Limits...
Oncogenic viral protein HPV E7 up-regulates the SIRT1 longevity protein in human cervical cancer cells.

Allison SJ, Jiang M, Milner J - Aging (Albany NY) (2009)

Bottom Line: Conversely, SIRT1 levels decrease following RNAi-mediated silencing of HPV E7 in SiHa cells.Silencing HPV E6 has no effect on SIRT1 but, as expected, causes marked accumulation of p53 protein accompanied by p53-mediated up-regulation of p21.Our discovery that HPV E7 up-regulates SIRT1 links a clinically important oncogenic virus with the multi-functional SIRT1 protein.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire Cancer Research P53 Research Unit, Department of Biology, University of York, York YO105DD, UK. ajm24@york.ac.uk

ABSTRACT
Senescence is blocked in human cervical keratinocytes infected with high risk human papillomavirus (e.g. HPV type16). Viral oncoproteins HPV E6 and HPV E7 access the cell cycle via cellular p53 and retinoblastoma proteins respectively. Previously we have shown that HPV E7, not HPV E6, is also responsible for cervical cancer cell survival (SiHa cells; HPV type16). We now present evidence that SIRT1, an aging-related NAD-dependent deacetylase, mediates HPV E7 survival function in SiHa cervical cancer cells. Moreover, HPV E7 up-regulates SIRT1 protein when expressed in primary human keratinocytes. Conversely, SIRT1 levels decrease following RNAi-mediated silencing of HPV E7 in SiHa cells. Silencing HPV E6 has no effect on SIRT1 but, as expected, causes marked accumulation of p53 protein accompanied by p53-mediated up-regulation of p21. However, p53 acetylation (K382Ac) was barely detectable. Since p53 is a known SIRT1 substrate we propose that elevated SIRT1 levels (induced by HPV E7) attenuate p53 pro-apoptotic capacity via its de-acetylation. Our discovery that HPV E7 up-regulates SIRT1 links a clinically important oncogenic virus with the multi-functional SIRT1 protein. This link may open the way for a more in-depth understanding of the process of HPV-induced malignant transformation and also of the inter-relationships between aging and cancer.

Show MeSH

Related in: MedlinePlus

HPV E7 enables SiHa cervical cancer cell survival via up-regulation of SIRT1 protein levels. (A)                                            Equal amounts of protein analysed by immunoblotting as indicated, upper                                            panels SiHa cells (50 μg protein). Bottom panel HCT116 cell positive control for p53 K382Ac                                            detection (40 μg protein, 2 minute exposure). Note that p53 K382Ac is undetectable                                            in SiHa cells (5 min exposure) and requires 2 h exposure for detection (†).                                            (B) Relative levels of SIRT1, SIRT1 S47P and SIRT1 S27P 48h                                            post-transfection with indicated siRNAs, mean of two experiments. (C)                                            Phase contrast images of SiHa cells post-transfection with the indicated                                            siRNAs. (D) Apoptotic SiHa cells 48h post-transfection with the                                            indicated siRNAs. (E) Primary human keratinocytes 48h                                            post-transfection with expression vectors for HPV E6 and HPV16 E7 and                                            equivalent samples immunoblotted for HPV E6, HPV E7, SIRT1, SIRT1 S27P and                                            SIRT1 S47P.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2806013&req=5

Figure 4: HPV E7 enables SiHa cervical cancer cell survival via up-regulation of SIRT1 protein levels. (A) Equal amounts of protein analysed by immunoblotting as indicated, upper panels SiHa cells (50 μg protein). Bottom panel HCT116 cell positive control for p53 K382Ac detection (40 μg protein, 2 minute exposure). Note that p53 K382Ac is undetectable in SiHa cells (5 min exposure) and requires 2 h exposure for detection (†). (B) Relative levels of SIRT1, SIRT1 S47P and SIRT1 S27P 48h post-transfection with indicated siRNAs, mean of two experiments. (C) Phase contrast images of SiHa cells post-transfection with the indicated siRNAs. (D) Apoptotic SiHa cells 48h post-transfection with the indicated siRNAs. (E) Primary human keratinocytes 48h post-transfection with expression vectors for HPV E6 and HPV16 E7 and equivalent samples immunoblotted for HPV E6, HPV E7, SIRT1, SIRT1 S27P and SIRT1 S47P.

Mentions: Previously we have shown that SIRT1 acts as a cancer-specific survival factor in a range of epithelial cancer cell lines [20]. This raised the possibility that SIRT1 might mediate the anti-apoptotic effects of HPV16 E7 [30]. Moreover SIRT1 protein levels are abnormally elevated in a range of human epithelial cell lines, including SiHa cervical cancer cells [25]. We now demonstrate that RNAi-mediated silencing of HPV E7 in SiHa cells down-regulates SIRT1 protein levels by ~50% 48h post-transfection with E7 siRNA (Figure 4A and 4B). In these same cells the levels of the pro-apoptotic E3 ligase Itch and anti-apoptotic c-FLIP (c-FLIPS and c-FLIPL) were unaffected by HPV E7 knock-down (data not shown) demonstrating that the reduction in SIRT1 protein levels reflects a selective effect of HPV E7 depletion. In contrast to HPV E7 depletion, the depletion of HPV E6 had no effect on levels of SIRT1 protein (Figure 4A and 4B). We conclude that HPV E7, but not HPV E6, is required to maintain the abnormally high levels of SIRT1 protein characteristically observed in SiHa cervical cancer cells. Our results thus identify the longevity protein SIRT1 as a novel cellular target of the viral oncogene HPV16 E7 in human cervical cancer cells.


