Efectos del Factor de crecimiento epidérmico sobre las características histológicas del riñón en ratas con Insuficiencia renal crónica / Effects of Epidermal growth factor on histological features of the kidney in rats with Chronic kidney failure

Introducción: la Insuficiencia renal crónica es un problema de salud pública, por lo que surge la necesidad de incrementar las acciones dirigidas a su prevención, diagnóstico e intervenciones terapéuticas eficaces; estudios recientes emplean el Factor de crecimiento epidérmico para evaluar su efecto reno-protector en variables funcionales; sin embargo se carece de estudios relacionados con los efectos de este producto sobre las características histológicas de riñones insuficientes. Objetivo: determinar los posibles efectos protectores y/o reparadores del Factor de crecimiento epidérmico humano recombinante sobre las características histológicas de riñones con insuficiencia renal crónica. Material y métodos: se trabajó con tres series, formadas por un grupo control y uno experimental cada una, de cinco animales, a los grupos experimentales se les realizó ablación quirúrgica de 5/6 de la masa renal. La serie A se conformó por animales controles, la B por los tratados con Factor de crecimiento epidérmico 24 horas antes del procedimiento y la C por los tratados con el bioproducto 24 horas después. Pasados 56 días del acto operatorio a los animales se les practicó la eutanasia y se procedió al estudio histológico del riñón. Resultados: en los animales de la serie A se observaron alteraciones histológicas en los corpúsculos y túbulos renales, en la serie B se observó que la mayor parte del parénquima renal presentó características normales y los de la serie C mostraron un daño renal incrementado. Conclusiones: El Factor de crecimiento epidérmico humano recombinante posee efecto reno-protector, sin embargo, no ofrece efecto reno-reparador(AU)

Introduction: Chronic Kidney Failure is a public health problem; therefore, the need for actions aimed at its prevention, diagnosis and effective therapeutic interventions is a most. Recent studies use the Epidermal Growth Factor to evaluate its reno-protective effect on functional variables; however, there are no studies related to the effects of this product on histological features of kidney in chronic failure. Objective: To determine the possible protective and / or repair effects of recombinant human epidermal growth factor on the histological features of kidneys in chronic renal failure. Material and methods: We worked with three series, each consisting of a control group and an experimental group of five animals. The experimental groups underwent 5/6 surgical ablation of the renal mass. Series A consisted of control animals, series B included those animals treated with Epidermal Growth Factor 24 hours before the procedure and series C was made up of those animals treated with the bioproduct 24 hours after the procedure. Fifty-six days after surgical act, euthanasia was practiced on the animals and the kidneys were histologically studied. Results: Histological alterations were observed in the renal corpuscles and tubules in the animals included in series A; in series B it was observed that most of the renal parenchyma presented normal characteristics and those in series C showed increased kidney damage. Conclusions: Recombinant human epidermal growth factor has a reno-protective effect; however, it does not offer a repair effect of acute kidney(AU)

Eficacia y seguridad de afatinib como tratamiento de primera línea en pacientes adultos con cáncer de pulmón de células no pequeñas avanzado (estadios IIIB/IV), con mutaciones activadoras del gen del receptor del factor de crecimiento epidérmico, con contraindicación a erlotinib por hipersensibilidad / Efficacy and safety of afatinib as first-line treatment in adult patients with advanced non-small cell lung cancer (stages IIIB/IV), with activating mutations of the epidermal growth factor receptor gene, with contraindication to erlotinib due to hypersensitivity

