Copper Uptake in Mammary Epithelial Cells Activates Cyclins and Triggers Antioxidant Response

The toxicologic effects of copper (Cu) on tumor cells have been studied during the past decades, and it is suggested that Cu ion may trigger antiproliferative effects in vitro. However, in normal cells the toxicologic effects of high exposures of free Cu are not well understood. In this work, Cu uptake, the expression of genes associated with cell cycle regulation, and the levels of ROS production and related oxidative processes were evaluated in Cu-treated mammary epithelial MCF10A nontumoral cells. We have shown that the Cu additive is associated with the activation of cyclin D1 and cyclin B1, as well as cyclin-dependent kinase 2 (CDK2). These nontumor cells respond to Cu-induced changes in the oxidative balance by increase of the levels of reduced intracellular glutathione (GSH), decrease of reactive oxygen species (ROS) generation, and accumulation during progression of the cell cycle, thus preventing the cell abnormal proliferation or death. Taken together, our findings revealed an effect that contributes to prevent a possible damage of normal cells exposed to chemotherapeutic effects of drugs containing the Cu ion

Blocking of Connexin-Mediated Communication Promotes Neuroprotection during Acute Degeneration Induced by Mechanical Trauma

Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We analyzed the role of Cx-mediated communication in a focal lesion induced by mechanical trauma of the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas. Cx36 and Cx43 exhibited distinct gene expression and protein levels throughout the neurodegeneration progress. Cx36 was observed close to TUNEL-positive nuclei, revealing the presence of this protein surrounding apoptotic cells. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour after lesion. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma in the retina.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Universidade Federal do ABC (UFABC)Universidade Federal do ABC (UFABC)Universidade de Sao Paulo (USP)Universidade de Sao Paulo (USP

Possible relation between cell coupling and cell cycle in the retina during neurodegeneration.

