RESUMEN
The trefoil protein TFF1 is expressed principally in the superficial cells of the gastric mucosa. It is a small protein and forms homo- and hetero-dimers via a disulphide bond through Cys58 which is located three amino acids from the C terminus. TFF1 is co-expressed with the secreted mucin MUC5AC in superficial cells of the gastric mucosa suggesting that it could be involved in the packaging or function of gastric mucus. We have previously shown that TFF1 co-sediments with mucin glycoproteins on caesium chloride gradients. To extend this observation we have now used gel filtration under physiological conditions, immunoprecipitation and Western transfer analysis to characterise the interaction of TFF1 with gastric mucin glycoproteins. We show that TFF1 co-elutes with MUC5AC but not MUC6 on gel filtration and that immunoprecipitation and Western transfer analysis confirms that TFF1 interacts with MUC5AC. We also demonstrate that the TFF1 dimer is the predominant molecular form bound to MUC5AC. Salt and chelators of divalent cations such as EDTA and EGTA disrupted the TFF1- MUC5AC interaction and increased the degradation of MUC5AC, whereas calcium increased the amount of TFF1 bound to MUC5AC. These data support the contention that TFF1 is pivotal in the packaging and function of human gastric mucosa.
Asunto(s)
Mucosa Gástrica/metabolismo , Mucinas/metabolismo , Proteínas/metabolismo , Calcio/metabolismo , Cromatografía en Gel , Citosol/metabolismo , Humanos , Inmunohistoquímica , Mucina 5AC , Mucina 5B , Unión Proteica , Factor Trefoil-1 , Proteínas Supresoras de TumorRESUMEN
TFF1 is one of three human trefoil proteins expressed principally in the gastrointestinal tract in normal tissues. TFF1 protects the gastric mucosa against damage as a result of its ability to facilitate reconstitution of damaged gastric mucosa and its involvement in the secretion and structure of gastric mucus. The most biologically active molecular form in cell culture and animal models tested is a dimer formed by a disulfide bond between two cysteine residues close to the C terminus of the protein. We have therefore developed an assay for this form of TFF1 which should facilitate its measurement in biological samples.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Disulfuros/química , Proteínas/inmunología , Dimerización , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas/análisis , Factor Trefoil-1 , Proteínas Supresoras de TumorRESUMEN
Novel therapies for the treatment of colitis are required. We therefore examined the potential value of the trefoil factor family 1 (TFF1) peptide and epidermal growth factor (EGF) alone and in combination. Effects of TFF1- Cys58 +/- EGF on an in vitro HT29 cell wounding model of restitution showed synergistic activity when used in combination. In addition, animals had colitis induced by adding 4% dextran sulphate sodium (DSS) to the drinking water for 7 days and they also received twice daily subcutaneous injections of test peptides. Treatment with TFF1-Cys58 alone (100 microg/kg) reduced histological colitis score by 22%, but the TFF1-Ser58 variant was ineffective. In a second study, TFF1-Cys58 reduced histological colitis score by 15%, EGF (600 microg/kg) by 26%, and an additive response (42% reduction) was demonstrated when used together (P < 0.01 versus either peptide given alone). Similar results were found using tissue myeloperoxidase (MPO) activity as a marker of inflammation. Where clinical risk/benefit seems justified, these initial studies suggest that combination therapy of systemic EGF and TFF peptides may prove useful for treatment of colitis in patients with disease extending beyond the reach of topical (enema) therapy.
