A toolkit for recombinant production of seven human EGF family growth factors in active conformation.

Epidermal growth factors (EGF) play a wide range of roles in embryogenesis, skin development, immune response homeostasis. They are involved in several pathologies as well, including several cancer types, psoriasis, chronic pain and chronic kidney disease. All members share the structural EGF domain, which is responsible for receptor interaction, thereby initiating transduction of signals. EGF growth factors have intense use in fundamental research and high potential for biotechnological applications. However, due to their structural organization with three disulfide bonds, recombinant production of these factors in prokaryotic systems is not straightforward. A significant fraction usually forms inclusion bodies. For the fraction remaining soluble, misfolding and incomplete disulfide bond formation may affect the amount of active factor in solution, which can compromise experimental conclusions and biotechnological applications. In this work, we describe a reliable procedure to produce seven human growth factors of the EGF family in Escherichia coli. Biophysical and stability analyses using limited proteolysis, light scattering, circular dichroism and nanoDSF show that the recombinant factors present folded and stable conformation. Cell proliferation and scratch healing assays confirmed that the recombinant factors are highly active at concentrations as low as 5 ng/ml.

The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles.

The effects of varying concentrations of EGF were evaluated in terms of in vitro follicular development and the mRNA expression levels of EGF, EGF-R, FSH-R and P450 aromatase. After 6 days, the addition of 50 ng/mL of EGF to the culture medium increased the antrum formation rates in comparison to cultured control and after 18 days of culture produced oocytes with higher rates of meiosis resumption when compared to the other treatments (P<0.05). The daily follicular growth rates in presence of EGF (50 or 100) were increased in comparison to the cultured control (P<0.05). Treatment with EGF 50 stimulated the expression of EGF mRNA but reduced EGF-R mRNA expression and estradiol secretion as compared to the cultured control (P<0.05). After 18 days of culture, the mRNA levels for FSH-R and P450 aromatase were greater than those of the non-cultured controls (P<0.05). In conclusion, the effects of EGF treatment on the mRNA levels for EGF, EGF-R, FSH-R, and P450 aromatase varied according to the stage of follicle development.

Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle.

FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells.

Gene expression in swine granulosa cells and ovarian tissue during the estrous cycle.

The components of the insulin-like growth factor (IGF) system appear to be involved in regulation of ovarian follicular growth and atresia in the pig. We investigated the expression pattern of mRNAs for IGF1 (IGF1), its binding proteins (IGFBP1, IGFBP2, IGFBP3, and IGFBP5), and epidermal growth factor in swine follicle cells and ovarian tissue throughout the estrous cycle using the real-time quantitative PCR technique. The results of gene expression were analyzed using linear regression with gene expression as a dependent variable and days of estrous cycle as an independent variable. Additionally, an analysis was made of the correlation of expression levels with plasma concentration of follicle-stimulating hormone, luteinizing hormone, estradiol-17ß, progesterone, and prolactin. Expression of mRNA of all of these genes was detected in granulosa cells and ovarian tissue. IGFBP3 mRNA showed a quadratic expression pattern (P ≤ 0.001) and was significantly and positively correlated with progesterone (r = 0.81; P ≤ 0.01) but negatively correlated with prolactin (r = -0.596; P ≤ 0.05). Expression of the other genes was unaffected by the stage of the estrous cycle. Real-time quantitative PCR effectively detected all transcripts, including the very low levels of IGFBP1 transcripts, and could be used for studies of follicle dynamics.

Clinical significance of EGFR, Her-2 and EGF in oral squamous cell carcinoma: a case control study.

BACKGROUND: The erbB receptors and their ligands are involved in the pathogenesis and progression of oral squamous cell carcinoma (OSCC). Although EGFR and Her-2 are frequently overexpressed in OSCC, few studies evaluated these proteins in saliva and their association with the tumor, which may represent potential usefulness in a clinical setting. METHODS: The levels of EGFR, Her-2, and EGF were evaluated in saliva of 46 patients with OSCC before and after the surgical removal of the lesion, as well as in matched healthy controls. The relationship of salivary levels and EGFR and Her-2 immunoexpression in tumor samples with clinicopathological features was analyzed. RESULTS: EGFR and Her-2 salivary levels did not show difference between to pre-surgery and control groups, however, both demonstrated an increase after surgical removal of the tumor. No association was detectable among receptor salivary levels, tissue expression and clinicopathological features. EGF levels in pre-surgery group were significantly lower when compared to the control group. CONCLUSIONS: EGFR and Her-2 were not considered to be valuable salivary tumor markers in OSCC, however, lower levels of EGF in saliva may suggest a higher susceptibility for OSCC development.

Blastocyst-endometrium interaction: intertwining a cytokine network.

