RESUMEN

In recent decades, there has been an increased interest in azo compounds with special optical and biological properties. In this work, we report the preparation of novel azo-compounds with two and three -N=N- double bonds, using the classical method of synthesis, diazotization and coupling. The compounds were characterized by 1H-NMR, 13C-NMR, FTIR, UV-VIS and fluorescence spectra. DFT calculations were employed for determining the optical parameters, polarizability α, the total static dipole moment µtot, the quadrupole moment Q and the mean first polarizability ßtot. All azo derivatives show strong fluorescence emission in solutions. The antioxidant and antifungal activities were determined and the influence of the number of azo bonds was discussed. The synthesized compounds exhibit remarkable efficiency in the growth reduction of standard and clinical isolated Candida strains, suggesting future applications as novel antifungal.

RESUMEN

The current challenges of a circular economy exert a high pressure on manufacturing companies that generate waste to track and implement policies to reduce them and eliminate the toxicity of residues. Hence, the purpose of this study is to analyze the waste management information disclosure linked to the financial performance of companies and test the moderating effect of internal and external variables. The average waste management information disclosure index shows a poor disclosure score for the analyzed period, however, the waste disclosure index after reaching a minimum threshold in 2019 recorded an encouraging increase at the end of 2021. Applying the fixed effects model, ordinary least squares, and two-stage least squares method, the results revealed a positive and statistically significant relationship between management information disclosure and the return on assets, while for the current ratio the connection has been invalidated. A statistically significant influence of the environmental-sensitive industry status, board size, and productivity on the moderating variables was found for the return on assets, while for current ratio, there was none. As for the alternative metrics of financial performance, the results showed that a higher degree of management information disclosure will increase the return on equity and earnings per share, while in the case of liquidity, the results are not conclusive.

Asunto(s)

Revelación , Administración de Residuos , Industrias , Comercio

RESUMEN

In the context of the new life-threatening COVID-19 pandemic caused by the SARS-CoV-2 virus, finding new antiviral and antimicrobial compounds is a priority in current research. Pyridine is a privileged nucleus among heterocycles; its compounds have been noted for their therapeutic properties, such as antimicrobial, antiviral, antitumor, analgesic, anticonvulsant, anti-inflammatory, antioxidant, anti-Alzheimer's, anti-ulcer or antidiabetic. It is known that a pyridine compound, which also contains a heterocycle, has improved therapeutic properties. The singular presence of the pyridine nucleus, or its one together with one or more heterocycles, as well as a simple hydrocarbon linker, or grafted with organic groups, gives the key molecule a certain geometry, which determines an interaction with a specific protein, and defines the antimicrobial and antiviral selectivity for the target molecule. Moreover, an important role of pyridine in medicinal chemistry is to improve water solubility due to its poor basicity. In this article, we aim to review the methods of synthesis of pyridine compounds, their antimicrobial and antiviral activities, the correlation of pharmaceutical properties with various groups present in molecules as well as the binding mode from Molecular Docking Studies.

Asunto(s)

Antiinfecciosos , Tratamiento Farmacológico de COVID-19 , Antibacterianos , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antivirales/química , Antivirales/farmacología , Humanos , Simulación del Acoplamiento Molecular , Pandemias , Piridinas/química , Piridinas/farmacología , SARS-CoV-2

RESUMEN

Anthocyanidins - the aglycone moiety of anthocyanins - are responsible for the antioxidant traits and for many of the health benefits brought by the consumption of anthocyanin-rich foods, but whether excessive anthocyanidins are deleterious to living organisms is still a matter of debate. In the present study we used the model eukaryotic microorganism Saccharomyces cerevisiae to evaluate the potential toxicity of cyanidin, one of the most prevalent anthocyanidins found in berries, grapes, purple vegetables, and red wine. We found that yeast cells lacking the transcription factors responsible for regulating the response to oxidative stress - Skn7 and Yap1 - exhibited different sensitivities to cyanidin. Cells lacking the transcription factor Skn7 were sensitive to low concentrations of cyanidin, a trait that was augmented by exposure to visible light, notably blue or green light. In contrast, the growth of yeast cells devoid of Yap1 was stimulated by low concentrations, but it was impaired by high cyanidin exposure. High, but not low cyanidin was shown to induce Yap1 translocation from cytosol to nucleus, probably by generating reactive oxygen species such as H2O2. Taken together, these observation suggested that Skn7 and Yap1 have complementary roles in adaptation to cyanidin stress, with Skn7 involved in adaptation to low concentrations and with Yap1 responsible for adaptation to high concentrations of cyanidin. The results imply that caution is needed when utilizing cyanidin-enriched supplements, especially when in combination with prolonged exposure to visible light.

