RESUMEN
Cystine, lanthionine, and cystathionine containing cyclic peptides incorporating the signature nuclear receptor (NR) box (LXXLL) motif have been synthesized and the abilities of these peptides to inhibit estrogen receptor (ER)-coactivator interactions have been determined. We found that helicity of these peptides directly correlated with their bioactivity. Cystathionine proved to be a redox-stable, isosteric replacement for the cystine disulfide. Cystathionine containing peptide 3 showed higher helical character and a lower inhibition constant (Ki, 7 nm) when compared with its cystine counterpart.
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Aminoácidos Sulfúricos/química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Receptores de Estrógenos/metabolismo , Sulfuros/química , Sulfuros/farmacología , Secuencias de Aminoácidos , Unión Competitiva , Estructura Molecular , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptidos Cíclicos/síntesis química , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Uncertainty about prognosis and treatment of axillary lymph node-negative patients with estrogen receptor (ER)-negative or ER-positive invasive breast tumors of 1 cm or less prompted the analysis of data from five National Surgical Adjuvant Breast and Bowel Project randomized clinical trials. METHODS: Two hundred thirty-five patients with ER-negative tumors and 1024 patients with ER-positive tumors were identified in these trials. Patients with ER-negative tumors received surgery alone or surgery and chemotherapy. Patients with ER-positive tumors received surgery alone; surgery and tamoxifen; or surgery, tamoxifen, and chemotherapy. End points were relapse-free survival (RFS), event-free survival, and overall survival. A result was considered to be statistically significant with a P value of.05 or less; all statistical tests were two-sided. RESULTS: The 8-year RFS of women with ER-negative tumors who received surgery alone or with chemotherapy was 81% and 90%, respectively (P = .06). Survival was similar in both groups (93% and 91%; P = .65). The 8-year RFS of women with ER-positive tumors was 86% after surgery alone, 93% when tamoxifen was added (P = .01), and 95% after the addition of tamoxifen and chemotherapy (P = .07 compared with tamoxifen). Survival in the three groups was 90%, 92% (P = .41), and 97%, respectively. The difference between the latter two groups was significant (P = .01). Regardless of ER status or treatment, overall mortality was 8%; one half of the deaths were related to breast cancer. Several covariates affected the risk of recurrence in ER-negative and ER-positive patients. Risk was greater in women with tumors of 1 cm than in those with tumors of less than 1 cm, in women aged 49 years or younger than in those aged 50 years or older, and in women with infiltrating ductal or lobular carcinoma than in those with other histologic tumor types. CONCLUSIONS: Chemotherapy and/or tamoxifen should be considered for the treatment of women with ER-negative or ER-positive tumors of 1 cm or less and negative axillary lymph nodes.
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Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Antineoplásicos Hormonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/cirugía , Canadá , Quimioterapia Adyuvante , Supervivencia sin Enfermedad , Moduladores de los Receptores de Estrógeno/uso terapéutico , Femenino , Humanos , Metástasis Linfática , Mastectomía/métodos , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Factores de Riesgo , Análisis de Supervivencia , Tamoxifeno/uso terapéutico , Resultado del Tratamiento , Estados UnidosRESUMEN
OBJECTIVE: Tumor invasion involves degradation of extracellular matrix. The urokinase plasminogen activation system participates in this process. Urokinase-type plasminogen activator (uPA), its receptor (uPAR), and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1), are proposed to be prognostic factors in some cancers. There are conflicting data regarding the prognostic role of this system in endometrial cancer. METHODS: To determine the prognostic value of the urokinase plasminogen activation system, contents of uPA, uPAR, and PAI-1 were measured in extracts of endometrial cancer tissue using ELISAs. uPA, uPAR, and PAI-1 levels were determined in 91, 54, and 92 extracts, respectively, and correlated with tumor histology, stage, grade, lymph node involvement, prevalence of metastasis, and recurrence as well as with estrogen (ER), progesterone (PR), epidermal growth factor (EGFR) receptor and HER-2/neu contents. RESULTS: Patients with cancers exhibiting advanced stage, high grade, unfavorable tumor histology, nodal involvement, recurrence, and lower PR levels determined by ligand binding had significantly higher uPA content than others. PAI-1 was significantly elevated in patients with advanced stage, high-grade tumor, recurrence, decreased ER content, and lower PR levels determined by ligand binding. uPAR did not show any relation to any of clinical and laboratory parameters. Elevated expression of PAI-1 was associated with significantly shorter disease-free (P = 0.005) and overall (P = 0.0003) survival. Multivariate analysis revealed that PAI-1 was a predictor of survival although stage was the strongest independent factor. CONCLUSION: Elevated uPA and PAI-1 levels appear to correlate with unfavorable prognosis in endometrial cancer.