Oncogenic viral protein HPV E7 up-regulates the SIRT1 longevity protein in human cervical cancer cells.

Allison SJ, Jiang M, Milner J - Aging (Albany NY) (2009)

HPV E7 enables SiHa cervical cancer cell survival via up-regulation of SIRT1 protein levels. (A)                                            Equal amounts of protein analysed by immunoblotting as indicated, upper                                            panels SiHa cells (50 μg protein). Bottom panel HCT116 cell positive control for p53 K382Ac                                            detection (40 μg protein, 2 minute exposure). Note that p53 K382Ac is undetectable                                            in SiHa cells (5 min exposure) and requires 2 h exposure for detection (†).                                            (B) Relative levels of SIRT1, SIRT1 S47P and SIRT1 S27P 48h                                            post-transfection with indicated siRNAs, mean of two experiments. (C)                                            Phase contrast images of SiHa cells post-transfection with the indicated                                            siRNAs. (D) Apoptotic SiHa cells 48h post-transfection with the                                            indicated siRNAs. (E) Primary human keratinocytes 48h                                            post-transfection with expression vectors for HPV E6 and HPV16 E7 and                                            equivalent samples immunoblotted for HPV E6, HPV E7, SIRT1, SIRT1 S27P and                                            SIRT1 S47P.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806013&req=5

Figure 4: HPV E7 enables SiHa cervical cancer cell survival via up-regulation of SIRT1 protein levels. (A) Equal amounts of protein analysed by immunoblotting as indicated, upper panels SiHa cells (50 μg protein). Bottom panel HCT116 cell positive control for p53 K382Ac detection (40 μg protein, 2 minute exposure). Note that p53 K382Ac is undetectable in SiHa cells (5 min exposure) and requires 2 h exposure for detection (†). (B) Relative levels of SIRT1, SIRT1 S47P and SIRT1 S27P 48h post-transfection with indicated siRNAs, mean of two experiments. (C) Phase contrast images of SiHa cells post-transfection with the indicated siRNAs. (D) Apoptotic SiHa cells 48h post-transfection with the indicated siRNAs. (E) Primary human keratinocytes 48h post-transfection with expression vectors for HPV E6 and HPV16 E7 and equivalent samples immunoblotted for HPV E6, HPV E7, SIRT1, SIRT1 S27P and SIRT1 S47P.
Mentions: Previously we have shown that SIRT1 acts as a cancer-specific survival factor in a range of epithelial cancer cell lines [20]. This raised the possibility that SIRT1 might mediate the anti-apoptotic effects of HPV16 E7 [30]. Moreover SIRT1 protein levels are abnormally elevated in a range of human epithelial cell lines, including SiHa cervical cancer cells [25]. We now demonstrate that RNAi-mediated silencing of HPV E7 in SiHa cells down-regulates SIRT1 protein levels by ~50% 48h post-transfection with E7 siRNA (Figure 4A and 4B). In these same cells the levels of the pro-apoptotic E3 ligase Itch and anti-apoptotic c-FLIP (c-FLIPS and c-FLIPL) were unaffected by HPV E7 knock-down (data not shown) demonstrating that the reduction in SIRT1 protein levels reflects a selective effect of HPV E7 depletion. In contrast to HPV E7 depletion, the depletion of HPV E6 had no effect on levels of SIRT1 protein (Figure 4A and 4B). We conclude that HPV E7, but not HPV E6, is required to maintain the abnormally high levels of SIRT1 protein characteristically observed in SiHa cervical cancer cells. Our results thus identify the longevity protein SIRT1 as a novel cellular target of the viral oncogene HPV16 E7 in human cervical cancer cells.

Bottom Line: Conversely, SIRT1 levels decrease following RNAi-mediated silencing of HPV E7 in SiHa cells.Silencing HPV E6 has no effect on SIRT1 but, as expected, causes marked accumulation of p53 protein accompanied by p53-mediated up-regulation of p21.Our discovery that HPV E7 up-regulates SIRT1 links a clinically important oncogenic virus with the multi-functional SIRT1 protein.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire Cancer Research P53 Research Unit, Department of Biology, University of York, York YO105DD, UK. ajm24@york.ac.uk

ABSTRACT
Senescence is blocked in human cervical keratinocytes infected with high risk human papillomavirus (e.g. HPV type16). Viral oncoproteins HPV E6 and HPV E7 access the cell cycle via cellular p53 and retinoblastoma proteins respectively. Previously we have shown that HPV E7, not HPV E6, is also responsible for cervical cancer cell survival (SiHa cells; HPV type16). We now present evidence that SIRT1, an aging-related NAD-dependent deacetylase, mediates HPV E7 survival function in SiHa cervical cancer cells. Moreover, HPV E7 up-regulates SIRT1 protein when expressed in primary human keratinocytes. Conversely, SIRT1 levels decrease following RNAi-mediated silencing of HPV E7 in SiHa cells. Silencing HPV E6 has no effect on SIRT1 but, as expected, causes marked accumulation of p53 protein accompanied by p53-mediated up-regulation of p21. However, p53 acetylation (K382Ac) was barely detectable. Since p53 is a known SIRT1 substrate we propose that elevated SIRT1 levels (induced by HPV E7) attenuate p53 pro-apoptotic capacity via its de-acetylation. Our discovery that HPV E7 up-regulates SIRT1 links a clinically important oncogenic virus with the multi-functional SIRT1 protein. This link may open the way for a more in-depth understanding of the process of HPV-induced malignant transformation and also of the inter-relationships between aging and cancer.

Show MeSH
Related in: MedlinePlus