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p16( INK4a) positively regulates cyclin D1 and E2F1 through negative control of AUF1.

Al-Khalaf HH, Colak D, Al-Saif M, Al-Bakheet A, Hendrayani SF, Al-Yousef N, Kaya N, Khabar KS, Aboussekhra A - PLoS ONE (2011)

Bottom Line: Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs.We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

ABSTRACT

Background: The cyclin-D/CDK4,6/p16(INK4a)/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined.

Methodology/principal findings: We have shown here that the tumor suppressor p16(INK4a) protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16(INK4a) stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16(INK4a), using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3'UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16(INK4a) -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.

Conclusion/significance: These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

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p16 modulates apoptosis through E2F1.(A) Western blots showing the expression of the indicated pro-and anti-apoptotic proteins in U2OS and EHI. (B) Western blots showing the expression of the indicated proteins in U2OS, U2OS cells stably transfected with plasmids encoding either scrambled sequence (ctl) or E2F1 (+). The numbers below the bands indicate the corresponding expression levels. (C) U2OS, EH1 and E2F1-expressing U2OS cells were either mock-treated or challenged with doxorubicin (2 µM) and then re-incubated for 72 hrs. Cells were then divided into two groups; one was used to analyze cell death by annexinV/PI flow cytometry. The numbers in the charts indicate the proportions of early and late apoptosis. (D) Histogram showing the proportions of apoptosis (early+late) induced by different doses of doxorubicin. The error bars represent standard deviation of three different experiments. (E) The second group of cells was used to assess the level of the indicated proteins by immunoblotting. (F) Graph showing the Bax/Bcl-2 ratio in the indicated cells after treatment with doxorubicin. Error bars represent standard deviation of at least three different experiments.
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pone-0021111-g008: p16 modulates apoptosis through E2F1.(A) Western blots showing the expression of the indicated pro-and anti-apoptotic proteins in U2OS and EHI. (B) Western blots showing the expression of the indicated proteins in U2OS, U2OS cells stably transfected with plasmids encoding either scrambled sequence (ctl) or E2F1 (+). The numbers below the bands indicate the corresponding expression levels. (C) U2OS, EH1 and E2F1-expressing U2OS cells were either mock-treated or challenged with doxorubicin (2 µM) and then re-incubated for 72 hrs. Cells were then divided into two groups; one was used to analyze cell death by annexinV/PI flow cytometry. The numbers in the charts indicate the proportions of early and late apoptosis. (D) Histogram showing the proportions of apoptosis (early+late) induced by different doses of doxorubicin. The error bars represent standard deviation of three different experiments. (E) The second group of cells was used to assess the level of the indicated proteins by immunoblotting. (F) Graph showing the Bax/Bcl-2 ratio in the indicated cells after treatment with doxorubicin. Error bars represent standard deviation of at least three different experiments.

Mentions: Next, we studied the effect of p16-dependent modulation of E2F1 level on the expression of apoptotic-regulatory proteins, known to be under the control of E2F1 [40]. Figure 8A shows that the levels of the pro-apoptotic proteins Bax and cleaved Caspase-3 are respectively 2.4 and 6.5 fold higher in EH1 as compared to U2OS. On the other hand, the expression levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and NF-κB were respectively 1.6, 2.6 and 18 fold lower in EH1 as compared to U2OS. This indicates that the expression level of various apoptosis proteins is modulated in a p16-dependent manner. To show that these variations are E2F1-dependent, E2F1 was ectopically introduced into the p16-defective U2OS cells and whole cell extracts were prepared from U2OS and U2OS expressing either the control plasmid or E2F1-expressing plasmid. Figure 8B shows that the expression level of E2F1 increased 3.4 fold in E2F1-expressing U2OS cells as compared to the control. Consequently, there was a clear correlation between the E2F1 expression level and the expression of the pro- and anti-apoptotic proteins. In E2F1-expressing U2OS cells, the expression levels of Bax and cleaved Caspase-3 were 2.2 and 2.9 fold higher than in the control cells, while the expression levels of Bcl-2, Bcl-xL and NF-κB were 2.5, 2.8 and 2.6 fold lower than in the control, respectively (Figure 8B). This suggests that p16-dependent modulation of the expression of the apoptotic genes could be mediated through E2F1.


p16( INK4a) positively regulates cyclin D1 and E2F1 through negative control of AUF1.

