RESUMO
BACKGROUND: As DNA methylation has been determined as an important diagnostic biomarker in cancer management, methods for the detection of quantitative DNA methylation which are high-throughput and low-cost are needed. MATERIALS AND METHODS: In this work, we introduce the solid-phase methylation-specific PCR (SPMSP) as a method for quantitative methylation analysis. SPMSP combines methylation-specific DNA amplification on a solid phase with subsequent colorimetric detection in an ELISA-based format. RESULTS: In contrast to existing methods for quantitative methylation analysis, SPMSP can be carried out with standard laboratory equipment, i.e. a thermocycler and an ELISA reader. With this method, DNA methylation in the promoter of the APC tumour suppressor gene was quantified in a set of colorectal carcinoma samples. CONCLUSION: As SPMSP can be modified to the analysis of other promoter sequences and is also adaptable to a high-throughput system, SPMSP offers a platform for methylation profiling with widespread applications in cancer research and management.
Assuntos
Colorimetria/métodos , Metilação de DNA , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Neoplasias Colorretais/genética , Humanos , Pessoa de Meia-IdadeRESUMO
The adenomatous polyposis coli (APC) protein is a key component of the WNT signalling pathway wherein it acts as a scaffolding protein in controlling the level of the proto-oncoprotein beta-catenin. Although APC has been shown to be genetically or epigenetically inactivated in a variety of carcinomas, little is known about its role in sarcoma. Liposarcomas (LPSs) are the second most common soft tissue sarcoma in adults. Despite different histology and malignancy, the myxoid and round-cell LPSs belong to one tumour entity characterized by a specific chromosomal translocation. We assessed the extent of genetic and epigenetic inactivation of the APC gene in myxoid/round-cell LPS. Sequencing of the mutation cluster region, the protein truncation test and a loss of heterozygosity (LOH) analysis did not reveal any genetic alterations of the APC gene in all of the liposarcoma samples. Methylation of the APC promoter was detected by methylation-specific PCR in 9 of 20 (45%) tumours. Analysis of APC expression by semiquantitative RT-PCR in a subset of the samples demonstrated that tumours with a methylated APC promoter showed a downregulation of the APC transcript. However, APC downregulation was not correlated with a stabilisation of the beta-catenin protein. Thus, the epigenetic regulation of the APC gene might play an important role in the pathogenesis of myxoid/round-cell LPS. However, the impact of APC methylation on liposarcoma development is quite likely not mediated through WNT signalling.