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Adenocarcinoma/metabolismo , Neoplasias Endometriales/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Receptores ErbB/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Análisis Multivariante , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Especificidad por Sustrato , Tasa de SupervivenciaRESUMEN
TNP-470 is a synthetic analogue of fumagillin that acts as a potent angiogenesis inhibitor. Recently, our laboratory demonstrated that systemic administration of TNP-470 (5.0 mg/kg) decreased the rate of cutaneous wound healing by greater than 20%. In this study, we tested the hypothesis that TNP-470 interferes with the wound repair-stimulating action of basic fibroblast growth factor (bFGF) by competing with endogenous bFGF for its binding sites on the receptor protein. The influence of TNP-470 was examined in vitro in a ligand competition assay of high- and low-affinity receptor binding to (125)I-bFGF in NIH/3T3 cells. Results demonstrated that recognition of (125)I-bFGF by low-affinity growth factor binding sites was significantly decreased (P < 0.01) in the presence of TNP-470. However, TNP-470 inhibition of radiolabeled bFGF binding to high-affinity sites was not significantly affected (P = 0.07). In view of recent studies demonstrating that the low-affinity receptors of bFGF were heparan sulfate proteoglycans, we suggest that the influence of TNP-470 on diminished wound healing is due to its direct recognition by these molecules.
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Inhibidores de la Angiogénesis/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Sesquiterpenos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Células 3T3 , Inhibidores de la Angiogénesis/metabolismo , Animales , Sitios de Unión/fisiología , Ciclohexanos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Radioisótopos de Yodo , Ratones , O-(Cloroacetilcarbamoil) Fumagilol , Receptores de Factores de Crecimiento de Fibroblastos/química , Sesquiterpenos/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Urokinase-type plasminogen activator (uPA), its receptor (uPAR) and inhibitor, plasminogen activator-type 1 (PAI-1) are proposed to be of prognostic significance in some cancers. To determine the prognostic value of the urokinase plasminogen activation system in ovarian cancer, levels of uPA, uPAR, and PAI-1 were measured in extracts of ovarian cancer tissue using ELISA tests. uPA and PAI-1 were determined in 70 tumor extracts and uPAR in 43 extracts. Levels were correlated with age, tumor histology, stage, grade, lymph node and metastatic status, residual disease, risk of recurrence, epidermal growth factor receptor (EGFR) expression, cathepsin D (Cath-D), and c-erbB-2 levels. uPA and uPAR did not exhibit correlation with any of these parameters. However, patients with high grade tumor, recurrence, and lower EGFR and Cath-D had significantly higher PAI-1 levels compared to those of others (P < 0.05). Kaplan-Meier plots of survival were compared. uPA and uPAR were not related to disease-free or overall survival. Although low PAI-1 appeared to predict a longer overall survival, the difference was not statistically significant. Multivariate analysis revealed that PAI-1 was a predictor for overall survival although it was not as strong as stage. These results suggest that elevated PAI-1 seems to be correlated with an unfavorable prognosis in ovarian cancer.
