Tumor suppressor proteins are believed to play a role in regulating cell cycle control during mammalian development. The E6 and E7 oncoproteins from human papillomavirus type 16 are known to affect cell growth control, at least in part, through their inactivation of cellular tumor suppressor gene products, p53 and Rb, respectively. Therefore, these viral proteins can serve as trans-dominant repressors of tumor suppressor gene function. To study the potential role of p53 and Rb in murine lens morphogenesis, we generated transgenic mice in which the expression of E6 or E7 was directed to the developing lens. Transgenic mice expressing E7 exhibited microphthalmia and cataracts, whereas transgenic mice expressing E6 exhibited cataracts without noticeable microphthalmia. Microscopic analysis of the lenses from neonatal and adult E7 transgenic mice revealed inhibition of lens fiber cell differentiation, induction of cell proliferation in spatially inappropriate regions of the lens, and apoptosis. Transgenic mice expressing a mutant E7 that is defective in Rb/pl07 binding exhibited normal eyes, suggesting that the activity of Rb and/or Rb-like proteins is required for the perturbation of lens development and induction of apoptosis in E7 mice. Microscopic analysis of lenses from E6 neonatal and adult transgenic mice indicated the presence of nuclei in elongated fiber cells, suggesting that E6 inhibits lens fiber cell denueleation. Furthermore, expression of E6 inhibited the apoptotic-like DNA degradation observed in the lenses of nontransgenic 15.5-day embryos. In lenses from neonatal E6 x E7 double transgenie mice, the level of apoptosis was reduced compared with that seen in lenses from neonatal E7 mice. In adult E6 x E7 double transgenic mice, lens tumors developed, whereas in E6 or E7 only transgenic mice, tumors did not. Taken together, these results point to specific roles in lens morphogenesis for Rb and p53 and to tile necessity of these tumor suppressor gene products in regulating exit from the normal cell division cycle in differentiating lens fiber cells.

Programmed cell death, or apoptosis, is a critical event in the development of multicellular organisms, and its perturbation is implicated in many diseases including cancer. The tumor suppressor protein p53 is known to mediate apoptosis induced by the DNA tumor virus oncoproteins, adenovirus EIA (AdE1A) and SV40 T antigen (SV40 Tag). We have recently demonstrated that the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) modulate apoptosis when expressed in the lens of transgenic mice. In this study we have identified the pathways that mediate E7 induction and E6 inhibition of apoptosis during different stages in the development of the lens. E7 transgenic mice made p53-null were only partially rescued in their apoptotic phenotype, indicating that both p53-dependent and -independent pathways mediate E7-induced apoptosis in the lens. The E6 transgene and p53-null genotype acted additively to reduce levels of apoptosis induced by E7 in neonatal lenses, indicating that E6 modulates apoptosis at least in part through p53-independent mechanisms. The partial reduction in E7-induced apoptosis by the p53-null genotype correlated with an increased incidence of lens tumors in adult E7 transgenic mice. Analyses of embryonic lenses at E13.5, E15.5, and E17.5 revealed a temporally distinct activation of p53-dependent and -independent apoptosis in the E7 lens. During the early stages of lens development, apoptosis was highly p53-dependent, whereas at later stages, apoptosis occurred through both p53-independent and -dependent pathways. This later time correlates temporally with the time of normal fiber cell denucleation, which can be inhibited by E6 through a p53-independent mechanism. These data suggest a similarity between the mechanism regulating E7-induced, p53-independent apoptosis and the apoptotic-like developmental process of fiber cell denucleation, and the mechanisms through which E6 suppresses both processes.

Apoptosis induced by the p53 tumor suppressor can attenuate cancer growth in preclinical animal models. Inactivation of the pRb proteins in mouse brain epithelium by the T121 oncogene induces aberrant proliferation and p53-dependent apoptosis. p53 inactivation causes aggressive tumor growth due to an 85% reduction in apoptosis. Here, we show that E2F1 signals p53-dependent apoptosis since E2F1 deficiency causes an 80% apoptosis reduction. E2F1 acts upstream of p53 since transcriptional activation of p53 target genes is also impaired. Yet, E2F1 deficiency does not accelerate tumor growth. Unlike normal cells, tumor cell proliferation is impaired without E2F1, counterbalancing the effect of apoptosis reduction. These studies may explain the apparent paradox that E2F1 can act as both an oncogene and a tumor suppressor in experimental systems.

Certain "high-risk" anogenital human papilomaviruses (HPVs) have been associated with the majority of human cervical carcinomas. In these cancers, two papillomaviral genes, E6 and E7, are commonly expressed. In this study we provide evidence that expression of the E6 and E7 genes from the high-risk HPV-16 in the skin of transgenic mice potentiated the development of preneoplastic lesions, and a high percentage of these epidermal lesions subsequently devel- In Situ Hybridization. In situ hybridizations were performed as described (12). The cRNA probes for type I epidermal-specific transglutaminase and al type IV collagen were synthesized in vitro from the template DNAs, pTG13 (13) and pCIV 1225 (14), respectively, using UTP [a-35S]. The E6 and E7 open reading frames from HPV-16 were independently cloned into pGEM3Z and each was used to generate cRNA probe, incorporating UTP [a-35S] and CTP [a-35S], and then combined for hybridization. Slides were exposed for 7 days at 4°C in the dark before developing.Single-Strand Conformation Polymorphism (SSCP) Analysis. For SSCP analysis, RNA/PCR products were generated using [a-32PJdATP during amplification, and products were resolved by gel electrophoresis using 0.5 x MDE gel mix with 0.5x Tris/borate/EDTA buffer and 10% glycerol (AT Biochem, Malvern, PA). RESULTS Incidence of Abnormal Skin and Skin Tumors in aAHPV16E6/E7 Transgenic Mice. In characterizing the patterns of transgene expression in tissues from neonatal aAHPV16E6/E7 transgenic mice, we noted expression of E6 and E7 in several nonlenticular tissues, particularly in one line of mice (11). Upon breeding this line of mice, line 19, we noted the frequent appearance of abnormal-looking skin, appearing at ==3 months of age. The incidence of abnormal skin was higher and appearance was earlier in mice homozygous for the transgene than in hemizygous mice (Table 1, 48% Abbreviations: HPV, human papillomavirus; SSCP, single-strand conformation polymorphism; PCNA, proliferating cell nuclear antigen; FITC, fluorescein isothiocyanate.§To whom reprint requests should be addressed.5583