RESUMEN
The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/fisiología , Proteína Oncogénica v-akt/antagonistas & inhibidores , Neoplasias de la Próstata/genética , ARN Interferente Pequeño/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Humanos , Masculino , FN-kappa B/genética , FN-kappa B/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/fisiología , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Células Tumorales Cultivadas , Quinasas p21 ActivadasRESUMEN
The cytokine hepatocyte growth factor/scatter factor (HGF/SF) protects epithelial and cancer cells against DNA-damaging agents via a pathway involving signaling from c-Met --> phosphatidylinositol-3- kinase --> c-Akt. However, the downstream alterations in gene expression resulting from this pathway have not been established. On the basis of cDNA microarray and semiquantitative RT-PCR assays, we found that MDA-MB-453 human breast cancer cells preincubated with HGF/SF and then exposed to Adriamycin (ADR), a DNA topoisomerase II inhibitor, exhibit an altered pattern of gene expression, as compared with cells treated with ADR only. [HGF/SF+ADR]-treated cells showed altered expression of genes involved in the DNA damage response, cell cycle regulation, signal transduction, metabolism, and development. Some of these alterations suggest mechanisms by which HGF/SF may exert its protective activity, e.g., up-regulation of polycystic kidney disease-1 (a survival-promoting component of cadherin-catenin complexes), down-regulation of 51C (an inositol polyphosphate-5-phosphatase), and down-regulation of TOPBP1 (a topoisomerase IIB binding protein). We showed that enforced expression of the cdc42-interacting protein CIP4, a cytoskeleton-associated protein for which expression was decreased in [HGF/SF+ADR]-treated cells, inhibited HGF/SF-mediated protection against ADR. The cDNA microarray approach may open up new avenues for investigation of the DNA damage response and its regulation by HGF/SF.
Asunto(s)
Neoplasias de la Mama/genética , Daño del ADN/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/farmacología , Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Doxorrubicina/farmacología , Perfilación de la Expresión Génica , Humanos , Proteínas Asociadas a Microtúbulos/fisiología , Antígenos de Histocompatibilidad Menor , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The cytokine hepatocyte growth factor/scatter factor (HGF/SF) has been found to protect a variety of epithelial and cancer cell types against cytotoxicity and apoptosis induced by DNA damage, but the specific apoptotic signaling events and the levels at which they are blocked by HGF/SF have not been identified. We found that treatment of MDA-MB-453 human breast cancer cells with adriamycin (also known as doxorubicin, a DNA topoisomerase IIalpha inhibitor) induced a series of time-dependent events, including the mitochondrial release of cytochrome c and apoptosis-inducing factor, mitochondrial membrane depolarization, activation of a set of caspases (caspase-9, -3, -7, -2, and -8), cleavage of poly(ADP-ribose) polymerase (PARP), and up-regulation of expression of the Fas ligand. All of these events were blocked by preincubation of the cells with HGF/SF. In contrast, the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone blocked some of these events (e.g. caspase-3 activation and PARP cleavage) but did not block cytochrome c release or mitochondrial depolarization. These findings suggest that HGF/SF functions, in part, upstream of the mitochondria to block mitochondrial apoptosis signaling, prevent activation of multiple caspases, and protect breast cancer cells against apoptosis.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Mitocondrias/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Antineoplásicos/farmacología , Factor Inductor de la Apoptosis , Caspasa 3 , Caspasas/metabolismo , Colorantes/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Grupo Citocromo c/metabolismo , Doxorrubicina/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas , Flavoproteínas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The tumor suppressor activity of the BRCA1 gene product is due, in part, to functional interactions with other tumor suppressors, including p53 and the retinoblastoma (RB) protein. RB binding sites on BRCA1 were identified in the C-terminal BRCT domain (Yarden and Brody, 1999) and in the N-terminus (aa 304-394) (Aprelikova et al., 1999). The N-terminal site contains a consensus RB binding motif, LXCXE (aa 358-362), but the role of this motif in RB binding and BRCA1 functional activity is unclear. In both in vitro and in vivo assays, we found that the BRCA1:RB interaction does not require the BRCA1 LXCXE motif, nor does it require an intact A/B binding pocket of RB. In addition, nuclear co-localization of the endogenous BRCA1 and RB proteins was observed. Over-expression of wild-type BRCA1 (wtBRCA1) did not cause cell cycle arrest but did cause down-regulation of expression of RB, p107, p130, and other proteins (e.g., p300), associated with increased sensitivity to DNA-damaging agents. In contrast, expression of a full-length BRCA1 with an LXCXE inactivating mutation (LXCXE-->RXRXH) failed to down-regulate RB, blocked the down-regulation of RB by wtBRCA1, induced chemoresistance, and abrogated the ability of BRCA1 to mediate tumor growth suppression of DU-145 prostate cancer cells. wtBRCA1-induced chemosensitivity was partially reversed by expression of either Rb or p300 and fully reversed by co-expression of Rb plus p300. Our findings suggest that: (1) disruption of the LXCXE motif within the N-terminal RB binding region alters the biologic function of BRCA1; and (2) over-expression of BRCA1 inhibits the expression of RB and RB family (p107 and p130) proteins.