RESUMEN

Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na+/K+-ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca2+-sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca2+ influx into the cytosol and that failing to pump Ca2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca2+ or Mn2+ uptake may be a way of controlling oleandrin toxicity.

Asunto(s)

Calcio/metabolismo , Cardenólidos/química , Glicósidos Cardíacos/química , Glicósidos Cardíacos/metabolismo , Manganeso/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Cardenólidos/farmacología , Glicósidos Cardíacos/farmacología , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Citosol/ultraestructura , Inhibidores Enzimáticos/metabolismo , Humanos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Fluorescencia

RESUMEN

One of the most important aspects of the detection of antioxidant compounds is developing a fast screening method. The screening of the overall relative antioxidant capacity (RAC) of several Romanian hydrosoluble plant extracts is the focus of this work. This is important because of the presence of increasing levels of reactive oxygen species (such as H2O2) generates oxidative stress in the human body. The consequences are a large number of medical conditions that can be helped by a larger consumption of plant extracts as food supplements, which do not necessarily contain the specified antioxidant contents. By exploiting the catalytic properties of gold nanoparticles, a specific and sensitive nanoparticle-based label-free electrochemical sensor was developed, where the working parameters were optimized for RAC screening of hydrosoluble plant extracts. First, electrochemical measurements (cyclic voltammetry and amperometry) were used to characterize different nanoparticle-based sensors, revealing the best performance of gold nanoparticle-based sensors, obtaining a RAC of 98% for lavender extracts. The sensing principle is based on the quenching effect of antioxidants for H2O2 amperometric detection, where the decrease in electrical signal suggests an increasing antioxidant capacity. The obtained results were expressed in terms of ascorbic acid and Trolox equivalents in order to be able to correlate our results with classical methods like chemiluminescence and UV-Vis spectrophotometry, where a correlation coefficient of 0.907 was achieved, suggesting a good correlation between electrochemistry and spectrophotometry. Considering these results, the optimized gold nanoparticle-based label-free sensor can be used as a simple, rapid alternative towards classical methods for relative antioxidant capacity detection of hydrosoluble plant extracts.

Asunto(s)

Técnicas Biosensibles/métodos , Nanopartículas del Metal/química , Extractos Vegetales/química , Antioxidantes , Oro/química , Humanos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/química

RESUMEN

Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn2+ surplus. Using cells expressing the Ca2+-sensitive photoprotein aequorin we found that Mn2+ augmented the Ca2+ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn2+-high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn2+ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn2+ activate TRPY1 in the response to oxidative stress.

Asunto(s)

Calcio/metabolismo , Haploinsuficiencia , Peróxido de Hidrógeno/toxicidad , Manganeso/farmacología , Estrés Oxidativo/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Canales Catiónicos TRPC/genética , Vacuolas/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Diploidia , Haploinsuficiencia/efectos de los fármacos , Heterocigoto , Mutación/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Vacuolas/efectos de los fármacos

RESUMEN

Epigallocatechin-3-O-gallate (EGCG), the main green tea component, is intensively studied for its anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer effects. In the present study, a screen on a Saccharomyces cerevisiae gene deletion library was performed to identify conditions under which EGCG had deleterious rather than beneficial effects. Two genes were identified whose deletion resulted in sensitivity to EGCG: FET3 and FTR1, encoding the components of the Fet3/Ftr1 high-affinity iron uptake system, also involved in Cu(I)/Cu(II) balance on the surface of yeast cells. The presence of EGCG in the growth medium induced the production of Cu(I), with deleterious effects on fet3Δ and ftr1Δ cells. Additionally, when combined, physiological surpluses of Cu(II) and EGCG acted in synergy not only against fet3Δ and ftr1Δ, but also against wild type cells, by generating surplus Cu(I) in the growth medium. The results imply that caution should be taken when combining EGCG-rich beverages/nutraceuticals with copper-rich foods.

