Beta human papillomavirus E6 expression inhibits stabilization of p53 and increases tolerance of genomic instability

Abstract

Infections with the beta genus of human papillomaviruses (β-HPVs) may contribute to the development of nonmelanoma skin cancers. However, β-HPV genomes are found at too low a copy number in tumors for the virus to be necessary for tumor maintenance. Instead, they are hypothesized to destabilize the host genome by allowing the persistence of mutations that can drive tumorigenesis independently of the viral genome. Supporting this premise is our previous finding that the expression of some β-HPV E6 proteins can attenuate p53 signaling in response to DNA damage. We show that β-HPV E6 proteins can prevent the stabilization of p53 in response to two types of genome-destabilizing events, aberrant mitosis and dysregulated centrosome duplication. The inability to stabilize p53 in response to these stimuli allows cells expressing HPV5, HPV8, or HPV38 E6 to remain proliferatively active, leading to further genome deterioration in a proportion of these cells. These phenotypes are lost by the introduction of a mutation into the p300 binding domain of HPV8 E6 or by the transfection of mutated p300 that is resistant to the degradation mediated by HPV5 or HPV8 E6. These findings expand the understanding of the role played by p300 in promoting the faithful resolution of mitotic figures as well as proper centrosome duplication. Finally, we describe a phenomenon by which binucleated cells are resolved via cytokinesis into two cells, each with one nucleus. These data support the hypothesis that β-HPV infections may promote tumorigenesis via genome destabilization.

Importance: The work described in this report provides support for the hypothesis that β-HPV infections may contribute to nonmelanoma skin cancer by increasing the likelihood that tumorigenic mutations are introduced into the host cell's genome. We demonstrate that expression of the E6 proteins from some of these viruses increases the tolerance of two genome-destabilizing events, aberrant cell division and dysregulated centrosome duplication. Typically, these mutagenic occurrences elicit the stabilization of the tumor suppressor p53, which prevents further propagation of cells containing these errors. We show that the expression of β-HPV E6 restricts this stabilization of p53, leading not only to continued cellular proliferation but also to further accumulation of similar mutagenic events. Finally, in addition to supporting a role for β-HPV infections in certain skin cancers, we present studies with a mutated β-HPV E6 protein suggesting that the histone acetyltransferase p300 plays a role in promoting genome stability during replication.

FIG 1

β-HPV E6 expression increases multinucleation. (A) Representative RT-PCR confirming HPV E6 expression. 36B4 was used as a loading control (L). Note that since different PCR primers were used to amplify each E6 protein, relative expression can only be confirmed using this approach; the results may not accurately reflect the relative expression of HPV E6 among the cell lines. (B) Overall (passages 5 through 25) mean percentage (from ≥60 independent experiments) of cells with two or more nuclei for each cell line. (C) Overall (passages 5 through 25) mean percentage (from ≥60 independent experiments) of cells with an abnormal number of nuclei (≥2) that are multinucleated (≥3 nuclei) for each cell line. (D) Mean percentage (from ≥15 independent experiments) of cells that are multinucleated in four passage (p) intervals. (E) Mean percentage of cells with an abnormal number of nuclei (≥2) that are multinucleated (≥3 nuclei) for HFKs expressing the vector control (L), HPV5 E6, or HPV8 E6. Filled bars, cells transfected with the p300A expression vector; open bars, cells transfected with the p300E expression vector. (F) Relative percentages of cells with >4N DNA content, a measure of polyploidy. The values for HFKs carrying the control LXSN vector are set to 1. Early-passage cells have been cultured for <15 passages, while late-passage cells have been grown for >15 passages. Error bars show the standard errors of the means. Asterisks indicate data points that are significantly different (P, ≤0.05) from the result for the corresponding vector control cell line.

FIG 2

β-HPV E6 expression promotes late-passage proliferation. (A) Equal numbers of cells from each indicated passage for each cell line were seeded, and proliferation rates were recorded over a 5-day period. Bars represent the mean growth index scores (from ≥3 independent experiments) for each of the cell lines. Early-passage cells were at passage 5, while late-passage cells were at passage 20. (B) Overall (passages 5 through 25) mean percentage (from ≥60 independent experiments) of cells staining positive for senescence-associated β-galactosidase (SA-Beta Gal). (C) Mean percentages (from ≥15 independent experiments) of cells staining positive for senescence-associated β-galactosidase for five passage intervals between p5 and p25. (D) Overall (passages 5 through 25) mean percentage (from ≥60 independent experiments) of binucleated cells staining positive for senescence-associated β-galactosidase. (E) Mean percentages (from ≥15 independent experiments) of binucleated cells staining positive for senescence-associated β-galactosidase for five passage intervals between p5 and p25. Error bars show the standard errors of the means. Asterisks indicate data points that are significantly different (P, ≤0.05) from the result for the corresponding vector control cell line.

