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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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Related in: MedlinePlus

Validation of mRNA levels.The mRNA levels of 11 genes coregulated by both p16 and AUF1 were analyzed by quantitative RT-PCR in HFSN1 cells expressing either p16-siRNA or control-siRNA, and are expressed as fold difference. The error bars represent standard deviation of three different values. *: The fold difference in the expression of these genes was less than 2 in the array data.
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pone-0021111-g007: Validation of mRNA levels.The mRNA levels of 11 genes coregulated by both p16 and AUF1 were analyzed by quantitative RT-PCR in HFSN1 cells expressing either p16-siRNA or control-siRNA, and are expressed as fold difference. The error bars represent standard deviation of three different values. *: The fold difference in the expression of these genes was less than 2 in the array data.

Mentions: The fact that p16 negatively controls the ubiquitous RNA-binding protein AUF1 and the transcription factor E2F1 suggests that p16 might control the expression of several genes. To test this hypothesis, we used oligonucleotide gene expression microarrays to identify genes whose expression is modulated in a p16-dependent manner. Therefore, total RNA was isolated from both p16-siRNA-expressing cells and their control counterparts and was hybridized to microarrays. The difference in gene expression was considered significant only when the variation was ≥2.00. Interestingly, the knocking-down of p16 modulated the expression of 2170 genes, with 1010 (46.5%) were up-regulated and 1160 (53.4%) were down-regulated. Most of these genes (1501) are involved in cell cycle regulation, and the others are implicated in various cancer-related mechanisms such as apoptosis, DNA repair, senescence, angiogenesis and aging. Importantly, the array data showed 4 fold increase in the expression of AUF1 in p16-deficient cells and 46 genes known to be AUF1 targets [38] are also under the control of p16 (Table 1). In addition, 33 genes known to be E2F1 targets [39] are also under the control of p16 (Table 2). To validate the microarray results, the expression level of 11 genes was assessed by quantitative RT-PCR. Figure 7 shows that the expression of all the analyzed genes is modulated in a p16-dependent manner, confirming the microarray data. This shows that the expression of a large number of genes is indeed under the control of the tumor suppressor p16 protein.


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)

Validation of mRNA levels.The mRNA levels of 11 genes coregulated by both p16 and AUF1 were analyzed by quantitative RT-PCR in HFSN1 cells expressing either p16-siRNA or control-siRNA, and are expressed as fold difference. The error bars represent standard deviation of three different values. *: The fold difference in the expression of these genes was less than 2 in the array data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140473&req=5

pone-0021111-g007: Validation of mRNA levels.The mRNA levels of 11 genes coregulated by both p16 and AUF1 were analyzed by quantitative RT-PCR in HFSN1 cells expressing either p16-siRNA or control-siRNA, and are expressed as fold difference. The error bars represent standard deviation of three different values. *: The fold difference in the expression of these genes was less than 2 in the array data.
Mentions: The fact that p16 negatively controls the ubiquitous RNA-binding protein AUF1 and the transcription factor E2F1 suggests that p16 might control the expression of several genes. To test this hypothesis, we used oligonucleotide gene expression microarrays to identify genes whose expression is modulated in a p16-dependent manner. Therefore, total RNA was isolated from both p16-siRNA-expressing cells and their control counterparts and was hybridized to microarrays. The difference in gene expression was considered significant only when the variation was ≥2.00. Interestingly, the knocking-down of p16 modulated the expression of 2170 genes, with 1010 (46.5%) were up-regulated and 1160 (53.4%) were down-regulated. Most of these genes (1501) are involved in cell cycle regulation, and the others are implicated in various cancer-related mechanisms such as apoptosis, DNA repair, senescence, angiogenesis and aging. Importantly, the array data showed 4 fold increase in the expression of AUF1 in p16-deficient cells and 46 genes known to be AUF1 targets [38] are also under the control of p16 (Table 1). In addition, 33 genes known to be E2F1 targets [39] are also under the control of p16 (Table 2). To validate the microarray results, the expression level of 11 genes was assessed by quantitative RT-PCR. Figure 7 shows that the expression of all the analyzed genes is modulated in a p16-dependent manner, confirming the microarray data. This shows that the expression of a large number of genes is indeed under the control of the tumor suppressor p16 protein.

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