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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.

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RNAi-mediated knock-down of HPV E6 and HPV E7 in SiHa and CaSki cells, and effects on p53 and retinoblastoma protein.  (A) mRNA qRT-PCR determinations 48h                                            post-transfection as indicated, mean ± s.d. of three determinations.                                            Asterix indicates differential effect of E7 siRNA on E6 mRNA levels in SiHa                                            versus CaSki cells. (B) Relative positions of siRNA sequences along                                            the bicistronic E6/E7 transcript. (C) Relative levels of E6 and E7                                            mRNAs 48h post-transfection of SiHa cells with the indicated siRNAs. (D,                                                    E) Immunoblots showing effects of E6 or E7 depletion on levels of p53,                                            p21 and hyperphosphorylated Rb (pRb*) in SiHa cells.
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Figure 1: RNAi-mediated knock-down of HPV E6 and HPV E7 in SiHa and CaSki cells, and effects on p53 and retinoblastoma protein. (A) mRNA qRT-PCR determinations 48h post-transfection as indicated, mean ± s.d. of three determinations. Asterix indicates differential effect of E7 siRNA on E6 mRNA levels in SiHa versus CaSki cells. (B) Relative positions of siRNA sequences along the bicistronic E6/E7 transcript. (C) Relative levels of E6 and E7 mRNAs 48h post-transfection of SiHa cells with the indicated siRNAs. (D, E) Immunoblots showing effects of E6 or E7 depletion on levels of p53, p21 and hyperphosphorylated Rb (pRb*) in SiHa cells.

Mentions: For this study we exploited the ability of RNAi to separately knockdown the viral oncogenes HPV16 E6 and HPV16 E7 in HPV16-positive SiHa cervical cancer cells. Individual knockdown of HPV E6 and E7 by their respective siRNAs was verified by quantitative RT-PCR (Figure 1A, left panel), thus confirming our previous observations [30]. This effect appears peculiar to SiHa cells since similar individual knock-down of E6 and E7 was not obtained for HPV16-positive CaSki cervical cancer cells in which E7 siRNA induced knock-down of both HPV E6 and E7 expression (Figure 1A, right panel, asterix).


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)

RNAi-mediated knock-down of HPV E6 and HPV E7 in SiHa and CaSki cells, and effects on p53 and retinoblastoma protein.  (A) mRNA qRT-PCR determinations 48h                                            post-transfection as indicated, mean ± s.d. of three determinations.                                            Asterix indicates differential effect of E7 siRNA on E6 mRNA levels in SiHa                                            versus CaSki cells. (B) Relative positions of siRNA sequences along                                            the bicistronic E6/E7 transcript. (C) Relative levels of E6 and E7                                            mRNAs 48h post-transfection of SiHa cells with the indicated siRNAs. (D,                                                    E) Immunoblots showing effects of E6 or E7 depletion on levels of p53,                                            p21 and hyperphosphorylated Rb (pRb*) in SiHa cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: RNAi-mediated knock-down of HPV E6 and HPV E7 in SiHa and CaSki cells, and effects on p53 and retinoblastoma protein. (A) mRNA qRT-PCR determinations 48h post-transfection as indicated, mean ± s.d. of three determinations. Asterix indicates differential effect of E7 siRNA on E6 mRNA levels in SiHa versus CaSki cells. (B) Relative positions of siRNA sequences along the bicistronic E6/E7 transcript. (C) Relative levels of E6 and E7 mRNAs 48h post-transfection of SiHa cells with the indicated siRNAs. (D, E) Immunoblots showing effects of E6 or E7 depletion on levels of p53, p21 and hyperphosphorylated Rb (pRb*) in SiHa cells.
Mentions: For this study we exploited the ability of RNAi to separately knockdown the viral oncogenes HPV16 E6 and HPV16 E7 in HPV16-positive SiHa cervical cancer cells. Individual knockdown of HPV E6 and E7 by their respective siRNAs was verified by quantitative RT-PCR (Figure 1A, left panel), thus confirming our previous observations [30]. This effect appears peculiar to SiHa cells since similar individual knock-down of E6 and E7 was not obtained for HPV16-positive CaSki cervical cancer cells in which E7 siRNA induced knock-down of both HPV E6 and E7 expression (Figure 1A, right panel, asterix).

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