FIG 3

β-HPV E6 expression increases the frequency of cells with abnormal numbers of centrosomes. (A) Representative images of cells stained with α-tubulin (red) and γ-tubulin (indicating centrosomes) (green) for each cell line. (Insets) Enlargements of areas positively stained with γ-tubulin. (B) Mean number of centrosomes (from ≥3 independent experiments) per cell in each cell line. (C) Distribution of centrosomes in cells. At least three independent experiments were performed. Error bars show the standard errors of the means. Asterisks indicate data points that are significantly different (P, ≤0.05) from the result for the corresponding vector control cell line.

FIG 4

β-HPV E6-expressing cells fail to stabilize p53 in response to binucleation. (A) Representative images of binucleated cells with nuclei (blue) and p53 (pink) stained. (B) Percentage of binucleated cells that are positive for p53 in each cell line. At least three independent experiments were performed. Error bars show the standard errors of the means. Asterisks indicate data points that are significantly different (P, ≤0.05) from the result for the corresponding vector control cell line. (C) Mean intensity of p53 staining in binucleated cells and overall p53 staining for each cell line. At least 15 independent experiments were performed. (D) Representative images of multinucleated cells with nuclei (blue) and p53 (pink) stained. (E) Representative images of multinucleated cells transfected with either p300A or p300E as indicated. Nuclei (blue) and p53 (pink) were stained.

FIG 5

β-HPV E6 expression abrogates p53 stabilization in mononucleated cells with ≥3 centrosomes. (A) Representative images of cells with more than 2 centrosomes. Nuclei were stained with DAPI (blue). Immunofluorescence was used to detect p53 (pink) and γ-tubulin (indicating centrosomes) (green). (Insets) Magnifications of the γ-tubulin-stained portions of the images. (B) Percentage of cells with more than 2 centrosomes that stained positive for p53 in each cell line. At least three independent experiments were performed. Error bars show the standard errors of the means. Asterisks indicate data points that are significantly different (P, ≤0.05) from the result for the corresponding vector control cell line. (C) Mean intensity (from ≥15 independent experiments) of p53 staining in cells with 3 or more centrosomes and overall p53 staining intensity in each cell line. (D) Representative images of cells with more than 2 centrosomes transfected with p300A or p300E as indicated. Nuclei were stained with DAPI (blue). Immunofluorescence was used to detect p53 (pink) and γ-tubulin (indicating centrosomes) (green). (Insets) Magnifications of the γ-tubulin-stained portions of the images.

FIG 6

Binucleated cells expressing β-HPV E6 continue to proliferate. (A) Representative images of binucleated cells stained with anti-α-tubulin (cytoplasm) and anti-Ki67 (proliferation) immunofluorescent antibodies. Nuclei were stained with DAPI. (B) Percentage of binucleated cells that were positive for Ki67 in each cell line. At least three independent experiments were performed. Error bars show the standard errors of the means. (C) Mean intensity (from ≥15 independent experiments) of Ki67 staining in binucleated cells as well as in the total cell population for each cell line. Asterisks indicate data points that are significantly different (P, ≤0.05) from the result for the corresponding vector control cell line. (D) Representative images of binucleated cells transfected with p300A or p300E as indicated and stained with anti-α-tubulin (cytoplasm) and anti-Ki67 (proliferation) immunofluorescent antibodies. Nuclei were stained with DAPI.

FIG 7

β-HPV E6 expression allows cells with more than 2 centrosomes to proliferate. (A) Representative images of cells with supernumerary centrosomes. DNA was stained with DAPI. Anti-γ-tubulin (centrosomes) and anti-Ki67 (proliferation) immunofluorescent antibodies were used to mark centrosomes. (Insets) Higher magnifications of the portions of the images stained with anti-γ-tubulin (centrosomes). (B) Frequency of positive staining for Ki67 expression among cells with 3+ centrosomes. At least 15 independent experiments were performed. Error bars show standard errors of the means. Asterisks indicate data points that are significantly different (P, ≤0.05) from the result for the corresponding vector control cell line. (C) Mean Ki67 staining intensity (from ≥15 independent experiments) both in the total cell population and in cells with supernumerary centrosomes. (D) Representative images of normal mitotic cells (cells carrying LXSN alone or expressing HPV Δ8 E6) and multipolar mitotic cells (cells expressing HPV5, -8, -38, or -16 E6). (E) Representative images of cells with supernumerary centrosomes transfected with p300A or p300E as indicated. DNA was stained with DAPI. Anti-γ-tubulin (centrosomes) and anti-Ki67 (proliferation) immunofluorescent antibodies were used to mark centrosomes. (Insets) Higher magnifications of the portions of the images stained with anti-γ-tubulin (centrosomes).