Al-Khalaf HH, Colak D, Al-Saif M, Al-Bakheet A, Hendrayani SF, Al-Yousef N, Kaya N, Khabar KS, Aboussekhra A - PLoS ONE (2011)

p16 modulates apoptosis through E2F1.(A) Western blots showing the expression of the indicated pro-and anti-apoptotic proteins in U2OS and EHI. (B) Western blots showing the expression of the indicated proteins in U2OS, U2OS cells stably transfected with plasmids encoding either scrambled sequence (ctl) or E2F1 (+). The numbers below the bands indicate the corresponding expression levels. (C) U2OS, EH1 and E2F1-expressing U2OS cells were either mock-treated or challenged with doxorubicin (2 µM) and then re-incubated for 72 hrs. Cells were then divided into two groups; one was used to analyze cell death by annexinV/PI flow cytometry. The numbers in the charts indicate the proportions of early and late apoptosis. (D) Histogram showing the proportions of apoptosis (early+late) induced by different doses of doxorubicin. The error bars represent standard deviation of three different experiments. (E) The second group of cells was used to assess the level of the indicated proteins by immunoblotting. (F) Graph showing the Bax/Bcl-2 ratio in the indicated cells after treatment with doxorubicin. Error bars represent standard deviation of at least three different experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140473&req=5

pone-0021111-g008: p16 modulates apoptosis through E2F1.(A) Western blots showing the expression of the indicated pro-and anti-apoptotic proteins in U2OS and EHI. (B) Western blots showing the expression of the indicated proteins in U2OS, U2OS cells stably transfected with plasmids encoding either scrambled sequence (ctl) or E2F1 (+). The numbers below the bands indicate the corresponding expression levels. (C) U2OS, EH1 and E2F1-expressing U2OS cells were either mock-treated or challenged with doxorubicin (2 µM) and then re-incubated for 72 hrs. Cells were then divided into two groups; one was used to analyze cell death by annexinV/PI flow cytometry. The numbers in the charts indicate the proportions of early and late apoptosis. (D) Histogram showing the proportions of apoptosis (early+late) induced by different doses of doxorubicin. The error bars represent standard deviation of three different experiments. (E) The second group of cells was used to assess the level of the indicated proteins by immunoblotting. (F) Graph showing the Bax/Bcl-2 ratio in the indicated cells after treatment with doxorubicin. Error bars represent standard deviation of at least three different experiments.
Mentions: Next, we studied the effect of p16-dependent modulation of E2F1 level on the expression of apoptotic-regulatory proteins, known to be under the control of E2F1 [40]. Figure 8A shows that the levels of the pro-apoptotic proteins Bax and cleaved Caspase-3 are respectively 2.4 and 6.5 fold higher in EH1 as compared to U2OS. On the other hand, the expression levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and NF-κB were respectively 1.6, 2.6 and 18 fold lower in EH1 as compared to U2OS. This indicates that the expression level of various apoptosis proteins is modulated in a p16-dependent manner. To show that these variations are E2F1-dependent, E2F1 was ectopically introduced into the p16-defective U2OS cells and whole cell extracts were prepared from U2OS and U2OS expressing either the control plasmid or E2F1-expressing plasmid. Figure 8B shows that the expression level of E2F1 increased 3.4 fold in E2F1-expressing U2OS cells as compared to the control. Consequently, there was a clear correlation between the E2F1 expression level and the expression of the pro- and anti-apoptotic proteins. In E2F1-expressing U2OS cells, the expression levels of Bax and cleaved Caspase-3 were 2.2 and 2.9 fold higher than in the control cells, while the expression levels of Bcl-2, Bcl-xL and NF-κB were 2.5, 2.8 and 2.6 fold lower than in the control, respectively (Figure 8B). This suggests that p16-dependent modulation of the expression of the apoptotic genes could be mediated through E2F1.

Bottom Line: Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs.We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

ABSTRACT

Background: The cyclin-D/CDK4,6/p16(INK4a)/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined.

Methodology/principal findings: We have shown here that the tumor suppressor p16(INK4a) protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16(INK4a) stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16(INK4a), using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3'UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16(INK4a) -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.

Conclusion/significance: These findings show that the cyclin-dependent kinase inhibitor p16( INK4a) is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

Show MeSH
Related in: MedlinePlus