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.
Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6 WT ) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6 ⌬146-151 , from the human keratin 14 (K14) promoter. The K14E6 ⌬146-151 mice exhibit a radiation response similar to that of the K14E6 WT mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6 ⌬146-151 mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia.Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital (27) and head and neck cancers (18,25). HPVs encode two oncogenes, E6 and E7, which are thought to contribute to cervical cancers because they are selectively induced in their expression in those cancers. E6 and E7 are multifunctional proteins best known for their abilities to bind and inactivate the p53 and pRb cellular tumor suppressors, respectively. E6 binds in a ternary complex with p53 and a cellular ubiquitin ligase, E6AP (10). This complex results in ubiquitination and degradation of p53 through the proteosome pathway (11). Thus, E6 abrogates p53 function. However, E6's oncogenic activities cannot be explained entirely by its effects on p53. E6's ability to transform cells does not always correlate with its ability to degrade p53; mutants of E6 that cannot induce p53 degradation are still able to immortalize mammary epithelial cells (13, 19), transform 3Y1 rat fibroblasts, and confer tumorigenicity onto 2 mouse fibroblast cells (14). Conversely, some E6 mutations that retain the ability to induce p53 degradation are unable to transform cells (5, 13). Studies with K14E6 WT mice also have identified p53-independent activities. Mice expressing the HPV type 16 (HPV-16) E6 protein in their epidermis (K14E6 WT ) develop epithelial hyperplasia (31). However, this phenotype has not been observed in...
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.
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 ⌬LXCXE ) 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 ⌬LXCXE 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.Human papillomaviruses (HPVs) are small DNA viruses and the causative agents of epithelial warts. The so-called "high-risk" HPVs, including HPV-16, infect the anogenital tract epithelium and are associated with almost all cases of cervical cancer, a leading cause of cancer mortality in women worldwide (64,69). Although the HPV genome usually exists extrachromosomally, many HPV-associated cancers contain HPV genomes integrated into the host DNA (20). These integrated genomes invariably contain intact viral E6 and E7 genes, and integration causes their increased expression (35). These data suggest that E6 and E7 contribute to the development of cervical cancers.HPV-16 E7 is a small nuclear phosphoprotein with potent transforming and tumorigenic properties. Coexpression of E6 and E7 is necessary and sufficient to transform primary human keratinocytes (48), and E7 acts robustly in a number of other in vitro transformation assays (6,44,52,62,63,65). E7-expressing cells exhibit genomic instability in culture (17,18,56,66,67) and have impaired cell cycle arrest responses to DNA damage (12,60). Additionally, expression of E7 in primary human keratinocytes or in transgenic mice results in abnormal centrosome synthesis, with associated multipolar mitoses and aneuploidy (3,(15)(16)(17). Previously, our laboratory generated mice transgenic for HPV-16 E7 under the control of the keratin 14 promoter, targeting E7 expression to the basal layer of stratified squamous epithelia such as the skin and cervical epithelium (30). Th...
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
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