RESUMEN

PURPOSE: Loss or dysfunction of trabecular meshwork (TM) cells has been associated with the development of pathologically elevated IOP, and it is conceivable that replacement of damaged TM cells could restore function to the TM. We propose that the use of TM-like cells derived from induced pluripotent stem cells (iPSCs) created from a patient's own dermal fibroblasts offers the best solution to this challenge. Here we demonstrate that mouse iPSCs can be induced to differentiate into TM-like cells suitable for autologous transplantation. METHODS: Directed induction of stem cell differentiation was achieved through coculture of mouse iPSCs with human TM cells for up to 21 days. The resultant TM-like cells (iPSC-TM) were characterized morphologically, immunohistochemically, and functionally. RESULTS: The iPSC-TM cells closely resembled cultured human TM cells morphologically and began to express many markers of TM cells while ceasing to express pluripotency markers such as Nanog, Oct4, and Sox2. Functionally, these cells developed the ability to phagocytose particles. Finally, exposure to dexamethasone or phorbol 12-myristate acetate caused a distinct increase in the production and secretion of myocilin and matrix metalloproteinase-3, respectively, behavior characteristic of TM cells. CONCLUSIONS: Our data demonstrate that iPSCs can be induced to assume a phenotype that resembles native TM cells in many important aspects. Not only do these cells represent a valuable research tool, but transplantation into glaucomatous eyes with elevated IOP may also restore function to the TM, resulting in re-establishment of IOP.

Asunto(s)

Glaucoma/patología , Células Madre Pluripotentes Inducidas/trasplante , Trasplante de Células Madre/métodos , Malla Trabecular/patología , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/patología , Glaucoma/cirugía , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos

RESUMEN

PURPOSE: Primary angle-closure glaucoma (PACG) in dogs is usually caused by the gradual collapse of the iridocorneal angle and cleft, eventually leading to aqueous humor (AH) outflow obstruction. The condition occurs in several breeds of dogs and the prognosis for affected animals is typically poor. We have identified several basset hound (BH) pedigrees, as well as unrelated cases with characteristic PACG that in many aspects recapitulates PACG in human patients. The goal of this study was to utilize the BH PACG model to characterize the genetics of PACG, and potentially discover genetic factors contributing to PACG in humans and animals. METHODS: We conducted a genome-wide logistic regression test for association using 37 PACG cases and 41 unaffected controls. Population stratification and cryptic relatedness were assessed using a multidimensional scaling analysis. The expression of two candidate genes within the target tissues of the BH eye was assessed by immunohistochemistry. RESULTS: We report significant associations at two novel loci, specifically BICF2P31912 in COL1A2 on chromosome 14 with a per-allele odds ratio (OR, 95% confidence interval [CI]) of 3.35 (1.73-6.51), P(genome)=3.6×10⁻4; and BICF2P893476 residing in proximity to RAB22A on chromosome 24 with a per-allele OR (95% CI) of 3.93 (1.78-8.66), P(genome)=4.9×10⁻4. COL1A2 and RAB22A demonstrated widespread expression throughout the eye and were prominently noted in the ciliary body (CB), trabecular meshwork (TM), and iris. CONCLUSIONS: Our finding of two genetic associations supports the potential segregation of PACG risk-conferring variants in the BH. The genetic associations identified may contribute to mechanisms underlying the pathogenesis of PACG, which remain to be elucidated.

Asunto(s)

Enfermedades de los Perros/genética , Sitios Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Glaucoma de Ángulo Cerrado/genética , Glaucoma de Ángulo Cerrado/veterinaria , Alelos , Animales , Cromosomas de los Mamíferos/genética , Enfermedades de los Perros/patología , Perros , Femenino , Glaucoma de Ángulo Cerrado/patología , Humanos , Inmunohistoquímica , Masculino , Linaje , Polimorfismo de Nucleótido Simple/genética

RESUMEN

PURPOSE: The degeneration of retinal ganglion cells (RGC) in the glaucomatous retina is accompanied by activation of the classical complement cascade. The purpose of this study was to evaluate whether complement component C1q binding and activation of the complement cascade in the glaucomatous retina requires the presence of immunoglobulins. METHODS: Experimental glaucoma was induced in normal mice and those carrying a targeted deletion of the RAG1 gene. Binding of C1q to RGC and accumulation of C3 and C5b-9 was investigated using immunohistochemical and proteomic approaches. Damage to the optic nerve and RGC was determined and compared between the two strains. Complement activation and accumulation were also evaluated in vitro using dissociated retinal cell cultures. RESULTS: C1q was detected in the RGC layer in both RAG1(-/-) and control mice with elevated IOP, but not in mice with normal IOP. Proteomic analysis of retinal membrane fractions indicated that C1q and C3 are membrane bound to a similar degree in RAG1(-/-) and control mice with elevated IOP. The absence of Ig does not affect the rate of axonal damage or RGC loss. Furthermore, cultured RGC maintained in serum-free media are also C1q and C3 immunoreactive, demonstrating that Ig is not required for C1q binding to damaged RGC. CONCLUSIONS: Our data demonstrate that lack of immunoglobulins and mature T/B cells does not influence the progression of glaucoma. Furthermore, immunoglobulins do not appear to be required for C1q binding and complement cascade activation on damaged RGC. These findings suggest that C1q recognizes an alternative binding partner expressed by stressed RGC.

