RESUMEN
PURPOSE: The interleukin 1 (IL-1) system plays an important role in human pathology and is involved in the local control of malignant disease. However, little is known about its expression in breast cancer and its correlation with prognostic parameters such as receptor status and grading. EXPERIMENTAL DESIGN: The expression of IL-1alpha and other IL-1 family members was analyzed by reverse transcription-PCR, ELISA, and immunohistochemistry in breast cancer cell lines, tumor-derived fibroblasts, and breast cancer tissue biopsies and compared with sex steroid receptor status and grading. RESULTS: In breast cancer cell lines, IL-1alpha and -beta gene expression was present in the phenotypically most malignant cell lines, whereas estrogen receptor (ER) alpha and progesterone receptor mRNA expression was confined to lines that exhibit a rather benign phenotype. Only the highly malignant receptor-negative tumor cell line MDA MB 231 expressed IL-1alpha protein, and none of the cell lines secreted IL-1beta. Biopsies from breast cancer tissue expressed various amounts of IL-1alpha, IL-1beta, and IL-1 receptor antagonist mRNA, but consistently high levels of IL-1tIR. IL-1alpha protein expression was detected in tumor cells and/or adjacent stroma in 88%, and epithelial protein expression was correlated with both poor differentiation (P = 0.002; r = 0.469) and decreasing epithelial ERalpha expression (P = 0.004; r = -0.387). Furthermore, stromal IL-1alpha was predominant in areas with low or absent ERalpha protein expression in neighboring tumor epithelium (P = 0.001; r = -0.457). CONCLUSION: We have demonstrated the presence of a functional IL-1 system in breast cancer and found that IL-1alpha is inversely correlated with local sex steroid receptor expression. We hypothesize that the unphysiological expression of IL-1alpha in less differentiated and ERalpha-negative tumors might contribute to their local invasiveness and malignant behavior.
Asunto(s)
Neoplasias de la Mama/metabolismo , Interleucina-1/biosíntesis , Interleucina-1/metabolismo , Esteroides/biosíntesis , Línea Celular Tumoral , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Fenotipo , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is a potent cytokine secreted primarily by activated cells from the monocyte/macrophage lineage which exhibits various antitumoral effects including the induction of apoptosis, necrosis, activation of lytic effector cells as well as upregulation of the expression of intercellular adhesion molecule-1 (ICAM-1) which is of decisive importance in the interaction with lymphokine activated killer cells. Previous studies from our laboratory have indicated impaired production of TNF-alpha by monocytes as well as decreased expression of ICAM-1 on monocytes derived from patients with various stages of breast cancer. METHODS: In the present experiments, we have assessed spontaneous as well as lipopolysaccharide (LPS)-induced production of TNF-alpha by as well as expression of ICAM-1 on monocytes derived from healthy females with germline mutations of BRCA1 and from healthy age-matched control females. RESULTS: We report that monocytes derived from healthy women with various germline mutations of BRCA1 had significantly decreased spontaneous (p = 0.03) and LPS-induced (p < 0.001) production of TNF-alpha, as compared to monocytes derived from healthy age-matched control females. In contrast, no difference in LPS- or TNF-alpha-induced production of interleukin-6 was found. Whereas unstimulated monocytes derived from healthy women with germline mutations of BRCA1 and from healthy control women had similar expression of ICAM-1, stimulation with cytokines TNF-alpha and/or interleukin-1 led to a significant increase of ICAM-1 expression on monocytes derived from control females only, but not from BRCA1 germline mutation carriers (p < 0.001). CONCLUSION: We conclude that the presence of germline mutations of BRCA1 was associated with a selective deficiency in spontaneous and LPS-induced production of TNF-alpha and of TNF-alpha-induced ICAM-1 expression on peripheral blood monocytes.
Asunto(s)
Neoplasias de la Mama/metabolismo , Genes BRCA1 , Mutación de Línea Germinal , Heterocigoto , Molécula 1 de Adhesión Intercelular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Análisis Mutacional de ADN , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-6/metabolismo , Persona de Mediana Edad , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
TSC1 and TSC2 are responsible for the tumor suppressor gene syndrome tuberous sclerosis (TSC). Mammalian TSC genes have been shown to be involved in cell cycle regulation. Recently, in Drosophila, these data have been confirmed and TSC genes have further been demonstrated to affect cell size control. Here we provide supporting data for the fact that the latter function is conserved in mammals. Human TSC1 and TSC2 trigger mammalian cell size reduction and a dominant-negative TSC2 mutant induces increased size. These effects occur in all cell cycle phases, are dependent on the activity of the phosphoinositide-3-kinase and are abolished by co-overexpression of a dominant-negative Akt mutant. Two independent naturally occurring and disease-causing mutations within the TSC2 gene eliminate tuberin's capacity to affect cell size control, emphasizing the relevance of this function for the development of the disease. The same mutations have earlier been shown not to affect tuberin's antiproliferative capacity. That the consequences of modulated TSC gene expression on cell proliferation and on cell size can be assigned to separable functions is further supported by two findings: A mutation within the TSC1 gene, earlier shown to still harbor anti-proliferative effects, was found to eliminate the cell size regulating functions. An important mammalian cell size regulator, c-Myc, was found to inhibit tuberin's antiproliferative capacity, but to have no effects on tuberin-dependent cell size control. To obtain further mechanistical insights, microarray screens for genes involved in TSC1- or TSC2-mediated cell size effects were performed. Antisense experiments revealed that the so observed regulation of the FK506-binding protein, FKBP38, plays a role in TSC gene-dependent cell size regulation. These data provide new insights into mammalian cell size regulation and allow a better understanding of the function of human TSC genes.
