RESUMEN
DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.
Asunto(s)
Metilación de ADN , ADN/análisis , ADN/metabolismo , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Adenocarcinoma/sangre , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Secuencia de Bases , Calcitonina/genética , Neoplasias del Colon/sangre , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/genética , ADN/genética , Cartilla de ADN/antagonistas & inhibidores , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Oligonucleótidos/genética , Sensibilidad y Especificidad , Sulfitos/metabolismo , Factores de TiempoRESUMEN
The role of promoter methylation in the process of cancer cell metastasis has, however, not yet been studied. Recently, methylation of the TPEF (transmembrane protein containing epidermal growth factor and follistatin domain) gene was reported in human colon, gastric, and bladder cancer cells. Using the Methylight assay, TPEF/HPP1 gene methylation was assessed in primary colorectal cancers (n = 47), matched normal colon mucosa, as well as in the liver metastasis of 24 patients with colorectal cancer, and compared to the methylation status of the TIMP-3, APC, DAPK, caveolin-2, and p16 genes. TPEF was frequently methylated in primary colorectal cancers (36 of 47) compared to the normal colon mucosa (1 of 21) (P < .0001), and TPEF mRNA expression in colon cancer cell lines was restored after treatment with 5-aza-2'-deoxycytidine. The p16 and APC genes were also frequently methylated in primary colorectal cancers (P < .02) compared to the normal colon mucosa. Interestingly, promoter methylation was significantly more frequent in proximal, nonrectal cancers (P < .05). Furthermore, a high degree of methylation of the TPEF gene was also observed in liver metastasis (19 of 24). In summary, we observed frequent TPEF methylation in primary colorectal cancers and liver metastases, indicating that epigenetic alterations are not only present in the early phases of carcinogenesis, but are also common in metastatic lesions. The high frequency of TPEF methylation in this series of colorectal cancers underscores the importance of epigenetic changes as targets for the development of molecular tests for cancer diagnosis.
Asunto(s)
Neoplasias Colorrectales/genética , Metilación de ADN , Neoplasias Hepáticas/genética , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Neoplasias Colorrectales/patología , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Humanos , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Regiones Promotoras GenéticasRESUMEN
Type I procollagen is a heterotrimer composed of two proalpha1(I) chains and one proalpha2(I) chain, encoded by the COL1A1 and COL1A2 genes, respectively. Mutations in these genes usually lead to dominantly inherited forms of osteogenesis imperfecta (OI) by altering the triple helical domains, but a few affect sequences in the proalpha1(I) C-terminal propeptide (C-propeptide), and one, which has a phenotype only in homozygotes, alters the proalpha2(I) C-propeptide. Here we describe four dominant mutations in the COL1A2 gene that alter sequences of the proalpha2(I) C-propeptide in individuals with clinical features of a milder form of the disease, OI type IV. Three of the four appear to interfere with disulfide bonds that stabilize the C-propeptide conformation and its interaction with other chains in the trimer. Cultured cells synthesized proalpha2(I) chains that were slow to assemble with proalpha1(I) chains to form heterotrimers and that were retained intracellularly. Some alterations led to the uncharacteristic formation of proalpha1(I) homotrimers. These findings show that the C-propeptide of proalpha2(I), like that of the proalpha1(I) C-propeptide, is essential for efficient assembly of type I procollagen heterotrimers. The milder OI phenotypes likely reflect a diminished amount of normal type I procollagen, small populations of overmodified heterotrimers, and proalpha1(I) homotrimers that are compatible with normal skeletal growth.