In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein / Células da glândula mamária ovina cultivadas in vitro expressam beta-lactoglobulina e beta-caseina

The expression of milk proteins in vitro is essential to exploit the mammary gland cells as a biological model. Enzymatic tissue disaggregation has been widely used to establish mammary cell culture, but its effect in long-term ovine mammary cell culture is not completely elucidated. This study aimed at comparing mechanical/enzymatic and mechanical dissociation methods to establish ovine mammary cell culture. We compared cellular differentiation induced by lactating ewe serum or fetal bovine serum based on the gene expression levels of milk proteins (beta-lactoglobulin, alpha s1-casein, and betacasein). Mechanically dissociated cells were positive immunostaining for cytokeratin 8.13, such as mammary epithelial cells. These cells are responsible for milk protein expression and they are low immunostaining for vimentin, mesenchymal marker. Mechanical/enzymatic dissociation cells were positive for vimentin. The fastest cell growth (cell/hour) was observed in the mechanical dissociation group cultured with 10% fetal bovine serum medium. Mechanically and mechanically/enzymatically derived cells were able to express beta-casein and beta-lactoglobulin, but not alpha s1-casein. The relative expression of beta-lactoglobulin was not affected by the tissue dissociation method or culture media, beta-casein relative expression was down regulated in mechanically dissociated cells cultured in the presence of lactating ewe serum, (P = 0.019). Beta-casein relative expression was also down regulated in mechanically/enzymatically dissociated cells cultured with fetal bovine serum (P = 0.021). In the present conditions, we conclude that mechanical dissociation followed by culture with 10% of fetal bovine serum was the most efficient method to induce milk proteins' mRNA expression by ovine mammary epithelial cells in vitro.(AU)

A expressão in vitro de proteínas do leite é essencial para explorar as células da glândula mamária como um modelo biológico. A desagregação tecidual via enzimática é amplamente utilizada para o estabelecimento cultivo de células mamárias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamária ovina ainda não é bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamária ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseína e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamárias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseína. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamárias ovinas in vitro.(AU)

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND: Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMEC US) or Swiss Holstein-Friesian (bMEC CH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA. RESULTS: The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS: The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Poros Nucleares y Función Celular en Epitelio Mamario / Nuclear Pores and Cellular Function

El presente artículo tiene como objetivo central evidenciar la interesante relación que se establece entre la función celular y el número de poros nucleares, relación que modula el activo intercambio nucleo-citoplasmatico en distintas etapas del ciclo celular de la estirpe HC11.

The main objective of this article is related to the study of different existing relationships between cellular function and the number of nuclear pores in order to explain the amount of nuclear-cytoplasmatic exchange through HC11 cell cycle stages.

El factor de crecimiento epidérmico y la diferenciación celular del epitelio mamario / Epidermal growth factor and mammary epithelial differentiation

El objetivo de este artículo es presentar una revisión relativa a evidenciar las características generales, estructura y funcionalidad de los factores de crecimiento con especial énfasis en precisar el rol que ejerce el factor de crecimiento epidérmico (EGF) como agente generador del proceso de diferenciación celular en el epitelio mamario.

The objective of this article is to present a review referred to general characteristics, such as structure and functionality of the growth factors, particularly those related to the Epidermal Growth Factor (EGF), as a responsible generator agent of cell differentiation at the mammary epithelial level.

Efeito da trimegestona sobre o tecido mamário de ratas castradas / Effect of trimegestone on mammary gland of castrated rats

OBJETIVO: Avaliar o efeito da trimegestona sobre a proliferação celular do tecido mamário de ratas castradas. MÉTODOS: Foram utilizadas 45 ratas adultas e virgens, da linhagem Wistar, submetidas à castração. Após o 60º dia da castração, confirmado o hipoestrogenismo, os animais foram divididos aleatoriamente em três grupos, conforme o tratamento proposto: controle (n=15) recebeu soro fisiológico 0,9 por cento; estrogênio (n=15) recebeu 17 beta-estradiol; e combinado (n=15) recebeu 17 beta-estradiol associado à trimegestona, todos por 60 dias consecutivos. Após o término do tratamento, procedeu-se a exérese das mamas inguinais, destinadas a análise morfométrica pela coloração de hematoxilina e eosina (HE) e imuno-histoquímica pela quantificação do anticorpo anti-PCNA no tecido mamário, seguido de eutanásia. Os parâmetros morfométricos avaliados foram: proliferação celular epitelial, atividade secretora e alteração do estroma mamário. Ocorreram nove óbitos durante o experimento. As variáveis foram submetidas à análise estatística adotando-se como significante p<0,05. RESULTADOS: Foram observadas alterações histológicas em 16/36 ratas, hiperplasia epitelial leve em 13/36, hiperplasia epitelial moderada em 3/36, não sendo encontrada hiperplasia epitelial severa. Encontrou-se fibrose no estroma em 10/36 e atividade secretora em 5/36 das ratas. Todas as variáveis do estudo morfométrico foram significantes comparando-se os grupos controle e estrogênio (p=0,03), e nenhuma foi significante na comparação dos grupos controle e combinado (p=0,4). A análise imuno-histoquímica não mostrou diferença entre os grupos. CONCLUSÃO: Os hormônios usados em ratas castradas aumentaram a proliferação de células mamárias, tanto o 17 beta-estradiol isolado quanto associado à trimegestona, porém este efeito parece ser menor quando se emprega a associação, o mesmo ocorrendo em relação à fibrose do estroma mamário.

