Disruption of the G1/S transition in human papillomavirus type 16 E7-expressing human cells is associated with altered regulation of cyclin E

Abstract

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F.

FIG. 1

Cyclin and CDK protein levels in asynchronous and G₀/G₁-arrested cells. Total-cell lysates were prepared from asynchronous HFKs and HFFs (A and B) or G₀/G₁-arrested fibroblasts (C) expressing the designated viral gene. Proteins (20 μg/lane) were fractionated on individual SDS-PAGE gels (12% polyacrylamide) and transferred to PVDF membranes. The membranes were probed with antibodies, as described in Materials and Methods, and were visualized by enhanced chemiluminescence. (D) The cells were fixed, processed for PI immunofluorescence, and monitored for their position in the cycle by FACScan analysis.

FIG. 2

CKI expression and CDK activity in proliferating cells. (A to C) Whole-cell lysates were prepared from retrovirally transduced asynchronous HFKs. Lysates (20 μg) were resolved on SDS-PAGE gels (12% polyacrylamide) and probed with monoclonal antibodies to p53 and p21 (A), p16 (B), and p27 (C). (D) Extracts were prepared from asynchronous HFKs expressing the indicated HPV oncogenes, immunoprecipitated with antibodies to cyclin A, cyclin E, CDK2, or CDC2 with protein A-Sepharose, washed, and tested for H1 histone kinase activity.

FIG. 3

mRNA expression of cyclins E, D1, and A in primary human keratinocytes. Total-cell RNA was extracted from asynchronous populations of HFKs expressing the high-risk HPV oncogenes. RNA (25, 10, and 5 μg) was used for hybridization with [α-³²P]UTP riboprobes for cyclins E, A and D1, respectively. Protected fragments and the internal loading control 36B4 were run on the same gel and exposed for the same length of time.

FIG. 4

Cell cycle progression following density and serum starvation arrest. HPV-16 E6, E7, cyclin E, and LXSN vector were retrovirally transduced into HFFs, and the cells were G₀/G₁ arrested by a combined growth to density and serum deprivation. The cells were restimulated to enter the cycle by being plated subconfluently in 10% FBS-supplemented media and harvested at 0, 6, 12, 18, 24, 30, and 36 h after release. (A) Cells were processed for PI immunofluorescence and monitored for cell cycle position by FACScan. (B to F) Cyclin E protein levels (B) and associated kinase activity (C) was determined, and protein lysates were probed for p53 (D), p21 (E), and p27 (F).

FIG. 5

Cell cycle arrest following serum starvation. HPV-16 E7- and vector-expressing HFFs were starved of serum for 48 h, restimulated with 10% FBS, and monitored at intervals for 30 h after release. (A to D) The cells were harvested and prepared for FACS analysis (A), protein lysates were immunoblotted for cyclin E (B) and cyclin A (C), and RNA samples were analyzed by RNase protection for cyclins E and A; 36B4 was used as a control (D). (E) RNA levels were quantitated by PhosphorImager analysis.

FIG. 6

Cyclin and CKI levels in different cell cycle compartments. Asynchronous HFKs expressing E7 or vector were harvested at 10⁶ cells/ml, stained with the DNA binding dye Hoechst 33342 (500 μg/ml), and counterstained with PI (100 μg/ml). G₁, S, and G₂/M cell populations were isolated at 6 × 10⁴ cells per phase. Protein lysates were probed with either anti-cyclin E, A, CDK2, p21, or p27 (A), and the amount of cyclin E was quantitated by PhosphorImager analysis (B).

FIG. 7

The CR1 and CR2 domains of E7 are required for increased cyclin E levels. Total-cell lysates were prepared from asynchronous HFK expressing the designated mutated E7 gene product. Proteins (20 μg/lane) were fractionated on SDS-PAGE gels (12% polyacrylamide), transferred to PVDF membranes, probed with the designated antibodies, and visualized by enhanced chemiluminescence.