Role of Cdc6 in re-replication in cells expressing human papillomavirus E7 oncogene

Abstract

The E7 oncoprotein of high-risk human papillomavirus (HPV) types induces DNA re-replication that contributes to carcinogenesis; however, the mechanism is not fully understood. To better understand the mechanism by which E7 induces re-replication, we investigated the expression and function of cell division cycle 6 (Cdc6) in E7-expressing cells. Cdc6 is a DNA replication initiation factor and exhibits oncogenic activities when overexpressed. We found that in E7-expressing cells, the steady-state level of Cdc6 protein was upregulated and its half-life was increased. Cdc6 was localized to the nucleus and associated with chromatin, especially upon DNA damage. Importantly, downregulation of Cdc6 reduced E7-induced re-replication. Interestingly, the level of Cdc6 phosphorylation at serine 54 (S54P) was increased in E7-expressing cells. S54P was associated with an increase in the total amount of Cdc6 and chromatin-bound Cdc6. DNA damage-enhanced upregulation and chromatin binding of Cdc6 appeared to be due to downregulation of cyclin-dependent kinase 1 (Cdk1) as Cdk1 knockdown increased Cdc6 levels. Furthermore, Cdk1 knockdown or inhibition led to re-replication. These findings shed light on the mechanism by which HPV induces genomic instability and may help identify potential targets for drug development.

Figure 1.

Upregulation of Cdc6 in E7-expressing cells. The steady-state levels of Cdc6 in PHKs and CE-HPV (A), NIKS (B) and RPE1 cells (C) expressing E7 or vector were examined by immunoblotting. Proliferation of E7-expressing RPE1 cells (D) and NIKS cells (E) was measured using a CCK8 assay. Cells were examined at 0, 12, 24, 36, 48 and 72h after seeding. Data are shown as the mean ± SD. A representative image of three experiments is shown. *P < 0.05, **P < 0.01.

Figure 2.

The downregulation of Cdc6 impairs the ability of E7 to induce polyploidy. RPE1 E7 cells were transfected with Cdc6 siRNAs, and DNA content/BrdU staining was analyzed by FACS. (A) Cdc6 protein levels were measured by immunoblotting 72h after si-CDC6-001 (20nM) transfection (left panel). For cell cycle analysis, bleomycin was added 24h post-transfection and incubated for an additional 48h. The cell cycle profiles are shown in the middle panel. The data are quantified and summarized in the right panel. (B) Regularly cultured RPE1 E7 cells were transfected with 500 pM si-CDC6-001 siRNA. Left panel, Cdc6 protein levels were measured by immunoblotting. A representative image from two independent experiments is shown. The cell cycle profiles are shown in the right panel; BrdU-positive cells are indicated. A histogram of the same profiles is shown below. (C) After si-CDC6-001 (500 pM) transfection, RPE1 E7 cells were treated with bleomycin. A representative image from two independent experiments of four determinants is shown (left panel). The data are summarized in the right panel. (D) After si-CDC6-002 transfection, the cells were treated with bleomycin and analyzed for Cdc6 expression (left panel) and cell cycle profiles (middle panel). A representative image from at least three independent experiments is shown, and the data are summarized (right panel). Error bars reflect the standard deviations of the mean. NS-siRNA, non-silencing siRNA.

Figure 3.

Cdc6 in E7-expressing cells is stabilized with bleomycin treatment, and its abundance is not cell cycle related. RPE1 cells expressing E7 or vector were treated with PBS or bleomycin (5 µg/ml for 48h). (A) Cdc6 (upper panel) and E7 (lower panel) levels were examined by immunoblotting. (B) After bleomycin treatment, the cells were incubated with 25 µg/ml cycloheximide (CHX) and harvested at the indicated times. The stability of Cdc6 was monitored using immunoblotting. The data from at least of two independent experiments are shown in the upper panel and summarized in the lower panel. (C) RPE1 E7 cells were blocked with 2.5M thymidine for 16h and then released to regular media and collected at the time points indicated. Cdc6 levels were determined (upper panel), and the cell cycle profile was analyzed. The data from a representative of four independent experiments are shown. AS, asynchronous.

Figure 4.

The localization and subcellular fractionation of Cdc6 in E7-expressing cells. RPE1 cells expressing E7 or vector were treated with bleomycin (3 µg/ml) for 48h. (A) Indirect immunofluorescence microscopy was performed to detect CDC6 and E7 expression and localization (green). 4,5-Diamidino-2-phenylindole dihydrochloride (blue) was used to counterstain the nucleus. Images were captured at ×40 magnification. The data from a representative of at least two independent experiments are shown. (B) The cells were fractionated as cytoplasmic extracts (CEs), soluble nuclear extracts (SNEs) and chromatin-bound fractions (CBEs). The total Cdc6 and Cdc6 S54P levels of each fraction were determined using immunoblotting. β-Tubulin, SP1 and histone H3 were used as loading controls for CE, SNE and CBE, respectively. The data from a representative of three independent experiments are shown in the left panel. The relative levels of total Cdc6 and Cdc6 S54P are summarized in the right panel.

Figure 5.

Cdk1 is involved in Cdc6 stability and polyploidy formation in E7-expressing cells. E7-expressing cells were transfected with 20nM si-Cdk1 or control siRNAs for 72h. (A) Downregulation of Cdk1 increased Cdc6 expression in E7-expressing RPE1 cells (left) or NIKS (right). Cdc6 levels were determined using immunoblotting. (B) Downregulation of Cdk1 induced polyploidy formation in E7-expressing NIKS. The cell cycle profile was analyzed using flow cytometry. The data from a representative experiment of two independent experiments are shown. (C) The Cdk1 inhibitor RO-3306 increased the Cdc6 level and efficiently induced polyploidy formation in RPE1 E7-expressing cells. RPE1 cells expressing E7 or vector were treated with 7.5 µM RO-3306 or dimethyl sulfoxide (DMSO) for 48h. Cdc6 levels were determined using immunoblotting, and the cell cycle profile was analyzed using flow cytometry. The data from at least three independent experiments are shown. NS-siRNA, non-silencing siRNA.