Asunto(s)
Proteína BRCA1/fisiología , Proteína de Retinoblastoma/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteína BRCA1/química , Sitios de Unión , Regulación hacia Abajo , Genes de Retinoblastoma , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Transactivadores/genética , Células Tumorales CultivadasRESUMEN
Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factor de Crecimiento de Hepatocito/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Proteínas Proto-Oncogénicas c-met/metabolismo , Androstadienos/farmacología , Animales , Apoptosis , Sitios de Unión , Western Blotting , Caspasa 3 , Caspasas/metabolismo , Caspasas/fisiología , División Celular , Línea Celular , Movimiento Celular , Supervivencia Celular , ADN/metabolismo , Daño del ADN , Fragmentación del ADN , Reparación del ADN , Perros , Doxorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Proteína Adaptadora GRB2 , Vectores Genéticos , Humanos , Modelos Biológicos , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/metabolismo , Transducción de Señal , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de Tiempo , Transfección , Wortmanina , Quinasas p21 ActivadasRESUMEN
Extracellular matrix (ECM) degrading matrix metalloproteinases (MMPs) lead to ECM turnover, a key event in cancer growth and progression. The tissue inhibitors of matrix metalloproteinases (TIMPs) limit the activity of MMPs, which suggests their use for cancer gene therapy. Here we report that systemic administration of naked TIMP-4 DNA significantly inhibited Wilms' tumor growth in nude mice. TIMP-4, whose expression was lost in Wilms' tumor, inhibited the growth of G401 Wilms' tumor cells at a concentration lower than those required for MMP inhibition. This inhibition was associated with internalization of exogenous recombinant TIMP-4. Electroporation-mediated intramuscular injection of TIMP-4 expression plasmid resulted in sustained plasma TIMP-4 levels and significant tumor suppression. Our data demonstrate a tumor suppressive effect of TIMP-4 against Wilms' tumor and the potential utility of intramuscular delivery of TIMP gene for treatment of kidney derived cancers.
Asunto(s)
Vacunas contra el Cáncer/farmacología , Inhibidores Tisulares de Metaloproteinasas/genética , Vacunas de ADN/farmacología , Tumor de Wilms/terapia , Adulto , Animales , División Celular , Niño , Humanos , Inyecciones Intramusculares , Riñón/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Plásmidos , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Inhibidores Tisulares de Metaloproteinasas/farmacología , Células Tumorales Cultivadas , Tumor de Wilms/enzimología , Tumor de Wilms/patología , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
The breast cancer susceptibility gene BRCA1 on chromosome 17q21 encodes an 1863 amino acid protein that is important for normal embryonic development. Germline mutations of this gene are linked to a significantly increased lifetime risk for breast and/or ovarian cancer, and recent studies suggest that the same may be true for prostate cancer. Several activities that may contribute to the tumor suppressor function of BRCA1 have been identified via in vitro and experimental animal studies. These include (i) regulation of cell proliferation; (ii) participation in DNA repair/recombination processes related to the maintenance of genomic integrity; (iii) induction of apoptosis in damaged cells; and (iv) regulation of transcription. A second breast cancer susceptibility gene (BRCA2) operates in some of the same molecular pathways as BRCA1, and mutations of this gene predispose to breast and ovarian cancer and probably to other tumor types, including prostate cancer. Finally, recent studies from our laboratory suggest that BRCA1 modulates proliferation, chemosensitivity, repair of DNA strand breaks, apoptosis induction, and expression of certain key cellular regulatory proteins (including BRCA2 and p300) in human prostate cancer cells. These activities are consistent with a putative prostate tumor suppressor function of BRCA1.