RESUMEN
This study was undertaken to establish the presence and characteristics of receptors for [D-Trp6]LH-RH on the membranes of human ovarian cancer. Specific binding of [125I, D-Trp6]LH-RH was found in 29 of 37 (78.4%) ovarian cancers and in 6 of 11 (54.5%) non-malignant human ovaries. Ligand binding was dependent on time and plasma membrane concentration in a fashion expected of a peptide hormone. Saturation, kinetic and displacement data were consistent with the presence of a highly specific, single class of non-cooperative binding site. On the basis of receptors affinity, LH-RH-receptor-positive ovarian cancers could be divided into two groups: high affinity group (Kd=2.71 +/- 0.60 nM; Bmax=0.46 +/- 0.07 pmol/mg membrane protein) comprising 55% of tumors, and low affinity group (Kd=78.0 +/- 19.6 nM; Bmax=9.44 +/- 2.68 pmol/mg membrane protein) which included 45% of tumors. LH-RH antagonist Cetrorelix showed an affinity to LH-RH receptors on ovarian cancers 14 times higher than the agonist [D-Trp6]LH-RH. Using 125I-epidermal growth factor, specific high affinity receptors were also detected in membranes from 13 of 24 (54%) ovarian cancers and 5 of 11 (45%) non-malignant ovaries. The demonstration of LH-RH receptors in human ovarian cancers provides a rationale for the use of therapeutic approaches based on LH-RH analogues in this malignancy. The probable involvement of growth factors in the development of ovarian cancers suggests the merit of trying a combined therapy based on analogs of LH-RH and somatostatin for this carcinoma.
Asunto(s)
Receptores ErbB/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Receptores LHRH/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Membrana Celular/química , Membrana Celular/metabolismo , Receptores ErbB/análisis , Femenino , Humanos , Cinética , Persona de Mediana Edad , Neoplasias Ováricas/química , Neoplasias Ováricas/clasificación , Ovario/metabolismo , Ovario/patología , Receptores LHRH/análisis , Pamoato de Triptorelina/metabolismoRESUMEN
Twenty N-terminal point mutations of the human estrogen receptor (hER) were constructed as ubiquitin fusion products and expressed under the control of the copper regulated promoter CUP1 in Saccharomyces cerevisiae. The objective of these studies was to overexpress hER in yeast and also to evaluate the functional properties of the N-terminal variants of hER. Fusion of the C-terminus of ubiquitin to the N-terminus of other proteins has been shown to increase the level of protein expression in yeast. Ubiquitin C-terminal hydrolases (UCHs) in yeast efficiently and precisely cleave at the junction with ubiquitin and render free hER with desired amino termini. The variant hER proteins, that were generated by mutating the N-terminus of hER, showed enormous differences in receptor protein levels and transactivation potential. All variant hER proteins were synthesized as 66 kDa species as identified by Western blotting with the exception of the proline-containing variant (Pro-ER). The UB-Pro-ER variant was cleaved inefficiently by UCHs in yeast. The UB-Pro-hER [correction of UB-Pro-hEr] variant also exhibited a different DNA band-shift profile compared to those of the other receptor variants and the wild-type. Val-, Thr-, and Lys-ER did not express, as measured by enzyme-immunoassay and Western blotting; nor did they transactivate a beta-galactosidase reporter gene in yeast. However, the Glu-ER was 50% more active in transactivation as compared to the wild-type. The results of the receptor content, DNA binding properties and transactivation analysis in yeast demonstrate that the N-terminal residue plays an important role in the structure and function of hER.
Asunto(s)
Receptores de Estrógenos/química , Secuencia de Aminoácidos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Regiones Promotoras Genéticas , Receptores de Estrógenos/genética , Proteínas Recombinantes , Saccharomyces cerevisiae , Relación Estructura-Actividad , Activación Transcripcional , Ubiquitinas/químicaRESUMEN
BACKGROUND: Hormone receptors and oncoproteins are receiving increased attention as possible prognostic factors in different carcinomas. Few data are available regarding quantification of their levels of expression in gynecologic malignancies. METHODS: Epidermal growth factor (EGF) receptor specific binding capacities and affinities were measured by ligand binding assay using [125I]EGF in a competition mode with Accufit software (Lundon Software, Inc., Middlefield, OH). HER-2/neu oncoprotein was extracted from membranes and measured using an enzyme-linked immunosorbent assay. Cathepsin D was measured by an immunoradiometric assay using cytosols for steroid receptor analyses. RESULTS: EGF receptors in 23 nonmalignant uteri ranged from undetectable to 50 fmol/mg membrane protein (median, 0), with dissociation constant values of 1.2 x 10(-9) M to 8.5 x 10(-10) M, compared with EGF receptors in 76 endometrial cancers that ranged from undetectable to 7674 fmol/mg (median, 52). HER-2/neu oncoprotein ranged from undetectable to 2.9 HER-2/neu units (HNU)/microg protein (median, 0.6) in 41 nonmalignant uteri and from undetectable to 5.8 HNU/microg protein (median, 2.5) in endometrial cancers (n = 53). Cathepsin D ranged from 5 to 32 pmol/mg cytosol protein (median, 11) in 42 nonmalignant uteri and 18 to 144 pmol/mg protein (median, 42) in 29 endometrial cancers. CONCLUSIONS: Determination of the frequency and levels of EGF receptors, HER-2/neu protein, and cathepsin D in uteri with and without cancer and the availability of reference materials developed in our laboratory, will allow evaluation of their prognostic value in cancers of the uterus.