Asunto(s)

Catequina/análogos & derivados , Ceruloplasmina/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Té/química , Catequina/química , Catequina/aislamiento & purificación , Catequina/farmacología , Ceruloplasmina/deficiencia , Cobre/metabolismo , Proteínas de Transporte de Membrana/deficiencia , Saccharomyces cerevisiae/genética , Té/metabolismo

RESUMEN

To respond to metal surpluses, cells have developed intricate ways of defense against the excessive metallic ions. To understand the ways in which cells sense the presence of toxic concentration in the environment, the role of Ca2+ in mediating the cell response to high Cu2+ was investigated in Saccharomyces cerevisiae cells. It was found that the cell exposure to high Cu2+ was accompanied by elevations in cytosolic Ca2+ with patterns that were influenced not only by Cu2+ concentration but also by the oxidative state of the cell. When Ca2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca2+ pool under Cu2+ stress was the vacuolar Ca2+ channel, Yvc1, also activated by the Cch1-mediated Ca2+ influx. Using yeast mutants defective in the Cu2+ transport across the plasma membrane, it was found that the Cu2+-dependent Ca2+ elevation could correlate not only with the accumulated metal, but also with the overall oxidative status. Moreover, it was revealed that Cu2+ and H2O2 acted in synergy to induce Ca2+-mediated responses to external stress.

Asunto(s)

Señalización del Calcio , Cobre/metabolismo , Saccharomyces cerevisiae/metabolismo , Calcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Cobre/toxicidad , Citosol/metabolismo , Peróxido de Hidrógeno/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

Lanthanides are a group of non-essential elements with important imaging and therapeutic applications. Although trivalent lanthanide ions (Ln³âº) are used as potent blockers of Ca²âº channels, the systematic studies correlating Ln³âº accumulation and toxicity to Ca²âº channel blocking activity are scarce. In this study, we made use of the eukaryotic model Saccharomyces cerevisiae to investigate the correlation between Ln³âº accumulation, their toxicity and their capacity to block the exogenous stress-induced Ca²âº influx into the cytosol. It was found that the Ln³âº blocked the Ca²âº entry into the yeast cells only when present at concentration high enough to allow rapid binding to cell surface. At lower concentrations, Ln³âº were taken up by the cell, but Ca²âº blockage was no longer achieved. At 1 mM concentration, all ions from the Ln³âº series could block Ca²âº entry into cytosol with the exception of La³âº, and to a lesser extent, Pr³âº and Nd³âº. The plasma membrane Ca²âº-channel Cch1/Mid1 contributed to La³âº and Gd³âº entry into the cells, with a significant preference for La³âº. The results open the possibility to obtain cells loaded with controlled amounts and ratios of Ln³âº.

Asunto(s)

Calcio/química , Elementos de la Serie de los Lantanoides/química , Saccharomyces cerevisiae/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Iones/farmacología , Elementos de la Serie de los Lantanoides/toxicidad , Modelos Biológicos , Saccharomyces cerevisiae/química

RESUMEN

To gain new insight into the antimicrobial potential of Ailanthus altissima Swingle, ethanol leaf extracts were evaluated for the antifungal effects against the model yeast Saccharomyces cerevisae. The extracts inhibited the yeast growth in a dose-dependent manner, and this effect could be augmented by heat shock, exposure to visible light or exposure to high concentrations of Ca(2+). Using transgenic yeast cells expressing the Ca(2+)-dependent photoprotein, aequorin, it was found that the leaf extracts induced cytosolic Ca(2+) elevation. Experiments on yeast mutants with defects in Ca(2+) transport demonstrated that the cytotoxicity of the A. altissima leaf extracts (AaLEs) was mediated by transient pulses of Ca(2+) ions which were released into the cytosol predominantly from the vacuole. The investigation of the antifungal synergies involving AaLEs may contribute to the development of optimal and safe combination therapies for the treatment of drug-resistant fungal infections.

