RESUMEN
Heterozygous loss of relatively large chromosomal regions is a hallmark of the inactivation of tumour suppressor genes. Searching for deletions in cancer genomes therefore provides an attractive option to identify new tumour suppressor genes. Here, we have performed a genome-wide survey for regions exhibiting allelic loss in 24 commercially available breast cancer cell lines and four breast cancer xenografts, using microsatellite analysis. The assembled allelotype revealed an average fractional allelic loss of 0.34. A total of 19 arms had low allelic loss frequencies (<25%) and 17 arms had moderate allelic loss frequencies (25-50%). Five chromosomal arms were deleted in more than half of the breast cancer samples (8p, 10q, 13q, 17p, and 17q). Three of these frequently lost chromosomal arms had not been identified as such by comparative genome hybridisation, illustrating the higher sensitivity of microsatellite analysis for the detection of allelic losses. As we present allelic loss data of individual samples, our allelotype should not only aid the identification of new breast cancer genes but also provides a baseline for myriad studies involving these breast cancer cell lines.
Asunto(s)
Neoplasias de la Mama/genética , Pérdida de Heterocigocidad/genética , Alelos , Línea Celular Tumoral , Femenino , Humanos , Repeticiones de Microsatélite/genética , Trasplante HeterólogoRESUMEN
Participation of E-cadherin in the Wnt signaling pathway was suggested because of the dual role of beta-catenin in cell adhesion and the Wnt signaling cascade. Whereas beta-catenin interacts at the cell membrane with the cell adhesion protein E-cadherin, in the nucleus it activates Wnt target genes through formation of transcriptionally active complexes with members of the Tcf/Lef family of transcription factors. Here, we analyzed by PCR and direct cycle sequencing 26 human breast cancer cell lines for alterations in the E-cadherin gene. Genetic alterations were identified in eight cell lines. Five cell lines had truncating mutations, whereas three cell lines had in-frame deletions in the gene transcript and expressed mutant E-cadherin proteins at the cell membrane. Involvement of E-cadherin in the Wnt pathway was evaluated through determination of the activity of a Tcf reporter gene, which had been transiently transfected into 15 breast cancer cell lines. None of six E-cadherin mutant cell lines and four cell lines that exhibit transcriptional silencing of the E-cadherin gene showed Tcf-mediated transcriptional activation. E-cadherin wild-type cell line DU4475 exhibited constitutive Tcf-beta-catenin signaling activity and was found to express truncated APC proteins. These results indicate that if cellular transformation occurred through mutation of E-cadherin, it is not mediated via constitutive activation of the Wnt signaling pathway.
Asunto(s)
Neoplasias de la Mama/genética , Cadherinas/genética , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Proteínas de Pez Cebra , Neoplasias de la Mama/metabolismo , Cadherinas/biosíntesis , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Expresión Génica , Silenciador del Gen , Genes Reporteros , Humanos , Luciferasas/genética , Factor de Unión 1 al Potenciador Linfoide , Mutación , Factores de Transcripción/genética , Activación Transcripcional/fisiología , Células Tumorales Cultivadas , Proteínas WntRESUMEN
BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Genes BRCA1/genética , Pérdida de Heterocigocidad , Neoplasias Ováricas/metabolismo , Regiones Promotoras Genéticas/genética , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metilación , Neoplasias Ováricas/genética , ARN , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Angiogenesis and synovial cell hyperplasia are characteristic features of rheumatoid arthritis (RA). Many growth and survival factors use receptors belonging to the tyrosine kinase family that share conserved motifs within the intracellular catalytic domains. To understand further the molecular basis of cellular hyperplasia in RA, we have used degenerate primers based on these motifs and RNA obtained from the synovium of a patient with RA to perform reverse transcriptase-polymerase chain reaction. We report detection of the receptor tyrosine kinase (RTK) Axl in RA synovium and we document the expression pattern of Axl in capillary endothelium, in vascular smooth muscle cells of arterioles and veins, and in a subset of synovial cells in RA synovial tissue. Gas6 (for growth arrest-specific gene 6), which is a ligand for Axl and is related to the coagulation factor protein S, was found in synovial fluid and tissue from patients with RA and osteoarthritis. Axl expression and function was studied in human umbilical vein endothelial cells (HUVECs). Gas6 bound to HUVECs; soluble Axl inhibited this binding. Exogenous Gas6 protected HUVECs from apoptosis in response to growth factor withdrawal and from TNFalpha-mediated cytotoxicity. These findings may reveal a new aspect of vascular physiology, which may also be relevant to formation and maintenance of the abnormal vasculature in the rheumatoid synovium.
Asunto(s)
Artritis Reumatoide/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas Oncogénicas/biosíntesis , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Artritis Reumatoide/enzimología , Artritis Reumatoide/patología , Northern Blotting , Western Blotting , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Inmunohistoquímica , Proteínas Oncogénicas/genética , Proteínas/fisiología , Proteínas Proto-Oncogénicas , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Líquido Sinovial/metabolismo , Membrana Sinovial/citología , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Tirosina Quinasa del Receptor AxlRESUMEN
GATA-1 is a tissue-specific DNA-binding protein containing two zinc-finger-like domains. It is expressed predominantly in erythrocytes. Consensus binding sites for GATA-1 have been found in the regulatory elements of all erythroid-specific genes examined. GATA-1 protein is required for erythroid differentiation beyond the proerythroblast stage. In this paper, we demonstrate that the overexpression of GATA-1 in murine erythroleukaemia (MEL) cells alleviates DMSO-induced terminal erythroid differentiation. Hence, there is no induction of globin gene transcription and the cells do not arrest in the G1 phase of the cell cycle. Furthermore, we demonstrate that expression of GATA-1 in non-transformed erythroid precursors also affects their proliferative capacity and terminal differentiation, as assayed by adult globin gene transcription. To gain insight into the mechanism of this effect, we studied the levels and activities of regulators of cell-cycle progression during DMSO-induced differentiation. A decrease in cyclin D-dependent kinase activity was observed during the induction of both control and GATA-1-overexpressing MEL cells. However, cyclin E-dependent kinase activity decreased more than 20-fold in control but less than 2-fold in GATA-1-overexpressing MEL cells upon induction. Thus GATA-1 may exert its effects by regulating cyclin E-dependent kinase activity. We also show that GATA-1 binds to the retinoblastoma protein in vitro, but not to the related protein p107, which may indicate that GATA-1 interacts directly with specific members of the cell-cycle machinery in vivo. We conclude that GATA-1 regulates cell fate, in terms of differentiation or proliferation, by affecting the cell-cycle apparatus.