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Catepsina D/análisis , Receptores ErbB/análisis , Leiomioma/patología , Receptor ErbB-2/análisis , Neoplasias Uterinas/patología , Útero/patología , Membrana Celular/patología , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Femenino , Humanos , Leiomioma/cirugía , Pronóstico , Radioinmunoensayo , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/metabolismo , Neoplasias Uterinas/cirugíaRESUMEN
We evaluated cathepsin D concentrations in 318 breast carcinoma specimens with a standardized IRMA and established distribution values of 5.9-217.8 nmol/g (median 51.8). Concentrations of cathepsin D did not correlate with age or with concentrations of HER-2/neu oncoprotein, estrogen receptor, or epidermal growth factor receptors. A significant correlation was observed between cathepsin D and progestin receptor (P = 0.009), but only in postmenopausal patients. In our role as a National Reference Laboratory for conducting interlaboratory comparisons of tumor markers, we evaluated cathepsin D assay proficiency by using control samples with intra- and interassay CVs of 2-8% and 10-13%, respectively. Human reference specimens containing known quantities of cathepsin D were developed to facilitate standardized testing. The IRMA procedure and the use of quality-assurance samples permits evaluation of the clinical significance of cathepsin D in human breast cancer trials.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/enzimología , Catepsina D/análisis , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento , Receptores ErbB/análisis , Femenino , Humanos , Ensayo Inmunorradiométrico/estadística & datos numéricos , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Control de Calidad , Receptor ErbB-2/análisis , Receptores de Progesterona/análisis , Reproducibilidad de los ResultadosRESUMEN
Overexpression of cathepsin D in several types of carcinoma in women appears to be associated with a poor clinical course. In this prospective investigation, cathepsin D levels in 170 specimens of normal and neoplastic human tissues were determined simultaneously by enzyme immunoassay (EIA) and immunoradiometric assay (IRMA) to allow comparisons in multicentric studies, such as cooperative clinical trials. Nonmalignant uteri and specially prepared reference powders were also evaluated. Linear regression analysis between the two assays for all specimens [EIA = 0.87(IRMA)-3.18] demonstrated a correlation coefficient (r) of 0.99 (P < 0.001). When malignancies were categorized by the tissue origin (i.e., breast, uterus, ovary, lymph node, and colon), highly significant correlations were also observed (regressions slopes ranged from 0.58 to 1.02). Intra- and interassay controls conducted for the new EIA procedure gave CV% ranging from 4.4 to 10.2, which was similar to the IRMA test for cathepsin D. The results of both assays correlated well and were highly reproducible. Either assay may be used with confidence that comparable cathepsin D values will be obtained in a wide range of tissue biopsies.
Asunto(s)
Catepsina D/análisis , Técnicas para Inmunoenzimas , Ensayo Inmunorradiométrico/métodos , Neoplasias/enzimología , Neoplasias de la Mama/enzimología , Neoplasias del Colon/enzimología , Neoplasias Endometriales/enzimología , Estudios de Evaluación como Asunto , Femenino , Humanos , Técnicas para Inmunoenzimas/estadística & datos numéricos , Ensayo Inmunorradiométrico/estadística & datos numéricos , Linfoma/enzimología , Neoplasias Ováricas/enzimología , Pronóstico , Reproducibilidad de los ResultadosRESUMEN
Surveillance colonoscopy and biopsy are inaccurate methods of predicting the likelihood of ulcerative colitis patients to develop colon carcinoma. We examined uPA and PAI-1 as potential markers for assessing these patients and those with familial polyposis who are at risk of developing colon cancer. For comparison, biopsies of normal colon and Crohn's disease were evaluated. We examined 77 colonic mucosa specimens taken from patients undergoing elective resection for benign and malignant colonic disease. uPA and PAI-1 were measured using a monoclonal antibody-based ELISA kit (American Diagnostica, Greenwich, CT) and expressed as ng/mg extract protein. Intra- and interassay controls of uPA gave CV = 3-4% and CV = 8-9%, respectively, while those for PAI-1 were 6-7% and 10-11%, respectively. The Mann-Whitney test showed that both uPA and PAI-1 expression were significantly higher in colon cancer, chronic ulcerative colitis, and Crohn's disease than in normal colon. uPA in familial polyposis samples was similar to that of normal colon, while PAI-1 was much lower than in normal colon. Neither patient age nor sex appeared to influence the expression of these potential markers in any tissue. The pattern of uPA and PAI-1 expression in normal, benign and malignant colon suggests these proteins deserve further consideration as markers for assessing colon carcinoma risk.
Asunto(s)
Enfermedades del Colon/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Poliposis Adenomatosa del Colon/metabolismo , Adolescente , Adulto , Anciano , Biomarcadores/análisis , Colitis Ulcerosa/metabolismo , Neoplasias del Colon/metabolismo , Enfermedad de Crohn/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Bombesin (BN) and its mammalian counterpart, gastrin-releasing peptide (GRP), are hormonally active peptides which appear to function as autocrine or paracrine growth factors in a variety of cells. As part of a long-term investigation of the relationship of peptide and steroid hormone receptors to breast cancer progression and treatment, we examined the binding of [125I-Tyr4]BN to membranes isolated from 100 human breast carcinomas. Thirty-three of these tumors expressed BN/GRP receptor levels of > 10 fmol/mg membrane protein. Two classes of [Tyr4]BN-binding sites were detected using Scatchard analyses of radioligand association data from hormone displacement curves. The high-affinity binding sites exhibited a mean dissociation constant (Kd1) of 2.1 nM and a mean specific binding capacity (Bmax1) of 237 fmol/mg membrane protein. The low affinity binding sites had a mean dissociation constant (Kd2) of 0.3 microM and a mean binding capacity (Bmax2) of 5.9 pmol/mg membrane protein. BN/GRP receptor expression in a breast carcinoma was unrelated to patient age. When the levels of BN/GRP receptors were compared to the content of the sex steroid receptors, a highly significant positive correlation (P < 0.005) was observed between the binding capacities of high-affinity [Tyr4]BN-binding sites and estrogen receptor levels and between the concentrations of low affinity [Tyr4]BN-binding sites and progestin receptor levels (P < 0.05). This represents the first report of these labile, regulatory proteins in biopsies of human breast carcinomas. Expression of specific receptor proteins for BN/GRP, potent mitogens, in a large number of human breast cancers suggests that they may be involved in tumor cell progression. The approach based on determination of BN/GRP receptors might be useful to guide a hormonal therapy with BN/GRP antagonists in some women with breast cancer.
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Bombesina/análisis , Neoplasias de la Mama/química , Receptores de Bombesina/análisis , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Unión Competitiva , Bombesina/metabolismo , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Menopausia , Persona de Mediana Edad , Receptores de Bombesina/metabolismo , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisisRESUMEN
Treatment of nude mice bearing xenografts of the human malignant glioma U87MG cell line with the steroid hormone antagonist RU486 for 4 weeks resulted in a significant and dose-dependent suppression of tumor volume and weight. Receptor analyses of tumor cytosol preparations demonstrated a single class of high affinity binding sites for dexamethasone, but the absence of receptors for progesterone. RU486 also nullified the stimulatory effect of dexamethasone on proliferation of U87MG cells in vitro. These results indicate that the growth of U87MG human malignant glioma is dependent on corticoids. The antiproliferative effect of RU486 appears to be due to the inhibition of binding of glucocorticoid hormones to their receptor proteins. Our results suggest a new therapy for some brain tumors, such as malignant gliomas based on the steroid hormone antagonist RU486.