The successful implantation of the blastocyst depends on adequate interactions between the embryo and the uterus. The development of the embryo begins with the fertilized ovum, a single totipotent cell which undergoes mitosis and gives rise to a multicellular structure named blastocyst. At the same time, increasing concentrations of ovarian steroid hormones initiate a complex signaling cascade that stimulates the differentiation of endometrial stromal cells to decidual cells, preparing the uterus to lodge the embryo. Studies in humans and in other mammals have shown that cytokines and growth factors are produced by the pre-implantation embryo and cells of the reproductive tract; however, the interactions between these factors that converge for successful implantation are not well understood. This review focuses on the actions of interleukin-1, leukemia inhibitory factor, epidermal growth factor, heparin-binding epidermal growth factor, and vascular endothelial growth factor, and on the network of their interactions leading to early embryo development, peri-implantatory endometrial changes, embryo implantation and trophoblast differentiation. We also propose therapeutical approaches based on current knowledge on cytokine interactions.

Blastocyst-endometrium interaction: intertwining a cytokine network

The successful implantation of the blastocyst depends on adequate interactions between the embryo and the uterus. The development of the embryo begins with the fertilized ovum, a single totipotent cell which undergoes mitosis and gives rise to a multicellular structure named blastocyst. At the same time, increasing concentrations of ovarian steroid hormones initiate a complex signaling cascade that stimulates the differentiation of endometrial stromal cells to decidual cells, preparing the uterus to lodge the embryo. Studies in humans and in other mammals have shown that cytokines and growth factors are produced by the pre-implantation embryo and cells of the reproductive tract; however, the interactions between these factors that converge for successful implantation are not well understood. This review focuses on the actions of interleukin-1, leukemia inhibitory factor, epidermal growth factor, heparin-binding epidermal growth factor, and vascular endothelial growth factor, and on the network of their interactions leading to early embryo development, peri-implantatory endometrial changes, embryo implantation and trophoblast differentiation. We also propose therapeutical approaches based on current knowledge on cytokine interactions.

Sobreexpresión del factor de crecimiento epidermico humano (HER2) en piezas operatorias de mujeres con cáncer de mamas en el Hospital Bertha Calderón en el período mayo a octubre del 2005 / Sobreexpresión of the factor of growth human epidermico (HER2) in women's operative pieces with cancer of you suckle in the Hospital Bertha Calderón in the period May to October of the 2005

El presente estudio se realizó con la intención de valorar el porcentaje de sobreexpresión del HER 2 a través del uso de Herceptest en 34 pacientes diagnósticadas con cáncer de mama en el Hospital Bertha Calderon Roque. Se realizó un estudio de tipo descriptivo de corte transversal cuyo universo y muestra lo constituyeron todas las pacientes que fueron mastectomizadas por cáncer de mama y que recibian apoyo de la fundación Ortiz Gurdián, durante los meses de mayo a octubre del 2005. Las pacientes una vez operadas se les enviaba la muestra al departamento de patología donde se fijaba en bloque de parafina y se realizaba posteriormente el estudio inmunohistoquímico, una vez obtenido el reporte el reporte, se procesó la información junto a otras variables clínicas en un sistema computarizado estadístico en salud (EPI INFO) los resultados se presentaban en tablas y gráficos de salida...

Modulation of gastrin and epidermal growth factor by pyrrolizidine alkaloids obtained from Senecio brasiliensis in acute and chronic induced gastric ulcers.

We investigated the antiulcerogenic activity of pyrrolizidine alkaloids (PAs) integerrimine, retrorsine, senecionine, usaramine and seneciplhylline, an alkaloidal extract obtained from Senecio brasiliensis. The PA extract demonstrated significantly activity in both, acute and chronic gastric ulcers on rats. The effects of PA extract were dose dependent. The mechanisms implicated on this activity were evaluated by determination of gastrin plasma levels in rats subjected to the acute treatment with PA extract and by expression of mRNA of Epidermal Growth Factor (EGF) after chronic treatment with this extract. The results showed that the PA extract increased both the levels of gastrin and the expression of EGF on these animals. Moreover, the histological examinations showed a reduction of exfoliation of superficial cells, hemorrhages and blood cell infiltration. We concluded that the PAs showed an important and qualitative antiulcerogenic activity mediated by increase in gastrin secretion and mRNA expression of EGF.

Fatty acid synthase is required for the proliferation of human oral squamous carcinoma cells.

