Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways

Abstract

A hallmark of human papillomavirus (HPV) associated carcinogenesis is the integration of the viral DNA into the cellular genome, usually accompanied by the loss of expression of the viral E2 gene. E2 binds to and represses the viral promoter directing expression of the E6 and E7 oncogenes. The re-introduction and expression of exogenous E2 in HPV-positive cancer cells results in cellular growth arrest, while growth in the context of exogenous E2 can be restored through the expression of exogenous E6 and E7. Here we examine the individual contributions of the viral E6 and E7 genes to this phenotype. E6 alone displays moderate activity, whereas both E7 and adenovirus E1A display high activity in reversing E2-mediated cellular growth suppression. Using defined mutants of E7 and E1A, we show that an intact retinoblastoma interaction domain is required for this function. In addition, we show that the E2-mediated growth arrest of HPV-positive cells results in cellular senescence, and implicate the cyclin/cdk inhibitor p21(CIP) as a downstream E2 effector in this phenotype.

Fig. 1. E2-mediated senescence in HPV-positive cell lines. HeLa, U2OS, C33A and Caski cells were co-transfected with expression vectors for the neo resistance gene and either expression vectors for BPV E2-TA, BPV E2-TR or HPV18 E2 or empty SVE vector (vector alone). At 20 days post-selection, the cells were stained for the senescence-specific β-galactosidase marker (SA-βGal) as described in Materials and methods.

Fig. 2. A functional E2 transactivation function is required for senescence induction. HeLa cells were co-transfected with a neomycin selection plasmid and either empty SVE vector (A) or expression vectors for wild-type HPV16 E2 (B), or the mutant HPV16 E2 proteins E39A or I73A (C and D). Senescence-specific staining was performed as described in the legend to Figure 3.

Fig. 3. Viral oncoproteins counteract E2-mediated senescence in HeLa cells. (A) HeLa cells were co-transfected with the neomycin plasmid and BPV E2-TA, either alone (1) or in the presence of wild-type 12S E1A (2), wild-type HPV16 E7 (3), the mutant ΔDLYC E7 (4), wild-type HPV16 E6 (5) or both HPV16 E6 and E7 (6). The cells were selected and stained for SA-βGal activity as described in Figure 1. (B) Cells that assumed the senescence-specific morphology were counted on days (d) 5, 7 and 9 post-selection. Three random fields were counted and averaged for each data point; the standard deviations are as indicated. Each field contained a minimum of 50 cells. The data are expressed as percent enlarged cells relative to total cell number.

Fig. 4. Induction of the cyclin/cdk inhibitor p21^CIP correlates with E2-mediated senescence. (A) HeLa cells were co-transfected with a puromycin selection plasmid and either E2-TA or E2-TR expression vectors. On the next day, the cells were split and placed under puromycin selection (0.4 µg/ml). At days 1–14 post-selection, the cells were harvested for preparation of whole-cell lysates and 50 µg of protein were separated by 12.5% SDS–PAGE. Expression of the p21^CIP protein as well as of p16^INK4A was detected by western blot analysis. (B) HeLa cells were co-transfected with the puromycin resistance plasmid and expression vectors for either BPV E2-TA, BPV E2-TR, HPV18 E2, HPV16 E2 and the two HPV16 E2 mutant proteins I73A and E39A. p21^CIP protein levels were measured on day 3 post-selection as described in (A).

Fig. 5. Expression of p21^CIP causes HeLa cell senescence in the absence of E2. (A) HeLa cells were transfected with the indicated expression vectors using either empty or p21^CIP-expressing RSV vector as well as neo^R plasmid. Two independent experiments are shown, in which the percentage of cells exhibiting the flat cell, senescent morphology was determined at 7 days. The data are expressed as percent flat cells relative to the total cell number. Three random fields were quantitated and averaged; the standard deviations are indicated. Each field contained a minimum of 100 cells. (B) In the same experiment, the SA-βGal-positive cells were counted over an area of 6 cm² at 15 days post-transfection. The average from the two independent experiments is shown by the bars, with the individual data indicated at the top.

Fig. 6. Microinjection of antisense p21^CIP oligonucleotides inhibits E2-mediated HeLa cell senescence. (A) Cells were left uninjected or injected cytoplasmically with E2-TA plasmid alone or in the presense of α-p21^CIP or control morpholino oligonucleotides. FITC-conjugated dextran served as a co-injection marker (left panel). Imaging was performed on day 7 post-injection. (B) Cells exhibiting the enlarged phenotype were counted on day 8 and are expressed as a percentage of the total imaged cells.