Cdc6 contributes to abrogating the G1 checkpoint under hypoxic conditions in HPV E7 expressing cells

Abstract

The human papillomavirus (HPV) plays a central role in cervical carcinogenesis and its oncogene E7 is essential in this process. We showed here that E7 abrogated the G1 cell cycle checkpoint under hypoxia and analyzed key cell cycle related proteins for their potential role in this process. To further explore the mechanism by which E7 bypasses hypoxia-induced G1 arrest, we applied a proteomic approach and used mass spectrometry to search for proteins that are differentially expressed in E7 expressing cells under hypoxia. Among differentially expressed proteins identified, Cdc6 is a DNA replication initiation factor and exhibits oncogenic activities when overexpressed. We have recently demonstrated that Cdc6 was required for E7-induced re-replication. Significantly, here we showed that Cdc6 played a role in E7-mediated G1 checkpoint abrogation under hypoxic condition, and the function could possibly be independent from its role in DNA replication initiation. This study uncovered a new function of Cdc6 in regulating cell cycle progression and has important implications in HPV-associated cancers.

Figure 1

HPV-16 E7 abrogates hypoxia-induced G1 arrest. (a) RPE1 E7 cells were treated with hypoxia mimic drug DFO or CoCl₂ at different concentrations for 72 hours, cell viability was determined using CCK8 assay. Data from a representative of 3 experiments are shown. (b) RPE1 vector and RPE1 E7 cells were incubated in hypoxic chamber at 1% oxygen or with 200 μM DFO for 8 hours, respectively. Cells were stained by PI and examined by flow cytometry. Data from a representative of 4 experiments are shown (Upper panel) and are summarized (Lower panel). (c) RPE1 vector and E7 expressing cells were treated with 200 μM DFO for 6 hours and labeled with 20 nM BrdU for 2 additional hours. Cells were stained with anti-BrdU antibody, counterstained with 7-AAD, analyzed by flow cytometry. Data from a representative of 4 experiments are shown (Upper panel) and the percentage of BrdU-positive cells was gated. Data are summarized (Lower panel). Error bars reflected the standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Hypoxia leads to G1 arrest in HPV-16 E7 knockdown cervical cancer cells. HPV-16 positive CaSki cells were transfected with siRNAs targeting E7 or E6 (siRNA198 or siRNA209). (a) Forty-eight hours later, the steady-state levels of p53 and pRb were examined by Immunoblotting (full-length blots are presented in Supplementary Figure 1). (b) Thirty-six hours post-transfection, the mRNA levels of HPV-16 E6 and HPV-16 E7 were determined by RT-qPCR. (c) Twenty-four hours post-transfection, cells were treated with 200 μM DFO for 8 hours. Cells were stained with PI and analyzed by flow cytometry. A representative experiment of 3 is shown (Upper panel) and summarized (Lower panel). (d) Twenty-four hours after transfection, cells were treated with 200 μM DFO for 6 hours and then labeled with BrdU for 2 additional hours. Cells were stained with anti-BrdU antibody, counterstained with 7-AAD, analyzed by flow cytometry. Data from a representative of 3 experiments are shown (Upper panel) and the percentage of BrdU-positive cells was gated. Mean percentage of BrdU positive CaSki cells are summarized (Lower panel). Error bars reflected the standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Expression of cell cycle-related proteins under hypoxic conditions. The steady-state levels of HIF-1α, p53, p21, p27, Cdk1, Cdk2, Cdk4, Cdk6 and cyclinA in RPE1 cells treated with 200 μM DFO (a) or 1% O₂ chamber (b) were examined by immunoblotting (Left panels) (original blots are presented in Supplementary Figure 2) and quantified (Right panels). A representative of 3 independent experiments is shown. (c) The mRNA level of HIF-1α, Cdk1 and Cdk2 were detected by RT-qPCR. GAPDH was used as a control. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Label-free mass spectrometry quantification of RPE1 vector and E7 expressing cells under hypoxia. (a) RPE1 vector and E7 expressing cells treated with 200 μM DFO were prepared for mass spectrometry. Functional classification of differentially expressed proteins that were quantified by M.S. (b) Expression level of Cdc6 in vector and E7 expressing cells under hypoxia was determined by M.S. (c) Verification of Cdc6 steady-state level in vector and E7 expressing cells treated in hypoxic chamber at 1% oxygen by immunoblotting (original blots are presented in Supplementary Figure 3). (d) Verification of Cdc6 mRNA level in vector and E7 expressing cells treated with 200 μM DFO by RT-qPCR. Error bars reflected the standard error of the mean. *P < 0.05.

Figure 5

Cdc6 is important for E7 expressing cells to alleviate G1 arrest under hypoxia. (a) Cdc6 in RPE1 E7 cells was knocked down by siRNA at indicated doses and analyzed by immunoblotting (Upper panel) (original blots are presented in Supplementary Figure 3) and RT-qPCR (Lower panel, 3 nM siRNA was used). (b) RPE1 E7 cells were transfected with either Cdc6 or negative control siRNA. Cells were treated with 200 μM DFO 24 hours post-transfection and cultured for an additional 8 hours, stained with PI and analyzed by flow cytometry. A representative experiment of 3 is shown (Upper panel) and summarized (Lower panel). (c) RPE1 E7 cells were transfected with either Cdc6 or negative control siRNA. Twenty-four hours later, cells were treated with 200 μM DFO for 6 hours and then labeled with BrdU for 2 additional hours. Cells were stained with anti-BrdU antibody, counterstained with 7-AAD, analyzed by flow cytometry. Data from a representative of 3 experiments are shown (Upper panel). Data are summarized (Lower panel). Error bars reflected the standard error of the mean. *P < 0.05; **P < 0.01.