Asunto(s)

Complemento C1q/metabolismo , Glaucoma/inmunología , Inmunoglobulinas/metabolismo , Síndromes de Inmunodeficiencia/inmunología , Células Ganglionares de la Retina/inmunología , Animales , Células Cultivadas , Complemento C1q/inmunología , Complemento C3/inmunología , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Glaucoma/metabolismo , Proteínas de Homeodominio/genética , Inmunoglobulinas/inmunología , Inmunohistoquímica , Síndromes de Inmunodeficiencia/metabolismo , Presión Intraocular/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Nervio Óptico/inmunología , Nervio Óptico/metabolismo , Nervio Óptico/patología , Proteómica , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología

RESUMEN

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.

Asunto(s)

Neoplasias de la Mama , Mama/metabolismo , Carcinoma in Situ/patología , Carcinoma Ductal/patología , Carcinoma/patología , Animales , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma Ductal/genética , Carcinoma Ductal/metabolismo , Colágeno , Progresión de la Enfermedad , Combinación de Medicamentos , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Laminina , Ratones , Ratones Desnudos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Procesos Neoplásicos , Proteoglicanos

RESUMEN

Osteopontin (OPN) is a tumor-associated, secreted phosphoprotein that has been implicated in breast cancer progression and metastasis. Research concerning how OPN functions in tumor progression has led to the identification of a limited number of genes that contribute functionally to OPN-induced cellular behaviors. Recent microarray analysis, comparing 21NT breast cancer cells transfected to constitutively overexpress OPN with control cells, revealed hyaluronan synthase 2 (HAS2) to be a gene highly up-regulated in OPN-overexpressing cells. In this study, we further examined the relationship between OPN and HAS2. We show that 21NT OPN-transfected cells express high levels of HAS2, which is associated with increased HA production and matrix retention and is necessary for tumor cell adhesion to bone marrow endothelial cells and anchorage-independent growth. Finally, stable transfection of antisense HAS2 into 21NT cells overexpressing OPN resulted in a reduction in HAS2 expression, HA production, and pericellular retention. Antisense-mediated down-regulation of HAS2 also resulted in a significant decrease in cellular proliferation and colony growth in soft agar. To our knowledge, this is the first report of the ability of OPN to regulate HAS2 expression and HA production in breast cancer cells and further illustrates a unique functional relationship by which enhanced HA production facilitates OPN-mediated cell behaviors.

Asunto(s)

Glucuronosiltransferasa/genética , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Sialoglicoproteínas/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Inducción Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Glucuronosiltransferasa/biosíntesis , Humanos , Hialuronano Sintasas , Ácido Hialurónico/biosíntesis , Osteopontina , Sialoglicoproteínas/metabolismo

RESUMEN

Tumor progression is a multistep process, which enables cells to evolve from benign to malignant tumors. This progression has been suggested to depend on six essential characteristics identified as the "hallmarks of cancer," which include: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Osteopontin (OPN) is an integrin-binding protein that has been shown to be associated with the progression of several cancer types, and to play an important functional role in various aspects of malignancy, particularly tissue invasion and metastasis. Here we studied genes regulated by OPN in a model of human breast cancer using oligonucleotide microarray technology by comparing the gene-expression profiles of 21NT mammary carcinoma cells transfected to overexpress OPN versus mock-transfected control cells. From over 12,000 human genes, we identified 99 known human genes differentially regulated by OPN whose expression changed by at least 1.5-fold and showed statistically significant differences in mean expression levels between groups. Functional classification of these genes into the hallmarks of cancer categories showed that OPN can affect the expression of genes involved in all six categories in this model. Furthermore, we were able to validate the expression of 18/19 selected candidate genes by quantitative real-time PCR, further supporting our microarray findings. This study provides the first evidence that OPN can lead to numerous gene expression changes that influence multiple aspects of tumor progression and malignant growth.

Asunto(s)

Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sialoglicoproteínas/farmacología , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Humanos , Modelos Biológicos , Osteopontina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética

RESUMEN

Osteopontin (OPN) is a glycosylated, secreted phosphoprotein that functions both as a cell attachment and chemotactic factor. Elevated expression of OPN confers enhanced metastatic ability on transformed cells, suggesting that OPN may contribute to the malignant progression of tumors. Migration of mammary carcinoma cells is stimulated by OPN via interactions with integrins and CD44 cell surface receptors. We hypothesized that OPN modulates specific CD44 isoform expression to facilitate breast cancer cell migration. The 21NT tumorigenic human breast cancer cell line was examined for regulation of CD44 expression at both the mRNA and protein levels in response to an engineered increase in OPN expression under CMV promoter control. Significant up-regulation of CD44s isoform mRNA expression was observed, but no change in CD44v6, v8, v9 or v10 mRNA levels. While there were elevated levels of CD44s, v6 and v9 protein at the cell surface, at the level of total cellular protein only CD44s and v6 were markedly increased. This suggests that OPN can regulate CD44 expression at both transcriptional and post-transcriptional (both amount and localization of protein) levels. To validate the functional consequence of OPN regulation of CD44 expression, we demonstrate that OPN-mediated cell migration was reduced by exposure to a anti-pan CD44 antibody, and to anti-CD44v6 and anti-CD44v9 function-blocking antibodies. Our data provide evidence that in 21NT cells OPN enhances CD44s mRNA expression, increases cell surface expression of CD44 variant forms without a change in mRNA levels, and stimulates cell migration.

Asunto(s)

Movimiento Celular/fisiología , Receptores de Hialuranos/genética , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , Sialoglicoproteínas/fisiología , Secuencia de Bases , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Membrana Celular/inmunología , Cartilla de ADN , Citometría de Flujo , Humanos , Metástasis de la Neoplasia , Osteopontina , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa