Establishment and validation of quantitative real-time pcr assay for aberrant methylation of 14-3-3û gene in breast and lung carcinoma

Background: 14-3-3Û gene has been shown to be responsible for G2 cell cycle checkpoint control by p53 in response to DNA damage in human cells. In order to increase the potential utility of 14-3-3Û gene as a molecular marker in tumor analysis and prognosis, we established and validated a quantitative real-time MSP assay and correlated our findings with the standard MSP assay. Materials and Methods: We examined the expression of 14-3-3 Û gene by reverse transcription PCR (RT-PCR) in breast and lung cancer cell lines and control non-malignant tissue samples. To elucidate the mechanism of gene silencing, we studied the methylation patterns in cell lines, tumors and non-malignant control tissues of breast and lung using previously reported MSP assay. For fluorescence based quantitative Real-Time PCR assay, we designed primers and probe specific to 14-3-3Û gene, validated the assay in cell lines and non-malignant control tissues of breast and lung and extended the study to primary tumors and corresponding nonmalignant tissues. Results: The concordances between the standard MSP assay and the real-time assay were 95-100%. The overall concordances between standard MSP and real-time assay in 60 cell lines were 97%. By real-time assay, the differences in methylation frequencies between malignant and non-malignant breast and between malignant and nonmalignant lung tissues; between NSCLC and SCLC cell lines between MSP (-) and MSP (+) samples and between MSP (+) and MSP (++) samples were statistically significant. The mean real-time values for MSP (-), MSP (+) and MSP (++) samples were 2, 28 and 53 respectively. Conclusion: We conclude that promoter methylation is a valid pathway for silencing of 14-3- 3Û gene in primary breast and lung carcinomas. The real-time assay to distinguish the extent and degree of methylation of 14- 3-3Û gene among malignant and non-malignant tissues would potentially enhance the utility of this marker in breast and lung cancer analysis and prognosis.