RESUMEN

In this study, we tested the hypothesis that ALYREF/THOC4, a poor prognostic factor in different cancer types, has potential as a drug target and prognostic biomarker for retinoblastoma (RB). Immunostaining (IHC), Western blot, and RT-qPCR analyses detected overexpression of ALYREF in the RB cell lines Y79, RB143, WERI-RB1, and RB116. IHC analysis on RB tumor array showed that 11/14 of RB tumors were ALYREF+ to varying degrees, with eight tumors at maximum 3+ intensity. The IHC analysis also detected ALYREF+ cells in normal retina, mainly in the inner nuclear and ganglion cell layer, while some tumor-bearing human eyes were ALYREF+ in the optic nerve suggesting a role in optic invasion/tumor invasion. The expression of ALYREF within the tumor itself, in the optic nerve, as well as in adjacent "normal" retina, suggest that this pattern of expression may lead to ALYREF being a potentially useful prognostic indicator for RB, as it is for other tumors. siRNA knockdown of ALYREF resulted in a 40 % decrease in cell growth in both WERI-RB1 and Y79 cells (p<0.05) and this was associated with decreased expression of mRNAs for the cell proliferation markers Ki67 and PCNA (p<0.005). These results suggest a role for ALYREF in RB cell growth regulation and its potential as both a target and a biomarker for tumor growth inhibition by anti-cancer therapies.

Asunto(s)

Proliferación Celular , Neoplasias de la Retina , Retinoblastoma , Retinoblastoma/patología , Retinoblastoma/metabolismo , Retinoblastoma/genética , Humanos , Neoplasias de la Retina/patología , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/genética , Línea Celular Tumoral , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética

RESUMEN

AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

Asunto(s)

Angiopatías Diabéticas/prevención & control , Ayuno/fisiología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Animales , Bovinos , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Células Cultivadas , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/terapia , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/metabolismo , Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Hipoglucemiantes/farmacología , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ratas , Retina/efectos de los fármacos , Retina/patología , Neuronas Retinianas/efectos de los fármacos , Neuronas Retinianas/metabolismo , Neuronas Retinianas/patología , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirtuina 1/efectos de los fármacos , Sirtuina 1/genética , Sirtuina 1/metabolismo

RESUMEN

Unilateral noise-induced hearing loss reduces the input to the central auditory pathway disrupting the excitatory and inhibitory inputs to the inferior colliculus (IC), an important binaural processing center. Little is known about the compensatory synaptic changes that occur in the IC as a consequence of unilateral noise-induced hearing loss. To address this issue, Sprague-Dawley rats underwent unilateral noise exposure resulting in severe unilateral hearing loss. IC tissues from the contralateral and ipsilateral IC were evaluated for acute (2-d) and chronic (28-d) changes in the expression of 84 synaptic plasticity genes on a PCR array. Arc and Egr1 genes were further visualized by in situ hybridization to validate the PCR results. None of the genes were upregulated, but many were downregulated post-exposure. At 2-d post-exposure, more than 75% of the genes were significantly downregulated in the contralateral IC, while only two were downregulated in the ipsilateral IC. Many of the downregulated genes were related to long-term depression, long-term potentiation, cell adhesion, immediate early genes, neural receptors and postsynaptic density. At 28-d post-exposure, the gene expression pattern was reversed with more than 85% of genes in the ipsilateral IC now downregulated. Most genes previously downregulated in the contralateral IC 2-d post-exposure had recovered; less than 15% remained downregulated. These time-dependent, asymmetric changes in synaptic plasticity gene expression could shed new light on the perceptual deficits associated with unilateral hearing loss and the dynamic structural and functional changes that occur in the IC days and months following unilateral noise-induced hearing loss.

