Full length HPV-16E6 was amplified by PCR from HPV type 16 complete genome, and then cloned in frame within the C terminus of the mammalian expression vector pEGFP-C1 (Clontech, CA, USA) and pcDNA4/To/myc-HisC (Invitrogen, CA, USA) respectively, producing plasmids pGFP-16E6 and pcDNA4/To/myc-HisC-16E6.

The human breast adenocarcinoma MCF-7 cells and human embryonic 293T kidney cells were maintained in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS). Human colon carcinoma HCT116 cells and HCT116 p53^−/− were maintained in DMEM (Gibco) supplemented with 10% FBS, at 37°C in a humidified atmosphere of 5% CO₂. The MCF-7 and 293T cells were seeded at approximately 30% confluency on glass coverslips in 12-well cell culture plates. The cells were transiently transfected with plasmid pGFP-16E6 and pGFP overnight using Lipofectamine 2000 transfection reagent (Invitrogen) following the recommendations of the manufacturer. The reagent:DNA ratio was 2:1.

The MCF-7 and 293T cells were grown on glass coverslips, transfected, and at 21 h post-transfection, coverslips were mounted on modified glass slides with 10% fetal calf serum-containing cell culture medium, and used immediately for imaging. Images of live cells were collected with a Leica confocal microscope (Leica Microsystems, Wetzler, Germany) at a magnification of ×400. Fluorescent images were analyzed using Leica Confocal Software (Leica Microsystems).

The cells were seeded on glass coverslips at a density of 100,000 or 200,000 cells/well. Following standard procedures, they were transfected with plasmid pGFP-16E6, pGFP, pcDNA4/To/myc-HisC-16E6 and pcDNA4/To/myc-HisC, respectively. After transfection, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. They were then rehydrated three times with cold PBS, permeabilized with 1% Triton X-100 for 5 min on ice, and rinsed with PBS and blocked. The pGFP and pGFP-16E6 transfected cells were incubated with a primary antibody against p53 (cell signaling: dilution, 1:500) overnight at 4°C. Subsequently, signal detection was performed using Cy3-conjugated goat anti-rabbit IgG (Sigma; dilution, 1:200) in blocking solution for 30 min at room temperature in the dark. Then, the cells were washed three times with PBS and examined by confocal microscopy.

The pcDNA4/To/myc-HisC and pcDNA4/To/myc-HisC-16E6 transfected cells were incubated with a primary antibody against His (Clontech, dilution 1:500) overnight at 4°C, the secondary antibody of HRP-conjugated goat anti-mouse IgG (Pierce, Rockford, IL; dilution, 1:500) in blocking solution for 30 min at room temperature. Reaction products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and the slides were counterstained with hematoxylin.

The pcDNA4/To/myc-HisC and pcDNA4/To/myc-HisC-16E6 transfected cells were also incubated with primary antibodies against p53 (cell signaling; dilution, 1:500) and against His (Clontech, dilution 1:500) overnight at 4°C, the secondary antibodies of FITC-conjugated goat anti-mouse IgG (Sigma; dilution, 1:200) and Cy3-conjugated goat anti-rabbit IgG (Sigma; dilution, 1:200) in blocking solution for 30 min at room temperature in the dark. Then, the cells were washed three times with PBS and examined by fluorescence microscopy.

Cells (10⁷) were lysed in RIPA buffer on ice for 20 min followed by 20 min of centrifugation at 16,000 × g. Cell lysates 0.5 ml (1 mg of protein) were clarified with 20 μl of Protein G PLUS-Agarose (Santa Cruz Biotechnology) for 1 h at room temperature. After centrifugation supernatants were incubated with 2 μg of anti-p53 rabbit antibody overnight at 4°C. Protein G-PLUS Agarose (20 μl) was then added to cell lysates and incubated for 2 h at room temperature. Beads were washed three times with PBS and proteins were eluted by the addition of 40 μl 1X electrophoresis sample buffer. Western blots were performed using anti-GFP mouse antibody (Clontech, JL, CA).

