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Oncogenic proteins E6 and E7 of high-risk human papillomaviruses (HPV's) either alone or in co-operation cause cell-cycle deregulation, cell-transformation and immortalization leading to cancer progression by abrogating the function of two most important cellular tumor suppressors p53 and pRb respectively. High-risk HPVE6 abrogates p53 functions by its degradation and/or by inhibiting its acetylation. Downregulation of E6 causes reactivation of dormant p53 pathways. Recently, for the first time we reported that in the presence of okadaic acid overexpressed p53 is activated by phosphorylation which inhibits tumor growth in mice. In the present study we observed that p53 overexpression has no effect on anchorage independent cell-growth and E6 nullifies its cell growth inhibitory effect. The overexpressed p53 is not associated with E6 and thus is not degraded in HPV-positive HeLa cells. Interestingly, we for the first time report that E6 expression de-activates activated p53 by inhibiting its phosphorylation. This prevents p53 from binding to p21 promoter restraining its cell-growth inhibitory functions.
Approximately 470,000 new cases of cervical cancer are diagnosed every year and approximately 230,000 women worldwide die, with the majority (~80%) of incidence occurring in developing countries. Human papillomavirus (HPV) infection is the main causative agent for cervical cancer. Reports suggest that 99.7% of cervical cancers harbor integrated HPV DNA in host cell genome (Walboomers et al., 1999). HPV presence is reported in 5-11% of oral cancers (Franceschi et al., 1996). In head and neck cancers the percentage of HPV infection is low and it accounts for 11-25% percent (Brandwein et al., 1994; Fouret et al., 1997; Gillison et al., 1999; Paz et al., 1997; Pintos et al., 1999; Snijders et al., 1996). In developed countries 50-70% of oropharyngeal and tonsillar carcinomas are associated with HPV infection (Gillison et al., 2000; Gillison and Shah, 2001). Papillomaviruses are also reported to be present in colon and 90% of anal cancers (Coutlee et al., 2006; Heideman et al., 2007; Parkin and Bray, 2006; Rubin et al., 2001). HPVs are classified in two categories, low risk which has less or no potential and high risk has potential to cause carcinogenesis. HPV 16 and 18 are high risk HPVs which accounts for more than 50% of cervical cancers and are considered as a major cause of other (head and neck as well as anal) cancers too.
The two onco-proteins of HPV, E6 and E7 cause transformation, immortalization and promote carcinogenesis primarily by binding to two important tumor suppressor's p53 and pRb, thereby completely deregulating cell cycle checkpoints (Bischof et al., 2005; Boyer et al., 1996; Scheffner et al., 1993; Werness et al., 1990). E6 and E7 alone can also immortalize, deregulate cell cycle and cause transformation of even primary cultures (Hawley-Nelson et al., 1989; Heck et al., 1992; Toussaint-Smith et al., 2004). E6 degrades p53 by E3 ubiquitin dependent as well as independent proteasomal degradation and it also inhibits p53 transactivity by inhibiting its acetylation (Camus et al., 2007; Scheffner et al., 1990; Thomas and Chiang, 2005). Inhibition of acetylation by E6 is facilitated by its ability to bind directly and degrade p300, an important acetyltransferase (Patel et al., 1999; Zimmermann et al., 1999). To completely abrogate p53 activity E6 also degrades bax, a major p53 downstream apoptosis inducer (Vogt et al., 2006). It has been reported that inhibition of E6 by its specific SiRNA reactivates dormant p53 pathways, though very little is known about the mechanism by which p53 functions are restored (Bousarghin et al., 2009; Butz et al., 2003; Jonson et al., 2008). There are studies suggesting reactivation of p53 pathways are mainly facilitated by enhancement in its acetylation and no report exist on the phosphorylation status of p53 as a consequence of silencing by E6. The role of E6 in phosphorylation of p53 is not well studied except one report which suggests that p53 is phosphorylated at multiple residues by transiently transfected E6 (Zhang et al., 2009). This study provides insight into the ability of viral protein to hijack the cellular machinery and describes the mechanism of inhibition of p53 transactivity independent of its degradation.
p53 induction is tightly regulated by Dox.