A sinalização no sistema nervoso pode ocorrer pelo acoplamento direto entre células, via canais de junção comunicantes (JCs). Estes canais permitem a passagem de moléculas de até 1 kDa, e são formados por subunidades proteicas denominadas conexinas (Cxs). A comunicação celular via JCs desempenha um importante papel durante o desenvolvimento e a sinalização visual. Além disso, o acoplamento celular provido pelas JCs tem sido relacionado a processos de sobrevivência/morte celular. Do mesmo modo, ciclinas e cinases dependentes de ciclinas (CDKs), além de seu papel clássico na regulação do ciclo e diferenciação celular, estão envolvidas em processos neurodegenerativos. Estudos recentes têm observado a reentrada no ciclo celular de células neuronais pós-mitóticas em apoptose. Neste contexto, analisamos a expressão gênica e proteica das Cxs e ciclinas em resposta às lesões no sistema visual, especificamente na retina. Estas análises foram realizadas após a indução de trauma mecânico, modelo experimental que permite a visualização do foco, penumbra e áreas adjacentes à lesão. Utilizando técnicas combinadas, como a reação em cadeia da polimerase em tempo real (PCR Real-Time), Western Blot e imuno-histoquímica, avaliamos a expressão espaço-temporal destes genes e as proteínas por eles codificadas, em diferentes tempos pós-lesão. Os resultados da PCR Real-Time revelaram uma ausência de modulação da expressão gênica de Cx36 para os diferentes tempos pós-lesão analisados. A Cx43 mostrou aumento dos transcritos, após três e sete dias pós-lesão. A ciclina D1 e B1 apresentaram modulação significativa após um, três e sete dias pós-lesão. As análises de imuno-histoquímica revelaram uma redistribuição da Cx36 em resposta à lesão em diferentes tempos pós-lesão. A Cx43, por sua vez, apresentou um aumento aparente de sua expressão no foco e zona de penumbra da lesão, nos período de um, três e sete dias pós-lesão. Em retinas, após um e três dias de lesão, a ciclina D1 encontrou-se presente em células próximas ao foco da lesão. Observamos a presença de ciclina B1 no foco da lesão após um dia de lesão. Por meio de análises de Western Blot não foi possível detectar alterações das diferentes proteínas estudadas nas retinas, em períodos variados de exposição à lesão. Os dados deste estudo sugerem que i) possivelmente, as células afetadas pela lesão se encontram acopladas; ii) expressam proteínas reguladoras do ciclo celular. Levando em consideração o conjunto de resultados, sugerimos que é possível induzir ou prevenir a reentrada do ciclo celular em células pós-mitóticas da retina, controlando o acoplamento mediado pelas proteínas formadoras das JC.Communication in the nervous system can occur directly between the cells through structures known as gap junction (GJ) channels. These channels allow the passage of small molecules up to 1 kDa and are composed of protein subunits named connexins (Cxs). Cell communication through GJ plays an important role during the development and visual signaling. Furthermore, cell coupling provided by the GJs has been related to processes of survival/cell death. Similarly, in addition to the classic role of cyclins and cyclins dependent kinases (CDKs) in the cell cycle regulation and differentiation, they are also involved in neurodegenerative processes. Recent studies have demonstrated the reentry of apoptotic post mitotic neurons in the cell cycle. In this context, we analyzed the gene and protein expression of Cxs and cyclins after lesions in the visual system, specifically in the retina. For this purpose, a mechanic trauma was induced in the retina, which represents a model that allows us to visualize the lesion focus, penumbra and adjacent areas. Using combined techniques, such as the real time polymerase chain reaction (real-time PCR), Western blot and immunohistochemistry, we evaluated the spatio-temporal expression of these genes and their encoded proteins at different times post-lesion. The real-time PCR revealed no modulation of the Cx36 gene expression for all the times post-lesion analyzed. Our results showed increase in the Cx43 transcripts one, three and seven days post-lesion. The immunohistochemistry analysis indicated redistribution of Cx36 in response to the lesion in different times. On the other hand, Cx43 presented evident increased expression in the focus and penumbra areas of the lesion one, three and seven days post-lesion. In our experiments we could observe that cyclin D1 is expressed in cells located close to the lesion focus one and three days post-lesion, while cyclin B1 is expressed in these cells only one day post-lesion. The Western blot analysis did not show changes on the protein levels evaluated in this study in any of the post-lesion times analyzed. Data obtained from this study suggest i) the cells affected by the lesion are possibly coupled by GJ; ii) these cells express protein regulators of cell cycle. Altogether, the results indicate that it is possible to induce or prevent the reentry of post mitotic cells of the retina in the cell cycle, by controlling cell coupling provided by GJ

Characterization of neuronal connexins in the post-natal development of rat hippocampus.

Durante o desenvolvimento pós-natal do hipocampo, as junções comunicantes (JC) formadas por conexinas (Cxs) neuronais participam na maturação da circuitaria hipocampal promovendo a regulação da atividade espontânea sincronizada neuronal. Neste estudo investigamos as duas Cxs neuronais mais abundantes no hipocampo, a Cx36 e Cx45, durante o desenvolvimento pós-natal. Identificamos mudanças nos níveis de transcritos e proteicos da Cx36 e Cx45 ao longo deste período. Nossos resultados revelaram que ambas as Cxs neuronais estão presentes nas sub-regiões do hipocampo e que sua distribuição é modulada em função da progressão do desenvolvimento. Atráves da avaliação dos níveis de atividade neuronal identificamos diferenças exercidas pelo bloqueio das JCs em hipocampo de neonatos e na segunda semana de vida. Nossos resultados mostram que as Cxs neuronais são reguladas durante o desenvolvimento pós-natal do hipocampo, assim como sua ação sobre sua excitabilidade, mostrando que as Cxs podem contribuir de forma distinta em periodo específicos do desenvolvimento hipocampal.Gap junctions (GJ) composed of neuronal connexins (Cx) play a significant role in the activity-dependent circuitry maturation promoting modulation of coherent spontaneous neuronal activity. Herein, we evaluate two major hippocampal neuronal Cxs, Cx36 and Cx45 during postnatal development. We identified changes in Cx36 and Cx45 transcript and protein levels during these developmental periods. Interestingly, immunofluorescence analyses showed that Cx36 and Cx45 are located in all hippocampal subregions. Also, neuronal Cx distribution in these sub-regions is modulated throughout postnatal development. Using electrophysiological recording, we identified changes imposed by GJ blocker over the hippocampal activity in neonates and two-week-old rats. Our finds demonstrate a regulation of neuronal Cxs and distinct GJ role in activity modulation during postnatal hippocampal development, which might be essential to physiological processes that govern proper hippocampal development