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Glioma/patología , Mifepristona/farmacología , Animales , División Celular/efectos de los fármacos , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Chronic ulcerative colitis and familial adenomatous polyposis are associated with an increased risk of colorectal carcinoma. Currently, there are no reliable methods to assess carcinoma risk. METHODS: Several prognostic factors known to be useful in breast carcinoma were determined in 102 specimens of colonic mucosa from 38 patients: 22 specimens from "normal," non-neoplastic colon, 49 from chronic ulcerative colitis, 10 from Crohn's colitis, 14 from familial adenomatous polyposis, four from mucosa adjacent to carcinoma, and three from colon carcinoma. Expression of estrogen receptor, progestin receptor, epidermal growth factor receptor, HER-2/neu (c-erb B-2) oncoprotein, and cathepsin D were determined. RESULTS: Epidermal growth factor receptor expression was higher in chronic ulcerative colitis, Crohn's colitis, familial adenomatous polyposis, and colon carcinoma and varied with location within the colon for chronic ulcerative colitis, Crohn's colitis, and familial adenomatous polyposis. Epidermal growth factor receptor expression in mucosa adjacent to carcinoma was similar to that in "normal" colon. CONCLUSION: Further analyses are needed to determine which parameters are related to and possibly predictive of increased carcinoma risk.
Asunto(s)
Catepsina D/análisis , Colon/química , Enfermedades del Colon/metabolismo , Proteínas Oncogénicas Virales/análisis , Receptores de Superficie Celular/análisis , Poliposis Adenomatosa del Colon/metabolismo , Colitis Ulcerosa/metabolismo , Neoplasias del Colon/química , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/etiología , Enfermedad de Crohn/metabolismo , Receptores ErbB/análisis , Humanos , Receptor ErbB-2 , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Factores de RiesgoRESUMEN
BACKGROUND: The S-phase fraction relates to proliferation, an important determinant of tumor behavior, and has been measured most accurately with the DNA precursor tritiated thymidine (TT). The TT labeling index (LI) is a strong stage-independent prognostic indicator for breast carcinoma. The thymidine analogue 5-bromodeoxyuridine (BrdU) is also incorporated into DNA and has the advantage over TT of immunohistochemical detectability rather than requiring autoradiography, but it is less well studied in breast carcinoma. This report demonstrates the equivalence of TT and BrdU LI and explores the relationships between LI and other biologic measurements. METHODS: The LI of 234 consecutive breast carcinomas were measured with TT as was a subsequent series of 450 cases with BrdU, both by incubation in vitro. RESULTS: The mean BrdU LI was 6.4 +/- 0.3% in comparison with 6.9 +/- 0.4% in the prior TT series. LI was unaffected by storage for 24 hours at 4 degrees C before labeling with BrdU. The BrdU and TT LI both correlated: (1) positively with tumor size, histologic type, nuclear size, the number of axillary metastases, the level of DNA ploidy, and the percent S-phase by flow cytometry and (2) negatively with the age of the patient and the levels of estrogen receptor and progesterone receptor measured either by ligand binding or by immunohistochemistry. CONCLUSIONS: BrdU labeling in vitro was an advantageous method for measuring S-phase fraction in breast carcinoma that produced results comparable to those from TT labeling. It should be equally effective for breast cancer kinetic classification and prognosis and is a suitable standard to evaluate newer methods for measuring cellular proliferation.