Induction of p53 is tightly regulated in a dose dependent manner by Dox (the inducer for Tet-On system) in HeLa cell derived Tet-On system wherein p53 can be expressed as per the experimental needs. The two-p53 expressing clones (HTet23p53 and HTet26p53) and one GFP expressing clone (HTet43GFP) are used in this study (Ajay et al., 2010). Dox in a dose dependent manner induces p53 protein in both HTet23p53 (Fig. 1A) and HTet26p53 (Fig. 1B) cells as compared to HTet43GFP cells (Fig. 1C). Densitometric analysis of these blots suggests that in comparison with non-induced state, in the presence of 2000 ng/ml Dox more than 5 fold increase in p53 level was achieved in both clones overexpressing p53 (Fig. 1D). No alteration in p53 level was detected in HTet43GFP cells (Fig. 1C) cells under identical experimental conditions.
p53 expression is time dependent.
To study the kinetics of p53 expression single dose of Dox (1000 ng/ml) was added for different time points (1 h to 48 h) and cell lysates were prepared. As shown in Fig. 2, p53 expression was initiated within 1 h of Dox addition and it increased progressively up to 48 h of incubation in p53 expressing HTet23p53 (Fig. 2A) and HTet26p53 cells (Fig. 2B). No significant alterations were detected in HTet43GFP (Fig. 2C) cells under identical experimental conditions. At 48 h, 5 fold increase in p53 protein expression was detected in Htet23p53 and HTet26p53 cells as compared to HTet43GFP cells (Fig. 2D).
Overexpressed p53 has no effect on anchorage independent growth.
The tumorigenic capability of p53 overexpressing cell was assessed by soft agarose assay which is a representative of in vivo tumor formation. Surprisingly, in HTet23p53 (Fig. 3A) as well as HTet26p53 (Fig. 3B) cells no decrease in anchorage independent growth after inducing p53 with 50 to 2000 ng/ml Dox was detected. HTet43GFP cells served as control (Fig. 3C). There was no significant difference in the colony number and size of p53 overexpressing clones as compared to GFP expressing clone. Dox treatment slightly decreased colony number in all the three cell lines which is likely due to long term Dox cytotoxicity.
Over expressed p53 is stable.
To perform tumor suppressor functions stability of p53 is essential. Therefore to ascertain that p53 over expressed is stable and it is not degraded by E6 cycloheximide (Chx) chase experiment was performed. Cells were subjected to Chx treatment for different time points to inhibit protein synthesis and then western blotted to detect p53. In the presence of Chx new protein synthesis is halted and p53 overexpressed with 1000 ng/ml Dox for 48 h did not significantly decrease in HTet23p53 and HTet26p53 cells (Fig. 4A and B) even after 6 h. Under these experimental conditions endogenous p53 in same cells or in HTet43GFP cells decreases to undetectable levels just within 1 h (Fig. 4A and B). From these experiments half calculated indicates that overexpressed p53 has a half life of about 6 h compared to that of less than 1 h for endogenous p53 (Fig. 4C).
Inhibition of proteasome promotes stability of endogenous p53 but not ectopically expressed p53.
p53 undergoes proteosomal degradation. Interestingly, inhibiting proteasomal degradation by two specific inhibitors MG132 and lactacystin did not stabilize overexpressed p53 protein in HTet23p53 as well as HTet26p53 cells upto 3 h. Surprisingly; treatment with these inhibitors increased the endogenous p53 protein stability in HTet23p53, HTet26p53 and HTet43GFP cells even after Chx chase for 1 h (Fig. 5A and B). Taken together these results suggest that overexpressed p53 is not a substrate for E6 mediated proteasomal degradation and endogenous p53 is stabilized in the presence of inhibitors.