Assessment of cell viability using quantification of lactate dehydrogenase (LDH) released by lesioned retinas treated with GJ blockers and openers.

<p>Retinas were removed after 1 day of binocular mechanical trauma. One retina was conditioned in medium containing PBS (control) whereas the other was conditioned with pharmacological agents (experimental), which were described to modulate cell coupling, either closing (cabenoxolone, CBX; quinine, Quin) or opening (trimethylamine hydrochloride, TMA) the GJ channels. LDH in the retinal explant medium was measured after 1, 2 and 4 hours of in vitro treatment, using the appropriate kit followed by quantification by spectrophotometric analysis. “Experimental” dots were shown as LDH quantification normalized by the respective control, with the horizontal bars representing the means. In some cases, experimental dots overlapped each other and/or the horizontal bar. We have tested two different concentrations of the pharmacological agents, i.e. 50 and 100 µM. We observed no changes in LDH release in retinal explants treated with (A) CBX 50 µM, (B) Quin 50 µM, (C) CBX+Quin 50 µM and (D) TMA 50 µM. On the other hand, we observed changes in LDH release when retinas were treated with the same drugs with a higher concentration (100 µM). (E) After incubating with CBX 100 µM for 4 h, we detected a significant decrease in the LDH release (−22%, <i>P</i><0.01). (F) Incubation with Quin 100 µM decreased LDH release after 1 h (−31%, <i>P</i><0.01), 2 h (−28%, <i>P</i><0.01) and 4 h (39%, <i>P</i><0.01). (G) CBX 100 µM and quinine 100 µM produced similar results: LDH release decreased after 1 h (−21%, <i>P</i><0.01), 2 h (−21%, <i>P</i><0.01) and 4 h (−16%, <i>P</i><0.01) when compared to controls. (H) We observed no significant changes in LDH release using TMA 100 µM in any of the analyzed time points. *<i>P</i><0.01 in paired T Test (n = 5).</p

TUNEL labeling in retinal explants treated with GJ blockers.

<p>Retinal explants were cultured for 4 hours with (A) PBS, (B) carbenoxolone (CBX), (C) quinine (Quin) and (D) CBX 100 µM + quinine 100 µM. Transverse sections of chick retina explants were submitted to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to characterize apoptotic spatial pattern. In each image it is possible to localize the focus of the lesion (arrows). (E–H) In order to determine whether GJ blockers caused changes in the distribution of apoptotic cells, we have counted the number of TUNEL- positive nuclei located as far as 150 µm away from the focus of the lesion. Values were plotted according to the distance of the focus, and were submitted to linear regression using the least square approach, generating mathematical parameters such as <i>R²</i>, <i>R</i> and also first order equation (<i>y  =  ax + b</i>). (I–L) The same procedure was undertaken using values from pixel analysis. <i>X–Y</i> axis bitmap analysis was used to view the pixel values in numeric format, where values correspond to the brightness of the pixels. (M) Considering the distribution of TUNEL-positive nuclei, the means of angular coefficient from first order equation (<i>a</i>) were calculated for each experimental condition (n = 3). When compared to the control (PBS), the means of <i>a</i> angular coefficient were higher for all evaluated conditions using GJ blockers (CBX, quinine and CBX + quinine). (N) Regarding values from bitmap pixel analysis, we observed that the means of <i>a</i> angular coefficient were higher for quinine and CBX + quinine conditions. *<i>P</i><0.01 and **<i>P</i><0.05 vs. PBS in Newman-Keuls pairwise comparisons after one-way ANOVA. Scale bar: 60 µm.</p

TUNEL labeling in retinas after mechanical lesions.