Asunto(s)

Pérdida Auditiva Provocada por Ruido , Colículos Inferiores , Estimulación Acústica , Animales , Vías Auditivas , Plasticidad Neuronal , Ratas , Ratas Sprague-Dawley

RESUMEN

The objective of this work was to develop a systems pharmacokinetics-pharmacodynamics (PK-PD) model that can characterize in vivo bystander effect of antibody-drug conjugate (ADC) in a heterogeneous tumor. To accomplish this goal, a coculture xenograft tumor with 50% GFP-MCF7 (HER2-low) and 50% N87 (HER2-high) cells was developed. The relative composition of a heterogeneous tumor for each cell type was experimentally determined by immunohistochemistry analysis. Trastuzumab-vc-MMAE (T-vc-MMAE) was used as a tool ADC. Plasma and tumor PK of T-vc-MMAE was analyzed in N87, GFP-MCF7, and coculture tumor-bearing mice. In addition, tumor growth inhibition (TGI) studies were conducted in all three xenografts at different T-vc-MMAE dose levels. To characterize the PK of ADC in coculture tumors, our previously published tumor distribution model was evolved to account for different cell populations. The evolved tumor PK model was able to a priori predict the PK of all ADC analytes in the coculture tumors reasonably well. The tumor PK model was subsequently integrated with a PD model that used intracellular tubulin occupancy to drive ADC efficacy in each cell type. The final systems PK-PD model was able to simultaneously characterize all the TGI data reasonably well, with a common set of parameters for MMAE-induced cytotoxicity. The model was later used to simulate the effect of different dosing regimens and tumor compositions on the bystander effect of ADC. The model simulations suggested that dose-fractionation regimen may further improve overall efficacy and bystander effect of ADCs by prolonging the tubulin occupancy in each cell type. SIGNIFICANCE STATEMENT: A PK-PD analysis is presented to understand bystander effect of Trastuzumab-vc-MMAE ADC in antigen (Ag)-low, Ag-high, and coculture (i.e., Ag-high + Ag-low) xenograft mice. This study also describes a novel single cell-level systems PK-PD model to characterize in vivo bystander effect of ADCs. The proposed model can serve as a platform to mathematically characterize multiple cell populations and their interactions in tumor tissues. Our analysis also suggests that fractionated dosing regimen may help improve the bystander effect of ADCs.

Asunto(s)

Efecto Espectador/efectos de los fármacos , Inmunoconjugados/farmacología , Inmunoconjugados/farmacocinética , Modelos Biológicos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica , Humanos , Distribución Tisular

RESUMEN

Retinoblastoma (RB) is an ocular tumor of early childhood. Current treatments attempt to preserve visual function, but may spare chemoresistant tumor cells. One potential therapeutic target for RB is HER2, (ERBB2), expressed in RB in truncated form. In this study, we tested the hypothesis that Her2 DNA and RNA are expressed in RB tumors and adjacent retina. We examined 24 human RB tumors as well as normal-appearing adjacent retinal tissues for Her2 DNA and RNA expression by in situ hybridization. We also examined 28 RB tumors for HER2 protein immunoreactivity. 21/22 RB tumors expressed Her2 DNA and 14/19 tumors expressed Her2 RNA. In 17 paired cases, there were three cases in which Her2 DNA was detected, but not RNA. We also saw Her2 RNA signal in six instances of "normal" adjacent retinal tissue. Heterogeneous HER2 protein expression in specific tumor regions also was confirmed by quantitative HER2 immunohistochemistry. In summary, Her2 DNA and RNA are expressed in many RB tumors, and in some adjacent ocular tissues, with hetereogenous protein expression throughout. These results may provide important insights regarding RB tumor progression, and drug targeting approaches designed to spare the eye, preserve vision and improve quality of life for RB patients.

RESUMEN

Historically, standard enzyme immunohistochemistry has been accomplished with brown (DAB, diaminobenzidine) substrate. This can become problematic in pigmented tissues, such as the retina, where brown pigment of retinal pigmented epithelial (RPE) cells can be easily confounded with brown substrate. Although immunofluorescence detection methods can overcome this challenge, fluorescence may fade over a period of weeks, while enzyme substrates allow for more long-lasting, archival results. In this report, we will describe a high-contrast enzyme immunohistochemistry method ideal for pigmented tissues that utilizes purple (VIP) substrate. We compared brown (DAB) and purple (VIP) substrates in enzyme immunohistochemistry experiments using human retina (paraffin sections) and monkey retinal pigmented epithelial cells (frozen sections), both containing brown pigmented cells. We compared substrates using several primary antibodies against markers that can be detected in the retina, including GFAP, VEGF, CD147 (EMMPRIN), RHO (rhodopsin) and PAX6. Methyl green was used as a counterstain for paraffin sections. A side-by-side comparison between DAB and VIP immunohistochemistry showed excellent contrast between pigmented cells and the purple VIP substrate in both human retinal tissue and monkey pigmented epithelial cells for all of the markers tested. This was a marked improvement over DAB staining in pigmented cells and tissues. For both paraffin sections and frozen sections of pigmented tissues, purple VIP substrate is an excellent alternative to brown DAB substrate and non-permanent immunofluorescence methods.