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Neoplasias de la Mama/patología , Bromodesoxiuridina/metabolismo , Carcinoma/patología , Fase S , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Criopreservación , Femenino , Humanos , Técnicas In Vitro , Timidina/metabolismoRESUMEN
We have used GR mouse mammary carcinomas as a model for evaluating the effect of estrogen receptor (ER) variants, present in the tumor cytosols, on the prognostic reliability of the receptor assay. Rapid, high resolution procedures were used for the assessment of wild-type and variant estrogen receptors in the cytosols of hormone-responsive and nonresponsive mammary tumors of GR mice. Two main ER types were resolved by high performance ion-exchange and size-exclusion chromatography: the wild-type receptor (II), and a fraction representing low molecular weight ER (I). ER types I and II both bound estradiol and both reacted with the anti-ER monoclonal antibodies H222 and D547 whose epitopes are in the C-terminal part of ER. The level of ER type II was higher in hormone-responsive than in hormone-nonresponsive tumors, whereas ER type I was about equally low in both types of tumor groups. ER types I and II both influence the standard ligand binding and antibody binding (Abbott EIA) test, but only the wild-type ER causes hormone-responsive growth of the tumor. These data suggest that prognostic clinical tests, currently in use for estimating ER in tumors of breast cancer patients, may be impaired by the presence of low molecular weight estrogen receptor variants in the tumor samples.
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Estradiol/metabolismo , Neoplasias Mamarias Experimentales/química , Receptores de Estrógenos/análisis , Animales , Anticuerpos Monoclonales/química , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Citosol/química , Femenino , Variación Genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Trasplante de Neoplasias , Pronóstico , Radioinmunoensayo , Receptores de Estrógenos/inmunología , Receptores de Estrógenos/metabolismoRESUMEN
We examined multiple samples of 65 primary breast carcinomas larger than 1 cm in diameter for thymidine labelling index (TLI), DNA index (DNAI, a measure of cellular DNA content by flow cytometry), and estrogen (ER) and progesterone (PgR) receptors by radioligand-binding. One or more axillary metastases were also assayed in 11 patients. Two to 15 samples were successfully assayed for TLI from 59 tumors, 2-31 samples for DNAI from 61 tumors, and 2-15 samples from 55 tumors for ER and PgR. Criteria for heterogeneity were excess inter-sample variance in comparison with intrasample variance at the p less than 0.05 level for TLI and DNAI, and variation of clinically significant magnitude in assay results for ER and PgR. Sixty-one percent of tumors were heterogeneous for TLI, 26% for DNAI, 24% for ER and 40% for PgR. High TLI disposed toward heterogeneity for TLI itself (p = 0.06), for ER (p = 0.04), and for PgR (p = 0.007). Young age favored heterogeneity for TLI (p = 0.12), ER (p = 0.002), and PgR (p = 0.04). Heterogeneity for DNAI was not related to age and TLI status but was more common in larger tumors (p = 0.08). After consideration of relationships between TLI, age, size, ER and PgR, TLI rather than age appears to be the more important determinant of heterogeneity for receptors. High TLI could lead to heterogeneity through increased numbers of cell divisions that favor emergence of variant stemlines, or by causing local vascular and humoral disparities through rapid growth. Regional heterogeneity can explain erroneous prognostic predictions in approximately 10% to 20% of breast carcinoma patients. We recommend multiple sampling of large breast carcinomas and analysis of axillary metastases for study of tumor markers.
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Neoplasias de la Mama/química , ADN/biosíntesis , Ploidias , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento , Aneuploidia , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , ADN/análisis , Femenino , Citometría de Flujo , Humanos , Persona de Mediana Edad , Timidina/metabolismoRESUMEN
The gene coding for the human estrogen receptor protein was expressed as a ubiquitin fusion under the control of the lambda PL promoter in Escherichia coli. Analysis of extracts by Western blot showed that intact receptor protein was produced only when PL promoter was depressed by nalidixic acid at 30 degrees C. To ascertain the intactness of the expressed protein, estrogen receptor in bacterial extracts was titrated with either 17 beta-[3H]estradiol or 17 beta-[125I]iodoestradiol, and the mass was quantified by enzyme immunoassay. Cell extracts contained 484 fmol of estrogen receptor per mg of soluble protein by titration with a Kd of 3.2 x 10(-10) M. The simultaneous analysis by enzyme immunoassay resulted in 487 fmol/mg of extract protein indicating that most of the expressed protein bound ligand with wild type properties. Ligand competition analysis demonstrated that expressed receptor contained a binding domain preferentially recognizing estrogenic substances identical with that of wild-type estrogen receptor. The E. coli-expressed estrogen receptor also associated specifically with estrogen response DNA elements. Full length receptor protein was detected by ligand binding and immunorecognition using size exclusion chromatography. Hydrophobic interaction chromatography showed a major isoform which exhibited both ligand binding and immuno-recognition identical with the estrogen receptor from human breast tumor tissues. These data indicate that estrogen receptor expressed in E. coli retained characteristics of the native protein found in human tissues.
