Role of beta-1 integrin in tumor growth and dissemination

SUMMARY Cancer is one of the leading causes of disease-related mortality. In most cases, death is due to the spread of cells from the primary tumor to distant sites causing formation of metastases. To become tumorigenic, cells should acquire ability, including self-sufficiency in growth signals, insensitivity to anti-growth signals, resistance to apoptosis, sustained angiogenesis, limitless replicative potential and tissue invasion and metastasis. Tumor progression depends, in part on the relationship between tumor cells and host tissue stroma, characterized by changes of tumor cell adhesion to their microenvironment and activation of a variety of extracellular proteases that play a role in ECM degradation. integrins are adhesion proteins implicated in tumorigenesis. Their main function is to mediate cell adhesion to the ECM or to other cells and to create a link between the ECM and the cytoskeleton. Tumor cells like normal cells use integrins to attach to ECM, migrate into surrounding tissues and derive survival and growth signals. Integrin-dependent adhesion and migration are thought to play an important role in tumor dissemination. A strategy was designed to address the role of β1 integrin tumor growth and dissemination. Murine mammary carcinoma (TA3) cells were stably transfected with a soluble β1 integrin construct, which is anticipated to play a dominant negative role, being able to associate with different α-subunits expressed on the cell surface but unable to transduce signals to the nucleus. Results from studies based on soluble β1 integrin TA3 transfectants showed that 1) the integrin expression pattern at the cell surface changed with an induction of α2β1 and α5β1 heterodimers; 2) adhesion to collagens, especially collagen I was increased; 3) tumor dissemination after intrape-ritoneal injection in syngeneic mice was abolished and 4) local growth after orthotopic injection was maintained but delayed. Taken together, the data presented here suggest that β1 integrin plays a potentially important role in the regulation of tumor behavior. RESUME Le cancer est une des principales causes de mortalité suite à une maladie. Dans la plupart des cas, la mort est la conséquence de la dissémination de cellules, provenant de la tumeur primaire, dans des endroits distants et causant la formation de métastases. Afin de devenir cancéreuse, une cellule doit acquérir certaines capacités, telles qu'une auto-suffisance en facteurs de croissance, une insensibilité aux facteurs empêchant la croissance cellulaire, une résistance à l'apoptose, une angiogénèse soutenue, un potentiel de réplication illimité et une capacité à pénétrer dans les tissus et à former des colonies métastatiques. La progression d'une tumeur dépend, en partie, de la relation entre les cellules tumorales et les cellules tissulaires de l'hôte. Cette relation est caractérisée par des modifications des cellules tumorales quant à leur adhésion au microenvironnement et à l'activation de protéases qui permettent de dégrader la matrice extracellulaire. Les intégrines sont des protéines impliquées dans le développement tumoral. Leur fonction principale est de réguler l'adhésion des cellules à la matrice extracellulaire, ou à d'autres cellules, et de créer un lien entre cette matrice extracellulaire et le cytosquelette. Les cellules tumorales utilisent également les intégrines pour se lier à la matrice extracellulaire, pour migrer dans les tissus adjacents et pour induire des signaux de croissance et de survie. Ces événements d'adhésion et de migration, qui dépendent des intégrines, jouent un rôle primordial dans la dissémination des cellules cancéreuses. Une stratégie a été élaborée afin de définir le rôle de l'intégrine β1 durant la croissance et la dissémination des cellules tumorales. Des cellules provenant d'un carcinome de la glande mammaire (TA3) ont été transfectées de manière stable avec un vecteur contenant la séquence codante de la partie extracellulaire de l'intégrine β1. L'intégrine tronquée doit être capable de se lier aux sous-unités α exprimées à la surface de la cellule, mais doit être incapable de transmettre un signal à l'intérieur de la cellule. Les résultats obtenus avec les cellules TA3 transfectées contenant l'intégrine β1 soluble montrent que I) le répertoire d'expression des intégrines à la surface de la cellule a changé en faveur des hétérodimères α2β1 et α5β1; 2) l'adhésion aux collagènes, particulièrement au collagène de type I a augmenté; 3) la dissémination des cellules tumorales après une injection intrapéritonéale est empêchée; 4) la croissance tumorale après une injection orthotopique est conservée mais retardée. Ces résultats montrent que l'intégrine β1 joue un rôle primordial dans la régulation du comportement tumoral.

Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.

PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes.

Multi-layer amniotic membrane (MLAM) transplantation for non traumatic corneal perforations or descemetoceles

Purpose:To describe the indications, the surgical procedure and the clinical outcome of MLAM in the treatment of non traumatic corneal perforations and descemetoceles . Methods:A prospective, non comparative, interventional case series of eight consecutive patients (mean age 59 years old, 6 men and 2 women) with non traumatic corneal perforations or descemetoceles.The surgery consisted in a MLAM transplantation of a cryopreservated human amniotic membrane. The series included: three active herpetic keratitis, one rosacea, one perforation of an hydrops, one cicatricial pemphigoid, one perforation after an abcess in a corneal graft and one perforation after protonbeamtherapy. The clinical outcome included: the follow-up, the integrity of the eye, corneal epithelialization, inflammation and neovascularization, and the integration of the MLAM. Stromal thickness was followed precisely with the slit lamp. A corneal graft was performed at one patient after the MLAM, allowing microscopic investigation of the removed MLAM integrated in the cornea. Results:The mean follow-up was 8.78 months (range 3.57 to 30.17). Amniotic membrane transplantation was successful and reduced inflammation in 7 patients out of 8 ,after one procedure.One patient who presented a large herpetic keratitis epithelial defect with corneal anaesthesia had his MLAM dissolved after two weeks with an aqueous leakage. Epithelium healed within 3 weeks above 7 MLAM and remained stable at 3 months in 7 out of 8 patients. MLAM opacification gradually disappeared over a few months, however, stromal layers filling in the corneal perforations or above the descemetoceles remained stable. Conclusions:MLAM transplantation is a safe, effective and useful technique to cure non traumatic corneal perforations and descemetoceles. It can be performed in emergency despite the presence of an active inflammation or infection. By facilitating epithelialization, reducing inflammation and neovascularization, it allows corneal surface reconstruction in patients with persistent epithelial defects and corneal melting that usually ends in a perforation. For full visual rehabilitation, a delayed penetrating keratoplasty is required.

Oligopotent stem cells are distributed throughout the mammalian ocular surface.

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency; treatments range from local medications to corneal transplants, and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals. Identification of slow cycling cells (label-retaining cells) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea; hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in marked opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium. Here we show that the corneal epithelium of the mouse can be serially transplanted, is self-maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. Furthermore, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones) with the capacity to generate individual colonies of corneal and conjunctival cells. Therefore, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. We propose a model that unifies our observations with the literature and explains why the limbal region is enriched in stem cells.

Lineage potential, plasticity and environmental reprogramming of epithelial stem/progenitor cells.

Recent evidence supports and reinforces the concept that environmental cues may reprogramme somatic cells and change their natural fate. In the present review, we concentrate on environmental reprogramming and fate potency of different epithelial cells. These include stratified epithelia, such as the epidermis, hair follicle, cornea and oesophagus, as well as the thymic epithelium, which stands alone among simple and stratified epithelia, and has been shown recently to contain stem cells. In addition, we briefly discuss the pancreas as an example of plasticity of intrinsic progenitors and even differentiated cells. Of relevance, examples of plasticity and fate change characterize pathologies such as oesophageal metaplasia, whose possible cell origin is still debated, but has important implications as a pre-neoplastic event. Although much work remains to be done in order to unravel the full potential and plasticity of epithelial cells, exploitation of this phenomenon has already entered the clinical arena, and might provide new avenues for future cell therapy of these tissues.

Non steroidal anti-inflammatory drugs and COX-2 inhibitors as anti-cancer therapeutics: hypes, hopes and reality.