PURPOSE: To evaluate the efect of trimegestone on the histological changes of the mammary tissue of castrated rats. METHODS: Forty-five virgin female Wistar rats were used after oophorectomy. Sixty days after surgery, with hypoestrogenisms confirmed, the experimental rats were randomly assigned to three groups of 15 animals each, when then the specific treatment for each group was started. The control group (C) and experimental groups 1 and 2 respectively received 0.9 percent saline solution, 17-beta-estradiol and 17-beta-estradiol in combination with trimegestone for 60 consecutive days. After the end of treatment , the inguinal mammary glands were removed, stained with hematoxylin and eosin (HE) for morphometry and examined by immunohistochemistry for the quantification of anti-PCNA antibody in the mammary tissue, followed by euthanasia. The morphometric parameters evaluated were: epithelium cell-proliferation, secretor activity and mammary stroma changes. There were nine deaths during the experiment. The variables were submitted to statistical analysis adopting the 0.05 level of significance. RESULTS:Histological changes were observed in 16/36 rats, mild epithelial hyperplasia in 13/36, moderate epithelial hyperplasia in 3/36, with no cases of severe epithelial hyperplasia. Stromal fibrosis was found in 10/36 and secretory activity in 5/36 rats. All morphometric variables were significant in the estrogen group compared to control (p=0.0361), although there were no difference between the group receiving combined treatment and the controls (p=0.405). The immunohistochemical analysis showed no difference between groups. CONCLUSIONS:The hormones administered to castrated rats, i.e., 17 beta-estradiol alone or in combination with trimegestone, increased the proliferation of breast cells, but this effect appeared to be lower in the combined treatment, the same occurring regarding fibrosis of the mammary stroma.

Variaciones nucleolares en la transformación celular del epitelio mamario / Nucleolar variations in the cell transformation of mammary epithelium

Antecedentes: En la medida que las células pertenecientes a la línea HC11 son transfectadas con el onco-gén ras, asumen distintas propiedades resultando en tipos celulares transformados, modificando tanto sus componentes como sus funciones celulares, los cuales pueden ser cuantificadas mediante técnicas morfométricas. Objetivo: Evidenciar en términos cuantitativos y morfológicos las variaciones experimentadas por los nucleolos pertenecientes a células mamarias de la línea HC11 con el decorrer del mecanismo de transformación celular. Método: Se estudió a nivel de la microscopia electrónica de transmisión los tipos celulares en proceso de transformación (Q6 GM), en comparación con células francamente neoplásicas (Q6 IM), cuantificando variaciones de los nucleolos y su relación con estructuras involucradas en síntesis proteica. Resultados: Se evidencian diferencias estadísticamente significativas en el área, volumen y longitud entre los nucleolos pertenecientes a estos tipos celulares. Conclusión: Las células del epitelio mamario en proceso neoplásico presentan un notable aumento de sus nucleolos y sus ribonucleoproteínas, constituyentes que generarán básicamente ribosomas libres, que sintetizarán proteínas para ser utilizadas en el decorrer de las mitosis sucesivas y desreguladas.

Background: As cells belonging to the HC11 line become transfected with the ras oncogene, they assume different properties resulting in transformed cell types, with modified cell components and functions. These may be quantified by morphometric techniques. Objective: To provide quantitative and morphological evidence of the variations occurring in the nucleoles of HC11 line mammary cells as the cell transformation mechanism takes its course. Method: Transmission electron microscopy was used to study cell types in the transformation phase (Q6 GM) as compared with frankly neoplastic cells (Q6 IM), quantifying the variations between the nucleoles and their relation to structures involved in protein synthesis. Results: Statistically significant differences were found between the nucleoles belonging to these cell types with respect to area, volume and length. Conclusion: The nucleoles of mammary epithelial cells in the process of neoplasia, present a notable increase, and their constituent ribonucleoproteins will basically generate free ribosomes, synthesising proteins available for use in successive, unregulated mitosis.