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Proteínas de Unión al ADN/genética , Receptores de Estrógenos/genética , Western Blotting , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Estradiol/metabolismo , Expresión Génica , Humanos , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitinas/genéticaRESUMEN
In view of advancements in treatment of certain hormone-dependent cancers with analogues of luteinizing hormone-releasing hormone (LH-RH), this study was undertaken to establish the presence and characteristics of receptors for [D-Trp6]LH-RH on the membranes of human endometrial cancer. Specific binding of [125I,D-Trp6]LH-RH was demonstrated in membrane preparations from 24 of 31 (77%) endometrial carcinomas and from 3 of 13 (23.1%) nonmalignant human endometrial specimens. Ligand binding was dependent on temperature, time, and plasma membrane concentration in a fashion expected of a peptide hormone. Mathematical analysis of the binding data showed that interaction of [125I,D-Trp6]LH-RH with the binding sites was consistent with the presence of a single class of high affinity, noncooperative receptors (Kd 9.88 +/- 4.59 x 10(-9) M; Bmax 0.70 +/- 0.14 x 10(-12) mol/mg membrane protein). The rates of association and dissociation were calculated to be 6.5 x 10(6) M-1 min-1 and 0.021 min-1, respectively. [125I,D-Trp6]LH-RH binding was not displaced by either unlabeled somatostatin or epidermal growth factor, but was displaced completely by native LH-RH. Using 125I-epidermal growth factor, specific, high-affinity receptors were also detected in membranes from 22 of 26 (85%) endometrial cancers and in all of 6 nonmalignant endometrial specimens (Kd 0.42 +/- 0.12 x 10(-9) M; Bmax 0.30 +/- 0.15 x 10(-12) mol/mg membrane protein). The potential functional role of the receptors for [D-Trp6]LH-RH in human endometrial carcinoma is not clear, but this finding provides a rationale for the use of therapeutic approaches based on LH-RH analogues in this malignancy.
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Adenocarcinoma/análisis , Antineoplásicos/metabolismo , Biomarcadores de Tumor/análisis , Receptores ErbB/análisis , Hormona Liberadora de Gonadotropina/análogos & derivados , Receptores LHRH/análisis , Neoplasias Uterinas/análisis , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Membrana Celular/análisis , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Femenino , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Cinética , Persona de Mediana Edad , Receptores LHRH/metabolismo , Pamoato de Triptorelina , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
Most investigators comparing ligand-binding procedures for quantifying estrogen and progestin receptors in human breast cancer with procedures employing monoclonal antibody-based methods have utilized an inappropriate variety of reaction conditions, including the elimination of sodium molybdate in the steroid-binding assays. We studied 197 biopsies of human breast cancer, comparing the results of simultaneous measurements of both estrogen and progestin receptors in identical cytosols by enzyme immunoassay and by radioligand binding using the commercially available kits developed by Abbott Laboratories and by DuPont/NEN Products, respectively. Regression analyses comparing the results from the two procedures indicated a linear relationship, with correlation coefficients ranging from 0.79 to 0.93 for both types of receptors over a wide range of data. Using the widely established cutoff value of 10 fmol/mg of cytosol protein for the ligand-binding method, and calculating sensitivity and specificity limits according to McNeil et al. (1975), an equivalent cutoff value of 15 fmol/mg of cytosol protein was determined for the enzyme immunoassay of these receptors. Endocrine status of the patient did not appear to alter the cutoff values of either estrogen or progestin receptors when determined by enzyme immunoassay. We recommend that these cutoff values be considered until the results of clinical correlations are completed.