RESUMEN

Membrane transporters can be major determinants in the targeting and effectiveness of pharmaceutical agents. A large number of biologically important membrane transporters have been identified and localized to a variety of tissues, organs and cell types. However, little is known about the expression of key membrane transporters in the inner ear, a promising site for targeted therapeutics, as well as a region vulnerable to adverse drug reactions and environmental factors. In this study, we examined the levels of endogenous membrane transporters in rat cochlea by targeted PCR array analysis of 84 transporter genes, followed by validation and localization in tissues by immunohistochemistry. Our studies indicate that several members of the SLC, VDAC and ABC membrane transporter families show high levels of expression, both at the RNA and protein levels in the rat cochlea. Identification and characterization of these membrane transporters in the inner ear have clinical implications for both therapeutic and cytotoxic mechanisms that may aid in the preservation of auditory function.

Asunto(s)

Cóclea/metabolismo , Perfilación de la Expresión Génica/métodos , Inmunohistoquímica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , Transporte Biológico , Regulación de la Expresión Génica , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley

RESUMEN

PURPOSE: Endoplasmic reticulum (ER)-resident chaperone protein p58(IPK) plays a vital role in regulation of protein folding and biosynthesis. The goal of this study was to examine the role of p58(IPK) in retinal neuronal cells under normal and stressed conditions. METHODS: Retinal expression of p58(IPK), retinal morphology, apoptosis, ER stress, and apoptotic gene expression were examined in p58(IPK) knockout (KO) and/or wild-type (WT) mice with or without intravitreal injection of N-methyl-D-aspartic acid (NMDA). In in vitro experiments, differentiated R28 retinal neuronal cells transduced with adenovirus encoding p58(IPK) (Ad-p58(IPK)) or control virus (Ad-LacZ) were exposed to tunicamycin (TM) or hydrogen peroxide (H2O2). Levels of ER stress, apoptosis, and cell survival were evaluated. RESULTS: Chaperone protein p58(IPK) is expressed predominantly in retinal ganglion cells (RGC), inner retinal neurons, and the photoreceptor inner segments. Mice lacking p58(IPK) exhibited increased CHOP expression and loss of RGCs with aging (8-10 months). Intravitreal injection of NMDA induced retinal ER stress and increased p58(IPK) expression in WT mice; this resulted in greater ER stress and enhanced RGC apoptosis in p58(IPK) KO mice. In cultured R28 cells, overexpression of p58(IPK) significantly reduced eIF2α phosphorylation, decreased CHOP expression, and alleviated the activation of caspase-3 and PARP. Overexpression of p58(IPK) also protected against oxidative and ER stress-induced cell apoptosis. Furthermore, p58(IPK) downregulated the proapoptotic gene Bax and upregulated the antiapoptotic gene Bcl-2 expression in stressed R28 cells. CONCLUSIONS: Our study has demonstrated a protective role of p58(IPK) in retinal neurons, which may act in part through a mechanism involving modulation of ER homeostasis and apoptosis, particularly under conditions of cellular stresses.

Asunto(s)

Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/genética , Neuronas/metabolismo , ARN/genética , Enfermedades de la Retina/genética , Células Ganglionares de la Retina/metabolismo , Animales , Apoptosis , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP40/biosíntesis , Immunoblotting , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , N-Metilaspartato/toxicidad , Neuronas/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/metabolismo , Células Ganglionares de la Retina/patología

RESUMEN

The R28 retinal precursor cell line was established 20 years ago, originating from a postnatal day 6 rat retinal culture immortalized with the 12S E1A (NP-040507) gene of the adenovirus in a replication-incompetent viral vector. Since that time, R28 cells have been characterized and used for a variety of in vitro and in vivo studies of retinal cell behavior, including differentiation, neuroprotection, cytotoxicity, and light stimulation, as well as retinal gene expression and neuronal function. While no cell culture is equivalent to the intact eye, R28 cells continue to provide an important experimental system for the study of many retinal processes.

