Differential regulation of Dlg1, Scrib, and Lgl1 expression in a transgenic mouse model of ocular cancer.

PURPOSE: Discs large (dlg), scribble (scrib), and lethal giant larvae (lgl) are major suppressor genes in Drosophila melanogaster. They encode proteins that regulate cell polarity and cell proliferation in Drosophila and mammals. However, their basic oncogenic roles have not yet been established in mouse epithelial ocular cancer. We evaluated the potential implication of these proteins in tumorigenesis of adenocarcinomas originating from the retinal pigmented epithelium of the Trp1/Tag transgenic mouse model. We examined the changes in the distribution and levels of these proteins in mouse ocular tissues from the Trp1/Tag mouse model. METHODS: The expression patterns of theses genes and their corresponding proteins in normal mouse ocular tissues were studied by in situ hibridization and immunohistofluorescence experiments. In addition, variations in mRNA and proteins levels and protein distributions for Dlg1, Scrib, and Lgl1 were analyzed in the ocular tissues from Trp1/Tag transgenic mouse model by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis, and immunohistofluorescence. RESULTS: We found that mouse Dlg1, Scrib, and Lgl1 are widely distributed in normal ocular tissues, particularly in retinal neurons. We found that the three proteins are mislocalized in retinal layers during ocular carcinogenesis. These mislocalizations were correlated to the early dysplastic stages of ocular tumorigenesis. Additionally, the mislocalization of each protein was associated with its downregulation. Decreased levels of these proteins may be considered as late-stage markers of the disease but also as markers of the invasive stage of this cancerous process. This downregulation may be involved in epithelial-mesenchymal transition in this mouse ocular tumoral model. This would be consistent with the downregulation of E-cadherin and upregulation of N-cadherin expression observed in this model. CONCLUSIONS: This is the first study to demonstrate the involvement of Dlg1, Scrib, and Lgl1 in a mouse with ocular adenocarcinoma and the simultaneous involvement of these proteins in the same cancer. Our results indicate that both the mislocalization and downregulation of these proteins may be involved together in ocular carcinogenesis.

ETS-1 and ETS-2 are upregulated in a transgenic mouse model of pigmented ocular neoplasm.

PURPOSE: Choroidal melanoma is the most common primary malignant ocular tumor in human adults. Relevant mouse models of human uveal melanoma still remain to be developed. We have studied the transgenic mouse strain, Tyrp-1-TAg, to try to gain insight into possible molecular mechanisms common to pigmented ocular neoplasms occurring spontaneously in the eyes of these mice and human choroidal melanoma. The role of two members of the ETS (E26 avian leukemia oncogene) family of transcription factors, ETS-1 and ETS-2, has been investigated in many cancers but has not yet been studied in ocular tumors. METHODS: This is the first study describing the production and distribution of ETS-1 and ETS-2 mRNAs and proteins using in situ hybridization and immunohistochemistry in murine ocular tissue sections of normal control eyes and tumoral eyes from mice of the same age. Using semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blots experiments, we compared changes in ETS-1 and ETS-2 expression, their protein levels, and the regulation of some of their target gene expressions at different stages of the ocular tumoral progression in the transgenic mouse model, Tyrp-1-TAg, with those in normal eyes from control mice of the same age. RESULTS: In normal control adult mouse eyes, ETS-1 was mostly present in the nuclei of all neuroretinal layers whereas ETS-2 was mostly localized in the cytosol of the cell bodies of these layers with a smaller amount present in the nuclei. Both were found in the retinal pigmentary epithelium (RPE). ETS-1 and ETS-2 mRNA and protein levels were much higher in the ocular tissues of Tyrp-1-TAg mice than in control ocular tissues from wild-type mice. This upregulation was correlated with tumor progression. We also demonstrated upregulation of ETS-1 and ETS-2 target expressions in Tyrp-1-TAg mice when comparing with the same target expressions in control mice. CONCLUSIONS: Our findings suggest that ETS-1 and ETS-2 are upregulated in ocular tumors derived from the retinal epithelium and may be involved in one or several signaling pathways that activate the expression of a set of genes involved in ocular tumor progression such as those encoding ICAM-1 (intercellular adhesion molecule-1), PAI-1 (Plasminogen activator inhibitor-1), MCP-1 (monocyte chemoattractant protein-1) and p16 (Cyclin dependent kinase inhibitor 2A).

In vivo adenovirus-mediated delivery of a uPA/uPAR antagonist reduces retinal neovascularization in a mouse model of retinopathy.

Diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Both diseases are characterized by pathological angiogenesis, which severely impairs vision. Extracellular proteinases play important roles in endothelial cell migration during angiogenesis. Amino-terminal fragment (ATF) is an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration. The angiostatic effect of ATF has been demonstrated in models of cancer, but has never been assessed in pathological retinal neovascularization. Endostatin also has angiostatic effects on tumor growth and retinal neovascularization. We used an adenoviral vector carrying the murine ATF (AdATFHSA) or endostatin gene coupled to human serum albumin (HSA) (AdEndoHSA) to increase the half-life of the therapeutic protein in the circulation. We induced retinopathy by exposing 7-day-old mice to high levels of oxygen. They were intravitreally injected with the vectors. Local injection of AdATFHSA or AdEndoHSA reduced retinal neovascularization by 78.1 and 79.2%, respectively. Thus, the adenovirus-mediated delivery of ATFHSA or EndoHSA reduces retinal neovascularization in a mouse model of hypoxia-induced neovascularization.