INTRODUCCIÓN: El cáncer de pulmón de células no pequeñas (CPCNP) es la causa más frecuente (85 % - 90 %) de tumores pulmonares malignos que generalmente afectan a adultos que fuman y que tienen ≥ 65 años. En Perú, en 2017, el cáncer de pulmón fue la segunda causa de muerte entre todos los cánceres, con una mortalidad anual de 8.8 muertes por cada 100,000 personas.  La terapia de primera línea del CPCNP avanzado (estadio IIIB/IV) depende del estado de las mutaciones conductoras oncogénicas, la expresión de PD-L1 y la histología. Así, para los casos en los que se detecta una mutación sensibilizante1 del receptor de factor de crecimiento epidérmico (EGFR, por sus siglas en inglés), se recomienda ofrecer una terapia dirigida contra el EGFR utilizando los inhibidores de la tirosina quinasa del EGFR (de aquí en adelante llamados TKI, por sus siglas en inglés) de primera línea (e.g., erlotinib, afatinib).  En el Seguro Social de Salud del Perú (EsSalud), los pacientes con CPCNP avanzado cuyos tumores poseen mutaciones positivas2 del EGFR disponen de erlotinib como tratamiento de primera línea según lo establecido en el Petitorio Farmacológico de EsSalud. Sin embargo, existe un grupo de pacientes que presentan eventos adversos (EA) cutáneos severos (grado 3 o mayor) asociados al tratamiento con erlotinib, quienes, en ciertos casos, requerirán de la discontinuación de uso del medicamento, a pesar del manejo óptimo de las toxicidades cutáneas (e.g., reducción de dosis). En este grupo de pacientes con hipersensibilidad a erlotinib (contraindicación de uso), los médicos especialistas de la institución sugieren el uso de un TKI alternativo: afatinib. OBJETIVO: Evaluar la mejor evidencia disponible sobre la eficacia y seguridad de afatinib, en comparación con la quimioterapia, en pacientes adultos con CPCNP avanzado, con mutaciones activadoras3 del gen del EGFR, con contraindicación a erlotinib por hipersensibilidad. TECNOLOGÍA SANITARIA DE INTERÉS: Afatinib: Afatinib es un inhibidor selectivo e irreversible de la actividad de la tirosina quinasa de los receptores de la familia ErbB: EGFR (ErbB1), HER2 (ErbB2), HER3 (ErbB3) y HER4 (ErbB4). Afatinib se une en forma covalente a los dominios de la tirosina quinasa de estos receptores e inhibe irreversiblemente la autofosforilación de la tirosina quinasa, lo que resulta en un bloqueo de las señales de los receptores ErbB (European Medicines Agency 2019). METODOLOGÍA: Se realizó una búsqueda sistemática de literatura con el objetivo de identificar evidencia sobre la eficacia y seguridad de afatinib, en comparación con la quimioterapia, en pacientes adultos con CPCNP avanzado, con mutaciones activadoras del gen del EGFR, con contraindicación a erlotinib por hipersensibilidad. Se utilizó la base de datos The Cochrane Library, PubMed, LILACS y el metabuscador TRIP Database, priorizándose evidencia proveniente de ensayos clínicos aleatorizados. Asimismo, se realizó una búsqueda dentro de bases de datos pertenecientes a grupos que realizan evaluaciones de tecnologías sanitarias y guías de práctica clínica, incluyendo el Scottish Medicines Consortium (SMC), el National Institute for Health and Care Excellence (NICE), la Canadian Agency for Drugs and Technologies in Health (CADTH), la Haute Autorité de Santé (HAS), el Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen (IQWiG), además de la Base Regional de Informes de Evaluación de Tecnologías en Salud de las Américas (BRISA) y páginas web de sociedades especializadas en cáncer de pulmón. Se hizo una búsqueda adicional en la página web de clinicaltrials.gov, para poder identificar ensayos clínicos en curso o que no hayan sido publicados para, de este modo, disminuir el riesgo de sesgo de publicación. La búsqueda sistemática se basó en una metodología escalonada, la cual consistió en la búsqueda inicial de estudios secundarios (tipo revisiones sistemáticas con o sin metaanálisis). RESULTADOS: Se realizó una búsqueda de la literatura con respecto a la eficacia y seguridad de afatinib, en comparación con la quimioterapia, en pacientes adultos con CPCNP avanzado, con mutaciones activadoras del gen del EGFR, con contraindicación a erlotinib por hipersensibilidad. Dado que no se identificaron estudios en una población específica de pacientes con contraindicación a erlotinib, se procedió a revisar la evidencia para la población general de pacientes con CPCNP avanzado, con mutaciones activadoras del gen del EGFR. CONCLUSIONES: Dado que no se identificaron estudios en la población específica de pacientes con contraindicación a erlotinib, se procedió a revisar la evidencia para la población general de pacientes con CPCNP avanzado, con mutaciones activadoras del gen del receptor del factor de crecimiento epidérmico. En líneas generales, todas las GPC y ETS basaron sus recomendaciones y/o conclusiones en los resultados de los estudios LUX-Lung 3 y/o LUX-Lung 6. La evidencia procedente de los estudios LUX-Lung 3 y LUX-Lung 6 muestra que afatinib comparado con la quimioterapia ofrece un beneficio clínico en términos de una mayor sobrevida global (aproximadamente 11 meses adicionales) en los pacientes con CPCNP metastásico, ECOG 0-1 y mutaciones del EGFR tipo Del19, sin tratamiento previo. Además, afatinib tuvo un perfil de seguridad similar al de la quimioterapia con cisplatino más pemetrexed y un mejor perfil de seguridad que la quimioterapia con gemcitabina más cisplatino, en términos de EA severos, EA serios y discontinuación debido a EA. A diferencia de las GPC identificadas, que recomendaron el uso de afatinib en la población general de pacientes con CPCNP y mutaciones positivas del EGFR, esta evaluación de la evidencia identificó que los pacientes con mutaciones Del19 serían el subgrupo con mayor probabilidad de beneficiarse del tratamiento con afatinib. Por otro lado, no se identificó evidencia directa que sustente el uso de afatinib en el grupo de pacientes previamente tratados. Sin embargo, tal como se menciona en el Dictamen Preliminar de Evaluación de Tecnología Sanitaria N° 041-SDEPFyOTSDETS-IETSI-2019: Eficacia y Seguridad de erlotinib en pacientes adultos con CPCNP, metastásico o irresecable, con mutación del gen del EGFR, tras fallo a al menos una línea de quimioterapia, se valora que la evidencia del uso de afatinib en el contexto de primera línea puede ser extrapolada al grupo de pacientes que han sido previamente tratados con quimioterapia sistémica y que han experimentado hipersensibilidad severa a erlotinib. Adicionalmente, es importante analizar el contexto de intercambio de TKI (de erlotinib a afatinib) debido a EA cutáneos severos como resultado de uma hipersensibilidad a erlotinib (contraindicación de uso) en pacientes con CPCNP avanzado y mutación del EGFR, ya que es el grupo específico de pacientes en quienes se propone el uso de afatinib en la institución. Así, si bien la evidencia sobre el intercambio de TKI debido a EA es limitada, algunas series de casos han sugerido que esta aproximación proporciona un efecto beneficioso en pacientes con CPCNP avanzado y mutaciones del EGFR. Además, el intercambio de erlotinib a afatinib podría justificarse biológicamente dada las diferencias en las estructuras químicas de erlotinib y afatinib, que podrían influir en los EA asociados con estos medicamentos. De este modo, teniendo en cuenta que los pacientes que recibirían un segundo TKI tendrían que haber demostrado no tener una mutación resistente a TKI, se estima que los pacientes que discontinúan el tratamiento con erlotinib debido a hipersensibilidad severa aún podrían beneficiarse de "cambiar" a un segundo TKI (afatinib). Con ello, y considerando la experiencia de uso de TKI a nivel institucional y la opinión favorable por parte de los médicos especialistas de la institución, el equipo evaluador del IETSI encuentra suficientes argumentos técnicos para aprobar el uso de afatinib en pacientes adultos con CPCNP avanzado, con mutaciones activadoras del gen del EGFR (Del19), con contraindicación a erlotinib por hipersensibilidad. Por lo expuesto, el IETSI aprueba el uso de afatinib en pacientes adultos con CPCNP avanzado, con mutaciones activadoras del gen del receptor del factor de crecimiento epidérmico, con contraindicación a erlotinib por hipersensibilidad, según lo establecido en el Anexo N° 1. La vigencia del presente dictamen preliminar es de un año a partir de la fecha de publicación. Así, la continuación de dicha aprobación estará sujeta a los resultados obtenidos de los pacientes que reciban este tratamiento, a los reportes de seguridad que puedan surgir durante farmacovigilancia activa y nueva evidencia que pueda surgir en el tiempo.