For each sample, 10⁶ cells were collected by centrifugation (1000 rpm for 5 min), washed once with ice cold PBS, and lysed in 100 μl RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 2.5 mM glycerophosphate, 1 mM PMSF, 10 mM NaF, and protease inhibitors (Complete Mini, Roche Diagnostics, Mannheim, Germany). Protein concentration was determined using the BCA reagents (Pierce). Samples (30 μg) were analyzed on 12% SDS polyacrylamide gels, transferred to PVDF membranes (Invitrogen), and blocked for 1 h at room temperature with 5% non-fat milk in TBS buffer [20 mM Tris-HCl (pH 7.5), 0.5 M NaCl]. The membranes were then incubated with the primary antibody overnight at 4°C. After three washes with TBS, the membranes were incubated with the secondary antibody for 30 min at room temperature. After three additional washes, the proteins were visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia, Piscataway, NJ, USA). The amounts of proteins were determined by densitometric scanning (Dinco and Rhenium Biological Imaging System BIS 202).

The primary antibodies used were: anti-p53 (Cell Signaling; dilution, 1:1,000), (the following were all from Santa Cruz Biotechnology) anti-bax (dilution 1:500), anti-bcl-2 (dilution 1:500), anti-Bak (dilution 1:500), anti-c myc (dilution 1:500), anti-cdc2 (dilution 1:500) and anti-β actin (dilution, 1:10,000). The blots were counterstained with goat anti-mouse or goat anti-rabbit IgG conjugated with HRP (Pierce).

The transfected cells were harvested after 24, 48, and 72 h by trypsinization, and apoptotic cells were assayed with the Annexin V-APC Apoptosis Detection kit (Bender Medsystems, Burlingame, CA). Briefly, 1×10⁶ cells in 100 μl binding buffer were stained with 5 μl Annexin V-APC and 10 μl PI (final concentration, 1 μg/ml) by mixing and incubating on ice for 10 min in the dark. The cells were analyzed by flow cytometry. The data were processed using the Cell Quest software.

All data were recorded as means ± standard deviation, and analyzed by the SPSS 11.0 software. Analysis of data was performed using one-way ANOVA for multiple comparisons. P-values <0.05 were considered statistically significant.

Viral E6 coding regions were inserted within the C terminus of the pGFP vector, producing plasmid pGFP-16E6. We transiently transfected pGFP-16E6 in MCF-7 and 293T cells, which allow E6 proteins to be expressed as GFP-16E6 fusion proteins. By confocal microscopy, we observed the subcellular localization of GFP-16E6 and GFP in viable cell images. The E6 fusion proteins may have low or high expression levels at different times, and this could affect the distribution of E6. Therefore, we observed the localization and expression of proteins from 6 to 72 h post-transfection. The results indicated that GFP-16E6 protein was expressed essentially in the nucleus from 6 h post-transfection. Its expression increased gradually, and reached its maximum expression level at 21 h (P<0.001). Then, it decreased gradually and disappeared after one week. During this whole period, no change was observed in protein localization. As control, we observed the expression of GFP alone. It exhibited a diffused signal, and was present in both the nucleus and cytoplasm from 6 h to one week post-transfection. In addition, its location did not change at any time (Fig. 1A).

The 293T cells were also used to study E6 localization. The cellular distributions of GFP and GFP-16E6 proteins were similar to those in MCF-7 cells (Fig. 1A). The analysis of relative fluorescence signal intensity of GFP-16E6 in different cell lines is shown in Fig. 1B.

Because high risk E6 can bind to p53 (15), we suspected that the GFP-16E6 and p53 might locate together. Using MCF-7 and 293T cells, we investigated endogenous wt p53 localization by immunocytofluorescence technique. The results showed that p53 protein was mainly located in the nuclei of pGFP transfected cells. In pGFP-16E6 transfected cells, the distribution of p53 protein was not changed, and it was located in the nuclei together with GFP-16E6. Fig. 2A shows representative images of the co-localization of GFP-16E6 and p53 proteins.

