RESUMEN
MicroRNAs (miRNAs) are small noncoding RNAs (ncRNAs, RNAs that do not code for proteins) that regulate the expression of target genes at the posttranscriptional or posttranslational level. Many miRNAs have conserved sequences between distantly related organisms, suggesting that these molecules participate in essential developmental and physiologic processes. miRNAs can act as tumor suppressor genes or oncogenes in human cancers. Mutations, deletions, or amplifications have been found in human cancers and shown to alter expression levels of mature and/or precursor miRNA transcripts. Moreover, a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Both miRNAs and UCRs are frequently located at fragile sites and genomic regions affected in various cancers, named cancer-associated genomic regions (CAGRs). Bioinformatics studies are emerging as important tools to identify associations and/or correlations between miRNAs/ncRNAs and CAGRs. ncRNA profiling has allowed the identification of specific signatures associated with diagnosis, prognosis, and response to treatment of human tumors. Several abnormalities could contribute to the alteration of miRNA expression profiles in each kind of tumor and in each kind of tissue. This review is focused on the miRNAs and ncRNAs as genes affecting cancer risk, and we provided an updated catalog of miRNAs and UCRs located at fragile sites or at cancer susceptibility loci. These types of studies are the first step toward discoveries leading to novel approaches for cancer therapies.
Asunto(s)
Biología Computacional , Genoma/genética , Neoplasias/genética , Neoplasias/terapia , ARN no Traducido/genética , Animales , Predisposición Genética a la Enfermedad , Humanos , MicroARNs/genéticaRESUMEN
FEZ1/LZTS1 is a tumor suppressor gene that is frequently altered in human cancers of different histotypes. We have reported previously that LZTS1 is downregulated in high-grade bladder cancer and that its restoration suppresses tumorigenicity in urothelial carcinoma cells. To further investigate the role of LZTS1 in the development of bladder cancer, we utilized heterozygous and nullizygous Lzts1 mice in a chemically induced carcinogenesis model. Fifty-eight mice consisting of 25 Lzts1(+/+), 17 Lzts1(+/-) and 16 Lzts1(-/-) were treated with N-butyl-N-(4-hydroxybutil) nitrosamine (BBN). Results showed that there was a significant increase in neoplastic lesions in the Lzts1(+/-) (82.3%) and Lzts1(-/-) (93.8%) versus Lzts1(+/+) (8.0%) mice after BBN treatment. No difference in cancer incidence between Lzts1(+/-) and Lzts1(-/-) was observed. Collectively, these findings indicate that loss of one or both LZTS1 alleles hampers the normal defenses of urothelial cells against carcinogens, favoring bladder cancer development. Therefore, LZTS1 may become an excellent target for gene therapy in advanced bladder carcinoma.
Asunto(s)
Butilhidroxibutilnitrosamina/toxicidad , Carcinógenos/toxicidad , Proteínas de Unión al ADN/deficiencia , Predisposición Genética a la Enfermedad , Proteínas del Tejido Nervioso/deficiencia , Neoplasias de la Vejiga Urinaria/genética , Animales , Cartilla de ADN , Proteínas de Unión al ADN/genética , Femenino , Genes Supresores de Tumor , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
OBJECTIVES: Micro-RNAs are a group of small noncoding RNAs with modulator activity of gene expression. Recently, micro-RNA genes were found abnormally expressed in several types of cancers. To study the role of the micro-RNAs in human kidney and bladder cancer, we analyzed the expression profile of 245 micro-RNAs in kidney and bladder primary tumors. METHODS AND MATERIALS: A total of 27 kidney specimens (20 carcinomas, 4 benign renal tumors, and 3 normal parenchyma) and 27 bladder specimens (25 urothelial carcinomas and 2 normal mucosa) were included in the study. Total RNA was used for hybridization on an oligonucleotide microchip for micro-RNA profiling developed in our laboratories. This microchip contains 368 probes in triplicate, corresponding to 245 human and mouse micro-RNA genes. RESULTS: A set of 4 human micro-RNAs (miR-28, miR-185, miR-27, and let-7f-2) were found significantly up-regulated in renal cell carcinoma (P < 0.05) compared to normal kidney. Human micro-RNAs miR-223, miR-26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203, miR-17-5p, miR-23a, and miR-205 were significantly up-regulated in bladder cancers (P < 0.05) compared to normal bladder mucosa. Of the kidney cancers studied, there was no differential micro-RNA expression across various stages, whereas with increasing tumor-nodes-metastasis staging in bladder cancer, miR-26b showed a moderate decreasing trend (P = 0.082). CONCLUSIONS: Our results show that different micro-RNAs are deregulated in kidney and bladder cancer, suggesting the involvement of these genes in the development and progression of these malignancies. Further studies are needed to clarify the role of micro-RNAs in neoplastic transformation and to test the potential clinical usefulness of micro-RNAs microarrays as diagnostic and prognostic tool.