Prominent beta-5 gene expression in the cardiovascular system and in the cartilaginous primordiae of the skeleton during mouse development.

The alpha v beta (alpha(v)beta5) heterodimer has been implicated in many biological functions, including angiogenesis. We report the beta5 gene expression pattern in embryonic and foetal mouse tissues as determined by Northern blotting and in situ hybridization. During the earliest stages, beta5 mRNA is widespread in the mesoderm. During later developmental stages, it remains mostly confined to tissues of mesodermal origin, although probable inductive effects trigger shifts of beta5 gene expression from some mesenchymatous to epithelial structures. This was observed in the teeth, skin, kidneys, and gut. Of physiological importance is the beta5 labeling in the developing cardiovascular and respiratory systems and cartilages. Furthermore, early beta5 gene expression was observed within the intra- and extraembryonic sites of hematopoiesis. This suggests a major role for beta5 in the hematopoietic and angiogenic stem cells and thus in the development of the vascular system. Later, the beta5 gene was expressed in endothelial cells of the vessels developing both by angiogenesis and vasculogenesis in the lung, heart, and kidneys. Moreover, the beta5 hybridization signal was detected in developing cartilages but not in ossified or ossifying bones. beta5-Integrin is a key integrin involved in angiogenesis, vasculogenesis, hematopoiesis, and bone formation.

Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis.

The RCS rat presents an autosomal recessive retinal pigment epithelium dystrophy characterized by the outer segments of photoreceptors being phagocytosis-deficient. A systematic genetic study allowed us to restrict the interval containing the rdy locus to that between the markers D3Mit13 and D3Rat256. We report the chromosomal localization of the rat c-mer gene in the cytogenetic bands 3q35-36, based on genetic analysis and radiation hybrid mapping. Using a systematic biocomputing analysis, we identified two strong related candidate genes encoding protein tyrosine kinase receptors of the AXL subfamily. The comparison of their expression patterns in human and mice tissues suggested that the c-mer gene was the best gene to screen for mutations. RCS rdy- and RCS rdy+ cDNAs were sequenced. The RCS rdy- cDNAs carried a significant deletion in the 5' part of the coding sequence of the c-mer gene resulting in a shortened aberrant transcript encoding a 20 amino acid peptide. The c-mer gene contains characteristic motifs of neural cell adhesion. A ligand of the c-mer receptor, Gas6, exhibits antiapoptotic properties.

Genomic characterization and embryonic expression of the mouse Bigh3 (Tgfbi) gene.

Mutations in human BIGH3 (TGFB1), a gene identified after treatment of an adenocarcinoma cell line with TGF-beta, have been observed in patients with granular Groenouw type I, Reis-Bücklers, Thiel-Behnke, Avellino, and Lattice type I and IIIa, six autosomal dominant corneal dystrophies linked to chromosome 5q. In order to gain insight into the physiological role of this gene, we characterized the genomic structure of the mouse Bigh3 and its expression in murine embryos. The gene spans 30 kb on mouse chromosome 13 and has 17 exons. Embryonic expression of Bigh3 is observed in the mesenchyme of the first and second branchial arches as early as dpc 11.5 and is particularly strong in the mesenchyme of numerous tissues throughout all the development stages. In fetal eye, the expression is first seen at 11.5 dpc in the mesenchyme surrounding the optic stalk, extends toward the sclera and choroid by 14.3 dpc and reaches the cornea by 17.5 dpc. Because the physiological role of BIGH3/Bigh3 is still largely unknown, embryonic expression in organs like heart, vessels, and intestine may help to identify new functions which could be searched for in patients and in knock-out animal models. The characterization of the murine structure is a prerequisite for the making of such models.

Corneal stromal wound healing in rabbits after 193-nm excimer laser surface ablation.

An argon fluoride excimer laser (193 nm) with a moving slit delivery system was used to perform anterior myopic keratomileusis in both eyes of 24 New Zealand white rabbits. Rabbits were killed immediately after ablation and at intervals up to 100 days. By slit-lamp microscopy, four rabbits at day 100 exhibited four clear corneas and four corneas had central, spotty, subepithelial haze. Light and electron microscopy documented corneal healing. In the early stages a transient acellular zone in the anterior stroma appeared over a period of three weeks, followed by an increased number of fibrocytes. In the corneas with opacification, focal areas of 20-microns-thick subepithelial scarring were present. An unexpected finding was transient damage to posterior stromal keratocytes and endothelial cells. The endothelium produced a layer of granular material that migrated anteriorly across Descemet's membrane. Immunochemistry at day 6 showed a marked staining for collagen IV, proteoglycans, fibronectin, and laminin.