels were detected in HTet43GFP (Figure 2C) cells under identical experimental conditions. At 48 h, 5-fold increase in p53 protein was detected in HTet23p53 and HTet26p53 cells as compared to HTet43GFP cells (Figure 2D).


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

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

p53 expression is regulated in a time dependent manner. (A, B and C) HTet23p53, HTet26p53 and HTet43GFP cells were treated with 1000 ng/ml Dox for indicated time points and western blots were performed for p53. (D) Fold induction was calculated by densitometric analysis of p53 in HTet23p53, HTet26p53 and HTet43GFP cells taking 0 h = 1 with normalization to β -Tubulin or GAPDH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: p53 expression is regulated in a time dependent manner. (A, B and C) HTet23p53, HTet26p53 and HTet43GFP cells were treated with 1000 ng/ml Dox for indicated time points and western blots were performed for p53. (D) Fold induction was calculated by densitometric analysis of p53 in HTet23p53, HTet26p53 and HTet43GFP cells taking 0 h = 1 with normalization to β -Tubulin or GAPDH.
Mentions: To study the kinetics of p53 expression single dose (1000 ng/ml) of Dox was added for different time durations (1 h to 48 h) and western blot was performed. As shown in Figure 2, p53 expression was initiated within 1 h of Dox addition and it increased progressively up to 48 h of incubation in HTet23p53 (Figure 2A) and HTet26p53 cells (Figure 2B). No changes in basal p53 levels were detected in HTet43GFP (Figure 2C) cells under identical experimental conditions. At 48 h, 5-fold increase in p53 protein was detected in HTet23p53 and HTet26p53 cells as compared to HTet43GFP cells (Figure 2D).

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