Inhibition of protein phosphatase 2A promotes cell death and E6 reverses it by inhibiting promoter occupancy of activated overexpressed p53.
To determine whether E6 plays a role in inhibiting activity, p53 was first activated by 5 nM OA which exclusively inhibits protein phosphatase 2A (PP2A). As a consequence of p53 activation cell growth was retarded in p53 overexpressing HTet23p53 and HTet26p53 cells as compared to p53 non overexpressing HTet23p53, HTet26p53 or HTet43GFP cells. Cell growth inhibitory effect was abolished by ectopic expression of HPV 18 E6 in OA treated p53 overexpressing cells (Fig. 6A). Overexpressed p53 was not associated with E6 and moreover ectopic expression of E6 did not further enhance this association as detected by co-immunoprecipitation experiment. (Fig. 6B). To confirm the presence of free p53 in the lysate subjected to immunoprecipitation by E6 antibody, second immunoprecipitation with p53 (FL-393) was done. Interestingly, ectopic expression of E6 did not significantly decrease the levels of p53 but it did drastically decrease the level of Ser46 phosphorylated p53 (Fig. 6C). Moreover, luciferase reporter driven by p21 promoter was activated by OA treatment and E6 overexpression inhibited promoter activation (Fig. 6D).
Overexpression of p53 causes cell death but E6 expression promotes cell survival in p53 and E6 null lung carcinoma cell line.
To study the direct functional interaction of E6 and p53 we transfected wild type-p53 and/or E6 in H1299 lung carcinoma cells, which are null for p53 and E6. p53 overexpression decreases cell survival by 25 percent and activation of p53 by OA further reduces cell survival by another 25 percent. Co-expression of HPV18E6 in p53 expressing cells treated with or without OA promotes survival (Fig. 7A). At protein level, as expected no p53 is detected at basal level. p53 protein is expressed in cells transfected with p53 plasmid with or without OA treatment. Interestingly, co-transfection with HPV18E6 construct not only reduces p53 levels, but it also diminishes OA induced phosphorylation at serine 46 residue of p53 (Fig. 7B).
It is known that in HPV positive cells p53 function is abrogated by E6 and even ectopic overexpression of p53 is not able to perform its tumor suppressor functions. In addition to facilitating its degradation, it is likely that E6 also may inhibit p53 transactivity, which is still poorly understood. It has been reported that inhibition of acetylation of p53 by inhibiting p300, a major acetyl transferase, may be responsible for inactivation by E6 because p53 acetylation is necessary for its transactivity (Patel et al., 1999; Zimmermann et al., 1999). No significant decrease in protein level following Chx treatment suggests that overexpressed p53 may not be degraded by ubiquitination pathway and it is likely that by yet unknown mechanism its activation is inhibited.
Activation of overexpressed p53 to cause cell growth inhibition is facilitated by its phosphorylation (Ajay et al., 2010, Mi et al., 2009). Over expressed p53 failed to get activated and we probed for the role that E6 might play in phosphorylation as very little is known about it. Majority of reports suggest the involvement of E6 mediated degradation of p53 as possible reason for its inactivation (Akutsu et al., 1996; Crook et al., 1994; Eichten et al., 2002). 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. However, proteasomal inhibition significantly increased the half life 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 to be understood. Surprisingly, ectopic expression of E6 also did not significantly decrease overexpressed p53 protein level which could be because of swamping out of the available E6 and/or E6AP. This proposition derives support from a report describing the involvement of an E6 associated protein (E6 AP) which forms a ternary complex essential for ubiquitination of p53 (Cooper et al., 2003). It is also possible that the post-translational modification as well as conformation of overexpressed p53 might be different and is therefore not recognized by the E6 and E6AP complex. Our finding that over expression of E6 does not facilitate p53 degradation is likely because of one of the above mentioned reasons. The involvement of specific post-translation alterations is consistent with our (Ajay et al., 2010) and others report (Mi et al., 2009; Jin et al., 2010). These reports clearly demonstrate that expressed p53 is activated only in the presence of PP2A inhibitor and that p53 phosphorylation at a key residue is very critical for specific DNA binding as well as promoter selection under various stress conditions. Interestingly, p53 phosphorylation is diminished by overexpression of E6 in HeLa cells (Fig. 6C) which indicates that E6 causes inactivation of p53 by inhibiting its phosphorylation. Also, E6 expression significantly inhibits p21 promoter occupancy of activated overexpressed p53 which has an impact on cell growth (Fig. 6D and 6A). To ascertain that these are indeed true and not as consequence of additive effect of other HPV protein too, the effect of E6 on p53 phosphorylation was investigated in non-HPV and p53 null H1299 cells. Very interestingly, E6 expression causes complete degradation of p53 in non OA treated cells whereas in OA treated cells E6 did not reduce p53 levels though it decreases Ser46 phosphorylated p53 level (Fig. 7B), which correlates with increased cell survival under these condition (Fig. 7A) .