Asunto(s)
Adenocarcinoma/genética , Genes BRCA1 , Neoplasias de la Próstata/genética , Adenocarcinoma/epidemiología , Adenocarcinoma/patología , Secuencias de Aminoácidos , Animales , Apoptosis/genética , Proteína BRCA1/química , Proteína BRCA1/fisiología , Proteína BRCA2 , Neoplasias de la Mama/etnología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular , División Celular , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 17/genética , Daño del ADN , Desarrollo Embrionario y Fetal/genética , Estrógenos , Femenino , Regulación de la Expresión Génica , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Humanos , Judíos/genética , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/patología , Síndromes Neoplásicos Hereditarios/genética , Neoplasias Ováricas/genética , Neoplasias de la Próstata/epidemiología , Neoplasias de la Próstata/patología , Estructura Terciaria de Proteína , Factores de Riesgo , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Tissue inhibitors of matrix metalloproteinase (TIMPs) are multifunctional proteins with both matrix metalloproteinase (MMP) inhibitory effects and growth-regulatory activity. TIMPs inhibit MMP activity, suggesting a use for cancer gene therapy. However, here we report that systemic administration of human TIMP-4 by electroporation-mediated i.m. injection of naked TIMP-4 DNA stimulates tumorigenesis of human breast cancer cells in nude mice. Consistent with tumor stimulation, TIMP-4 up-regulates Bcl-2 and Bcl-X(L) protein. TIMP-4 also inhibits apoptosis in human breast cancer cells in vitro and mammary tumors in vivo. A synthetic MMP inhibitor BB-94 did not have such antiapoptotic effect. Analysis of TIMP-4 expression in human mammary specimens indicates that TIMP-4 protein is increased in mammary carcinoma cells compared with normal mammary epithelial cells. These data indicate an antiapoptotic activity in breast cancer cells and a tumor-stimulating effect of TIMP-4 when administrated systemically.
Asunto(s)
Neoplasias de la Mama/genética , Mama/fisiología , Transformación Celular Neoplásica/genética , ADN/administración & dosificación , Inhibidores Tisulares de Metaloproteinasas/fisiología , Animales , Apoptosis/genética , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/patología , Supervivencia Celular/genética , ADN/genética , Electroporación , Femenino , Terapia Genética , Humanos , Inyecciones Intramusculares , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Plásmidos/administración & dosificación , Plásmidos/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Conejos , Inhibidores Tisulares de Metaloproteinasas/biosíntesis , Inhibidores Tisulares de Metaloproteinasas/genética , Trasplante Heterólogo , Proteína bcl-X , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
UNLABELLED: P73, a new p53 homologue, has been recently identified as a candidate tumor suppressor gene. PURPOSE: We studied the alterations in p73 in a panel of human cancer cell lines treated with the chemotherapeutic agent, Adriamycin (ADR), in comparison with the changes in p53. METHODS: P73 and p53 mRNA and protein were determined using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. ADR cytotoxicity was examined by a trypan blue dye exclusion assay. RESULTS: The cell lines bearing wild-type p53 were more susceptible to ADR than the cell lines bearing mutant p53. ADR treatment resulted in a significant accumulation of p53 protein and mRNA expression in the wild-type p53 cell lines and caused little (slight increase) or no influence on p53 expression in the cell lines with p53 mutation and deletion. However, in striking contrast to the alterations in p53, a decline in p73 at both the protein and mRNA levels was observed in all the cell lines examined following ADR treatment. Further studies indicated that this p53-independent downregulation of p73 was induced by ADR in a dose- and time-dependent manner. Moreover, the p73 protein decline was abrogated by the presence of proteasome inhibitors. CONCLUSIONS: Our findings revealed that although p73 shares a similar structural and functional composition with p53, there is a significant difference in the mechanisms that govern the responses of p53 and p73 to ADR-induced DNA damage.