Neuron-Like Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

PURPOSE: Mesenchymal stem cells (MSCs) are multipotent and give rise to distinctly differentiated cells from all three germ layers. Neuronal differentiation of MSC has great potential for cellular therapy. We examined whether the cluster of mechanically made, not neurosphere, could be differentiated into neuron-like cells by growth factors, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: BMSCs grown confluent were mechanically separated with cell scrapers and masses of separated cells were cultured to form cluster BMSCs. As described here cluster of BMSCs were differentiated into neuron-like cells by EGF, HGF, and VEGF. Differentiated cells were analyzed by means of phase-contrast inverted microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and immunocytochemistry to identify the expression of neural specific markers. RESULTS: For the group with growth factors, the shapes of neuron-like cells was observable a week later, and two weeks later, most cells were similar in shape to neuron-like cells. Particularly, in the group with chemical addition, various shapes of filament structures were seen among the cells. These culture conditions induced MSCs to exhibit a neural cell phenotype, expressing several neuro-glial specific markers. CONCLUSION: bone marrow-derived mesenchymal stem cells (BMSCs) could be easily induced to form clusters using mechanical scraping, not neurospheres, which in turn could differentiate further into neuron-like cells and might open an attractive possibility for clinical cell therapy for neurodegenerative diseases. In the future, we consider that neuron-like cells differentiated from clusters of BMSCs are needed to be compared and analyzed on a physiological and molecular biological level with preexisting neuronal cells, and studies on the possibility of their transplantation and differentiation capability in animal models are further required.

Isolation, culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85 +/- 4.6%), and a correspondingly low proportion K3 positive cells (15 +/- 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean +/- SE, n=10). Cultures reached full confluency after 17.3 +/- 1.2 days when the medium was supplemented with human EGF, while 21.7 +/- 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.

Effect of Recombinant Human Epidermal Growth Factor Against Cutaneous Scar Formation in Murine Full-thickness Wound Healing

A visible cutaneous scar develops from the excess formation of immature collagen in response to an inflammatory reaction. This study examined the role of epidermal growth factor (EGF) in the formation of cutaneous scars. Twenty Crl:CD-1 (ICR) mice were used and 2 full-thickness skin wounds were made on the dorsum of each mouse. One of the wounds was treated with recombinant human EGF by local application and the other was treated with saline for control until complete healing was achieved. The EGF-treated group's wounds healed faster than the control group's. The width of the scar was smaller by 30% and the area was smaller by 26% in the EGF-treated group. Inflammatory cell numbers were significantly lower in the EGF-treated group. The expression of transforming growth factor (TGF)-beta1 in the EGF-treated group was increased. It was observed that the amount of collagen in the EGF-treated group was larger than the control group. In the EGF-treated group, the visible external scars were less noticeable than that in the control group. These results suggest that EGF can reduce cutaneous scars by suppressing inflammatory reactions, decreasing expression of TGF-beta1, and mediating the formation of collagen.

A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

Effects of FGF-2 and EGF removal on the differentiationof mouse neural precursor cells

Cell therapy for neurological disorders has advanced, and neural precursor cells (NPC) may become the ideal candidates for neural transplantation in a wide range of diseases. However, additional work has to be done to determine either the ideal culture environment for NPC expansion in vitro, without altering their plasticity, or the FGF-2 and EGF mechanisms of cell signaling in neurospheres growth, survival and differentiation. In this work we evaluated mouse neurospheres cultured with and without FGF-2 and EGF containing medium and showed that those growth factors are responsible for NPC proliferation. It is also demonstrated that endogenous production of growth factors shifts from FGF-2 to IGF-1/PDGFb upon EGF and FGF-2 withdrawal. Mouse NPC cultured in suspension showed different patterns of neuronal localization (core versus shell) for both EGF and FGF-2 withdrawal and control groups. Taken together, these results show that EGF and FGF-2 removal play an important role in NPC differentiation and may contribute to a better understanding of mechanisms of NPC differentiation. Our findings suggest that depriving NPC of growth factors prior to grafting might enhance their chance to effectively integrate into the host.

As terapias celulares para doenças neurológicas têm avançado e células precursoras neurais (NPC) surgem como candidatas ideais para o transplante de células neurais em muitas doenças. No entanto, trabalhos adicionais devem ser feitos para determinar o ambiente de cultivo ideal para a expansão in vitro das NPC, sem alterar sua plasticidade, e os mecanismos de sinalização celular do fator de crescimento epidérmico (EGF) e fator de crescimento de fibroblasto 2 (FGF-2) no crescimento, sobrevivência e diferenciação da neuroesfera. Nesse trabalho avaliamosNPCcultivadas na presença e na ausência de FGF-2 e EGF e mostramos que esses fatores de crescimento são responsáveis pela proliferação das NPC. Também foi demonstrado que a produção endógena de fatores de crescimento alterna de FGF-2 a fator de crescimento de insulina 1 (IGF-1) e fator de crescimento derivado de plaquetas b (PDGFb) após remoção de EGF e FGF-2. NPC de camundongo cultivadas em suspensão mostraram padrões de localização neuronal distintos (centro versus borda) tanto no grupo controle como no grupo sem EGF e FGF-2. Juntos, esses resultados mostram que a remoção de EGF e FGF-2 exerce importante ação na diferenciação de NPC e possivelmente contribui para melhor compreensão dos mecanismos envolvidos na diferenciação. Nossos achados sugerem que, privando as NPC de fatores de crescimento antes do transplante, talvez aumente as chances de que as células efetivamente se integrem ao hospedeiro.

Akt is involved in the inhibition of cell proliferation by EGF

Axin is a negative regulator of the Wnt/beta-catenin pathway and is involved in the regulation of axis formation and proliferation. Involvement of Axin in the regulation of other signaling pathways is poorly understood. In this study, we investigated the involvement of Akt in growth regulation by Axin in L929 fibroblasts stimulated by EGF. Akt activity was increased by EGF treatment and Ras activation, respectively. Both the EGF- and Ras-induced Akt activations were abolished by Axin induction, as revealed by both Western blot and immunocytochemical analyses. The proliferation and Akt activation induced by EGF were decreased by Axin induction, and the effects of EGF were abolished by treatment of an Akt-specific inhibitor. Therefore, Axin inhibits EGF-induced proliferation of L929 fibroblasts by blocking Akt activation.

Tyrosine phosphatase and cytochrome P450 activity are critical in regulating store-operated calcium channels in human fibroblasts

Diverse signaling pathways have been proposed to regulate store-operated calcium entry (SOCE) in a wide variety of cell types. However, it still needs to be determined if all of these known pathways operate in a single cell type. In this study, we examined involvement of various signaling molecules in SOCE using human fibroblast cells (HSWP). Bradykinin (BK)-stimulated Ca2+ entry, previously shown to be via SOCE, is enhanced by the addition of vanadate, an inhibitor of tyrosine phosphatases. Furthermore, SOCE is regulated by cytochrome P-450, as demonstrated by the fact that the products of cytochrome P-450 activity (14,15 EET) stimulated SOCE while econazole, an inhibitor of cytochrome P450, suppressed BK-stimulated Ca2+ entry. In contrast, Ca2+ entry was unaffected by the guanylate cyclase inhibitor LY83583, or the membrane permeant cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP). Neither nitric oxide donors nor phorbol esters affected BK-stimulated Ca2+ entry. SOCE in HSWP cells is primarily regulated by tyrosine phosphorylation and the cytochrome P-450 pathway, but not by cyclic GMP, nitric oxide, or protein kinase C. Thus, multiple pathways do operate in a single cell type leading to the activation of Ca2+ entry and some of these signaling pathways are more prominently involved in regulating calcium entry in different cell types.

Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

Effect of beta-mercaptoethanol or epidermal growth factor supplementation on in vitro maturation of canine oocytes collected from dogs with different stages of the estrus cycle

Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.

The effect of epidermal growth factor & platelet-derived growth factors on wound healing process.

BACKGROUND & OBJECTIVES: Various growth factors play significant roles during the process of wound healing. A systematic study was carried out to evaluate the effects of topically applied epidermal growth factor (EGF) and platelet-derived growth factors (PDGF-BB, PDGF-AB, PDGF-AA) in various concentrations on wound healing. Various combinations of these growth factors were also studied to find the best combination for wound healing. METHODS: Wounds were created on a mouse model. Various concentrations and combinations of EGF and PDGFs were applied topically and the effects on wound healing were monitored visually. RESULTS: EGF, PDGF-AB and PDGF-BB when applied alone or in combination, healed the wounds in significantly less time and with less scar tissue formation compared to the controls. The best results were obtained with a combination of EGF (10 ng) and PDGF-BB (15 ng). PDGF-AA even at the maximum concentration of 20 ng did not enhance the wound repair process. A combination of PDGF-AA (20 ng) with EGF (10 ng) did not enhance the wound repair process either. INTERPRETATION & CONCLUSION: Topically applied EGF, PDGF-AB and PDGF-BB enhanced the wound repair process and had a role in decreasing the formation of scar tissue. PDGF-AA either alone or in combination with EGF did not enhance the wound repair process significantly. It appears that PDGF-AA might be limiting the EGF-mediated wound repair and such observation has not been previously reported in studies on wound healing using topically applied growth factors.

Phosphorylation of glycogen synthase kinase-3beta at serine-9 by phospholipase Cgamma1 through protein kinase C in rat 3Y1 fibroblasts

Phospholipase Cgamma1 (PLCgamma1) plays an important role in controlling cellular proliferation and differentiation. PLCgamma1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mechanisms underlying PLCgamma1-induced cell proliferation are not fully understood. Here we show that overexpression of PLCgamma1 highly phosphorylated glycogen synthase kinase-3beta (GSK-3beta) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3beta phosphorylation by PLCgamma1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLCgamma1-transfected cells as compared with vector-transfected cells. Since GSK-3beta is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLCgamma1-mediated GSK-3beta phosphorylation may function as a regulation of cyclin D1 accumulation in PLCgamma1-overexpressing cells.

Lessons from tumor and immunocompetent cells. The quantitative engagement of ligand-receptor interactions modulates stop-and-go behavior as well as proliferation

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.

Effect of short-term ethanol on the proliferative response of Swiss 3T3 cells to mitogenic growth factors

Both adaptive and deleterious responses of cells to ethanol are likely triggered by short-term interactions of the cells with ethanol. Many studies have demonstrated the direct effect of ethanol on growth factor-stimulated cell proliferation. Using Swiss 3T3 cells whose growth was inhibited by ethanol in a concentration-dependent manner, we further investigated the molecular mechanisms of acute ethanol treatment by examining its effect on EGF- and PDGF-mediated cellular signaling systems for the mitogenic function. Tyrosine autophosphorylation of the growth factor receptors was partially prevented by ethanol in intact cells. When ethanol was included before or after EGF stimulation, no effect on the receptor signaling was observed. Here we also report that ethanol inhibits activation of ERK induced by both EGF and PDGF. EGF-induced JNK activation was reduced but PDGF-induced rapid JNK activation was delayed by the addition of ethanol. The balance between its inhibitory and stimulatory effect on the signaling molecules might determine the rate of cell growth.

Perturbation of EGF-induced MAP kinase activation by TGF-ß1

TGF-ß1 regulates both cellular growth and phenotypic plasticity important for maintaining a growth advantage and increased invasiveness in progressively malignant cells. Recent studies indicate that TGF-ß-1 stimulates the conversion of epitheliod to fibroblastoid phenotype which presumably leads to the inactivation of growth-inhibitory effects by TGF-ß1 (Portella et al. (1998) Cell Growth and Differentiation, 9: 393-404). Therefore, the investigation of TGF-ß1 signaling that leads to altered growth and migration may provide novel targets for the prevention of increased cell growth and invasion. Although much attention has been paid to TGF-ß1 responses in epithelial cells, the above studies suggest that examination of signal transduction pathways in fibroblasts are important as well. Data from our laboratory are consistent with the concept that TGF-ß1 can act as a regulatory switch in density-dependent C3H 10T1/2 fibroblasts capable of either promoting or delaying G1 traverse. The regulation of this switch is proposed to occur prior to pRb phosphorylation, namely prior to activation of cyclin-dependent kinases. The current study is concerned with the evaluation of a key cyclin (cyclin D1) which activates cdk4 and p27KIP1 which in turn inhibit cdk2 in the proliferative responses of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and their modulation by TGF-ß1. Although the molecular events that lead to elevation of cyclin D1 are not completely understood, it appears likely that activation of p42/p44MAPK kinases is involved in its transcriptional regulation. TGF-ß1 delayed EGF- or PDGF-induced cyclin D1 expression and blocked the induction of active p42/p44MAPK. The mechanism by which TGF-ß1 induces a block in p42/p44MAPK activation is being examined and the possibility that TGF-ß1 regulates phosphatase activity is being tested

The effects of laminin on the characteristics and differentiation of neuronal cells from epidermal growth factor-responsive neuroepithelial cells

Many extracellular matrix molecules are expressed in the embryonic nervous system and there is some evidence that they are important regulators of neural development. Of these molecules, laminin appears to be the most potent, affecting virtually all neurons of the peripheral and central nervous system. This study was undertaken to investigate the effects of laminin on the proliferation and differentiation of cultured neuroepithelial cells taken from fetal rat forebrains (embryonic day 17-19). The results are summarized as follows. 1) Neuroepithelial cells cultivated in epidermal growth factors containing serum-free medium subsequently differentiated into neurons, astrocytes, and oligodendrocytes. 2) Neuronal cells derived from neuroepithelial cells were immunoreactive for gamma-aminobutyric acid (GABA) or substance P, but were not for serotonin and tyrosine hydroxylase. 3) In western blot analysis, the phosphorylated neurofilament content in neuronal cells was higher in culture on laminin than in culture on poly-L-lysine (PLL). 4) The proliferation rate of GABAergic neurons was higher in culture on laminin than in culture on PLL. These results suggest that GABAergic and substance P-ergic neurons can be differentiated from neuroepithelial cells and that laminin promotes the differentiation of neuronal cells from neuroepithelial cells and the increased proliferation rate of GABAergic cells.