Non-steroidal anti-inflammatory drugs (NSAIDs) and specific inhibitors of cyclooxygenase (COX)-2, are therapeutic groups widely used for the treatment of pain, inflammation and fever. There is growing experimental and clinical evidence indicating NSAIDs and COX-2 inhibitors also have anti-cancer activity. Epidemiological studies have shown that regular use of Aspirin and other NSAIDs reduces the risk of developing cancer, in particular of the colon. Molecular pathology studies have revealed that COX-2 is expressed by cancer cells and cells of the tumor stroma during tumor progression and in response to chemotherapy or radiotherapy. Experimental studies have demonstrated that COX-2 over expression promotes tumorigenesis, and that NSAIDs and COX-2 inhibitors suppress tumorigenesis and tumor progression. Clinical trials have shown that NSAIDs and COX-2 inhibitors suppress colon polyp formation and malignant progression in patients with familial adenomatous polyposis (FAP) syndrome. Recent advances in the understanding of the cellular and molecular mechanisms of the anti-cancer effects of NSAIDs and COX-2 inhibitors have demonstrated that these drugs target both tumor cells and the tumor vasculature. The therapeutic benefits of COX-2 inhibitors in the treatment of human cancer in combination with chemotherapy or radiotherapy are currently being tested in clinical trials. In this article we will review recent advances in the understanding of the anti-tumor mechanisms of these drugs and discuss their potential application in clinical oncology.

Characterization of the effects of radiation therapy on the tumour microenvironment and their consequences on cancer progression

RESUME La radiothérapie est utilisée avec succès pour le traitement d'un grand nombre de pathologies tumorales (1). Cependant, les récidives post-actiniques sont associées à un risque accru de développer des métastases régionales et à distance (2, 3). La prise en charge de ce type de patients demeure insatisfaisante à l'heure actuelle, principalement parce que les mécanismes physio-pathologiques sous- sous-jacents restent mal compris. Etant donné le rôle primordial du stroma dans la progression tumorale (4) et l'importance des effets de la radiothérapie sur le micro-environnement des tumeurs (5), nous avons émis l'hypothèse que la radiothérapie pouvait engendrer des modifications stromales susceptibles de contribuer à l'émergence d'un phénotype tumoral plus agressif. Nous avons observé que l'exposition préalable d'un environnement tumoral à des radiations ionisantes engendre une inhibition locale et à long terme de l'angiogenèse. Cette inhibition conduit à la création d'un environnement tumoral hypoxique favorisant l'invasion et la métastatisation tumorale. Les mécanismes sous-jacents impliquent l'activation de gènes prométastatiques sous le contrôle du facteur de transcription HIF-1, ainsi que la sélection hypoxique de cellules hautement invasives et métastatiques. Par des analyses de profile d'expression génétique ainsi que par des analyses fonctionnelles, nous avons identifié la protéine matri-cellulaire CYR61 ainsi que ses partenaires d'interaction, les intégrines aVb5/aVb3, comme médiateurs importants de ces effets. De plus, une corrélation significative a également été trouvée entre le niveau d'expression de CYR61 et le taux d'hypoxie dans un grand nombre de carcinomes mammaires chez l'humain. Une association a aussi été observée entre le niveau d'expression de CYR61 et le pronostic de patientes souffrant d'un cancer du sein traité par chimiothérapie adjuvante. Globalement ces résultats identifient l'interaction entre la protéine CYR61 et ses récepteurs aVb5/aVb3 comme un mécanisme important du processus de métastatisation et en font une cible thérapeutique potentielle pour le traitement de patients souffrant d'une récidive tumorale après un traitement de radiothérapie. Finalement, bien que l'inhibition de l'angiogenèse soit locale dans ce cas particulier, nos résultats justifient une surveillance particulière des patients souffrant d'une pathologie tumorale et étant au bénéfice d'un traitement inhibiteur de l'angiogenèse. SUMMARY Radiotherapy is successfully used to treat a large variety of tumours (1 ). However, cancer patients experiencing local recurrent disease after radiation therapy are at increased risk of developing regional and distant metastasis (2, 3). The clinical management of this condition represents a difficult and challenging issue, mainly because the underlying physio-pathological mechanisms remain poorly understood. Given the well established role of the tumour stroma in promoting cancer progression (4) and since radiotherapy is known to persistently alter the tumour microenvironment (5), we hypothesized that ionising radiations may generate stromal modifications contributing to the metastatic spread of relapsing tumours. Here, we report that irradiation of the prospective tumour microenvironment promotes tumour invasion and metastasis through a mechanism of local and sustained impairment of angiogenesis leading to both HIF-1 dependent activation of pro-metastatic genes and hypoxia-mediated selection of highly metastatic tumour cell variants. Through gene expression profiling and functional experiments, we identified the matricellular signalling protein CYR61 and its interaction partners aVb5/ aVb3 integrins as critical mediators of these effects. Furthermore, we found a significant correlation between CYR61 expression and the hypoxic status of a large number of human mammary carcinomas. A positive correlation between increased levels of CYR61 expression and shorter relapse free survival was also identified in breast cancer patients treated with adjuvant chemotherapy. Together, these results identify CYR61 and aVb5/aVb3 integrins as critical mediators of metastasis and potential therapeutic targets to improve outcome in patients with post-radiation tumour recurrences. Finally, although inhibition of angiogenesis is local in this setting, our data warrant close monitoring of tumour progression in patients under anti-angiogenic therapy.

