Anne E. Griep

The PDZ Ligand Domain of the Human Papillomavirus Type 16 E6 Protein Is Required for E6's Induction of Epithelial Hyperplasia In Vivo

ABSTRACT 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 HPVs oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6WT) 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. E6s interaction with the PDZ partners leads to their degradation. Additionally, E6s 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, E6Δ146-151, from the human keratin 14 (K14) promoter. The K14E6Δ146-151 mice exhibit a radiation response similar to that of the K14E6WT mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6Δ146-151 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.

Requirement of PDZ-containing proteins for cell cycle regulation and differentiation in the mouse lens epithelium.

ABSTRACT The roles of PDZ domain-containing proteins such as Dlg and Scrib have been well described for Drosophila; however, their requirement for mammalian development is poorly understood. Here we show that Dlg, Scrib, MAGI1, MAGI3, and MPDZ are expressed in the mouse ocular lens. We demonstrate that the increase in proliferation and defects in cellular adhesion and differentiation observed in epithelia of lenses that express E6, a viral oncoprotein that can bind to several PDZ proteins, including the human homologs of Dlg and Scrib, is dependent on E6s ability to bind these proteins via their PDZ domains. Analyses of lenses from mice carrying an insertional mutation in Dlg (dlggt ) show increased proliferation and proliferation in spatially inappropriate regions of the lens, a phenotype similar to that of lenses expressing E6. The results from this study indicate that multiple PDZ domain-containing proteins, including Dlg and Scrib, may be required for maintaining the normal pattern of growth and differentiation in the lens. Furthermore, the phenotypic similarities among the Drosophila dlg mutant, the lenses of dlggt mice, and the lenses of E6 transgenic mice suggest that Dlg may have a conserved function in regulating epithelial cell growth and differentiation across species.

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 (pRbΔLXCXE) 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 E7s 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 RbΔLXCXE 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 p19ARF. 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.

Disruption of Retinoblastoma Protein Family Function by Human Papillomavirus Type 16 E7 Oncoprotein Inhibits Lens Development in Part through E2F-1

ABSTRACT Complexes between the retinoblastoma protein (pRb) and the transcription factor E2F-1 are thought to be important for regulating cell proliferation. We have shown previously that the E7 oncoprotein from human papillomavirus type 16, dependent upon its binding to pRb proteins, induces proliferation, disrupts differentiation, and induces apoptosis when expressed in the differentiating, or fiber, cells of the ocular lenses in transgenic mice. Mice that carry a null mutation inE2F-1 do not exhibit any defects in proliferation and differentiation in the lens. By examining the lens phenotype in mice that express E7 on an E2F-1 null background, we now show genetic evidence that E7’s ability to alter the fate of fiber cells is partially dependent on E2F-1. On the other hand,E2F-1 status does not affect E7-induced proliferation in the undifferentiated lens epithelium. These data provide genetic evidence that E2F-1, while dispensible for normal fiber cell differentiation, is one mediator of E7’s activity in vivo and that the requirement for E2F-1 is context dependent. These data suggest that an important role for pRb-E2F-1 complex during fiber cell differentiation is to negatively regulate cell cycle progression, thereby allowing completion of the differentiation program to occur.

Cell-autonomous requirements for Dlg-1 for lens epithelial cell structure and fiber cell morphogenesis

Cell polarity and adhesion are thought to be key determinants in organismal development. In Drosophila, discs large (dlg) has emerged as an important regulator of epithelial cell proliferation, adhesion, and polarity. Herein, we investigated the role of the mouse homolog of dlg (Dlg‐1) in the development of the mouse ocular lens. Tissue‐specific ablation of Dlg‐1 throughout the lens early in lens development led to an expansion and disorganization of the epithelium that correlated with changes in the distribution of adhesion and polarity factors. In the fiber cells, differentiation defects were observed. These included alterations in cell structure and the disposition of cell adhesion/cytoskeletal factors, delay in denucleation, and reduced levels of α‐catenin, pERK1/2, and MIP26. These fiber cell defects were recapitulated when Dlg‐1 was disrupted only in fiber cells. These results suggest that Dlg‐1 acts in a cell autonomous manner to regulate epithelial cell structure and fiber cell differentiation. Developmental Dynamics 238:2292–2308, 2009.

Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.

Deregulated cell cycle control in lens epithelial cells by expression of inhibitors of tumor suppressor function

Previous studies have shown that cell cycle proteins such as retinoblastoma protein (pRB) are essential for cell cycle withdrawal in differentiating lens cells. However, little is known about which factors are critical for cell cycle control in the lens epithelial cells. Here we use the K14 promoter to direct expression of E6 and E7, oncogenes from human papillomavirus type 16, which are known to bind and inactivate p53 and pRB, as molecular tools to study cell cycle regulation in the lens epithelium of transgenic mice. Expression of either gene resulted in increased proliferation and apoptosis, and in the case of E6, a unique epithelial phenotype characterized by multilayering and intercellular vacuoles was observed. Lenses from mice expressing E7 mutants, which are defective in inactivating pRB proteins, were normal and the lens phenotype in the E6 mice was p53-independent. Thus, cell proliferation in the lens epithelium is controlled by multiple factors including, but not necessarily limited to, the pRB family.

ROLE OF PAPILLOMAVIRUS ONCOGENES IN HUMAN CERVICAL CANCER : TRANSGENIC ANIMAL STUDIES

Abstract Human papillomaviruses are believed to be etiologic agents for the majority of human cervical carcinoma, a common cancer that is a leading cause of death by cancer among women worldwide. In cervical carcinoma, a subset of papillomaviral genes, namely E6 and E7, are expressed. In vitro tissue culture studies indicate that HPV E6 and E7 are oncogenes, and that their oncogenicity is due in part to their capacity to inactivate cellular tumor suppressor genes. The behavior of E6 and E7 in vitro and the genetic evidence from analysis of human cancers suggest that the E6 and E7 genes play a significant role in the development of cervical cancer. This hypothesis is now being tested using animal models. In this review, we summarize our current knowledge of the oncogenicity of papillomavirus genes that has been generated through their study in transgenic mice.

Requirement for Dlgh-1 in planar cell polarity and skeletogenesis during vertebrate development.

The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP), appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg) plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP). Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.

Studies on the relation of DNA synthesis to retinoic acid-induced differentiation of F9 teratocarcinoma cells☆

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.