Role of Cdk1 in the p53-independent abrogation of the postmitotic checkpoint by human papillomavirus E6

Abstract

Specific types of human papillomavirus (HPV) are strongly associated with the development of cervical carcinoma. The HPV E6 oncoprotein from HPV degrades p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have previously identified a p53-independent function of E6 in attenuating the postmitotic G1-like checkpoint that can lead to polyploidy, an early event during cervical carcinogenesis that predisposes cells to aneuploidy. How E6 promotes cell cycle progression in the presence of p53 and its target, p21, remains a mystery. In this study, we examined the expression of cell cycle-related genes in cells expressing wild-type E6 and the mutant that is defective in p53 degradation but competent in abrogating the postmitotic checkpoint. Our results demonstrated an increase in the steady-state levels of G1- and G2-related cyclins/Cdks in E6-expressing keratinocytes. Interestingly, only Cdk1 remained active in E6 mutant-expressing cells while bypassing the postmitotic checkpoint. Furthermore, the downregulation of Cdk1 impaired the ability of both wild-type and mutant E6 to induce polyploidy. Our study thus demonstrated an important role for Cdk1, which binds p21 with lower affinity than Cdk2, in abrogating the postmitotic checkpoint in E6-expressing cells. We further show that E2F1 is important for E6 to upregulate Cdk1. Moreover, reduced nuclear p21 localization was observed in the E6 mutant-expressing cells. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets.

Importance: HPV infection is strongly associated with the development of cervical carcinoma. HPV encodes an E6 oncoprotein that degrades the tumor suppressor p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have recently demonstrated a p53-independent abrogation of the postmitotic checkpoint by HPV E6 that induces polyploidy. However, the mechanism is not known. In this study, we provide evidence that Cdk1 plays an important role in this process. Previously, Cdk2 was thought to be essential for the G1/S transition, while Cdk1 only compensated its function in the absence of Cdk2. Our studies have demonstrated a novel role of Cdk1 at the postmitotic G1-like checkpoint in the presence of Cdk2. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets.

FIG 1

Expression of postmitotic checkpoint-related proteins in cells expressing HPV E6 and E6 mutant F2V. Total protein extracts from NIKS cells expressing vector, HPV-16 E6, or F2V that were treated with nocodazole (50 ng/ml for 48 h) (A) or DMSO (B) were resolved by SDS-PAGE and blotted with antibodies against p53, Cdk1, Cdk2, Cdk4, cyclin A2, and cyclin B1. β-Tubulin was used as the loading control. Data from a representative experiment of at least three independent samples are shown. Not all experiments were performed at the same time. (C) Quantification of the protein expression levels from the experiment whose results are shown in panel A. (D) Quantification of the protein expression levels from the experiment whose results are shown in panel B. Error bars reflect the standard deviations of the means. NOC, nocodazole. *, P < 0.05; **, P < 0.01.

FIG 2

Kinase activities of postmitotic checkpoint-related proteins. Total protein extracts of NIKS cells or PHKs expressing HPV-16 E6, mutant F2V, or vector were immunoprecipitated with anti-Cdk1, anti-Cdk2, anti-Cdk4, anti-cyclin A2, anti-cyclin B1, anti-cyclin D1, and anti-cyclin E1 antibodies. In vitro kinase assays were performed with the full-length pRb as a substrate. Autoradiograms showing levels of phosphorylated pRb from experiments that are representative of at least three experiments are provided. (A) Kinase activities of cyclins/Cdks in asynchronous NIKS cells. (B) Top, quantification of the kinase activities from the experiment whose results are shown in panel A; bottom, Western blot assay of the immunoprecipitates showing the levels of Cdk1. (C) Kinase activities of Cdk1, Cdk2, and Cdk4 in NIKS cells treated with nocodazole for 48 h. (D) Quantification of kinase activities from the experiment whose results are shown in panel C. (E) Kinase activities of Cdk1 in PHKs with or without nocodazole treatment. (F) Quantification of kinase activities from the experiment whose results are shown in panel E. Error bars reflect the standard deviations of the means. NOC, nocodazole. *, P < 0.05; **, P < 0.01.