Asunto(s)
Colitis/tratamiento farmacológico , Colon/patología , Factor de Crecimiento Epidérmico/farmacología , Proteínas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran/toxicidad , Sinergismo Farmacológico , Células HT29 , Humanos , Lipopolisacáridos , Masculino , Mucinas , Proteínas Musculares , Péptidos , Peroxidasa/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Factor Trefoil-1 , Factor Trefoil-2 , Proteínas Supresoras de TumorRESUMEN
Breast cancer screening is important for the early detection of breast cancer. Tumors that become symptomatic in the screening interval are known as interval cancers but the reasons for their rapid progression are unknown. Estrogen receptor expression is lower in interval cancers suggesting that they may have reduced hormonal responsiveness. To investigate this hypothesis we have measured the expression of the estrogen receptor and three estrogen-responsive genes (cathepsin D, progesterone receptor, and TFF1) in screen-detected and interval breast cancers. The expression of the protease cathepsin D was not associated with estrogen receptor in either group of tumor. Progesterone receptor expression was highly correlated with that of the estrogen receptor in both groups of tumors but it was not expressed at significantly different levels in the two groups of tumors. Expression of TFF1, a cellular motogen, was correlated with estrogen receptor in screen-detected but not interval cancers and was expressed at markedly higher levels in interval breast tumors, the group that expresses lower levels of estrogen receptor. Interval cancers are characterized by high levels of expression of TFF1 and/or Ki67 suggesting that cell migration and cell division play important roles in the rapid progression of interval cancers. The observation that TFF1 expression in interval cancers tends to be estrogen-independent and that interval cancers have reduced estrogen receptor expression suggests they may have a reduced response to hormone therapy.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas/metabolismo , Neoplasias de la Mama/diagnóstico , Catepsina D/metabolismo , Estrógenos/fisiología , Femenino , Humanos , Antígeno Ki-67/metabolismo , Tamizaje Masivo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Análisis de Regresión , Factor Trefoil-1 , Proteínas Supresoras de TumorRESUMEN
The trefoil factor family protein, TFF1, forms a homodimer, via a disulphide linkage, that has greater activity in wound healing assays than the monomer. Having previously determined a high-resolution solution structure of a monomeric analogue of TFF1, we now investigate the structure of the homodimer formed by the native sequence. The two putative receptor/ligand recognition domains are found to be well separated, at opposite ends of a flexible linker. This contrasts sharply with the known fixed and compact arrangement of the two trefoil domains of the closely related TFF2, and has significant implications for the mechanism of action and functional specificity of the TFF of proteins.
Asunto(s)
Mucinas , Proteínas Musculares , Neuropéptidos , Proteínas/química , Secuencia de Aminoácidos , Calcio/farmacología , Cisteína/química , Dimerización , Disulfuros/química , Sustancias de Crecimiento/química , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Concentración Osmolar , Péptidos/química , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas/genética , Soluciones , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: TFF2, a member of the trefoil factor family of proteins, is a glycosylated protein of 106 amino acids. It is secreted by gastric antral and pyloric glands and by Brunner's glands of the duodenum. TFF2 is found in high concentrations around sites of ulceration. It stimulates cell motility and is probably the principal cytoprotective trefoil peptide in the stomach. AIMS: To determine if production of TFF2 follows a circadian rhythm and to measure changes in secretion of TFF2 in response to food intake and during sleep. SUBJECTS: Young healthy adults were recruited. They were asymptomatic and were not receiving medication. The 24 hour regimen was designed to allow normal stimulation of gastric secretion in response to food intake and sleep. Gastric juice was collected two hourly via a nasogastric tube. METHODS: Glycosylated and non-glycosylated TFF2 proteins were measured by quantitative western transfer analysis. The results were analysed statistically using SPSS software. RESULTS: There was a dramatic diurnal variation in the concentration of TFF2. The mean concentration was lowest in the early evening (0.29 microg/ml), increased gradually during the evening, and then sharply during the night to reach 7.9 microg/ml. The ratio of glycosylated to non-glycosylated TFF2 varied and was higher during the night than in the afternoon. pH, total protein, and pepsin concentrations in gastric juice did not vary significantly over 24 hours. CONCLUSION: The data suggest that diurnal variations in TFF2 secretion occur independently of pepsin and gastric acid secretion. The concentration of glycosylated TFF2 in the gastric lumen falls in response to food intake. TFF2 secretion increases during inactivity and sleep. These results suggest that secretion of TFF2 in the stomach is highest during the night and that the cytoprotective effects of TFF2 on the gastric mucosa occur mainly during sleep.