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Proteínas de Unión al ADN/genética , Doxorrubicina/farmacología , Genes p53/fisiología , Proteínas Nucleares/genética , Cisteína Endopeptidasas , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Genes Supresores de Tumor , Humanos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/análisis , Factores de Tiempo , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
The BRCA1 gene was previously found to inhibit the transcriptional activity of the estrogen receptor [ER-alpha] in human breast and prostate cancer cell lines. In this study, we found that breast cancer-associated mutations of BRCA1 abolish or reduce its ability to inhibit ER-alpha activity and that domains within the amino- and carboxyl-termini of the BRCA1 protein are required for the inhibition. BRCA1 inhibition of ER-alpha activity was demonstrated under conditions in which a BRCA1 transgene was transiently or stably over-expressed in cell lines with endogenous wild-type BRCA1 and in a breast cancer cell line that lacks endogenous functional BRCA1 (HCC1937). In addition, BRCA1 blocked the expression of two endogenous estrogen-regulated gene products in human breast cancer cells: pS2 and cathepsin D. The BRCA1 protein was found to associate with ER-alpha in vivo and to bind to ER-alpha in vitro, by an estrogen-independent interaction that mapped to the amino-terminal region of BRCA1 (ca. amino acid 1-300) and the conserved carboxyl-terminal activation function [AF-2] domain of ER-alpha. Furthermore, several truncated BRCA1 proteins containing the amino-terminal ER-alpha binding region blocked the ability of the full-length BRCA1 protein to inhibit ER-alpha activity. Our findings suggest that the amino-terminus of BRCA1 interacts with ER-alpha, while the carboxyl-terminus of BRCA1 may function as a transcriptional repression domain. Oncogene (2001) 20, 77 - 87.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Genes BRCA1/fisiología , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/fisiología , Sitios de Unión/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Mapeo Cromosómico , Receptor alfa de Estrógeno , Genes BRCA1/genética , Glutatión Transferasa/genética , Humanos , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Germ-line mutations of the breast cancer susceptibility gene 1 (BRCA1) confer a high risk for breast and ovarian cancer in women and prostate cancer in men. The BRCA1 protein contributes to cell proliferation, cell cycle regulation, DNA repair and apoptosis; however, the mechanisms underlying these functions of BRCA1 remain largely unknown. Here, we showed that, in Du-145 human prostate cancer cells, enhanced expression of BRCA1 resulted in constitutive activation of signal transducer and activator transcription factor 3 (STAT3) tyrosine and serine phosphorylation. Moreover, Janus kinase 1 (JAK1) and JAK2, the upstream activators of STAT3, were also activated by BRCA1. Immunoprecipitation assay showed that BRCA1 interacted with JAK1 and JAK2. Blocking STAT3 activation using antisense oligonucleotides significantly inhibited cell proliferation and triggered apoptosis in Du-145 cells with enhanced expression of BRCA1. These findings indicate that BRCA1 interacts with the components of the JAK-STAT signaling cascade and modulates its activation, which may provide a new critical survival signal for the growth of breast, ovarian and prostate cancers in the presence of normal BRCA1.