Fatty acid synthase (FAS) is the enzyme responsible for the endogenous synthesis of saturated long-chain fatty acids from the precursors acetyl-CoA and malonyl-CoA. A growing body of evidence indicates that FAS is over expressed in several human cancers, such as prostate, breast, bladder, liver, lung, melanoma and oral squamous cell carcinoma (SCC). In the present study we used human oral SCC cell lines (SCC-4, -9, -15 and -25) as a model to investigate the role of FAS in the pathogenesis of oral cancer. RT-PCR and western blot experiments demonstrated that FAS is differentially expressed by the four oral SCC cell lines, with the highest production in SCC-9 followed by SCC-25. FAS expression in SCC-4 and -15 was similarly lower than the other cell lines. Proliferation curves and immunocytochemistry for PCNA and Ki-67 demonstrated that SCC-25 has the highest proliferative potential. In addition, the specific inhibitor of FAS activity cerulenin was able to significantly reduce the proliferation of oral SCC cells. Expression of androgen receptor was low in SCC-4, -9 and -15 and undetectable in SCC-25, whereas EGFR and c-erb-B2 were expressed in high amounts by the four cell lines. Immunocytochemical reactions showed that SCC-25 expresses higher levels of EGF compared to the other three cell lines. Finally, oral SCC cells exposed to nanomolar concentrations of exogenous EGF presented a reduction in the FAS protein levels concomitant with a decrease in their proliferation rates. Taken together, our results indicate that FAS is expressed in an apparently androgen-independent fashion in oral SCC cells and it is necessary for their proliferation.

Bioquímica del líquido quístico mamario, III: niveles del factor de crecimiento epidérmico, gonadotrofina coriónica y andrógenos conjugados / Biochemistry of breast cyst fluids, III: levels of NA, K, androgens, epidermal growth factor and human chorionic gonadotropin

En la última década se ha implementado una serie de análisis bioquímicos que permiten identificar varios tipos de líquidos quísticos (LQs). En el presente trabajo se confirma la presencia de polipéptidos y esteroides conjugados -como el factor de crecimiento epidérmico (FCE), el sulfato de dehidroepiandrosterona (S-DHEA) y el androstano-3Ó, 17ß-diol glucurónido (3Ó-Adiol G)- a veces en concentraciones muy elevadas con respecto a los niveles encontrados simultáneamente en el plasma circulante. Como contraste, la concentración del cortisol apenas alcanza a un 20 por ciento del normalmente hallado en el plasma. Se demuestra además que la concentración intraquística del 3Ó-Adiol G se correlaciona positiva y significativamente con la del S-DHEA (r = 0,8744, p < 0,0001) y con el FCE (r = 0,8949, p < 0,0001), con amplia variabilidad en los resultados. Se establece también una correlación negativa entre el 3Ó-Adiol G y el cociente Na/K (r = - 0,6592, p = 0,0001). Por último, se determinan los niveles de la gonadotrofina coriónica (hCG), utilizando un sistema automatizado de quimioluminiscencia, demostrándose que esta glicoproteína se encuentra en cantidades determinables (> 1,1 mUI/ml) en el 73,8 por ciento de los LQs analizados. En el 57,4 por ciento los niveles superan a los encontrados normalmente en el plasma que oscilan entre < 1,1 mUl/ml y 5,5 mUl/ml. En un 4,9 por ciento las concentraciones resultan significativamente elevadas, alcanzando hasta las 1.000 mUl/ml. Se demuestra una correlación negativa con alta significación estadística entre los valores normalizados de la hCG con los niveles del S-DHEA, del 3Ó-Adiol G y del FCE y una correlación positiva con el cociente NA/K. Se discute la posibilidad de que el FCE, los esteroides conjugados y la hCG puedan ser sintetizados de novo en el tejido epitelial que recubre las paredes del quiste

Bioquímica del líquido quístico mamario, III: niveles del factor de crecimiento epidérmico, gonadotrofina coriónica y andrógenos conjugados / Biochemistry of breast cyst fluids, III: levels of NA, K, androgens, epidermal growth factor and human chorionic gonadotropin

En la última década se ha implementado una serie de análisis bioquímicos que permiten identificar varios tipos de líquidos quísticos (LQs). En el presente trabajo se confirma la presencia de polipéptidos y esteroides conjugados -como el factor de crecimiento epidérmico (FCE), el sulfato de dehidroepiandrosterona (S-DHEA) y el androstano-3O, 17ß-diol glucurónido (3O-Adiol G)- a veces en concentraciones muy elevadas con respecto a los niveles encontrados simultáneamente en el plasma circulante. Como contraste, la concentración del cortisol apenas alcanza a un 20 por ciento del normalmente hallado en el plasma. Se demuestra además que la concentración intraquística del 3O-Adiol G se correlaciona positiva y significativamente con la del S-DHEA (r = 0,8744, p < 0,0001) y con el FCE (r = 0,8949, p < 0,0001), con amplia variabilidad en los resultados. Se establece también una correlación negativa entre el 3O-Adiol G y el cociente Na/K (r = - 0,6592, p = 0,0001). Por último, se determinan los niveles de la gonadotrofina coriónica (hCG), utilizando un sistema automatizado de quimioluminiscencia, demostrándose que esta glicoproteína se encuentra en cantidades determinables (> 1,1 mUI/ml) en el 73,8 por ciento de los LQs analizados. En el 57,4 por ciento los niveles superan a los encontrados normalmente en el plasma que oscilan entre < 1,1 mUl/ml y 5,5 mUl/ml. En un 4,9 por ciento las concentraciones resultan significativamente elevadas, alcanzando hasta las 1.000 mUl/ml. Se demuestra una correlación negativa con alta significación estadística entre los valores normalizados de la hCG con los niveles del S-DHEA, del 3O-Adiol G y del FCE y una correlación positiva con el cociente NA/K. Se discute la posibilidad de que el FCE, los esteroides conjugados y la hCG puedan ser sintetizados de novo en el tejido epitelial que recubre las paredes del quiste (AU)