Asunto(s)
Ritmo Circadiano , Jugo Gástrico/química , Sustancias de Crecimiento/análisis , Mucinas , Proteínas Musculares , Neuropéptidos , Péptidos/análisis , Adulto , Ingestión de Alimentos/fisiología , Electroforesis en Gel de Poliacrilamida , Femenino , Glicosilación , Sustancias de Crecimiento/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Masculino , Pepsina A/análisis , Péptidos/metabolismo , Proteínas/análisis , Sueño/fisiología , Estadísticas no Paramétricas , Factor Trefoil-2 , Factor Trefoil-3RESUMEN
Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. The mechanism underlying the increased proliferation could involve the induction of components of the insulin-like growth factor signal transduction pathway by estrogen. In this study we have examined the regulation of the expression of insulin receptor substrate-1, a major intracellular substrate of the type I insulin-like growth factor receptor tyrosine kinase. Estradiol increased insulin receptor substrate-1 mRNA and protein levels at concentrations consistent with a mechanism involving the estrogen receptor. Insulin receptor substrate-1 was not induced significantly by the antiestrogens tamoxifen and ICI 182,780, but they inhibited the induction of insulin receptor substrate-1 by estradiol. Analysis of tyrosine-phosphorylated insulin receptor substrate-1 showed that the highest levels were found in cells stimulated by estradiol and insulin-like growth factor-I, whereas low levels were found in the absence of estradiol irrespective of whether type I insulin-like growth factor ligands were present. Insulin receptor substrate-2, -3, and -4 were not induced by estradiol. These results suggest that estrogens and antiestrogens may regulate cell proliferation by controlling insulin receptor substrate-1 expression, thereby amplifying or attenuating signaling through the insulin-like growth factor signal transduction pathway.
Asunto(s)
Estrógenos/fisiología , Fosfoproteínas/metabolismo , Regulación hacia Arriba , Proteínas Adaptadoras Transductoras de Señales , Northern Blotting , División Celular , Relación Dosis-Respuesta a Droga , Estradiol/metabolismo , Antagonistas de Estrógenos/farmacología , Moduladores de los Receptores de Estrógeno/metabolismo , Estrógenos/metabolismo , Humanos , Immunoblotting , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Fosforilación/efectos de los fármacos , ARN/metabolismo , Receptor IGF Tipo 1/metabolismo , Tamoxifeno/farmacología , Factores de Tiempo , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
BACKGROUND: TFF2, a member of the trefoil factor family (TFF) of peptides, is a secreted protein of 106 amino acids that is expressed in mucous neck cells of the fundus and glands at the base of the antrum in normal human stomach. TFF2 is also detected at high concentrations around sites of ulceration. It is protective against mucosal damaging agents and stimulates cell motility. AIMS: To measure the expression of TFF2 in normal human stomach and its secretion into gastric juice. METHODS: TFF2 cDNA was amplified by reverse transcription polymerase chain reaction from gastric mucosa and sequenced. Gastric juice or cytosol, prepared from gastric mucosa, was obtained from individuals with macroscopically normal stomachs. TFF2 concentrations were measured by quantitative western transfer analysis. RESULTS: Sequencing of TFF2 cDNA revealed a single amino acid change from the published sequence. Significant amounts of 12 kDa TFF2 were detected in human gastric juice. Larger quantities of a protein of higher apparent molecular mass were also detected. This was shown to be N-glycosylated TFF2 using the endoglycosidase, peptide-N-Gycosidase F. The majority of TFF2 in normal gastric mucosa was also glycosylated. CONCLUSIONS: Human TFF2 is glycosylated via an N-linkage, presumably on Asn(15) which forms part of the single consensus site for N-glycosylation in human TFF2. The glycosylation may be of functional significance. Future studies of human TFF2 should use antibodies raised against the correct amino acid sequence. Biological studies should be performed with recombinant protein of the correct sequence, and the biological consequences of glycosylation investigated.