Asunto(s)

Técnicas de Cultivo de Célula/historia , Retina/citología , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Historia del Siglo XX , Historia del Siglo XXI , Fármacos Neuroprotectores/farmacología , Neurotoxinas/toxicidad , Ratas , Retina/efectos de los fármacos , Retina/fisiología

RESUMEN

PURPOSE: To evaluate the effect of bevacizumab on the mitochondrial function of human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28) and human microvascular endothelial (HMVEC) cells in culture. MATERIALS AND METHODS: ARPE-19 and R28 cells were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab. The HMVEC cultures were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab or 1 mg/ml of immunoglobulin G (control). Mitochondrial function assessed by mitochondrial dehydrogenase activity (MDA) was determined using the WST-1 assay. RESULTS: Bevacizumab doses of 0.125 to 1 mg/ml for 5 days did not significantly affect the MDA of ARPE-19 cells. Bevacizumab treatment at 0.125 and 0.25 mg/ml (clinical dose) did not significantly affect the MDA of R28 cells; however, 0.50 and 1 mg/ml doses significantly reduced the R28 cell mitochondrial function. All doses of bevacizumab significantly reduced the MDA of proliferating and non-proliferating HMVEC. CONCLUSION: Bevacizumab exposure for 5 days was safe at clinical doses in both ARPE-19 and R28 retinal neurosensory cells in culture. By contrast, bevacizumab exposure at all doses show a significant dose-dependent decrease in mitochondrial activity in both the proliferating and non-proliferating HMVEC in vitro. This suggests a selective action of bevacizumab on endothelial cells at clinical doses.

Asunto(s)

Anticuerpos Monoclonales Humanizados/farmacología , Endotelio Vascular/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Vasos Retinianos/ultraestructura , Inhibidores de la Angiogénesis/farmacología , Animales , Animales Recién Nacidos , Bevacizumab , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Humanos , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Degeneración Macular/patología , Microscopía de Contraste de Fase , Células Fotorreceptoras de Vertebrados/diagnóstico por imagen , Células Fotorreceptoras de Vertebrados/metabolismo , Ratas , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismo , Ultrasonografía , Factor A de Crecimiento Endotelial Vascular

RESUMEN

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs), but little is known about SSEA-5 expression in other primitive tissues (e.g., human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors, cell lines, teratomas, and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors, such as embryonal carcinoma. In tumor cell lines, SSEA-5 was highly immunoreactive in Capan-1 cells, while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs, with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use.

RESUMEN

PURPOSE: Retinoblastoma (RB), an intraocular tumor of childhood, is commonly associated with mutations in the RB1 gene. RB116 is a novel, early passage RB cell line that has not been previously characterized. In this study, we examined RB116 for the expression of RB1 and tested the hypothesis that RB116 cells would express stem cell markers as well as retinal progenitor cell markers. We compared RB116 cells with other well known RB cell lines, including Y79 and WERI-RB27. METHODS: We evaluated expression of RB1 in RB116 cells by sequencing, multiplex ligation-dependent probe amplification, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), western immunoblot, and immunocytochemistry. Next, RB116 cells, along with Y79 and WERI-RB27 cells, were examined for expression of stem cell markers (ABCG2, Nanog, Oct3/4, ALDH1A1) and retinal progenitor markers (PAX6, CHX10) by quantitative immunocytochemistry. Immunocytochemical findings were accompanied by PCR analysis. RESULTS: RB116 cells expressed RB1 at the mRNA and protein levels, with no mutations detected by either sequencing analysis, or gene dosage abnormalities detected by multiplex ligation-dependent probe amplification. The RB1 protein was immunoreactive in RB116 cells with an atypical perinuclear localization. RB116 cells also expressed stem cell markers, with 3%-5% of cells immunopositive for ABCG2, Oct3/4 and ALDH1A1, with at least 18% of cells immunoreactive to Nanog. These findings were confirmed by RT-PCR. Small percentages of RB116 cells also exhibited immunoreactivity to retinal progenitor markers PAX6 (9.8%) and CHX10 (1.2%). Expression of mRNAs for these markers was confirmed by qRT-PCR. CONCLUSIONS: RB116 cells demonstrate RB1 expression accompanied by atypical perinuclear localization. RB116 cells also express primitive stem cell and retinal progenitor cell markers. Further studies on the phenotypes of both RB1-positive and RB1-negative human RB cells may be important in assessing differentiation potential of these cells, as well as designing targeted differentiation therapies.

