Examination of the pRb-dependent and pRb-independent functions of E7 in vivo

Abstract

High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRb(DeltaLXCXE)) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the Rb(DeltaLXCXE) mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19(ARF). These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed p19-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins.

FIG. 1.

Proliferation and differentiation in Rb^WT/WT and Rb^Δ/Δ epidermis. Shown are immunohistochemistry and immunofluorescence images of Rb^WT/WT (top row), K14E7Rb^WT/WT (second row), Rb^Δ/Δ (third row), and K14E7Rb^Δ/Δ (bottom row) ear epidermis from 21-day-old mice. Left column: BrdU immunohistochemistry is brown with hematoxylin counterstain. Middle column: immunofluorescence stain for keratin 14 (K14, red) and keratin 10 (K10, green) with DAPI nuclear counterstain. Right column: immunofluorescence stain for filaggrin (red) with DAPI nuclear counterstain.

FIG. 2.

pRb inactivation is required for E7-induced cell cycle progression. Basal and suprabasal keratinocytes in ear epidermal sections from 21-day-old mice were quantified as BrdU positive or BrdU negative for four to six mice per genotype.

FIG. 3.

E7 induces a pRb-independent delay in keratinocyte terminal differentiation. Epidermal hyperplasia was measured as the ratio of suprabasal to basal cells in ear epidermis. (A) E7 induces a major increase in epithelial thickness in Rb^WT/WT mice, and a minor thickening of both Rb^Δ/Δ and Rb^Δ/WT epidermis (P = 0.025 for Rb^Δ/Δ versus E7Rb^Δ/Δ and P = 0.004 for Rb^Δ/WT versus E7Rb^Δ/WT). (B) E7 expression induces more severe hyperplasia than that resulting from pRb loss alone. P < 0.005 for K14CreRb^f/f vs K14E7Rb^f/f. Data are shown from 28-day-old mice; similar results were seen at 21 and 56 days. (C and D) pRb-independent hyperplasia in K14E7 mice results primarily from expansion of the filaggrin-negative suprabasal cell layer. (C) P = 0.028 for Rb^Δ/Δ versus E7Rb^Δ/Δ filaggrin-negative layer. A significant expansion of the filaggrin-negative layer was also seen in Rb^Δ/WT versus E7Rb^Δ/WT (not shown, P = 0.034). (D) P < 0.04 for K14E7Rb^f/f versus K14CreRb^f/f in both filaggrin-negative and filaggrin-positive cell layers.

FIG. 4.

E7 overcomes DNA damage-induced cell cycle arrest in a pRb-dependent manner. We exposed 21-day-old mice to 0 or 5 Gy ionizing radiation, BrdU was injected 24 h later, and mice were sacrificed 1 hour later. BrdU incorporation in ear epidermis was quantified.

FIG. 5.

E7 induces p21 in a pRb-dependent manner. Ear epidermal sections from 21-day-old mice were stained for p21 (brown) and counterstained with hematoxylin. (A) E7 induces p21 in Rb^WT/WT but not Rb^Δ/Δ epidermis. (B) Inactivation of the Rb gene and expression of E7 similarly induce p21 in epidermis.

FIG. 6.

p19- and p53-independent induction of p21. Ear epidermal sections from 8-day-old mice of the indicated genotypes were stained for p21 (A and B) or p53 (C) in mice of the indicated genotypes. Brown precipitate indicates positive staining, with blue hematoxylin counterstain.