RESUMEN
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
BACKGROUND: The finding of hemizygous or homozygous deletions at band 14 on chromosome 13 in a variety of neoplasms suggests the presence of a tumor-suppressor locus telomeric to the RB1 gene. METHODS: We studied samples from 216 patients with various types of sporadic tumors or idiopathic pancytopenia, peripheral-blood samples from 109 patients with familial cancer or multiple cancers, and control blood samples from 475 healthy people or patients with diseases other than cancer. We performed functional studies of cell lines lacking ARLTS1 expression with the use of both the full-length ARLTS1 gene and a truncated variant. RESULTS: We found a gene at 13q14, ARLTS1, a member of the ADP-ribosylation factor family, with properties of a tumor-suppressor gene. We analyzed 800 DNA samples from tumors and blood cells from patients with sporadic or familial cancer and controls and found that the frequency of a nonsense polymorphism, G446A (Trp149Stop), was similar in controls and patients with sporadic tumors but was significantly more common among patients with familial cancer than among those in the other two groups (P=0.02; odds ratio, 5.7; 95 percent confidence interval, 1.3 to 24.8). ARLTS1 was down-regulated by promoter methylation in 25 percent of the primary tumors we analyzed. Transfection of wild-type ARLTS1 into A549 lung-cancer cells suppressed tumor formation in immunodeficient mice and induced apoptosis, whereas transfection of truncated ARLTS1 had a limited effect on apoptosis and tumor suppression. Microarray analysis revealed that the wild-type and Trp149Stop-transfected clones had different expression profiles. CONCLUSIONS: A genetic variant of ARLTS1 predisposes patients to familial cancer.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Cromosomas Humanos Par 13 , Genes Supresores de Tumor , Mutación de Línea Germinal , Neoplasias/genética , Polimorfismo Genético , Factores de Ribosilacion-ADP/metabolismo , Animales , Deleción Cromosómica , Codón sin Sentido , Metilación de ADN , Análisis Mutacional de ADN , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pancitopenia/genética , Linaje , ARN Mensajero/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
PURPOSE: WWOX (WW domain containing oxidoreductase) is a tumor suppressor gene that maps to the common fragile site FRA16D. We showed previously that WWOX is frequently altered in human lung and esophageal cancers. The purpose of this study was to delineate more precisely the role of WWOX in pancreatic carcinogenesis. EXPERIMENTAL DESIGN: We analyzed 15 paired pancreatic adenocarcinoma samples and 9 pancreatic cancer cell lines for WWOX alterations. Colony assay and cell cycle analysis were also performed to evaluate the role of the WWOX as a tumor suppressor gene. RESULTS: Loss of heterozygosity at the WWOX locus was observed in 4 primary tumors (27%). Methylation analysis showed that site-specific promoter hypermethylation was detected in 2 cell lines (22%) and treatment with the demethylating agent 5-aza-2'-deoxycytidine demonstrated an increase in the expression of WWOX. In addition, 2 primary tumor samples (13%) showed promoter hypermethylation including the position of site-specific methylation. Transcripts missing WWOX exons were detected in 4 cell lines (44%) and in 2 tumor samples (13%). Real-time reverse transcription PCR revealed a significant reduction of WWOX expression in all of the cell lines and in 6 primary tumors (40%). Western blot analysis showed a significant reduction of the WWOX protein in all of the cell lines. Furthermore, transfection with WWOX inhibited colony formation of pancreatic cancer cell lines by triggering apoptosis. CONCLUSION: These results indicate that the WWOX gene may play an important role in pancreatic tumor development.