Asunto(s)

Ailanthus/química , Antifúngicos/química , Calcio/química , Calor , Luz , Extractos Vegetales/química , Saccharomyces cerevisiae/efectos de los fármacos , Hojas de la Planta/química

RESUMEN

Carbamazepine (CBZ) is a recalcitrant xenobiotic pharmaceutical pollutant highly stable in soil and wastewater during treatment. The biodegradation of CBZ using streptomycetes has been few studied up to now. Sixteen newly filamentous bacteria belong to genus Streptomyces spp. isolated from different Romanian soil samples and three strains from a collection of microorganisms (MIUG) were morphologically characterized, tested based on their resistance against CBZ toxicity and then selected as agents for bioremediation. Five Streptomyces spp. strains coded MIUG 4.88, MIUG 4.89, SNA, LP1 and LP2 showed CBZ tolerance at all of the tested concentrations, i.e. 0.05, 0.2, 1, 5 and 8 mg L⁻¹. Two of these (MIUG 4.89 and SNA strains) were selected based on their resistance to target compound and were then assessed for CBZ biodegradation. The strain Streptomyces MIUG 4.89 showed an interesting efficiency for CBZ removal, with a yield of 35% when it was cultivated in submerged conditions on a minimal medium supplemented with 5 g L⁻¹ glucose. This ability was linked to extracellular laccase production. These results are promising for the use of these filamentous bacteria as bioremediation agents.

Asunto(s)

Anticonvulsivantes/metabolismo , Carbamazepina/metabolismo , Streptomyces/metabolismo , Biodegradación Ambiental , Biomasa , Lacasa/metabolismo , Microbiología del Suelo , Streptomyces/crecimiento & desarrollo , Streptomyces/aislamiento & purificación

RESUMEN

Blueberries (Vaccinium corymbosum L.) are a rich source of antioxidants and their consumption is believed to contribute to food-related protection against oxidative stress. In the present study, the chemoprotective action of blueberry extracts against cadmium toxicity was investigated using a cadmium-hypersensitive strain of Saccharomyces cerevisiae. Four varieties of blueberries were used in the study, and it was found that the extracts with high content of total anthocyanidins exhibited significant protective effect against the toxicity of cadmium and H2O2. Both the blueberry extracts and pure cyanidin exhibited protective effects against cadmium in a dose-dependent manner, but without significantly interfering with the cadmium accumulation by the yeast cells. The results imply that the blueberry extracts might be a potentially valuable food supplement for individuals exposed to high cadmium.

Asunto(s)

Arándanos Azules (Planta)/química , Cadmio/toxicidad , Frutas/química , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad

RESUMEN

The Ca(2+) -dependent response to oxidative stress caused by H(2)O(2) or tert-butylhydroperoxide (tBOOH) was investigated in Saccharomyces cerevisiae cells expressing transgenic cytosolic aequorin, a Ca(2+) -dependent photoprotein. Both H(2)O(2) and tBOOH induced an immediate and short-duration cytosolic Ca(2+) increase that depended on the concentration of the stressors. Sublethal doses of H(2)O(2) induced Ca(2+) entry into the cytosol from both extracellular and vacuolar sources, whereas lethal H(2)O(2) shock mobilized predominantly the vacuolar Ca(2+). Sublethal and lethal tBOOH shocks induced mainly the influx of external Ca(2+), accompanied by a more modest vacuolar contribution. Ca(2+) transport across the plasma membrane did not necessarily involve the activity of the Cch1p/Mid1p channel, whereas the release of vacuolar Ca(2+) into the cytosol required the vacuolar channel Yvc1p. In mutants lacking the Ca(2+) transporters, H(2)O(2) or tBOOH sensitivity correlated with cytosolic Ca(2+) overload. Thus, it appears that under H(2)O(2)-induced or tBOOH-induced oxidative stress, Ca(2+) mediates the cytotoxic effect of the stressors and not the adaptation process.