Concepts of metastasis in flux: the stromal progression model.

The ability of tumor cells to leave a primary tumor, to disseminate through the body, and to ultimately seed new secondary tumors is universally agreed to be the basis for metastasis formation. An accurate description of the cellular and molecular mechanisms that underlie this multistep process would greatly facilitate the rational development of therapies that effectively allow metastatic disease to be controlled and treated. A number of disparate and sometimes conflicting hypotheses and models have been suggested to explain various aspects of the process, and no single concept explains the mechanism of metastasis in its entirety or encompasses all observations and experimental findings. The exciting progress made in metastasis research in recent years has refined existing ideas, as well as giving rise to new ones. In this review we survey some of the main theories that currently exist in the field, and show that significant convergence is emerging, allowing a synthesis of several models to give a more comprehensive overview of the process of metastasis. As a result we postulate a stromal progression model of metastasis. In this model, progressive modification of the tumor microenvironment is equally as important as genetic and epigenetic changes in tumor cells during primary tumor progression. Mutual regulatory interactions between stroma and tumor cells modify the stemness of the cells that drive tumor growth, in a manner that involves epithelial-mesenchymal and mesenchymal-epithelial-like transitions. Similar interactions need to be recapitulated at secondary sites for metastases to grow. Early disseminating tumor cells can progress at the secondary site in parallel to the primary tumor, both in terms of genetic changes, as well as progressive development of a metastatic stroma. Although this model brings together many ideas in the field, there remain nevertheless a number of major open questions, underscoring the need for further research to fully understand metastasis, and thereby identify new and effective ways of treating metastatic disease.

The microenvironment in T-cell lymphomas: Emerging themes

Peripheral T-cell lymphomas (PTCLs) are heterogeneous and uncommon malignancies characterized by an aggressive clinical course and a mostly poor outcome with current treatment strategies. Despite novel insights into their pathobiology provided by recent genome-wide molecular studies, several entities remain poorly characterized. In addition to the neoplastic cell population, PTCLs have a microenvironment component, composed of non-tumor cells and stroma, which is quantitatively and qualitatively variable, and which may have an effect on their pathological and clinical features. The best example is provided by angioimmunoblastic T-cell lymphoma (AITL), a designation reflecting the typical vascularization and reactive immunoblastic content of the tumor tissues. In this disease, a complex network of interactions between the lymphoma cells and the microenvironment exists, presumably mediated by the neoplastic T cells with follicular helper T-cell properties. A better understanding of the crosstalk between neoplastic T or NK cells and their microenvironment may have important implications for guiding the development of novel therapies.

Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein.

Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.

Harnessing epithelial stem cell potency

Increase in potency of adult stem/progenitor cells holds great expectations for regenerative medicine; reprogramming is achieved by manipulating the genome or indirectly by manipulating the microenvironment. However, the genetic approach, which can result in lineage conversion up to ground pluripotent embryonic state, will certainly face strict regulatory constraints and consequently translation to the clinic may be difficult. Manipulating stem cell fate without altering the genome of adult stem cells is a promising alternative. My laboratory has demonstrated that non hairy squamous epithelia e.g. the cornea, the oral cavity, the oesophagus, the vagina, contain clonogenic stem cells that can respond to skin morphogenetic signals and form epidermis, cycling hair follicles and sebaceous glands. This capacity is maintained in serial transplantation, crosses primary germ line boundaries and is intrinsic to the stem cells, as cells which have never been exposed to cell culture behave in a similar fashion. Even more surprising, the thymus contains a population of clonogenic epithelial cells of endodermal origin that maintain a thymic identity in culture and have the capacity to incorporate into a thymic network, but can acquire the functionality of bona fide multipotent stem cells of the skin when exposed to proper developmental signals. Thymic epithelial cells exposed to a skin microenvironment exhibit a down-regulation or silencing of transcription factors important for thymic function. Hence, it is possible to reveal unsuspected potency and even to robustly reprogram stem cells by solely manipulating the microenvironment.