Asunto(s)

Línea Celular Tumoral/metabolismo , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Células Madre/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular Tumoral/citología , Efecto Fundador , Expresión Génica , Humanos , Inmunohistoquímica , ARN Mensajero/genética , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/metabolismo , Retinoblastoma/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citología

RESUMEN

AIM: To explore the molecular pathophysiology that might explain the epidemiologic association between cigarette smoke and age-related macular degeneration (AMD) by examining the effects of hydroquinone (HQ), a toxic compound present in high concentration in cigarette smoke-related tar, on human retinal pigment epithelial cells (ARPE-19), rat retinal neurosensory cells (R-28), and human microvascular endothelial cells (HMVEC). MATERIALS AND METHODS: ARPE-19, R-28, and HMVEC were treated for 24 h with four different concentrations of HQ (500 µM, 200 µM, 100 µM, 50 µM). Cell viability, caspase-3/7 activation, DNA laddering patterns, and lactate dehydrogenase (LDH) levels were analyzed. RESULTS: At 50 µM HQ, R-28 cells showed a significant decrease in cell viability compared with the dimethyl sulfoxide (DMSO)-treated controls. At the 100-500 µM concentrations, all three cell lines showed significant cell death (P < 0.001). In the ARPE-19, R-28, and HMVEC cultures, the caspase-3/7 activities were not increased at any of the HQ concentration. CONCLUSION: Our findings suggest that the mechanism of cell death in all three cell lines was through non-apoptotic pathway. In addition, neuroretinal R-28 cells were more sensitive to HQ than the ARPE-19 and HMVEC cultures.

Asunto(s)

Endotelio Vascular/efectos de los fármacos , Hidroquinonas/toxicidad , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Supervivencia Celular , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Humanos , Degeneración Macular/inducido químicamente , Degeneración Macular/genética , Mutágenos/toxicidad , Ratas , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/patología

RESUMEN

Tyrosine nitration is an important sequel of cellular signaling induced by reactive oxygen species. Cisplatin is an anti-neoplastic agent that damages the inner ear through reactive oxygen species and by the formation of DNA adducts. This study reveals a correlation between cisplatin-mediated hearing loss and nitroxidative modification of cochlear proteins and is the first to report nitration of Lmo4. Cisplatin induced a dose-dependent increase in hearing loss in Wistar rats. A 10-15-dB decrease in distortion product amplitude and massive loss of outer hair cells at the basal turn of the cochlea was observed 3 days post-treatment after a 16 mg/kg dose. Cisplatin induced nitration of cellular proteins within the organ of Corti, spiral ganglion, and stria vascularis, which are known targets of cisplatin ototoxicity. Nitration of a 76-kDa cochlear protein correlated with cisplatin dose. The nitrated protein was identified as Lmo4 (LIM domain only 4) by MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) mass spectrometry and confirmed by reciprocal immunoprecipitation and immunoblotting. Co-localization of nitrotyrosine and Lmo4 was particularly high in outer hair cell nuclei after cisplatin treatment. Cochlear levels of Lmo4 were decreased in rats treated with cisplatin. In vitro studies supported the repression of Lmo4 in nitroxidative conditions and the induction of apoptosis upon repression of Lmo4. Inhibition of cochlear protein nitration prevented cisplatin-induced hearing loss. As Lmo4 is a transcriptional regulator that controls the choice between cell survival and cell death, these results support the hypothesis that nitration of Lmo4 influences cisplatin-induced ototoxicity.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos/toxicidad , Cisplatino/toxicidad , Cóclea/efectos de los fármacos , Proteínas con Dominio LIM/metabolismo , Nitratos/metabolismo , Animales , Western Blotting , Cóclea/metabolismo , Masculino , Ratas , Ratas Wistar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones Subcelulares/metabolismo