Asunto(s)

Calcio/metabolismo , Estrés Oxidativo/fisiología , Aequorina/genética , Aequorina/fisiología , Citosol/efectos de los fármacos , Citosol/metabolismo , Cartilla de ADN , Peróxido de Hidrógeno/farmacología , Cinética , Mutación , Estrés Oxidativo/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Sistemas de Mensajero Secundario/fisiología , Vacuolas/efectos de los fármacos , Vacuolas/fisiología , terc-Butilhidroperóxido/farmacología

RESUMEN

Hepatic stellate cell transdifferentiation, epithelial-mesenchymal cell transition, and the ductular reaction each contribute to the development of hepatic fibrosis in cholestatic liver diseases. Inhibitors of mammalian target of rapamycin have antifibrotic properties. We evaluated the hypothesis that the antifibrotic action of rapamycin is due to attenuated myofibroblast proliferation in addition to an inhibitory effect on epithelial-mesenchymal transition and the ductular reaction. Hepatic fibrosis was induced by bile duct ligation, and rodents received 1.5 mg/kg/day rapamycin by subcutaneous infusion for 21 days. The expression of various markers of hepatic fibrosis, stellate cell transactivation, epithelial-mesenchymal transition, and the ductular reaction was compared between treated and untreated animals. Hepatic fibrosis, hepatic procollagen type 1 messenger RNA, and alpha-smooth muscle actin expression were significantly reduced in treated animals. Hepatic stellate cell procollagen expression and proliferation were also reduced by rapamycin. The following markers of epithelial-mesenchymal transition--vimentin protein expression, S100 calcium binding protein A4 and transforming growth factor beta 1 messenger RNA, and the mothers against decapentaplegic homolog signaling pathway--were all reduced after rapamycin treatment. The intensity of the ductular reaction was reduced by rapamycin as assessed by histopathological scoring and by reduced cytokeratin 19 expression. Rapamycin caused a reduction in hepatic progenitor cell proliferation. Together, these data show that multiple profibrogenic pathways are activated in an animal model of cholestasis and that rapamycin attenuates epithelial-mesenchymal transition and the ductular reaction as well as hepatic stellate cell activation.

Asunto(s)

Fibrinógeno/metabolismo , Cirrosis Hepática/patología , Sirolimus/farmacología , Animales , Diferenciación Celular , Proliferación Celular , Epitelio/patología , Fibrosis , Inmunosupresores/farmacología , Hígado/patología , Masculino , Mesodermo/metabolismo , Modelos Biológicos , Ratas , Ratas Wistar , Células Madre/metabolismo

RESUMEN

Recently, E2F function has expanded to include the regulation of differentiation in human epidermal keratinocytes (HEKs). We extend these findings to report that in HEKs, Sp1 is a differentiation-specific activator and a downstream target of E2F-mediated suppression of the differentiation-specific marker, transglutaminase type 1 (TG-1). Deletion of elements between -0.084 to -0.034 kb of the TG-1 promoter disabled E2F1-induced suppression of promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and Sp3 bound this region. Protein expression analysis suggested that squamous differentiation was accompanied by increased Sp1/Sp3 ratio. Cotransfection of proliferating HEKs or the squamous cell carcinoma (SCC) cell line, KJD-1/SV40, with an E2F inhibitor (E2Fd/n) and Sp1 expression plasmid was sufficient to activate the TG-1 promoter. The suppression of Sp1 activity by E2F in differentiated cells appeared to be indirect since we found no evidence of an Sp1/E2F coassociation on the TG-1 promoter fragment. Moreover, E2F inhibition in the presence of a differentiation stimulus induced Sp1 protein. These data demonstrate that (i) Sp1 can act as a differentiation stimulus, (ii) E2F-mediated suppression of differentiation-specific markers is indirect via Sp1 inhibition and (iii) a combination of E2F inhibition and Sp1 activation could form the basis of a differentiation therapy for SCCs.