Asunto(s)
Jugo Gástrico/química , Mucosa Gástrica/química , Sustancias de Crecimiento/metabolismo , Mucinas , Proteínas Musculares , Neuropéptidos , Péptidos/metabolismo , ADN Complementario/análisis , Glicosilación , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Factor Trefoil-2 , Factor Trefoil-3RESUMEN
BACKGROUND: TFF1 is a 6.5 kDa secreted protein that is expressed predominantly in normal gastric mucosa. It is coexpressed with mucins and it can form dimers via a free carboxy terminal cysteine residue. AIMS: To investigate the molecular forms of TFF1 that are present in normal human stomach and the association of the different molecular forms with mucus. SUBJECTS: All subjects had macroscopically normal stomachs at gastroscopy. None had a significant past medical history. METHODS: TFF1 was detected in normal gastric mucosa and adherent mucus by western transfer analysis after electrophoresis on reducing and non-reducing polyacrylamide gels. In some instances, proteins were fractionated by caesium chloride density gradient centrifugation prior to detection of TFF1. The location of TFF1 in gastric mucosa with an intact adherent mucus layer was assessed by immunohistochemistry. RESULTS: Three different molecular forms of TFF1 were detected: TFF1 monomer, TFF1 dimer, and a TFF1 complex with an apparent molecular mass of about 25 kDa. TFF1 was present at higher concentrations than realised previously. The TFF1 complex was present in the adherent mucus gel layer but while its interaction with mucin was destabilised by caesium chloride, the interaction between mucin and the TFF1 dimer was resistant to caesium chloride. CONCLUSIONS: Most of TFF1 in normal human gastric mucosa is present in a complex that is stabilised by a disulphide bond. TFF1 is intimately associated with mucus. The high concentration, colocalisation, and binding of TFF1 to gastric mucus strongly implicate TFF1 in gastric mucus function.
Asunto(s)
Mucosa Gástrica/química , Proteínas/química , Western Blotting , Disulfuros/química , Electroforesis en Gel de Poliacrilamida , Humanos , Moco/química , Conformación Proteica , Proteínas/análisis , Factor Trefoil-1 , Proteínas Supresoras de TumorRESUMEN
Three promoters have been identified for the human estrogen receptor-alpha gene. The positions of promoters A and B are known whereas that of the recently identified promoter C is not. Cloning and hybridization experiments demonstrated that promoter C is located more than 21 kb upstream of promoter A. The use of the three promoters was examined in estrogen receptor-positive breast cancer cell lines, cell lines derived from other malignancies, and some normal tissues by RT-PCR and transient transfection. All estrogen-responsive breast cancer cell lines used all three promoters, apart from ZR-75 cells, which did not use promoter B in one of two sublines examined. Cell lines derived from other malignancies and other normal tissues that express lower levels of estrogen receptor-alpha showed more selective promoter usage. This suggests that the level of expression of estrogen receptor-alpha is determined by the number of promoters used, rather than the selective use of specific promoters. We also show that promoter C is used more widely than suggested by others. Analysis of a series of estrogen receptor-positive primary breast tumors showed that all three promoters were used in all the tumors. All three promoters were modulated by estrogen in estrogen-responsive breast cancer cell lines: all three promoters were down-regulated by estrogen in MCF-7 cells in which estrogens reduce receptor expression whereas all promoters used were upregulated in T47D, ZR-75, and EFM-19 cells in which estrogens increase receptor expression. This suggests that it is the repertoire of transcription factors present within a cell rather than the selective use of a specific promoter that determines whether estrogen receptor-alpha expression is increased or decreased by estrogen.