Asunto(s)
Proteína BRCA1/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Transactivadores/metabolismo , Apoptosis , Proteína BRCA1/genética , Western Blotting , División Celular , Supervivencia Celular , Proteínas de Unión al ADN/genética , Activación Enzimática , Genes BRCA1/genética , Humanos , Interleucina-6/farmacología , Janus Quinasa 1 , Janus Quinasa 2 , Masculino , Oligonucleótidos Antisentido/genética , Fosforilación , Pruebas de Precipitina , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Unión Proteica , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , Transactivadores/genética , Transfección , Células Tumorales CultivadasRESUMEN
Unregulated expression of wild-type BRCA1 (wtBRCA1) confers an altered phenotype in cultured human prostate cancer cells, characterized by chemosensitivity, susceptibility to apoptosis, decreased DNA repair activity, and alterations of key cell regulatory proteins. We now report that the expression of truncated or mutant full-length BRCA1 genes can abrogate certain phenotypic characteristics and/or confer the opposite phenotype to the wild-type BRCA1 gene. In particular, several carboxyl-terminal truncated BRCA1 proteins conferred chemoresistance, decreased susceptibility to apoptosis, and decreased ability to suppress in vivo tumor growth. These truncated BRCA1 proteins also blocked the ability of ectopically expressed wtBRCA1 to induce chemosensitivity and to inhibit estrogen receptor transcriptional activity. Studies using epitope-tagged truncated proteins confirmed their expression, nuclear localization, and functionality. On the other hand, in cells with no endogenous wild-type BRCA1 (HCC1937 human breast cancer cells), the wtBRCA1 gene enhanced cellular DNA repair activity and rendered the cells resistant to DNA damage; while truncated BRCA1 proteins blocked the wtBRCA1-induced chemoresistance. Our findings suggest that truncated BRCA1 proteins can inhibit the function of wild-type BRCA1. They raise the possibility that some inherited BRCA1 mutations may actively promote oncogenesis by blocking the function of the remaining wild-type BRCA1 allele, although this hypothesis remains to be proved.
Asunto(s)
Proteína BRCA1/antagonistas & inhibidores , Genes BRCA1 , Mutación , Neoplasias de la Próstata/patología , Apoptosis , Proteína BRCA1/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , División Celular , Daño del ADN , Reparación del ADN , ADN de Neoplasias/análisis , Resistencia a Antineoplásicos , Receptor alfa de Estrógeno , Femenino , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Fenotipo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores de Estrógenos/antagonistas & inhibidores , Transfección , Células Tumorales CultivadasRESUMEN
Mutations of the human breast cancer susceptibility gene 1 (BRCA1) confers a risk for breast, ovarian and prostate cancers and BRCA1 exerts multiple biological functions. Using Western blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, we have determined the expression of endogenous BRCA1 protein and mRNA in forty-three human tumor cell lines established from eleven types of human tumor tissues. BRCA1 was differentially expressed in tumor cell lines. No significant association was found between BRCA1 expression and the p53 gene status of cell lines. The disruption of wild-type p53 by either the human papillomavirus E6 oncogene or the mutant p53 gene (143Ala-->Val) did not cause any significant alteration in basal level of BRCA1 expression, while the knockout of p21 (-/-) by homologous recombination assay and Blocking Gadd45 expression by constitutive antisense expression slightly increased BRCA1 protein expression. Therefore, although the functional significance of the differential expression in human tumor cells is currently unknown, the present data provide a valuable background for further study of BRCA1 in tumor cell lines.
Asunto(s)
Proteína BRCA1/genética , Genes p53/genética , Proteína BRCA1/biosíntesis , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Estrogen, via its binding to the estrogen receptor (ER), plays an important role in breast cancer cell proliferation and tumor development. Indole-3-carbinol (I3C), a compound occurring naturally in cruciferous vegetables, exhibits a potent antitumor activity via its regulation of estrogen activity and metabolism. This study was designed to determine the effect of I3C on the potential to inhibit the ER-alpha. Using a reporter gene driven by the estrogen receptor, I3C (10-125 micromol/L) significantly repressed the 17ss-estradiol (E2)-activated ER-alpha signaling in a dose-dependent manner. I3C and breast cancer susceptibility gene 1 (BRCA1) synergistically inhibited transcriptional activity of ER-alpha. Moreover, I3C down-regulated the expression of the estrogen-responsive genes, pS2 and cathepsin-D, and up-regulated BRCA1. The inhibitory effects of I3C did not contribute to its cytotoxic effects because these activities were observed at less than toxic concentrations. These results further suggest that antitumor activities of I3C are associated not only with its regulation of estrogen activity and metabolism, but also its modulation of ER transcription activity.