Curcuminoid-phospholipid complex induces apoptosis in mammary epithelial cells by STAT-3 signaling

Curcumin (from the rhizome of Curcuma longa) is well documented for its medicinal properties in Indian and Chinese systems of medicine where it is widely used for the treatment of several diseases. Epidemiological observations are suggestive that curcumin consumption may reduce the risk of some form of cancers and provide other protective biological effects in humans. These biological properties have been attributed to curcuminoids that have been widely studied for their anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects. In this study we have investigated on the effect of a curcumin phospholipid complex on mammary epithelial cell viability. HC11 and BME-UV cell lines, validated models to study biology of normal, not tumoral, mammary epithelial cells, were used to analyse these effects. We report that curcumin acts on STAT-3 signal pathway to reduce cell viability and increase apoptosis evaluated by the the amount of activated caspase 3. Further it reduces MAPK and AKT activations. JSI-124, a STAT-3 inhibitor (100 nM) was able to block the negative effect of curcumin on cell viability and caspase 3 activation. Finally the negative effect of cucumin on cell viability has been impaired in STAT-3i HC11, where STAT-3 protein was greatly reduced by shRNA-interference. These results indicate that curcumin presents a potential adverse effect to normal mammary epithelial cells and that it has a specific effect on signal trasduction in mammary epithelium.

Cell-cell communication between mouse mammary epithelial cells and 3T3-L1 preadipocytes: effect on triglyceride accumulation and cell proliferation

Interaction between parenchyma and stroma is essential for organogenesis, morphogenesis, and differentiation. Mammary gland has being the chosen model for developmental biologist because the most striking changes in morphology and function take place after birth. We have demonstrated a regulation of triglyceride accumulation by protein factors synthesized by normal mouse mammary gland epithelial cells (NMMG), acting on a cell line, 3T3-L1, long used as a model for adipogenesis. In this paper, we demonstrate that this inhibitory effect seems to be shared by other cells of epithelial origin but not by other cell types. We found a regulation of cell proliferation when NMMG cells are cultured in the presence of conditioned media from Swiss 3T3 or 3T3-L1 cells. We found a possible point of regulation for the mammary factor on a key enzyme of the lipid metabolic pathway, the glycerol-3-phosphate dehydrogenase. The inhibitory factor seems to have an effect on this enzyme's activity and reduces it. The results presented herein contribute to the understanding of cell-cell communication in a model of a normal mammary gland.

Variaciones mitocondriales en el transcurso de la diferenciación celular del epitelio mamario / Mitochondrial variations during the cellular differentiation in the mammary epithelium

El proceso de diferenciación representa un complejo mecanismo mediante el cual las células, variando notablemente su morfología, adquieren una forma determinada, tamaño específico, cierta polaridad y constancia de constituyentes celulares, producto de la activación génica que permiten que dicha célula diferenciada cumpla con una función en forma óptima. Estas modificaciones pueden ser precisadas utilizando técnicas morfométricas que dan cuenta de las variaciones que caracterizan el mecanismo. Células normales de epitelio mamario de rata en cultivo, estimuladas a proliferar con el factor de crecimiento epidérmico (EGF) origina la célula HC11 GM. Ellas son inducidas a diferenciarse con inducciones hormonales de dexametasona, insulina y prolactina, generándose la célula HC11 IM. Se estudió con microscopía electrónica de transmisión estos tipos celulares con énfasis en las mitocondrias, determinando variaciones del organelo generador de energía, en lo relacionado a variables tanto cuantitativas como morfológicas, precisando así su rol en la actividad metabólica de cada tipo celular.

Apoptosis en células mamarias transfectadas con el oncogen ras / Apoptosis in mammary epithelium transfected with ras oncogen

La muerte celular programada o apoptosis, es un proceso activo que involucra gasto energético y control génico desencadenado tanto por agentes internos como producto de señales externas a la célula, las cuales inducen a desviar el transcurso normal de desarrollo mediante la ejecución de un nuevo programa genético optando por el suicidio celular. Células normales de epitelio mamario de ratas HC11 GM en proceso de proliferación fueron transfectadas con el oncogen ras generando el tipo celular transformado Qey produciendo múltiples modificaciones cuanti y cualitativas de sus componentes celulares. Esta transformación generada por el ras determina finalmente la aparición de un fenotipo neoplásico, sin embargo, algunas células, producto de estas profundas modificaciones provenientes del oncogen, derivan al proceso apoptótico las cuales son evidenciadas y caracterizadas en este trabajo con ayuda de la microscopía óptica.