Epidermal growth factor and transforming growth factor alpha mRNA expression in human breast cancer biopsies; analysis in relation to estradiol, progesterone and EGF receptor content.

Among the peptide growth factors active in breast glandular cell proliferation epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are thought to play a major role in tumour development. They operate through binding to and activation of a common membrane receptor, defined as EGF-R. Their production is modulated by hormones and local growth factors. After it was shown by previous investigation in this laboratory that EGF-R could be detected in 90% of the tumours, but was masked by endogenous ligand in 36% of them, the question was raised as to the level of the ligand's expression in tumour tissue biopsies. Therefore, we investigated the expression of EGF and TGF alpha mRNA in 146 breast cancer biopsies by slot blot analysis using specific 32P-labelled probes. The data were correlated with sex steroids and EGF receptor content. Our results showed that EGF and TGF alpha coexisted in all tumour samples, and that their level of mRNA expression was similar in half of the tumours. Northern blot and polymerase chain reaction (PCR) analysis validated these findings. A significant direct correlation was found between the level of TGF alpha/EGF mRNA expression and the ER/progesterone receptor (PGR) content. TGF alpha and EGF mRNA levels were significantly higher in ER+ (P = 0.0015 and P = 0.0001, respectively) and in PGR+ tumours (P < 0.005 and P = 0.0001) than in their negative counterparts. Moreover, TGF alpha mRNA expression negatively correlated with the number of EGF-R binding sites measured by the standard method (P = 0.02), and it was significantly related to the number of sites occupied by endogenous ligand. In conclusion, it was shown that TGF alpha and EGF mRNA were coexpressed in all the tumour biopsies tested and that their level was higher in the hormone receptor positive than in negative samples. The correlation between the presence of ER/PGR sites, high level of TGF alpha/EGF mRNA and EGF-R occupancy by endogenous ligand is in favour of ER mediated control of TGF alpha and EGF production.

Oral human recombinant epidermal growth factor in the treatment of patients with duodenal ulcer.

The epidermal growth factor has been shown to be mucoprotective and to accelerate healing of gastroduodenal ulcers in animals. A prospective, positively controlled clinical trial was conducted. Seventy five patients with duodenal ulcer were randomly distributed in three groups to receive oral human recombinant epidermal growth factor in 1% carboxymethyl cellulose at two different doses (450 mg or 600 mg/day), or cimetidine. Treatment was administered up to a maximum of 6 weeks. The most important assessment criteria was the proportion of patients healed after 2, 4 and 6 weeks of treatment determined by endoscopy. Treatment with both doses of epidermal growth factor showed a long-term healing effect in 76.5% at 6 weeks vs 92.5% with cimetidine (p = N.S.). The evolution of the clinical symptoms was similar in the three groups. Adverse reactions were not detected in any of the patients included in this study. To our knowledge, this is the first report on the oral use of epidermal growth factor in humans.

Epidermal growth factor in human milk: daily production and diurnal variation during early lactation in mothers delivering at term and at premature gestation.

Epidermal growth factor is a polypeptide that stimulates proliferation and differentiation of a variety of cell types, including the developing intestinal epithelium; it is the agent in human milk that induces mitosis in human fibroblast culture. We systematically evaluated the EGF content of milk from 20 women delivering prematurely and from 11 women delivering at term. In preterm mothers, the concentration of EGF was 70 +/- 5 ng/ml (mean +/- SEM), with no significant change during seven weeks of lactation. EGF concentration in milk of term mothers was 68 +/- 19 ng/ml (mean +/- SEM). No diurnal variation in the concentration was found. Total EGF content was closely correlated with the volume of milk expressed, suggesting a passive transport from the circulation. These observations confirm that a substantial amount of EGF is present in human milk and that EGF concentrations are not affected by duration of gestation, time of day, or duration of lactation.