To conclude, the results presented here provide insight into differential regulation of endogenous and exogenous p53 and the role HPV E6 plays in its phosphorylation and activation. These findings imply that replacement of degraded, mutated p53 protein or functionally inactivated p53 with the wild-type one will have significant therapeutic importance only when its activation is also achieved simultaneously.
Materials and methods
Doxycycline (Dox) and cycloheximide was purchased from Sigma (St. Louis, MO). Tet-system approved serum and Hygromycin solution (50 mg/ml) was purchased from BD (Mountain View, CA). G418 was purchased from (USB, OH). Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), ß-Tubulin (rabbit polyclonal) and ß-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MG132 and Lactacystin were purchased from Calbiochem (CA). Okadaic acid (OA) was purchased from Invitrogen Corporation. Phospho-p53 Ser46 antibody, (rabbit polyclonal) was purchased from Cell Signaling Technology (Denvers, MA). HRP conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.
HeLa and H1299 cell lines were purchased from American Type Culture Collection (Mannas, VA) and maintained in our in-house National Cell Repository, National Centre for Cell Science (NCCS), Pune, India. Dox inducible cell lines were developed by utilizing BD-TetOn system and stably transfecting with pTetOn, pTREp53 or pBIEGFP. Dulbecco's minimum essential medium (DMEM), was purchased from Invitrogen Corporation. All these inducible cell lines were regularly cultured in DMEM, supplemented with 10% Tet system approved fetal bovine serum at 370C with 5% CO2. Inducible cell lines were maintained in 100 µg/ml of G418 and 50 µg/ml of hygromycin.
Plasmids and transfection.
pTet-On, pTRE, pTK-Hyg and pBIEGFP were purchased from BD. pC53-SN3 was a kind gift from Dr. Bert Vogelstein, John Hopkins, Baltimore, MD USA. p53 fragment of pC53-SN3 was sub-cloned in BamH1 site of pTRE and was renamed as pTREp53. p21Luciferase was kind gift from Dr. Bert Vogelstein. pFLAG-HPV 18 E6 plasmid was kind gift from Dr. McCance DJ, University of Rochester, USA.
Transfections were performed by Lipofectamine 2000 (Invitrogen) as per manufacture's protocols.
Soft agarose assay.
Culture dish was layered with 1 ml of 0.7% agarose. Five thousand HTet23p53, HTet26p53 and HTet43GFP cells were plated in 0.5% low melting agarose (FMC Bioproducts, ME) containing DMEM and 10% FBS with or without Dox and incubated after layering with 1 ml complete medium. Medium containing indicated concentration of Dox was changed every 4th day. After 30 days plates were stained with 0.1% crystal violet for 1 h and photographed under a microscope. Colonies of more than 50 cells were counted and graph was plotted from the average of two independent fields from each plates.
Cell proliferation assay.