<p>Transverse sections of lesioned chick retinas were submitted to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, green) to characterize the apoptosis spatial pattern, and counterstained with propidium iodide (PI, red). A–C, after 6 h of the mechanical trauma, we could observe TUNEL-positive nuclei in the focus of the lesion (arrows). D–F, in 24 h-lesioned retinas, we observed a peak in the number of the TUNEL-positive cells, mainly located in the focus of the lesion. These cells were mainly distributed in the inner nuclear layer (INL), but also in the outer nuclear layer (ONL). Notice that some cells located in the lower part of the image were actually from the INL and displaced by the mechanical trauma. G–I, after 3 days, we observed a decrease in the number of TUNEL-positive cells. Moreover, these cells were located away from the focus, in the penumbra area (arrowheads), evidencing the secondary cell death. In 7-day lesioned retinas, we typically observed very few or no TUNEL-positive cells in retinal transverse sections. Scale bar: 60 µm.</p

TUNEL labeling in retinas from eyes injected with GJ blockers.

<p>Right after mechanical trauma in the retina, eyes were injected with (A) PBS, (B) carbenoxolone (CBX), (C) quinine (Quin) and (D) CBX 500 µM + Quin 500 µM. Animals were euthanized 1 day post-lesion, and transverse sections of retinas were submitted to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to characterize apoptotic spatial pattern. In each image it is possible to localize the focus of the lesion (arrows). (E–H) In order to determine whether GJ blockers caused changes in the distribution of apoptotic cells, we have counted the number of TUNEL- positive nuclei located as far as 150 µm away from the focus of the lesion. Values were plotted according to the distance of the focus, and were submitted to linear regression using the least square approach, generating mathematical parameters such as <i>R²</i>, <i>R</i> and also first order equation (<i>y  =  ax + b</i>). (I–L) The same procedure was undertaken using values from pixel analysis. (M) Considering the distribution of TUNEL-positive nuclei, the means of angular coefficient from first order equation (<i>a</i>) were calculated for each experimental condition (n = 3). When compared to the control (PBS), the means of <i>a</i> angular coefficient were higher for Quin 500 µM and CBX 500 µM + Quin 500 µM. (N) Regarding values from bitmap pixel analysis, we observed that the mean of <i>a</i> angular coefficient was higher for retinas from eyes injected with Quin 500 µM. *<i>P</i><0.05 vs. PBS in Newman-Keuls pairwise comparisons after two-way ANOVA. Scale bar: 60 µm.</p

Connexin43 (Cx43) immunolabeling in transverse sections of the chick retina after mechanical lesions.

<p>We determined spatial distribution of Cx43 protein (green) in retinas counterstained with propidium iodide (PI, red) in control retinas(A–C) and after 1- (D–F), 3- (G–I) and 7-days (J–L) of the mechanical lesion. Control retinas showed a pattern of Cx43 distribution similar to the that described in previous studies, with punctate labeling located in the ganglion cell layer (GCL), and fine punctate labeling located in the inner plexiform layer (IPL) and in the outer plexiform layer (OPL). In 1-day lesioned retinas, we observed an increased labeling for Cx43 in the focus of the lesion. Outsized and brighter punctate labeling was seen in the ganglion cell layer, surrounding large nuclei of presumptive ganglion cells (arrows). Notice that some puncta located in the center of the image correspond to labeling in the GCL. In addition, in the upper part of the image, Cx43 labeling was seen in the pigmented epithelium, as described in previous studies. After 3 days, Cx43 punctate labeling was seen in the vicinity of the lesion focus, in the penumbra area, within the GCL (arrowheads), and also in the inner part of the inner nuclear layer. In 7-day lesioned retinas, we observed a dusty punctate labeling in the focus of the lesion, with a disorganized pattern. Away from the focus, the typical connexin punctate immunolabeling was seen in the GCL. Scale bar: 60 µm.</p