In the present study, we observed p53 and E6 protein located together, it was necessary to determine whether GFP-16E6 could interact with p53 in vivo. We investigated the potential role of the interaction between endogenous wt p53 and E6 protein by performing immunoprecipitation assay with anti-p53 antibodies. Then, by western blot analyses with anti-GFP antibodies it was shown that p53 interacted with GFP-16E6 protein. Moreover, as a control, in GFP expressing cells, only the GFP protein that lacked the E6 protein was unable to interact with p53 (Fig. 2B). Therefore, in present study, we observed that p53 could interact with HPV-16E6 protein in vivo. This is supported by a report that p53 has binding sites for high risk HPV-E6 (15).

It has been extensively shown that p53 was degraded by 26S proteasome via the ubiquitin pathway (16). Since E6 interaction with p53, we next to determine whether the overexpressed E6 affected this process of p53 degradation. At 24 h post-transfection, the GFP and GFP-16E6 expressing cells were treated with MG132, a potent inhibitor of 26S, and then examined p53 level by immunoblotting. The ubiquitination of p53 was readily detected in both GFP and GFP-16E6 expressing cells upon MG132 treatment. The results showed that the ubiquitination of p53 was significantly lower in GFP-16E6 cells than GFP control cells (Fig. 3). Thus, p53 was increased in 24 h in GFP-16E6 expressing cells, which was partly due to the decreased ubiquitin-mediated degradation of p53.

With the overexpression of oncoprotein E6, we next asked whether it can promote cell apoptosis along with the expression of p53. By Annexin V and PI double-staining combined with flow cytometry, we observed obvious apoptosis in GFP-16E6 expressing MCF-7 cells. Apoptosis occurred at 12 h post-transfection, and it increased gradually. From 12 to 72 h post-transfection, apoptosis was prominent compared with GFP alone expressing cells (P<0.001). For 293T cells, we obtained similar result to that in MCF-7 cells (Fig. 4A).

By western blotting, we examined p53 level in GFP-16E6 cells at 12, 24, 48 and 72 h post-transfection. The result indicated, for GFP-16E6 expressed cells, p53 was increased in 24 h post-transfection, and then it degraded gradually at later times. Accordingly, the other apoptosis associated proteins, such as bax, Bak, c-myc and cdc2 were increased and the bcl-2 was decreased compared with GFP control cells (Fig. 5). On the other hand, p53 in GFP-16E6 expressing MCF-7 cells was degraded more than 293T cells. The various activity of E6 to target and degrade p53 was partly due to p53 conformation in different cells (17). Thus, our data indicated the GFP-16E6 could induce apoptosis in wt p53 cell lines.

Our result showed there was obvious apoptosis induced by E6 along with the expression of p53. To further investigate whether p53 was necessary for apoptosis, we transfected pGFP-16E6 in both HCT116 and HCT116 p53^−/− cells. We observed, in the context of HPV-16E6, there was obvious apoptosis in HCT116 cells, whereas there was no obvious apoptosis in HCT116 p53^−/− cells (Fig. 4B). Therefore, we concluded p53 was necessary for GFP-16E6 induced apoptosis.

To avoid the possible effect of GFP-fusion protein on E6-p53 binding and the degradation of p53, we used His with HPV-16E6 fusion protein for further research. By immunocytochemistry stain, we observed the His-16E6 expression in both MCF-7 and 293T cells (Fig. 6). Furthermore, using immonofluorescent technique, we clearly observed His-16E6 was mainly located in the nuclei together with p53 (Fig. 7A). Comparing with pcDNA4/To/myc-HisC control cells, the p53 protein expression level was increased in 24 h along with the His-16E6 expression (Fig. 7B).