RESUMEN

Ototoxicity is a dose-limiting side effect of chemotherapeutic treatment with cisplatin. In a series of experiments on neonatal rat cochlear organotypic cultures, the extent of damage induced by a broad range of cisplatin treatment concentrations was examined. Paradoxically, it was found that hair cell loss was greater following 48 h exposure to low (10, 50 and 100 µM) versus high (400 and 1000 µM) concentrations of cisplatin; these findings indicate that hair cells possess intrinsic resistance to high levels of extracellular cisplatin. Using cisplatin conjugated to Alexa Fluor 488, it was found that cisplatin is readily taken up by hair cells at low concentrations, but is largely excluded at high concentrations. Recent studies indicate that the major influx of cisplatin into hair cells occurs via the copper transporter, Ctr1, whereas ATP7A and ATP7B are copper pumps responsible for cisplatin sequestration and efflux. Using immunolabeling procedures for these copper trafficking proteins, it was found that Ctr1 and ATP7B were localized in the hair cells, whereas ATP7A showed extensive labeling in the pillar cells in the organ of Corti. Additional experiments confirmed the protective effect of copper sulfate and cimetidine in attenuating cisplatin-induced hair cell loss. However, because neither copper sulfate nor cimetidine provided complete protection against cisplatin, and high levels of copper sulfate itself were found to be ototoxic, it is suggested that future therapeutic efforts may benefit from a combination of pharmacological treatments which seek to not only limit the uptake of cisplatin into cochlear cells but also increase its efflux.

Asunto(s)

Antineoplásicos/toxicidad , Cisplatino/toxicidad , Cóclea/efectos de los fármacos , Células Ciliadas Auditivas/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Animales , Animales Recién Nacidos , Antineoplásicos/metabolismo , Transporte Biológico , Proteínas de Transporte de Catión/metabolismo , Cimetidina/farmacología , Cisplatino/metabolismo , Cóclea/metabolismo , Cóclea/patología , Sulfato de Cobre/farmacología , Transportador de Cobre 1 , ATPasas Transportadoras de Cobre , Citoprotección , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Microscopía Fluorescente , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley

RESUMEN

PURPOSE: To evaluate the effects of Triesence® (TRI), a new preservative-free triamcinolone approved by the U.S. Food and Drug Administration (FDA) for intraocular use, on human retina pigment epithelial (ARPE-19) and rat neurosensory (R28) cells in culture. METHODS: ARPE-19 and R28 cell cultures were treated 24 h with 1,000, 500, 200, or 100 µg/mL of crystalline (cTRI) or 1,000, 500, or 200 µg/mL of solubilized (sTRI). TRI was solubilized by centrifuging the drug, discarding the supernatant containing the vehicle and then resuspending the drug pellet in an equivalent amount of Dimethyl sulfoxide to achieve the same concentration as the commercial preparation. Percentage of cell viability (CV) was evaluated by a trypan blue dye-exclusion assay. The mitochondrial membrane potential (ΔΨm) was analyzed with the JC-1 assay. The caspase-3/7 activity was measured by a fluorochrome assay. RESULTS: In the ARPE-19 cultures, the cTRI caused a decrease in CV at 1,000 µg/mL (13.03±6.51; P<0.001), 500 µg/mL (28.87±9.3; P<0.001), 200 µg/mL (54.93±5.61; P<0.001), and 100 µg/mL (82.53±0.65; P<0.005) compared with the untreated controls (96.98±0.16). In R28 cultures, the cTRI treatment also reduced CV values significantly (P<0.001) for the 1,000 µg/mL (22.73±2.44), 500 µg/mL (34.63±1.91), 200 µg/mL (58.70±1.39), and 100 µg/m (75.33±2.47) compared with the untreated controls (86.08±3.54). Once the TRI was solubilized (sTRI), the CV and ΔΨm remained similar to the untreated controls for both ARPE-19 and R28 cells. The sTRI treatment with 1,000, 500, and 200 µg/mL increased in caspase-3/7 activity in ARPE-19 cells (P<0.01) and in R28 cells (P<0.05) compared with dimethyl sulfoxide equivalent controls. CONCLUSION: The crystalline form of TRI (cTRI) can cause a significant decrease in CV to cultured retinal cells. Once the TRI is solubilized (sTRI), at the same concentrations, the cells remain viable with no decrease in CV or ΔΨm. The sTRI can, however, increase caspase-3/7 activity, thus suggesting some degree of apoptosis.