Asunto(s)
Biomarcadores de Tumor , Perfilación de la Expresión Génica/instrumentación , Neoplasias Renales/genética , MicroARNs/aislamiento & purificación , Análisis por Micromatrices/instrumentación , Neoplasias de la Vejiga Urinaria/genética , Animales , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Renales/patología , Neoplasias Renales/cirugía , Masculino , Ratones , MicroARNs/clasificación , MicroARNs/metabolismo , Análisis por Micromatrices/métodos , Hibridación de Ácido Nucleico/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Estudios Retrospectivos , Transcripción Genética/genética , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/cirugíaRESUMEN
Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.
Asunto(s)
Carcinoma/genética , Leucemia/genética , ARN no Traducido/química , Secuencia de Bases , Análisis por Conglomerados , Secuencia Conservada , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/fisiología , Datos de Secuencia Molecular , Oncogenes/fisiología , Análisis de Secuencia de ARNRESUMEN
ARLTS1 is a newly characterized tumor suppressor gene located at chromosome 13q14.3 and involved in the pathogenesis of various types of tumors: two single-nucleotide polymorphisms, one of them responsible for protein truncation, were found statistically associated with familial malignancies, whereas DNA hypermethylation and genomic deletions have been identified as a mechanism of ARLTS1 down-regulation in sporadic cancers. We found that in a large portion of lung carcinomas (37%) and in all analyzed lung cancer cell lines, ARLTS1 is strongly down-regulated due to DNA methylation in its promoter region. After its restoration by adenoviral transduction, ARLTS1-negative A549 and H1299 cells underwent apoptosis and inhibition of cell growth. Furthermore, ARLTS1 reexpression significantly reduced the ability of A549 and H1299 to form tumors in nude mice. Finally, we identified approximately 650 transcripts differentially expressed after restoration of ARLTS1 expression in A549 cells, suggesting that various pathways involved in cell survival, proliferation, signaling, and development mediate the effects of wild-type ARLTS1 in a lung cancer system.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Factores de Ribosilacion-ADP/biosíntesis , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Secuencia Conservada , Metilación de ADN , Regulación hacia Abajo , Terapia Genética/métodos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Alineación de SecuenciaRESUMEN
MicroRNAs (miRNAs) are short 19- to 24-nt RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. Abnormal expression of miRNAs has been observed in several human cancers, and furthermore, germ-line and somatic mutations in human miRNAs were recently identified in patients with chronic lymphocytic leukemia. Thus, human miRNAs can act as tumor suppressor genes or oncogenes, where mutations, deletions, or amplifications can underlie the development of certain types of leukemia. In addition, previous studies have shown that miRNA expression profiles can distinguish among human solid tumors from different organs. Because a single miRNA can simultaneously influence the expression of two or more protein-coding genes, we hypothesized that miRNAs could be candidate genes for cancer risk. Research in complex trait genetics has demonstrated that genetic background determines cancer susceptibility or resistance in various tissues, such as colon and lung, of different inbred mouse strains. We compared the genome positions of mouse tumor susceptibility loci with those of mouse miRNAs. Here, we report a statistically significant association between the chromosomal location of miRNAs and those of mouse cancer susceptibility loci that influence the development of solid tumors. Furthermore, we identified distinct patterns of flanking DNA sequences for several miRNAs located at or near susceptibility loci in inbred strains with different tumor susceptibilities. These data provide a catalog of miRNA genes in inbred strains that could represent genes involved in the development and penetrance of solid tumors.