Asunto(s)
Estrógenos/metabolismo , Regiones Promotoras Genéticas , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/genética , Carcinoma/genética , Clonación Molecular , Receptor alfa de Estrógeno , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Elementos de Respuesta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Breast cancer screening facilitates the early detection of breast cancer, although a significant number of tumors still arise in the interval between screening. The objective of this study was to measure the expression of five markers of proven prognostic significance in symptomatic breast cancer (estrogen receptor, progesterone receptor, p53, Ki67, and c-erbB2) in screen-detected and interval breast cancers to identify biological markers that may be associated with the emergence of symptomatic breast cancer in the screening interval. The expression of estrogen receptor, progesterone receptor, p53, Ki67, and c-erbB2 was assessed in a series of 51 true interval and 84 screened-detected invasive tumors by immunohistochemistry. Interval cancers tended to be of higher histological grade and were of larger pathological size than screen-detected cancers. Expression of estrogen receptor was 1.7-fold lower (P<0.001), whereas expression of p53 was 2.5-fold (P<0.01), Ki67 2.4-fold (P<0.001), and c-erbB2 3.6-fold higher (P<0.01) in true interval cancers compared with screen-detected invasive cancers. There was no significant difference in progesterone receptor expression. The most important differences identified by multiple logistic regression analysis were in the expression of Ki67 and c-erbB2. The differences in the expression of these markers were more important than clinical features such as pathological grade and size. Using the logistic regression model, 83% of the tumors analyzed in this study could be correctly assigned as interval or screen-detected tumors on the basis of Ki67 and c-erbB2 expression. The importance of high expression of Ki67 in interval cancers compared with screen-detected cancers suggests that tumors may become symptomatic in the screening interval as a result of increased levels of cell proliferation. The inclusion of c-erbB2 in the regression equation suggests that this growth factor receptor may play a significant role in stimulating the rapid growth of interval cancers.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Antígeno Ki-67/análisis , Receptor ErbB-2/análisis , Anciano , Femenino , Humanos , Inmunohistoquímica , Modelos Logísticos , Persona de Mediana Edad , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/análisisRESUMEN
TFF1 is a 60-amino acid peptide produced in normal gastric mucosa which forms dimers spontaneously. Tumours of patients with gastric cancer usually have reduced TFF1 levels and disruption of the TFF1 gene causes animals to develop gastric adenomas and carcinomas. The effect of normal sequence human recombinant TFF1 and an analogue (Cys(58)-->Ser(58)), which is unable to dimerize, on the proliferation and morphology of the human gastric adenocarcinoma cell line AGS was therefore investigated. Proliferation, assessed by total cell number and [methyl-(3)H]thymidine incorporation, was reduced by dimeric TFF1 in a dose-dependent manner. Monomeric TFF1 also reduced proliferation but was less potent than the dimeric form. It is concluded that TFF1 may be an important controller of gastric cell proliferation, that dimerization of TFF1 is important in this effect, and that the reduced levels of TFF1 seen in gastric cancer may be of clinical relevance.
Asunto(s)
Adenocarcinoma/patología , Inhibidores de Crecimiento/farmacología , Proteínas/farmacología , Neoplasias Gástricas/patología , División Celular/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Persona de Mediana Edad , Proteínas Recombinantes/farmacología , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Human pS2 (trefoil factor family 1, TFF1), a 60-amino acid member of the trefoil peptide family, forms dimers via Cys58 and may stimulate gut repair. The effects of dimeric pS2-TFF1 and monomeric pS2-TFF1 (Cys58 replaced by Ser58) were compared in models of wound healing. Rats given dimeric pS2-TFF1 at 25 and 50 micrograms/kg per h had 50 per cent and 70 per cent reduction in gastric damage induced respectively by indomethacin (20 mg/kg subcutaneously) and restraint (P < 0.01). Monomeric pS2-TFF1, at the same doses, was significantly less effective at reducing injury (about half the amount of protection, P < 0.01 vs. same doses of dimeric). The rate of migration of cells at the leading edge of wounded monolayers of the human colonic cell line HT29 was increased by addition of dimeric or monomeric forms of pS2-TFF1 (0.65-325 micrograms/ml). Dimeric pS2-TFF1 had a greater effect than the monomeric form at all doses tested (P < 0.05). Cell migration induced by pS2-TFF1 was blocked by a pS2-TFF1 antibody, but not by a transforming growth factor beta neutralizing antibody. pS2-TFF1 did not influence cell proliferation as assessed by thymidine incorporation. The increased biological effects of dimeric pS2-TFF1 might be due to direct interaction of Cys58 with a putative trefoil receptor or, more likely, dimerization of pS2-TFF1 might stabilize the interaction with its receptor. This may involve a bivalent interaction of residues on the surfaces of the two trefoil domains.
