Recapitulation of the effects of the human papillomavirus type 16 E7 oncogene on mouse epithelium by somatic Rb deletion and detection of pRb-independent effects of E7 in vivo

Abstract

Although the human papillomavirus (HPV) E7 oncogene is known to contribute to the development of human cervical cancer, the mechanisms of its carcinogenesis are poorly understood. The first identified and most recognized function of E7 is its binding to and inactivation of the retinoblastoma tumor suppressor (pRb), but at least 18 other biological activities have also been reported for E7. Thus, it remains unclear which of these many activities contribute to the oncogenic potential of E7. We used a Cre-lox system to abolish pRb expression in the epidermis of transgenic mice and compared the outcome with the effects of E7 expression in the same tissue at early ages. Mice lacking pRb in epidermis showed epithelial hyperplasia, aberrant DNA synthesis, and improper differentiation. In addition, Rb-deleted epidermis (i.e., epidermis composed of cells with Rb deleted) exhibited centrosomal abnormalities and failed to arrest the cell cycle in response to ionizing radiation. Transgenic mice expressing E7 in skin display the same range of phenotypes. In sum, few differences were detected between Rb-deleted epidermis and E7-expressing epidermis in young mice. However, when both E7 was expressed and Rb was deleted in the same tissue, increased hyperplasia and dysplasia were observed. These findings indicate that inactivation of the Rb pathway can largely account for E7's phenotypes at an early age, but that pRb-independent activities of E7 are detectable in vivo.

FIG. 1.

Cre-mediated inactivation of pRb in 21-day-old murine epidermis. (A) Schematic of the floxed Rb allele before and after recombination. Cre expression under the K14 promoter limits recombination to stratified squamous epithelia. (B) pRb immunohistochemistry of ear epidermal sections. (C) Quantification of pRb inactivation. Keratinocytes from Rb^f/f or K14CreRb^f/f sections stained for pRb were counted either as pRb positive (brown) or negative (blue).

FIG. 2.

Hyperplasia in E7-expressing and Rb-deleted epidermis. (A) BrdU immunohistochemistry of ear epidermal sections from mice injected with BrdU 1 h prior to sacrifice. (B) Quantification of BrdU incorporation. Basal and suprabasal cells were counted as BrdU positive (brown) or BrdU negative (blue), and the percentage of keratinocytes in each layer incorporating BrdU was calculated (basal cells are in contact with the basement membrane, but suprabasal cells are not). Increases in DNA synthesis in K14CreRb^f/f (Cre-f/f) and K14E7 (E7) basal and suprabasal layers were statistically significant compared to Rb^f/f (f/f) and K14CreRb^f/wt (Cre-f/+) mice (P < 0.03). Differences between K14CreRb^f/f and K14E7 mice were not statistically significant.

FIG. 3.

Disruption of DNA damage-induced cell cycle arrest by E7 expression or Rb deletion. Mice were exposed to 0 or 5 Gy of ionizing radiation 24 h prior to BrdU injection and sacrificed 1 h after BrdU administration. Epidermal sections were stained for BrdU incorporation and counted as in Fig. 2. Only Rb^f/f (f/f) and K14CreRb^f/wt (Cre-f/+) mice exhibited significant decreases in BrdU incorporation in response to ionizing radiation (P < 0.05). Cre-f/f, K14CreRb^f/f mice; E7, K14E7 mice.

FIG. 4.

E7 expression or pRb inactivation disrupts epithelial differentiation. Ear epidermal sections of K14E7, K14CreRb^f/f, and control mice were stained for the basal layer marker K14 (red) and the suprabasal layer marker K10 (green) and then counterstained with DAPI. For K10-stained sections, the white line represents the basement membrane.

FIG. 5.

Increased phenotype severity in K14E7K14CreRb^f/f mice and phenotypic variation between epidermal sites. Hematoxylin-and-eosin-stained sections from dorsal skin and ear of 21-day-old mice are shown. K14E7 and K14CreRb^f/f mice have pronounced hyperplasia in ear epidermis but relatively little hyperplasia in dorsal skin epidermis. K14E7K14CreRb^f/f mice, however, exhibit pronounced hyperplasia at both epidermal sites. Also shown are examples of dysplastic lesions common in K14E7K14CreRb^f/f mice but not seen in other genotypes.

FIG. 6.

Centrosome abnormalities in E7-expressing and Rb-deleted epidermis. Ear epidermal sections from 21-day-old animals were stained for gamma-tubulin and counterstained with DAPI. The fraction of cells containing >2 centrosomes was counted for three to seven mice per genotype. K14E7, K14CreRb^f/f, and K14E7K14CreRb^f/f sections all had increases over Rbf/f sections (P < 0.05), but the former three genotypes did not differ from one another.