Asunto(s)
Mapeo Cromosómico , Predisposición Genética a la Enfermedad , MicroARNs/genética , Neoplasias/genética , Animales , Secuencia de Bases , Diferenciación Celular , Bases de Datos como Asunto , Ratones , Ratones Endogámicos , MicroARNs/química , Datos de Secuencia Molecular , Regiones Promotoras GenéticasRESUMEN
Human adenocarcinomas commonly harbor mutations in the KRAS and MYC proto-oncogenes and the TP53 tumor suppressor gene. All three genetic lesions are potentially pro-angiogenic, as they sustain production of vascular endothelial growth factor (VEGF). Yet Kras-transformed mouse colonocytes lacking p53 formed indolent, poorly vascularized tumors, whereas additional transduction with a Myc-encoding retrovirus promoted vigorous vascularization and growth. In addition, VEGF levels were unaffected by Myc, but enhanced neovascularization correlated with downregulation of anti-angiogenic thrombospondin-1 (Tsp1) and related proteins, such as connective tissue growth factor (CTGF). Both Tsp1 and CTGF are predicted targets for repression by the miR-17-92 microRNA cluster, which was upregulated in colonocytes coexpressing K-Ras and c-Myc. Indeed, miR-17-92 knockdown with antisense 2'-O-methyl oligoribonucleotides partly restored Tsp1 and CTGF expression; in addition, transduction of Ras-only cells with a miR-17-92-encoding retrovirus reduced Tsp1 and CTGF levels. Notably, miR-17-92-transduced cells formed larger, better-perfused tumors. These findings establish a role for microRNAs in non-cell-autonomous Myc-induced tumor phenotypes.
Asunto(s)
MicroARNs/metabolismo , Neoplasias/irrigación sanguínea , Neovascularización Patológica/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , Animales , Línea Celular , Línea Celular Transformada , Transformación Celular Viral , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Medios de Cultivo Condicionados/análisis , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias/patología , Neovascularización Patológica/genética , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , ARN Neoplásico/metabolismo , Retroviridae/genética , Células Madre/citología , Trombospondina 1/genética , Trombospondina 1/metabolismo , Trasplante Homólogo , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The basis of eukaryotic complexity is an intricate genetic architecture where parallel systems are involved in tuning gene expression, via RNA-DNA, RNA-RNA, RNA-protein, and DNA-protein interactions. In higher organisms, about 97% of the transcriptional output is represented by noncoding RNA (ncRNA) encompassing not only rRNA, tRNA, introns, 5' and 3' untranslated regions, transposable elements, and intergenic regions, but also a large, rapidly emerging family named microRNAs. MicroRNAs are short 20-22-nucleotide RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. MicroRNAs are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the cytoplasmic Dicer, a member of the RNase III enzyme family. A recent analysis of the genomic location of human microRNA genes suggested that 50% of microRNA genes are located in cancer-associated genomic regions or in fragile sites. This review focuses on the possible implications of microRNAs in post-transcriptional gene regulation in mammalian diseases, with particular focus on cancer. We argue that developing mouse models for deleted and/or overexpressed microRNAs will be of invaluable interest to decipher the regulatory networks where microRNAs are involved.
Asunto(s)
Expresión Génica , MicroARNs/genética , Animales , HumanosRESUMEN
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the world. The TCL1 gene, responsible for prolymphocytic T cell leukemia, is also overexpressed in human B cell malignancies and overexpression of the Tcl1 protein occurs frequently in CLL. Aging transgenic mice that overexpress TCL1 under control of the mu immunoglobulin gene enhancer, develop a CD5+ B cell lymphoproliferative disorder mimicking human CLL and implicating TCL1 in the pathogenesis of CLL. In the current study, we exploited this transgenic mouse to investigate two different CLL-related issues: potential treatment of CLL and characterization of neoplasms that accompany CLL. We successfully transplanted CLL cells into syngeneic mice that led to CLL development in the recipient mice. This approach allowed us to verify the involvement of the Tcl1/Akt/mTOR biochemical pathway in the disease by testing the ability of a specific pharmacologic agent, rapamycin, to slow CLL. We also showed that 36% of these transgenic mice were affected by solid malignancies, in which the expression of the Tcl1 protein was absent. These findings indicate that other oncogenic mechanism(s) may be involved in the development of solid tumors in Emu-TCL1 transgenic mice.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Proto-Oncogénicas/genética , Sirolimus/farmacología , Animales , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas/biosíntesisRESUMEN
BACKGROUND: MicroRNA expression profiles can be used to distinguish normal B cells from malignant B cells in patients with chronic lymphocytic leukemia (CLL). We investigated whether microRNA profiles are associated with known prognostic factors in CLL. METHODS: We evaluated the microRNA expression profiles of 94 samples of CLL cells for which the level of expression of 70-kD zeta-associated protein (ZAP-70), the mutational status of the rearranged immunoglobulin heavy-chain variable-region (IgV(H) ) gene, and the time from diagnosis to initial treatment were known. We also investigated the genomic sequence of 42 microRNA genes to identify abnormalities. RESULTS: A unique microRNA expression signature composed of 13 genes (of 190 analyzed) differentiated cases of CLL with low levels of ZAP-70 expression from those with high levels and cases with unmutated IgV(H) from those with mutated IgV(H) . The same microRNA signature was also associated with the presence or absence of disease progression. We also identified a germ-line mutation in the miR-16-1-miR-15a primary precursor, which caused low levels of microRNA expression in vitro and in vivo and was associated with deletion of the normal allele. Germ-line or somatic mutations were found in 5 of 42 sequenced microRNAs in 11 of 75 patients with CLL, but no such mutations were found in 160 subjects without cancer (P<0.001). CONCLUSIONS: A unique microRNA signature is associated with prognostic factors and disease progression in CLL. Mutations in microRNA transcripts are common and may have functional importance.