Asunto(s)
Tamaño de la Célula/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas/fisiología , Proteínas Represoras/fisiología , Proteínas de Unión a Tacrolimus/fisiología , Sustitución de Aminoácidos , Animales , Ciclo Celular/fisiología , División Celular/fisiología , Cromonas/farmacología , ADN Complementario/genética , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Dominantes , Células HeLa/citología , Humanos , Morfolinas/farmacología , Mutación Missense , Oligodesoxirribonucleótidos Antisentido/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-myc/fisiología , Ratas , Proteínas Recombinantes de Fusión/fisiología , Proteínas Represoras/genética , Transfección , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
Breast cancer is one of the most frequent malignancies affecting women. The human breast cancer gene 1 (BRCA1) gene is mutated in a distinct proportion of hereditary breast and ovarian cancers. Tumourigenesis in individuals with germline BRCA1 mutations requires somatic inactivation of the remaining wild-type allelle. Although, this evidence supports a role for BRCA1 as a tumour suppressor, the mechanisms through which its loss leads to tumourigenesis remain to be determined. Neither the expression pattern nor the described functions of human BRCA1 and murine breast cancer gene 1 (Brca1) can explain the specific association of mutations in this gene with the development of breast and ovarian cancer. Investigation of the role of Brca1 in normal cell differentiation processes might provide the basis to understand the tissue-restricted properties.
Asunto(s)
Neoplasias de la Mama/genética , Diferenciación Celular/fisiología , Genes BRCA1 , Animales , Neoplasias de la Mama/metabolismo , División Celular/fisiología , Femenino , Regulación de la Expresión Génica , Humanos , Mutación , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Receptores de Estrógenos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.
Asunto(s)
Proteína BRCA1/fisiología , Glándulas Mamarias Animales/citología , Animales , Proteína BRCA1/genética , Diferenciación Celular , División Celular , Línea Celular , Línea Celular Transformada , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Células HeLa , Humanos , Cinética , Glándulas Mamarias Animales/metabolismo , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mutación , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Células Tumorales CultivadasRESUMEN
Tissue remodeling is a key element in the local invasion and metastasis of malignant breast tumors. The degradation of extracellular matrix that is associated with this process is thought to be mediated by a number of Zn2+-dependent matrix metalloproteinases (MMPs). In most cases these enzymes are not produced by the malignant epithelium itself but by adjacent breast stroma, suggesting an important role for cell-cell interactions. We have analyzed Gelatinase A (MMP-2) and Gelatinase B (MMP-9) gene expression in a panel of six breast cancer cell lines and six primary cultures of stromal cells deriving from breast cancer biopsies. With one exception we did not detect MMP-2 or MMP-9 gene expression in any of the established tumor cell lines. Conversely, tumor stroma-derived fibroblasts expressed MMP-2 mRNA. although no MMP-9 mRNA was seen in RNase protection assays. When fibroblasts were cultured in the presence of media conditioned by MCF-7 tumor cells, MMP-2 enzyme production increased but MMP-9 activity remained undetectable. However, when fibroblasts and MCF-7 tumor cells were co-cultured together, MMP-9 was induced. These observations were confirmed by immunocytochemical analysis of co-cultures of MCF-7 and tumor-derived fibroblasts in which MMP-2 and MMP-9 protein expression was confined to stromal cells adjacent to MCF-7 tumor cells. No MMP-2 or MMP-9 staining was detected in monocultures of the two respective cell types. We conclude that MMP-2 expression is present in the stroma of malignant tumors and is increased by paracrine stimulation mediated by soluble factors. In contrast, MMP-9 expression tumor-derived fibroblasts requires direct contact with malignant tumor epithelium.
Asunto(s)
Neoplasias de la Mama/enzimología , Comunicación Celular , Fibroblastos/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Mama/patología , Células Epiteliales/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Células del Estroma/fisiología , Células Tumorales Cultivadas/metabolismoRESUMEN
Tuberous sclerosis is an autosomal dominant disease affecting approximately 1 in 6,000 individuals. It is caused by mutations in either TSC1 on chromosome 9q34, which encodes hamartin, or TSC2 on chromosome 16p13.3, which encodes tuberin. The growths, named hamartomas, characteristically occur in different organs of patients and are speculated to result from defects in proliferation control. The observation that hamartin and tuberin can interact in vivo suggests that they might function in the same complex. Here we show that hamartin can affect proliferation control independent of the presence of functional tuberin and that binding to hamartin is not essential for tuberin to affect proliferation. Ectopic expression of hamartin negatively regulates proliferation to a similar extent in tuberin-positive and tuberin-negative cells; this is accompanied by binding to tuberin and upregulation of endogenous p27 in tuberin-positive cells and is without effects on p27 expression in the latter. Our data show for the first time that TSC proteins possess separable functions. We further demonstrate that hamartin can deregulate proliferation control by different mechanisms depending on the presence of tuberin. Besides an overlap in many features of patients with TSC1 and TSC2 mutations, data has accumulated that provides evidence for specific clinical differences. This study provides new insights into the cellular roles of TSC proteins and initiates a discussion of whether separable functions of these proteins might be associated with the clinical differences of TSC1- and TSC2-associated disease.