El Factor de Crecimiento Epidérmico (EGF). Su aplicación en la Córnea / The epidermal growth factor (EGF). Its application on the cornea

Se revisan los aspectos de la localización y concentración del EGF en el organismo, sus efectos biológicos, así como, los aspectos farmacológicos vinculados con la córnea. Su efecto biológico consiste en la modulación de la proliferación celular, cuyas células dianas las constituyen las epiteliales, queratocitos y fibroblastos. Se han preparado diversas formas farmacéuticas tales como: colirio, oleosa, uniones a metilcelulosa e hialurinamo de sodio, entre otras. Se hace énfasis en que, el EGF no se difunde a través de las córneas intactas. Se ha empleado en el tratamiento de quemaduras corneales, úlceras, queratitis, heridas, erosiones epiteliales, etcétera, con resultados promisorios, por lo que posee potencialidades de convertirse en una parte integral de la generación próxima de medicamentos oftálmicos

Effects of epidermal growth factor on the [3H]-thymidine uptake in the SK-N-SH and SH-SY5Y human neuroblastoma cell lines

The studies on the factors that regulate the biology of the neuroblastoma cell lines may ofter important information on the development of tissues on organs that derive from the neural crest. In the present paper we study the action of epidermal growth factor (EGF) on two human neuroblastoma cell lines: SK-N-SH which is composed at least of two cellular phenotypes (neuroblastic and melanocytic/glial cells), and its pure neuroblastic subclone SH-SY5Y. The results show that EGF (10 ng/ml) significantly stimulates the incorporation of [3H]-thymidine in the SK-N-SH cells only in the presence of fetal bovine serum (FBS) (control = 58285 + 9327 cpm; EGF= 75523 + 4457; p<0.05). Such effect is not observed in the presence of a chemical defined medium, that is, in the absence of FBS (control = 100997 + 4375; EGF = 95268 + 4683; NS). In the SH-SY5Y cells the EGF does not modify the incorporation of [3H]thymidine either in the presence of 10 percent of BFS (control = 113838 + 6978; EGF = 119434 + 9411; NS) or in its absence (control = 46197 + 3335; EGF = 44472 + 3493; NS). The results here reported suggest that: a) EGF may effect the proliferation of cells derived from a primary human neuroblastoma; b) this is evident by the EGF-induced increase of [3H]-thymidine incorporation in SK-N-SH cells; c) it is required the presence of other growth factors, present in the FBS, for the mitogenic action to be accomphished; d) since the pure neuroblastic SH-SY5Y cell line are refractory to the EGF, the effects observed in SK-N-SH cells probably occur on the melanocytic/glial cell subpopulation.

Collagenase-IV in human trophoblast invasion and differentiation.

Trophoblast cells are unique with respect to their functions and responsibilities. These cells demonstrate three sequential phenotypes, proliferation and invasion into the endometrium, differentiation to form syncytia and endocrine secretions. Equipped with these properties placental trophoblasts are endowed with a variety of functions, like implantation of the blastocyst to the endometrium, providing nutrition to the developing embryo and also transmitting extraordinary array of signals for the embryonic development. Experimental evidences and logical extrapolation suggest that these functions are precisely controlled by growth factors, cytokines and hormones produced either by the trophoblast themselves or by the utero-placental unit. Any error in this control mechanism has extremely adverse consequences. The cells also synthesize a large number of enzymes, amongst which collagenase type IV secretion is involved in digestion of underlying basement membrane necessary for the process of invasion. Our results implicate the enzyme in the functional differentiation of the trophoblast as well. Inhibitors to this enzyme inhibit trophoblast differentiation as monitored by secretion of hCG and progesterone, the two markers of trophoblastic differentiation. In contrast, BeWo cells, a choriocarcinoma cell line which does not differentiate spontaneously, undergo increased proliferation when challenged with EGF. The results indicate the possibility of invasive and differentiative phenotypes to be coupled. Exact molecular involvements in this coupling process are looked into.