Asunto(s)

Retina/efectos de los fármacos , Triamcinolona/toxicidad , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Ratas , Retina/citología , Retina/enzimología , Triamcinolona/administración & dosificación

RESUMEN

Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3'-untranslated region (3'UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2-) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3'UTR.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/biosíntesis , Regulación Neoplásica de la Expresión Génica , MicroARNs/biosíntesis , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Luciferasas de Renilla/fisiología , MicroARNs/fisiología

RESUMEN

The current study was aimed at investigating the effect of HIV-1 protein Tat on the retinal neurosensory cell line R28. Exposure of Tat resulted in induction of pro-inflammatory mediators such as CXCL10 and TNF-α in addition to the activation marker GFAP in these cells. Conditioned media from Tat-treated R28 cells was able to induce monocyte migration, an effect that was blocked by CXCR3 antagonist. Complementary studies in the HIV-1 Tat-transgenic mice, showed a complete absence of the nuclear layer and the outer photoreceptor segments of the retina with a concomitant increase in glial activation. These findings lend support to the observation in post-HAART era of increased incidence of immune response-mediated retinal degeneration. These findings have direct relevance to diseases such as immune response uveitis and patients recovering from CMV retinitis.

Asunto(s)

Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/virología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Línea Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL10/biosíntesis , Inmunohistoquímica , Ratones , Ratones Transgénicos , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesis , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología

RESUMEN

Understanding the mechanisms regulating expression of retinal ganglion cell (RGC) specific and axon-guidance genes during development and in retinal stem cells will be critical for successful optic nerve regeneration. Müller glia have some characteristics of retinal stem cells but in mammals have demonstrated limited potential to differentiate into RGCs. Chromatin remodeling through histone deacetylation and DNA methylation are a potential mechanism for silencing genes necessary for neuronal differentiation of glial cells. We investigated DNA methylation as a mechanism for regulating expression of mouse EphA5, one member of a large family of ephrin receptor genes that regulate patterning of the topographic connections of RGCs during visual system development. We analyzed spatial and age-related patterns of EphA5 promoter methylation by bisulfite sequencing and mRNA expression by quantitative RT-PCR in the mouse retina. The CpG island in the EphA5 promoter was hypomethylated in the retina and showed no change in overall methylation with age, despite a decline in EphA5 mRNA expression levels in the adult retina. In the nasal retina of post-natal day 0 mice, there was a modest, but statistically significant increase in methylation. Increased methylation corresponded with lower levels of receptor mRNA expression in the nasal retina. We cloned the EphA5 promoter and found that site-specific differences in methylation could preferentially activate or repress promoter activity in transient transfections of rat retinal progenitor cells (R28) using luciferase assays. In sphere cultures generated by EGF/FGF2 stimulation of conditionally immortalized mouse Müller glia (ImM10), EphA5 promoter was hypermethylated and EphA5 mRNA was not detected. Demethylation using 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation of the EphA5 promoter and re-expression of the EphA5 mRNA. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. The robust up-regulation of EphA5 in ImM10 cells following demethylation suggests that modulation of chromatin structure may be a useful approach for promoting expression of silenced developmental genes and increasing the neurogenic potential of Müller glia.

Asunto(s)

Metilación de ADN , Receptor EphA5/metabolismo , Retina/metabolismo , Animales , Células Cultivadas , Ratones , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Regulación hacia Arriba

RESUMEN

Retinoblastoma (RB), an intraocular tumor of childhood, contains small subpopulation(s) of stem-like cells expressing the ABCG2 drug transporter that can efflux standard chemotherapies. Since chemo-resistant stem-like cells appear to be a driving force in tumor progression and metastasis for a variety of cancers, innovative treatment strategies are necessary to eradicate these rare cell populations. Terminal differentiation, as a means to deplete the pool of stem-like cells in RB, is an intriguing approach to cancer therapeutics. However, the full extent of RB differentiation remains unknown. Differentiation of RB cells has been examined in response to a variety of different agents, including retinoic acid/sodium butyrate, Pigment Epithelial-Derived Factor, as well as Succinylated Concanavalin A. RB cells exhibit morphologic and phenotypic responses to these differentiating agents, although the permanence of these effects is questionable due to reversibility. Further study of differentiation programs may lead to new approaches in the design of strategies to combat the initiation and progression of RB in vivo.

Asunto(s)

Diferenciación Celular , Células Madre Neoplásicas/patología , Retinoblastoma/patología , Antineoplásicos/farmacología , Butiratos/farmacología , Concanavalina A/farmacología , Proteínas del Ojo/farmacología , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Retinoblastoma/fisiopatología , Serpinas/farmacología , Tretinoina/farmacología , Células Tumorales Cultivadas