RESUMEN
Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 13/genética , Neoplasias Esofágicas/genética , Genes Supresores de Tumor , Análisis por Micromatrices , Trombospondinas/genética , Alelos , Línea Celular Transformada , Línea Celular Tumoral , Segregación Cromosómica , Metilación de ADN/efectos de los fármacos , Desoxicitidina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano/genética , Humanos , Ácidos Hidroxámicos/farmacología , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite/genética , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombospondinas/metabolismo , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
The group 2 LIM domain protein, Cysteine-rich intestinal protein 2 (CRIP2) was found to play an important role in esophageal squamous cell carcinoma (ESCC) tumorigenesis. Subcellular fractionation studies show that CRIP2 is expressed in the nucleus. Real-time quantitative PCR shows CRIP2 expression is down-regulated in ESCC tissues and cell lines. Functional studies reveal that CRIP2 reduces colony formation, growth, and invasion abilities. Furthermore, over-expression of CRIP2 induces apoptosis through induction of active caspases 3 and 9 proteins. In conclusion, this study shows CRIP2 plays an important role in the development of ESCC.