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. 2015 Oct 27;6(33):34979-91.
doi: 10.18632/oncotarget.5512.

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma

Yuezhen Xue  1   2 , Shen Yon Toh  1 , Pingping He  1 , Thimothy Lim  3 , Diana Lim  4 , Chai Ling Pang  5 , Jean-Pierre Abastado  5 , Françoise Thierry  1
Affiliations

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma

Yuezhen Xue et al. Oncotarget. .

Abstract

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.

Keywords: DNA damage response; HPV16-E2; cell cycle; cervical intraepithelial neoplasia; prophase.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. SiHa and A549 cells that express E2 are arrested in prophase
A. Flow cytometric analysis of cell cycle distribution of SiHa cells infected with recombinant adenoviruses expressing GFP, GFP-16E2, GFP-DBD or GFP-TAD of HPV16 E2, 6 hours post thymidine release and their protein levels analysed at the same time points by Western blot (lower panel). B. As in (A) on cells harvested 22 hours post thymidine release. H3p labeling density plots corresponding to the same samples are shown in the lower panels. Yellow arrows point to high-intensity H3p staining indicative of metaphasic cells while red arrow points to lower intensity H3p staining indicative of prophase cells. C. Immunofluorescent staining visualizes H3p (red) and 16E2 or GFP (green) in SiHa cells infected with GFP and GFP-16E2 recombinant adenoviruse, the cells were seeded on coverslips and harvested 22 hours post thymidine release. Yellow arrows: metaphasic nuclei, red arrows: prophasic nuclei. Scale bar = 50 μm. D. Flow cytometric analysis of A549 cells infected with GFP either treated (Noco) or not by nocodazole, or GFP-16E2 recombinant adenoviruses in synchronized cells 16 h after release from thymidine block. DNA content (blue) and H3p (red) were co-labeled (NT = not transduced). H3p staining density plots were done on the same samples and are shown in the lower panels together with percentage of cells in mitosis. Black arrow: high-intensity H3p staining indicative of metaphasic cells, red arrow: lower intensity H3p staining indicative of prophase cells. E. Confocal microscopy visualizes H3p (red) and 16E2 (green) in A549 cells. Yellow arrows: metaphasic cell, red arrows: prophasic nuclei. Scale bar = 10 μm. F. Time-lapse microscopy was performed to observe cell cycle progression in synchronized A549 cells infected with GFP- or GFP-16E2-recombinant adenoviruses. Typical mitotic division for GFP-expressing cells became evident 12–13 h after thymidine release. E2-expressing cells do not divide within the 29 h of observation.
Figure 2
Figure 2. E2 induces a DNA damage signal in A549 cells arrested in prophase
A. Western blot analyses of lysates from A549 cells expressing GFP-16E2, GFP-18E2, HPV18E2 transactivation domain (GFP-18E2TAD) or the DNA binding domain (GFP-18E2DBD), probed with antibodies against GFP, γH2AX, Chk2T68 and ATMS1981. B. Confocal microscopy of E2-expressing A549 cells double labeled with anti-γH2AX (light blue) and anti-Chk2T68 (red). White arrows: co-localization of the DDR markers, red arrows: DDR marker partial co-localization with 16E2 in infected nuclei. C. As in (B) except the cells were co-labeled with anti-H3p (red) and anti-γH2AX (light blue). Inset show higher magnification of double stained infected nuclei with distinct punctate signals for the H3p and γH2AX markers D. Expression of H3p (red) and γH2AX (green) in A549 cells treated with increasing doses of etoposide. Scale bar = 10μm.
Figure 3
Figure 3. Expression of E2 induces the mitotic activator Cdc25CT48 and coincides with the induction of DDR markers
A. Thymidine synchronized A549 cells were infected with GFP or GFP-16E2 and released at various time points between 0–16 h, as indicated (T0-T16) for cell cycle analyses by flow cytometry. B. Western blot detection of protein levels of the same samples at the same time points. The activated cell cycle regulators Cdc25CT48 and H3p were used as markers of mitotic entry. Chk2T68 and γH2AX were used as markers of DDR signal activation. C. Thymidine synchronized A549 cells infected with GFP were treated by nocodazole for 16 hours (NR0) and were then released from nocodazole for 5 hours and harvested (NR5) for cell cycle analysis by flow cytometry. D. Western blot detection of the same proteins as in (B) as indicated.
Figure 4
Figure 4. Transcripts of DDR components are less abundant in SCC than in CIN3 samples
Digital quantification of viral and cellular mRNAs was performed by NanoString analysis in n = 28 microdissected cervical lesions, including samples from normal cervix (NorCx) and HPV16-associated CIN2, CIN3 and SCC lesions. Quantified viral genes were 16E6E7 and 16E1^E4, while the cellular gene transcripts analyzed included the CDKN2A (p16INK4) surrogate marker of E7 expression, proliferative marker MKI67, mitotic gene AURKB, and DDR components ATM and CHK2. For each category of lesions the transcript levels were plotted after log 2 transformation. The indicated p-values were calculated by 1-way ANOVA. The horizontal bars represent the mean values for each group.
Figure 5
Figure 5. Prophase cells are more common in the areas of HPV16-associated CIN3 lesions that co-express E2 and p16
A. Prophase arrest in CIN3 with E2 and p16 co-expression. Consecutive sections of CIN3 lesions were labeled for 16E2, p16 INK4 and, H3p. In E2-positive regions, prophase cells were abundant. HPV DNA was detected by amplified-ISH. B. Co-labeling of the mitotic marker H3p (red) and DDR marker γH2AX (green) in CIN3 tissue samples. C. Similar as (A) another lesion with early M phase arrest in CIN3 with E2 and p16 co-expression. High magnification images in Inset illustrate that prophase cells host low levels of HPV DNA replication. Yellow arrows: metaphasic cells; red arrows: prophasic cells; blue arrows: HPV DNA. Scale bar = 50 μm.
Figure 6
Figure 6. Metaphase but not prophase patterns of H3p expression in HPV16 associated SCC lesions
Serial sections from SCC samples were labeled for 16E2, p16 INK4, H3p, and HPV DNA. High expression of p16INK4 was observed in the absence of E2, while amplified-ISH of the contiguous section shows integration of HPV DNA visualized as punctate brown signals (Blue arrow). H3p stained by IHC or IF staining shows mostly metaphasic cells (yellow arrows). Lower panel shows co-labeling for H3p (red) and γH2AX (green). Scale bar = 50μm.
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