Asunto(s)
Mucosa Gástrica/lesiones , Proteínas/metabolismo , Cicatrización de Heridas , Análisis de Varianza , Animales , Antiinflamatorios no Esteroideos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Dimerización , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Células HT29 , Humanos , Técnicas In Vitro , Indometacina , Masculino , Proteínas/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/farmacología , Factor Trefoil-1 , Proteínas Supresoras de Tumor , Cicatrización de Heridas/efectos de los fármacosRESUMEN
There is increasing evidence for interactions between steroid and growth factor signalling pathways. Oestrogens modulate the responsiveness of breast epithelial cells to insulin-like growth factors (IGFs), and this may be the mechanism by which oestrogens modulate cell proliferation. Oestrogens appear to act at several points in the IGF signal transduction pathway. Despite earlier studies suggesting that breast epithelial cells do not synthesize IGF-I, we have shown by PCR that IGF-I is expressed and that its expression is regulated by oestrogen. IGF-II is expressed at markedly higher levels than IGF-I and is also regulated by oestrogen, consistent with it being an oestrogen-regulated autocrine growth factor. Oestrogens regulate the expression of IGF binding proteins and the type I IGF receptor. The biological significance of oestrogen regulation of IGF binding protein expression is not clear. Experiments in which the type I IGF receptor has been constitutively overexpressed have suggested that oestrogen regulation of the receptor is not involved in mediating the effects of oestrogen on cell proliferation. Recent studies have started to assess the effects of oestrogen on the expression of components of the IGF signal transduction pathway, and have shown that the expression of insulin receptor substrate-1, the principal substrate for the tyrosine kinase of the type I IGF receptor, is regulated by oestradiol.
Asunto(s)
Mama/metabolismo , División Celular/fisiología , Estrógenos/farmacología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Transducción de Señal , Mama/citología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismoRESUMEN
The single-domain human trefoil proteins [pNR-2/pS2 and human intestinal trefoil factor (hITF)] have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain. The seventh does not form part of the trefoil domain and is located three residues from the C-terminus. The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE. pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not. Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds. To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2. Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein. Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond. These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.
Asunto(s)
Cisteína/química , Mucinas , Proteínas Musculares , Neuropéptidos , Proteínas/química , Secuencia de Aminoácidos , Western Blotting , Dimerización , Disulfuros/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Glutatión/farmacología , Sustancias de Crecimiento/química , Humanos , Datos de Secuencia Molecular , Péptidos/química , Conformación Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de Sulfhidrilo/farmacología , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The three human trefoil proteins pS2, human intestinal trefoil factor (hITF), and human spasmolytic polypeptide (hSP) are expressed principally in the mucosa of the gastrointestinal tract. They are also expressed in a variety of other normal tissues and tumours. This review discusses the pattern of expression of trefoil proteins in cancer, current views on the biological functions of trefoil proteins, and the way in which the expression of trefoil proteins may influence the behaviour of cancer cells.