FIG 3

Expressions and activities of HPV-16 E6 and F2V in RPE1 cells. (A) Total RNA isolated from RPE1 cells expressing HPV-16 E6, F2V, or vector was subjected to RT-PCR using HPV-16 E6 primers. β-Actin was used as a control. (B) Total protein extracts from RPE1 cells expressing HPV-16 E6, F2V, or vector treated with or without nocodazole were collected, resolved by SDS-PAGE, and blotted with antibodies against p53, p21, and β-tubulin. (C) RPE1-E6, F2V, and vector control cells were treated with DMSO or nocodazole, and the DNA content was detected by flow cytometry. Polyploid cells are indicated as 8C. Results representative of three experiments are shown. (D) Percentages of polyploid cells from three experiments are summarized in a histogram format. Error bars reflect the standard deviations of the means. NOC, nocodazole. *, P < 0.05; **, P < 0.01.

FIG 4

Downregulation of Cdk1 impairs the ability of E6 mutant F2V to induce polyploidy. (A) Total protein extracts from RPE1-vector, RPE1-E6, and RPE1-F2V cells were analyzed by Western blotting for Cdk1. (B) siRNAs efficiently knock down Cdk1 and Cdk2. RPE1-F2V cells were transfected with two siRNAs specific to Cdk1 and Cdk2, respectively, for 36 h, and the levels of Cdk1 and Cdk2 were measured by Western blotting. β-Tubulin was used as a loading control in the experiments whose results are shown in panels A and B. Data from one representative experiment of three are shown. (C, D) Downregulation of Cdk1 impairs the ability of F2V and E6 to induce polyploidy. RPE1-F2V (C) and RPE1-E6 cells (D) were transfected with siRNAs targeting Cdk1 (Cdk1-beck siRNA) or Cdk2 (Cdk2-beck siRNA), and 36 h later, the cells were treated with nocodazole at 50 ng/ml for an additional 48 h and analyzed by flow cytometry. Polyploid cells are indicated as 8C. Results representative of three experiments are shown. (E) Quantification of the percentages of polyploid cells from the experiments whose results are shown in C and D, as well as results from experiments using Cdk1-sp2 siRNA and Cdk2-tetsu siRNA. Error bars reflect the standard deviations of the means. NOC, nocodazole. **, P < 0.01.

FIG 5

Pharmacological inhibition of Cdk1 reduces E6-induced polyploidy. (A) F2V- and E6-expressing NIKS cells were treated with DMSO or 3 μM purvalanol A for 24 h. In parallel experiments, cells were treated with nocodazole for 24 h, followed by treatment with purvalanol A or DMSO for an additional 24 h in the presence of nocodazole before being analyzed by flow cytometry. Polyploid cells are indicated as 8C. Data from one representative experiment of four are shown. (B) Percentages of polyploid cells from the experiment whose results are shown in panel A are summarized in a histogram format. Error bars reflect the standard deviations of the means. Purv A, purvalanol A; NOC, nocodazole. *, P < 0.05; **, P < 0.01.

FIG 6

Role of E2F1 in regulation of Cdk1 in E6-expressing cells. (A and B) Total protein extracts from NIKS (A) and RPE1 (B) cells expressing vector, E6, and F2V were analyzed for E2F1 by Western blotting. β-Tubulin was used as a loading control. (C) RPE1-F2V cells were transfected with siRNA specific to E2F1 for 36 h, and the levels of E2F1 and Cdk1 were measured by Western blotting. β-Tubulin was used as a loading control. Bottom, quantification of the relative protein levels in the experiments whose results are shown in panels A to C.

FIG 7

Cellular localization of the p21 protein in E6 mutant-expressing cells. (A) NIKS cells expressing F2V or vector were treated with nocodazole, and cytoplasmic (C) and nuclear (N) fractions were prepared and immunoblotted with antibodies specific for p21, β-tubulin, GAPDH (cytoplasmic protein marker), or SP1 (nuclear marker). Fifty micrograms of the 800 μg of cytoplasmic proteins and 10 μg of the 80 μg of nuclear proteins were loaded. Data from one representative experiment of three are shown. (B) Quantification of steady-state levels of cytoplasmic and nuclear p21 in F2V or vector cells in the experiment whose results are shown in panel A. Error bars reflect the standard deviations of the means. NOC, nocodazole. **, P < 0.01.