Asunto(s)
Expresión Génica , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , MicroARNs , Mutación , Proteínas Tirosina Quinasas/metabolismo , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Reordenamiento Génico , Genes de Inmunoglobulinas , Genes Supresores de Tumor , Mutación de Línea Germinal , Humanos , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , MicroARNs/análisis , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Mutación Puntual , Pronóstico , Análisis de Secuencia de ADN , Proteína Tirosina Quinasa ZAP-70RESUMEN
The FHIT gene plays important roles in cancer development, including lung cancers, in which the Fhit protein is frequently lost. To determine if Fhit-deficient mice exhibit increased susceptibility to carcinogen-induced lung cancer, mice were treated with the pulmonary carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone. Wild-type and Fhit-deficient animals did not exhibit significantly different frequencies of lung lesions, but Fhit-/- mice showed significantly increased average tumor volume (1.62 mm3) and multiplicity in tumor-bearing mice, compared with wild-type mice (0.70 mm3). Tumors of Fhit-/- mice were all carcinomas, whereas Fhit+/+ mice did not develop carcinomas. To determine if Fhit absence, in combination with deficiency of an additional 3p tumor suppressor, would affect the frequency of tumor induction, we examined the spontaneous and dimethylnitrosamine-induced tumor phenotype of Fhit-/-Vhl+/- mice. Whereas no spontaneous lung tumors were observed in Fhit-/- or Vhl+/- mice, 44% of Fhit-/-Vhl+/- mice developed adenocarcinomas by 2 years of age. Dimethylnitrosamine (6 mg/kg body weight) induced lung tumors (adenomas and carcinomas) in 100% of Fhit-/-Vhl+/- mice and adenomas in 40% of Fhit-/- mice by 20 months of age. Thus, double deficiency in murine homologues of 3p suppressor genes, including haploinsufficiency of Vhl, predisposes to spontaneous and induced lung cancers, showing that Fhit-deficient mice will be useful, in combination with other 3p tumor suppressors, in recapitulating a pattern of lung cancer development similar to the human pattern; such double- or triple-deficient mice will be excellent lung cancer prevention and therapy models.
Asunto(s)
Ácido Anhídrido Hidrolasas/deficiencia , Cocarcinogénesis , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/deficiencia , Proteínas Supresoras de Tumor/deficiencia , Ubiquitina-Proteína Ligasas/deficiencia , Ácido Anhídrido Hidrolasas/genética , Alelos , Animales , Carcinógenos , Femenino , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Predisposición Genética a la Enfermedad , Haploidia , Neoplasias Pulmonares/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Nitrosaminas , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
PURPOSE: The human TESTIN (TES) gene is a putative tumor suppressor gene in the fragile chromosomal region FRA7G at 7q31.1/2 that was reported to be altered in leukemia and lymphoma cell lines. In this report, we investigated the effect of TES gene expression in vivo to evaluate a possible role of TES gene in human cancer. EXPERIMENTAL DESIGN: We have analyzed the expression of TES gene in a panel of 25 breast tumors and 17 cell lines of breast, colon, and uterine cancers. Furthermore, to evaluate the potential of TES gene therapy, we studied the effects of adenoviral TES transduction (Ad-TES) in cell lines with undetectable TES expression (T47D and MES-SA) as well as in MCF-7 cell line where TES expression is normal. RESULTS: Twenty-five percent of primary breast tumor samples as well as the breast cancer cell line T47D and the uterine sarcoma cell line MES-SA were negative or displayed low levels of TES. After TES restoration by Ad-TES transduction, T47D and MES-SA cell lines underwent apoptosis. Furthermore, TES expression significantly reduced the tumorigenic potential of both T47D and MES-SA in nude mice, whereas the untreated cells and Ad-GFP-infected cells showed tumor growth in vivo. The TES-positive cell line control (MCF-7) was not affected by TES expression and did not show a reduction of tumorigenicity in nude mice after infection with Ad-TES. CONCLUSIONS: Ad-TES expression inhibit the growth of breast and uterine cancer cells lacking of TES expression through caspase-dependent and caspase-independent apoptosis, respectively, suggesting that Ad-TES infection should be explored as a therapeutic strategy.