Asunto(s)
Neoplasias de la Mama/genética , Antagonistas de Estrógenos/farmacología , Indoles/farmacología , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/etiología , Catepsina D/efectos de los fármacos , Catepsina D/genética , Catepsina D/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Receptor alfa de Estrógeno , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes BRCA1/efectos de los fármacos , Inhibidores de Crecimiento/metabolismo , Humanos , Proteínas/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , Receptores de Estrógenos/genética , Factor de Transcripción Sp1/efectos de los fármacos , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/fisiología , Transcripción Genética/efectos de los fármacos , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
We previously identified and characterized a novel tumor growth inhibitor and a fatty acid-binding protein in human mammary gland and named it the mammary-derived growth inhibitor-related gene (MRG). Here, the effects of MRG on mammary gland differentiation and its interaction with omega-3 polyunsaturated fatty acids (omega-3 PUFAs) on growth inhibition were investigated. MRG protein expression was associated with human mammary gland differentiation, with the highest expression observed in the differentiated alveolar mammary epithelial cells from the lactating gland. Overexpression of MRG in human breast cancer cells induced differentiation with changes in cellular morphology and a significant increase in the production of lipid droplets. Treatment of mouse mammary gland in organ culture with MRG protein resulted in a differentiated morphology and stimulation of beta-casein expression. Treatment of human breast cancer cells with the omega-3 PUFA docosahexaenoic acid resulted in a differential growth inhibition proportional to their MRG expression. MRG-transfected cells or MRG protein treated cells were much more sensitive to docosahexaenoic acid-induced growth inhibition than MRG-negative or untreated control cells. Our results suggest that MRG is a candidate mediator of the differentiating effect of pregnancy on breast epithelial cells and may play a major role in omega-3 PUFA-mediated tumor suppression.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Ácidos Docosahexaenoicos/farmacología , Inhibidores de Crecimiento/farmacología , Mama/citología , Mama/metabolismo , Mama/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras/biosíntesis , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Ácidos Docosahexaenoicos/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Proteína 3 de Unión a Ácidos Grasos , Proteínas de Unión a Ácidos Grasos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/metabolismo , Humanos , Lactancia/fisiologíaRESUMEN
Indole-3-carbinol (I3C) is a promising phytochemical agent in chemoprevention of breast cancer. Our present study is the first description of I3C that significantly inhibits the cell adhesion, spreading and invasion associated with an up-regulation of PTEN (a tumor suppressor gene) and E-cadherin (a regulator of cell-cell adhesion) expression in T47-D human breast cancer cells. Therefore, I3C exhibits anti-cancer activities by suppressing breast tumor cell growth and metastatic spread. Metastatic breast cancer is a devastating problem, clinical application of I3C as a potent chemopreventive agent may be helpful in limiting breast cancer invasion and metastasis.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/patología , Indoles/farmacología , Proteínas Supresoras de Tumor , Neoplasias de la Mama/prevención & control , Cadherinas/análisis , Adhesión Celular , Movimiento Celular , Femenino , Humanos , Invasividad Neoplásica , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Células Tumorales CultivadasRESUMEN
Epidemiological studies suggest that moderate alcohol consumption increases the risk of breast cancer, and that alcohol combined with estrogen replacement therapy may synergistically enhance the risk. However, the mechanism(s) of alcohol-induced mammary cancer is unknown. In human breast cancer cell lines, we found that ethanol (EtOH) caused a dose-dependent increase of up to 10- to 15-fold in the transcriptional activity of the liganded estrogen receptor (ER-alpha), but did not activate the nonliganded receptor. Significant stimulation of ER-alpha activity was observed at EtOH concentrations comparable with or less than blood alcohol levels associated with intoxication and at doses below the threshold for in vitro cytotoxicity. These findings may be explained, in part, by an EtOH-induced down-regulation of the expression of BRCA1, a potent inhibitor of ER-alpha activity, and, in part, by a modest increase in the ER-alpha levels. Our findings suggest that inactivation of BRCA1 and increased estrogen-responsiveness might contribute to alcohol-induced breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Etanol/efectos adversos , Receptores de Estrógenos/fisiología , Transducción de Señal/efectos de los fármacos , Proteína BRCA1/biosíntesis , Proteína BRCA1/genética , Proteína BRCA1/fisiología , Neoplasias de la Mama/etiología , Neoplasias de la Mama/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Receptor alfa de Estrógeno , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes BRCA1/efectos de los fármacos , Genes BRCA1/genética , Humanos , Neoplasias Hormono-Dependientes/etiología , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/efectos de los fármacos , Transducción de Señal/fisiología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Células Tumorales CultivadasRESUMEN