The programmed cellular death, is an active process that involves energy and genie control, triggered so much by internal agents as well as by external cell signals which induce to turn aside the normal course of development by means of the execution of a new genetic program and entering the cellular suicide. Normal HC11GM cells of mammary ephitelium of rats in proliferation process were transfected with ras oncogen generating the cellular Q.6 transformed type and producing multiple qualitative and cuantitative modifications of their cellular components. This transformation generated by ras, finally determines the appearance of a neoplasic phenotype, nevertheless, some cells produced by these deep modifications from the oncogen, enter the apoptotic process which are demonstrated and characterized in this work with the help of optical microscopy.

Drástica disminución de Beta 1 integrina caracteriza la transformación celular del epitelio mamario / Beta 1 integrin decrease determine the cellular transformation of mammary epithelium

El proceso biológico de transformación es el resultado de numerosos y complejos mecanismos traducidos en cambios sustanciales que involucran tanto a la ultraestructura, como a la bioquímica y fisiología celular, los cuales pueden ser visualizados mediante la utilización de técnicas morfométricas que expresan la correspondiente información cuantitativa. Células en cultivo de epitelio mamario de rata tanto normales, estimuladas a proliferar con el factor de crecimiento epidérmico (EGF), como transformadas, producto de la transfección con el oncogén ras, originan el grupo celular HC11 y HC11 ras, respectivamente. Se estudió a nivel de microscopía electrónica de transmisión en los tipos celulares descritos, precisando datos morfométricos concomitantes al decorrer de la transformación, evaluando específicamente las fracciones volumétricas de Beta 1 integrina, glicoproteínas receptoras presentes en la superficie celular involucradas en la adhesión tanto a componentes de la matriz extra-celular (MEC) como a la interacción célula-célula. La diferente expresión de estas moléculas determinará en los tipos celulares descritos, distintas características fisiológicas inherentes al mecanismo de transformación.

Las células mamarias en proceso de diferenciación disminuyen su volumen citoplasmático y nuclear / Mammalian cell differentiation process decreasing their cytoplasmatic and nuclear volume

Las modificaciones experimentadas en el proceso biológico de diferenciación celular determinan cambios complejos y fundamentales en la ultraestructura, la bioquímica y la fisiología celular que pueden ser apreciadas claramente utilizando técnicas morfométricas, las cuales traducidas en información cuantitativa permiten evidenciar los cambios que conlleva este mecanismo. Células normales de epitelio mamario de rata, mantenidas en cultivo, estimuladas a proliferar con el factor de crecimiento epidérmico, originan el grupo celular HC11 GM. Estas células normales y proliferantes son inducidas a diferenciarse mediante inducciones de hormonas lactogénicas: dexametasona, prolactina e insulina, determinándose la generación de un tipo celular diferenciado: HC11 IM. Estudiamos a nivel de microscopía electrónica estos tipos celulares, procurando datos morfométricos discriminantes a lo largo de la diferenciación, diagnosticando las fracciones volumétricas de componentes celulares, determinando así mismo la variación en el área de las células involucradas, precisando de este modo, nuevos marcadores en el padrón de modificación que caracteriza este proceso.

Hypoxia activates signal transducers and activators of transcription 5 (STAT5) and increases its binding activity to the GAS element in mammary epithelial cells

STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.

Distribución de mastocitos del estroma de la glándula mamaria de perra (Canis familiaris), en periodos activo e inactivo / Distribution of mast cells in mammary gland stroma, during the active and inactive periods in the bitch (Canis familiaris)

Los mastocitos (MC) son células del tejido conjuntivo que participan activamente en los mecanismos de comunicación paracrina mediante la liberación de diversos mediadores químicos contenidos en sus gránulos. El presente estudio se efectuó con el propósito de evaluar la distribución de los MC en la glándula mamaria de la perra (Canis familiaris), en periodos activos e inactivo. Las muestras de tejido mamario se obtuvieron a partir de perras adultas. Dichas muestras se procesaron siguiendo el método de inclusión en parafina y los cortes histológicos obtenidos fueron teñidos con azul de tolouidina para posteriormente efectuar la cuantificación de MC por mm², considerando para este fin el estroma interalveolar y el estroma interlobulillar del tejido mamario. Durante el periodo activo de la glándula mamaria, la población de MC presentes en el estroma disminuye significativamente respecto del periodo inactivo (P< 0.05). Estos resultados en conjunto sugieren que los MC presentes en el estroma de la glándula mamaria de perra, participan activamente en los cambios proliferativos que experimentan el tejido conjuntivo laxo areolar, dependiendo del estadio funcional de la glándula