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/terapia , Neoplasias del Colon/terapia , Proteínas de Homeodominio/uso terapéutico , Transducción Genética , Proteínas Supresoras de Tumor/uso terapéutico , Neoplasias Uterinas/terapia , Adenoviridae/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Caspasas/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Proteínas del Citoesqueleto , Femenino , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Proteínas con Dominio LIM , Ratones , Ratones Desnudos , Proteínas de Unión al ARN , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Sarcoma Experimental/terapia , Células Tumorales Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The fragile histidine triad (FHIT) gene located on chromosome 3p14.2 is frequently deleted in human tumors. We have previously reported deletions at the FHIT locus in 50% of bladder carcinoma derived cell lines and reduced expression in 61% of primary transitional carcinomas of the urinary bladder. To additionally investigate the role of FHIT alterations in the development of bladder cancer, we used heterozygous and nullizygous Fhit-deficient mice in a chemically induced carcinogenesis model. Results showed that 8 of 28 (28%) and 6 of 13 (46%) of the Fhit -/- and +/-, respectively, versus 2 of 25 (8%) Fhit +/+ mice developed invasive carcinoma after treatment with N-butyl-N-(4-hydroxybutyl) nitrosamine. To explore the possibility of a FHIT-based gene therapy for bladder cancer, we studied the effects of restored Fhit protein expression on cell proliferation, cell kinetics, and tumorigenicity in BALB/c nude mice, with human SW780 Fhit-null transitional carcinoma derived cells. In vitro transduction of SW780 Fhit-negative cells with adenoviral-FHIT inhibited cell growth, increased apoptotic cell population, and suppressed s.c. tumor growth in nude mice. These findings suggest the important role of Fhit in bladder cancer development and support the effort to additionally investigate a FHIT-based gene therapy.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Carcinoma de Células Transicionales/genética , Proteínas de Neoplasias/genética , Neoplasias de la Vejiga Urinaria/genética , Adenoviridae/genética , Animales , Butilhidroxibutilnitrosamina/farmacología , Línea Celular , Línea Celular Tumoral , Femenino , Citometría de Flujo , Terapia Genética/métodos , Genotipo , Heterocigoto , Humanos , Immunoblotting , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Factores de TiempoRESUMEN
Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673-676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253-258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Leucemia Linfocítica Crónica de Células B/genética , MicroARNs/análisis , Linfocitos B , Deleción Cromosómica , Cromosomas Humanos Par 13 , Genes de Inmunoglobulinas , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Mutación , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Pronóstico , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/genética , ARN Neoplásico/análisis , Proteína Tirosina Quinasa ZAP-70RESUMEN
MicroRNAs (miRNAs) are a class of small noncoding RNA genes recently found to be abnormally expressed in several types of cancer. Here, we describe a recently developed methodology for miRNA gene expression profiling based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes. We used these microarrays to obtain highly reproducible results that revealed tissue-specific miRNA expression signatures, data that were confirmed by assessment of expression by Northern blots, real-time RT-PCR, and literature search. The microchip oligolibrary can be expanded to include an increasing number of miRNAs discovered in various species and is useful for the analysis of normal and disease states.