Hepatocyte growth factor (HGF), also known as scatter factor (SF), plays an important role in cell:cell adhesion, cell proliferation, motility, and invasiveness of epithelial cells and tumor cells. In this study, we examined the effects of HGF on these types of biological activities and chemosensitivity in Chinese hamster ovary (CHO) cells by stable transfection of the HGF gene. HGF-transfected clones produced very high titers of HGF protein, whereas control vector-transfected clones did not produce detectable HGF protein. HGF-transfected clones showed modestly increased proliferation rates and became more resistant to cell death and apoptosis caused by two anticancer drugs, adriamycin (ADR) and camptothecin (CPT), compared to controlvector-transfected clones. Furthermore, HGF-transfected clones also exhibited increased activities of cell adhesion, migration, and invasion. The current study is the first demonstration that overexpression of the HGF gene affects chemosensitivity and cell metastasis behaviors, suggesting that HGF signaling pathway is a promising new target of therapeutic intervention of tumors.
Asunto(s)
Movimiento Celular/fisiología , Resistencia a Antineoplásicos/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células CHO , Camptotecina/farmacología , Adhesión Celular/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Cricetinae , Perros , Doxorrubicina/farmacología , TransfecciónRESUMEN
Indole-3-carbinol (I3C) is a compound occurring naturally in cruciferous vegetables and has been indicated as a promising agent in preventing breast cancer development and progression. In the present study we have investigated the effect of I3C on the cell migration and invasion behavior in estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB-468 human breast cancer cell lines. Both MCF-7 and MDA-MB-468 were poorly invasive cell lines and exhibited modest invasion and migration capacity in the presence of fibronectin as the chemoattractant. I3C (50 or 100 microM) elicited a significant inhibition of in vitro cell adhesion, migration, and invasion as well as in vivo lung metastasis formation in both cell lines. I3C also suppressed the 17beta-estradiol stimulated migration and invasion in estrogen-responsive MCF-7 cells. These results indicate that anti-invasion and antimigration activities of I3C occur via estrogen-independent and estrogen-dependent pathways. Moreover, I3C significantly caused a dose-dependent increase in E-cadherin, three major catenins (alpha, beta, and gamma-catenin) and BRCA1 expression. Our current finding is the first demonstration that I3C can activate the function of invasion suppressor molecules associated with the suppression of invasion and migration in breast cancer cells. Thus, clinical application of I3C may contribute to the potential benefit for suppression of breast cancer invasion and metastasis.
Asunto(s)
Anticarcinógenos/farmacología , Proteína BRCA1/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Cadherinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Genes BRCA1/genética , Indoles/farmacología , Transactivadores , Regulación hacia Arriba , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Desmoplaquinas , Relación Dosis-Respuesta a Droga , Estradiol/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Invasividad Neoplásica , Trasplante de Neoplasias , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Células Tumorales Cultivadas , alfa Catenina , beta Catenina , gamma CateninaRESUMEN
Recent studies have elucidated some of the molecular and cellular mechanisms that determine the sensitivity or resistance to ionizing radiation. These findings ultimately may be useful in devising new strategies to improve the therapeutic ratio in cancer treatment. Despite the rapid advances in knowledge of cellular functions that affect radiosensitivity, we still cannot account for most of the clinically observed heterogeneity of normal tissue and tumor responses to radiotherapy, nor can we accurately predict which individual tumors will be controlled locally and which patients will develop more severe normal tissue damage after radiotherapy. However, several candidate genes for which deletion or loss of function mutations may be associated with altered cellular radiosensitivity (e.g., ATM, p53, BRCA1, BRCA2, DNA-PK) have been identified. Some of the differences in normal tissue sensitivity to radiation may stem from mutations with milder effects, heterozygosity, or polymorphisms of these genes. Finally, molecular mechanisms linking genetic instability, radiosensitivity, and predisposition to cancer are being unraveled.