Asunto(s)

Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp1/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transglutaminasas/biosíntesis , Biomarcadores de Tumor/análisis , Regulación hacia Abajo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Ensayo de Cambio de Movilidad Electroforética , Humanos , Queratinocitos , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

The inhibition of E2F has been demonstrated to be important in the initiation of squamous differentiation by two independent manners: promotion of growth arrest and the relief of the differentiation-suppressive properties of E2Fs. E2F6 is reported to behave as a transcriptional repressor of the E2F family. In this study, we examined the ability of E2F6 to act as the molecular switch required for E2F inhibition in order for keratinocytes to enter a terminal differentiation programme. Results demonstrated that whilst E2F6 was able to suppress E2F activity in proliferating keratinocytes, it did not modulate squamous differentiation in a differentiated keratinocyte. Furthermore, inhibition of E2F, by overexpressing E2F6, was not sufficient to sensitise either proliferating keratinocytes or the squamous cell carcinoma cell line, KJD-1/SV40, to differentiation-inducing agents. Significantly, although E2F6 could suppress E2F activity in proliferating cells, it could not inhibit proliferation of KJD-1/SV40 cells. These results demonstrate that E2F6 does not contain the domains required for modulation of squamous differentiation and imply isoform-specific functions for individual E2F family members.

Asunto(s)

Carcinoma de Células Escamosas/metabolismo , Factores de Transcripción/fisiología , Bromodesoxiuridina/farmacología , Diferenciación Celular , Proliferación Celular , Separación Celular , Células Cultivadas , Colorantes/farmacología , ADN Complementario/metabolismo , Factor de Transcripción E2F6 , Citometría de Flujo , Genes Reporteros , Humanos , Queratinocitos/metabolismo , Microscopía Fluorescente , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/química , Transfección

RESUMEN

The AP-2 transcription factor family is presumed to play an important role in the regulation of the keratinocyte squamous differentiation program; however, limited functional data are available to support this. In the present study, the activity and regulation of AP-2 were examined in differentiating human epidermal keratinocytes. We report that (1) AP-2 transcriptional activity decreases in differentiated keratinocytes but remains unchanged in differentiation-insensitive squamous cell carcinoma cell lines, (2) diminished AP-2 transcriptional activity is associated with a loss of specific DNA-bound AP-2 complexes, and (3) there is an increase in the ability of cytoplasmic extracts, derived from differentiated keratinocytes, to phosphorylate AP-2 alpha and AP-2 beta when cells differentiate. In contrast, extracts from differentiation-insensitive squamous cell carcinoma cells are unable to phosphorylate AP-2 proteins. Finally, the phosphorylation of recombinant AP-2 alpha by cytosolic extracts from differentiated keratinocytes is associated with decreased AP-2 DNA-binding activity. Combined, these data indicate that AP-2 trans-activation and DNA-binding activity decrease as keratinocytes differentiate, and that this decreased activity is associated with an enhanced ability to phosphorylate AP-2 alpha and beta.

Asunto(s)

Proteínas de Unión al ADN/genética , Queratinocitos/metabolismo , Factores de Transcripción/genética , Western Blotting , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Inmunohistoquímica , Queratinocitos/citología , ARN Mensajero/metabolismo , Factor de Transcripción AP-2 , Factores de Transcripción/metabolismo

RESUMEN

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation.

Asunto(s)

Antígenos de Diferenciación/metabolismo , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Queratinocitos/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Biomarcadores , Proteína Quinasa CDC2/genética , División Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes Reporteros/genética , Humanos , Queratina-14 , Queratinocitos/citología , Queratinas/genética , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína smad7 , Transactivadores/genética , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética

RESUMEN

BACKGROUND: Normal keratinocytes (KC) and neoplastic cells derived from a head and neck lesion (SCC-25) were grown as organotypic raft cultures to mimic in vivo architecture in the absence of contaminating cell types. Alterations in gene expression between normal keratinocytes and a head and neck squamous cell carcinoma (HNSSC) cell line (SCC-25) were analysed using gene arrays. MATERIALS AND METHODS: RNA from the organotypic raft cultures were used to probe four gene arrays. Gene expression alterations between the normal and neoplastic cells were identified and analysed using both fold differences and 2-tailed t-test. Four genes from different functional groups were used for immunohistochemical staining of patient tumours to confirm the gene array data. RESULTS: Statistical analysis of the array data revealed 124 significantly altered genes between normal and neoplastic HNSCC cells. These gene expression alterations are associated with a variety of different functional groups and indicate the complexity of gene de-regulation associated with HNSCC. CONCLUSION: This study identified many novel gene alterations associated with HNSCC. The significantly altered gene alterations belong in a variety functional groups including: growth control, apoptosis and detoxication and present new targets for investigating the molecular basis of HNSCC formation.