Cell proliferation was determined by methylthiazole tetrazolium (MTT) assay. Cells were seeded at the density of 7,500 per well into 96 well plates and allowed to adhere for 24 h. Cells were transfected with pCDNA3 or HPV 18 E6 plasmid construct by Lipofectamine 2000 reagent. Eighteen hour post transfection cells were washed thrice with DMEM and treated with Dox in the presence of absence of OA and further incubated for 48 h. Fifty microliter of MTT (1 mg/ml) was added to each well and incubated for 4 h at 370C. Hundred microliter of 2-propanol was added and incubated in shaking condition at room temperature for 10 min. Absorbance was taken at 570 nm using 630 nm as reference filter. Absorbance given by untreated cells was considered as 100% cell survival.
Preparation of whole cell lysate and western blotting.
Following indicated treatments, cells were washed thrice with ice-cold phosphate buffered saline (PBS) and lysed in ice-cold lysis buffer (50 mM Tris-Cl, pH 7.5, with 120 mM NaCl, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 1% NP-40 and protease inhibitor cocktail (Roche Diagnostics, Germany). Equal amount of protein was resolved on SDS-PAGE and western blotting was preformed as described earlier (Singh et al., 2007). Where ever possible blots were stripped by incubating the membranes at 500C for 30 min in stripping buffer (62.5 mM Tris-Cl pH 6.7, 100 mM mercaptoethanol, 2% SDS) with intermittent shaking. Membranes were washed thoroughly with TBS and reprobed with required antibodies. Otherwise gels run in duplicates were probed for the desired proteins by western blotting and then compiled.
Cycloheximide chase assay.
HTet23p53, HTet26p53 and HTet43GFP cells grown in a 35 mm plate were treated with 1000 ng/ml of Dox for 48 h and then 100 µg/ml cycloheximide (Chx) was added. Cells were further incubated for indicated time points and processed for western blotting. For inhibitor experiments cells were treated with MG132 or Lactacystin 10 µM each 48 h prior to Chx addition and harvested after indicated time points after Chx addition.
After indicated treatment cells were harvested and lysed in RIPA buffer. Equal amount of protein (400 µg) was taken and lysates were pre-cleared with 50 µl protein A/G plus agarose (Invitrogen Corporation) for 30 min. Agarose beads were pelleted and supernatant was incubated with anti-E6 goat polyclonal antibody overnight at 40C in an IP rotator. Fifty microliter protein A/G plus agarose was then added in antibody-antigen complex with gentle shaking for 4-5 hours at 40C. The immune complex bound to protein A/G plus agarose was separated by centrifugation at 4000 rpm and supernatant was immunoprecipitated with anti-p53 goat polyclonal antibody and as described above. Target as well as its associated proteins was disrupted from protein A/G plus agarose beads by adding SDS gel sample buffer, resolved on SDS PAGE and processed for western blotting (Singh et al., 2007) with mouse monoclonal E6 and p53 antibodies.
Semi-confluent HTet23p53 and HTet26p53 cells plated in a 12 well plate were co- transfected with pEGFPC1 and p21Luciferase plasmids by Lipofectamine2000. Eighteen hour post-transfection 1000 ng/ml Dox was added with or without OA and further incubated for 48 h. Luciferase assay was performed as per manufacturer's protocol (Amersham Biosciences). GFP reading was taken as an internal control for normalization of transfection efficiency and graphs were plotted Luciferase reading/GFP.
Data are expressed as the mean of three independent experiments. Statistical comparisons are made using paired t test (SPSS Inc, USA) and P value < 0.05 was considered as significant.
Conflict of interest
Acknowledgements. We would like to thank Dr. G. C. Mishra, Director, NCCS for being supportive and giving all the encouragement to carry out this work. We also thank Department of Biotechnology, Government of India, for providing research grant. A.K.A thanks Indian Council for Medical Research. A.S.M. thanks Council of Scientific and Industrial Research for fellowship. Support from other group members and all technical staff of NCCS is also duly acknowledged.