Asunto(s)
Sustancias de Crecimiento/fisiología , Mucinas , Proteínas Musculares , Proteínas de Neoplasias/fisiología , Neoplasias/metabolismo , Neuropéptidos , Péptidos/fisiología , Proteínas/fisiología , Neoplasias de la Mama/metabolismo , Estrógenos/fisiología , Femenino , Humanos , Metástasis de la Neoplasia , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Proteínas Supresoras de TumorRESUMEN
Human intestinal trefoil factor (hITF) is a small cysteine-rich protein expressed in the gastrointestinal (GI) tract. Its sequence is related to that of other trefoil peptides including the pNR-2/pS2 protein, which is regulated by oestrogen in breast cancer. This study was designed to investigate whether hITF is expressed in human carcinoma cells. cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) of gastric mucosal RNA and sequenced, establishing that this mRNA is expressed in the stomach. Expression of hITF was detected in a proportion of cell lines derived from malignancies of the GI tract, in hepatocellular carcinoma cells, and at highest levels in a small cell lung carcinoma cell line. Amongst breast cancer cell lines, it was expressed in all the oestrogen-responsive but in none of the oestrogen-nonresponsive breast cancer cell lines. The possibility that hITF expression in breast cells is controlled by oestradiol was then tested. Oestradiol treatment increased hITF expression between three- and ten-fold in the oestrogen-responsive breast cancer cell lines, demonstrating that, like pNR-2/pS2, hITF is regulated by oestrogen in breast cancer cells. Tamoxifen inhibited the induction of hITF expression by oestradiol but tamoxifen alone was a partial oestrogen agonist for hITF expression. These results show that hITF is expressed, sometimes ectopically, in several human malignancies, which suggests that trefoil peptides may have a more general role in tumourigenesis than hitherto appreciated. That the expression of hITF is regulated by oestrogen in breast cancer cells suggests that hITF expression may provide a novel marker for oestrogen responsiveness in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Sustancias de Crecimiento/genética , Neoplasias Intestinales/metabolismo , Neoplasias Pulmonares/metabolismo , Mucinas , Proteínas Musculares , Neuropéptidos , Péptidos/genética , ARN Mensajero/análisis , Neoplasias Gástricas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Carcinoma Hepatocelular/metabolismo , Carcinoma de Células Pequeñas/metabolismo , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Femenino , Mucosa Gástrica/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas/genética , Tamoxifeno/farmacología , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Ligands for the type I insulin-like growth factor (IGF) receptor interact with oestrogens to control the proliferation of oestrogen responsive breast cancer cells. The aim of this study was to determine the involvement of ligands for the type I IGF receptor in the regulation of oestrogen receptor (OR) expression by oestrogens and antioestrogens in these cells. Oestrogen decreased OR mRNA levels in MCF-7 cells whereas it increased them in T47D, EFM-19 and ZR-75 cells. In MCF-7 cells, IGF-I and insulin lowered further OR expression in the presence of oestrogen. In the presence of IGF-I or insulin, the induction of progesterone receptor mRNA by oestradiol was considerably attenuated in MCF-7 cells, showing that the enhanced down-regulation of OR mRNA levels influenced the expression of oestrogen-regulated genes. The oestrogen agonist activity of the antioestrogens tamoxifen and ICI 182 780 for the down-regulation of OR expression in MCF-7 cells was modulated by type I IGF receptor ligands. Overall these experiments show that OR expression is differentially regulated by oestrogen in individual oestrogen-responsive breast cancer cell lines. Ligands for the type I IGF receptor can modulate regulation of OR expression by oestrogens and antioestrogens principally in cells in which oestrogens down-regulate OR expression.
Asunto(s)
Estradiol/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Receptores de Estrógenos/biosíntesis , Receptores de Progesterona/biosíntesis , Transcripción Genética/efectos de los fármacos , Neoplasias de la Mama , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/farmacología , Cinética , ARN Mensajero/biosíntesis , Receptor IGF Tipo 1/fisiología , Tamoxifeno/farmacología , Células Tumorales CultivadasRESUMEN
Trefoil proteins contain a conserved domain of distinctive structure. Three human trefoil proteins have been described to date of which the human spasmolytic polypeptide (hSP) and pNR-2/pS2 proteins have a similar pattern of expression in normal tissues. The genes encoding these two proteins were isolated from a human DNA library. Preliminary experiments suggested that some recombinants contained both genes. Southern hybridisation showed that all the recombinants were derived from a single stretch of DNA spanning 45 kb and suggested that the hSP gene was located downstream of the pNR-2/pS2 gene. Further experiments demonstrated that the two genes are transcribed in the same direction and that the distance between the 3' end of the pNR-2/pS2 gene and the 5' end of the hSP gene is 12.5 kb. The close linkage of these two genes is evidence that they have evolved by gene duplication and that their similar pattern of expression in normal tissues could result from the retention of common regulatory elements.