The alternatively spliced domain TnFnIII A1A2 of the extracellular matrix protein tenascin-C suppresses activation-induced T lymphocyte proliferation and cytokine production.

Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A-D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.

The zebrafish is an animal model in ophthalmic diseases : Fleck corneal dystrophy and Schorderet-Munier syndrome

RAPPORT DE SYNTHÈSE : Pip5k3 : Pip5k3 is a kinase responsible for fleck corneal dystrophy when mutated. It is a well conserved gene that has only been characterized in human and mouse. Characterization of pip5k3 in zebrafish was necessary before using it as a model. The protein is 70 % similar to the human homologue. The full coding sequence encompasses 6303 by and presented four isoforms. They were differentially expressed during development. All the analyzed organs of the adult zebrafish expressed pip5k3. The adult eye expressed pip5k3 in the cornea, lens, ganglion cell layer (GCL), inner nuclear layer (INL) and outer limiting membrane (OLM). During development, pip5k3 was first uniformly expressed before to be restricted to the head region and to the somites. The expression of pip5k3 in the cornea of the larval eye could make possible the study of fleck corneal dystrophy on this animal. NkxS-3 : NKXS-3 is a transcription factor responsible for a new oculo-auricular syndrome in human when mutated. This recessive disorder is characterized by defects in ear lobule and multiple defects in eye, including microphthalmia and cataract. During development, the zebrafish expressed nkx5-3 in the lens, in the anterior retina and in otic vesicles. Knockdown experiments partially phenocopied the human disease. Microphthalmia and cataract were reproduced, but zebrafish showed also defects in the cartilage of the jaw associated with a microcephaly and fins abnormalities. The retinal cell differentiation was delayed, possibly linked with the delayed expression of at`h5 and crx also observed in morphants. Shh, a regulator of ath5, was normally expressed in morphant. Overexpression of nkx5-3 lead to an anophthalmia, suggesting a role at the early organogenesis of the eye. All the phenotypes observed in morphants and embryos overexpressing nkx5-3 suggest a potential involvement of the FGF and hedgehog signaling pathways.

Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice.

PURPOSE: Despite ubiquitous expression of the keratoepithelin (KE) protein encoded by the transforming growth factor beta induced/beta induced gene human clone 3 (TGFBI/BIGH3) gene, corneal dystrophies are restricted to the cornea, and no other tissues are affected. We investigated the role of TGFBI/BIGH3 in Groenouw corneal dystrophies by generating transgenic mice overexpressing TGFBI/BIGH3 containing the R555W mutation. METHODS: Transgenic animals expressing the Groenouw mutation of human TGFBI/BIGH3 were generated using lentiviral vectors. The line expressed TGFBI/BIGH3 containing the R555W mutation under the control of the phosphoglycerate kinase (PGK) promoter. Expression of the transgene was monitored by Southern and western blotting and by RT-PCR. Electroretinogram analysis was performed and four mice were subjected to complete necroscopy. RESULTS: Transgene expression was observed in different organs although without specific expression in the cornea. The overall morphology of the transgenic animals was not severely affected by KE overexpression. However, we observed an age-dependent retinal degeneration both functionally and histologically. Female-specific follicular hyperplasia in the spleen and increased levels of lipofuscin in the adrenal gland were also seen in transgenic animals. CONCLUSIONS: Cellular degeneration in the retina of transgenic animals suggest that perturbation of the transforming growth factor beta (TGFbeta) family regulation may affect photoreceptor survival and may induce possible accelerated aging in several tissues. No corneal phenotype could be observed, probably due to the lack of transgene expression in this tissue.