Asunto(s)
Perfilación de la Expresión Génica , Genoma , Genómica/métodos , MicroARNs/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de Oligonucleótidos/genética , Adulto , Animales , Northern Blotting , Células Cultivadas , Feto/metabolismo , Genoma Humano , Células HeLa , Humanos , Macrófagos/metabolismo , Ratones , MicroARNs/genética , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Especificidad de Órganos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
Effective treatment of malignant carcinomas requires identification of proteins regulating epithelial cell proliferation. To this end, we compared gene expression profiles in murine colonocytes and their c-Myc-transformed counterparts, which possess enhanced proliferative potential. A surprisingly short list of deregulated genes included the cDNA for clusterin, an extracellular glycoprotein without a firmly established function. We had previously demonstrated that in organs such as skin, clusterin expression is restricted to differentiating but not proliferating cell layers, suggesting a possible negative role in cell division. Indeed, its transient overexpression in Myc-transduced colonocytes decreased cell accumulation. Furthermore, clusterin was down-regulated in rapidly dividing human keratinocytes infected with a Myc-encoding adenovirus. Its knockdown via antisense RNA in neoplastic epidermoid cells enhanced proliferation. Finally, recombinant human clusterin suppressed, in a dose-dependent manner, DNA replication in keratinocytes and other cells of epithelial origin. Thus, clusterin appears to be an inhibitor of epithelial cell proliferation in vitro. To determine whether it also affects neoplastic growth in vivo, we compared wild-type and clusterin-null mice with respect to their sensitivity to 7, 12-dimethylbenz(a)anthracene /12-Otetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis. We observed that the mean number of papillomas/mouse was higher in clusterin-null animals. Moreover, these papillomas did not regress as readily as in wild-type mice and persisted beyond week 35. The rate of progression toward squamous cell carcinoma was not altered, although those developing in clusterin-null mice were on average better differentiated. These data suggest that clusterin not only suppresses epithelial cell proliferation in vitro but also interferes with the promotion stage of skin carcinogenesis.
Asunto(s)
Genes myc/fisiología , Glicoproteínas/fisiología , Queratinocitos/citología , Queratinocitos/metabolismo , Chaperonas Moleculares/fisiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Transformación Celular Neoplásica , Clusterina , Regulación hacia Abajo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/fisiología , Ratones , Ratones Noqueados , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Chaperonas Moleculares/farmacología , Proteínas Recombinantes/farmacología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/prevención & control , Trombospondina 1/antagonistas & inhibidores , Trombospondina 1/biosíntesis , Trombospondina 1/genéticaRESUMEN
A large number of tiny noncoding RNAs have been cloned and named microRNAs (miRs). Recently, we have reported that miR-15a and miR-16a, located at 13q14, are frequently deleted and/or down-regulated in patients with B cell chronic lymphocytic leukemia, a disorder characterized by increased survival. To further investigate the possible involvement of miRs in human cancers on a genome-wide basis, we have mapped 186 miRs and compared their location to the location of previous reported nonrandom genetic alterations. Here, we show that miR genes are frequently located at fragile sites, as well as in minimal regions of loss of heterozygosity, minimal regions of amplification (minimal amplicons), or common breakpoint regions. Overall, 98 of 186 (52.5%) of miR genes are in cancer-associated genomic regions or in fragile sites. Moreover, by Northern blotting, we have shown that several miRs located in deleted regions have low levels of expression in cancer samples. These data provide a catalog of miR genes that may have roles in cancer and argue that the full complement of miRs in a genome may be extensively involved in cancers.
Asunto(s)
Sitios Frágiles del Cromosoma/genética , Genoma Humano , MicroARNs/genética , Neoplasias/genética , Mapeo Cromosómico , Clonación Molecular , Bases de Datos de Ácidos Nucleicos , Genes Homeobox , Marcadores Genéticos , Humanos , Pérdida de Heterocigocidad , Familia de Multigenes , PubMedRESUMEN
The fragile histidine triad (FHIT) gene at chromosome 3p14.2 is a tumor suppressor gene that is altered mainly by deletion in a large fraction of human tumors, including breast cancers. To evaluate the potential of FHIT gene therapy in this type of cancer, we have studied the biological effects of adenoviral FHIT transduction (Ad-FHIT) in breast cancer cell lines. The results showed that, after FHIT restoration in BT-549, MDA-MB-436, and HCC1806 cells, they underwent apoptosis by activation of the intrinsic pathway. In all three cell lines infected with Ad-FHIT, we have found activation of caspase-2, which is required for permeabilization of mitochondria, release of cytochrome c, and apoptosis. Furthermore, Fhit overexpression produces alteration in cell cycling properties, as well as reduction of the tumorigenic potential in nude mice.
Asunto(s)
Ácido Anhídrido Hidrolasas , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Neoplasias/genética , Adenoviridae/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Ciclo Celular/genética , División Celular/genética , Femenino , Terapia Genética/métodos , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/fisiología , Transducción Genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In various studies of sporadic breast cancers, 40-70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.