Detection of methylated CDO1 in plasma of colorectal cancer; a PCR study.

Keishi Yamashita,Masahiko Watanabe,Mina Waraya,Takeo Sato,Takatoshi Nakamura,Natsuya Katada,David Sidransky,Myoung Sook Kim,Diego Calvisi

doi:10.1371/journal.pone.0113546

Abstract

BackgroundCysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1) in colorectal cancer (CRC). In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP) and extensive analysis of the PCR reaction.MethodsDNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from “positive control” DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined.Results(1) The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2) DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3) By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20%) of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9%) or CA19-9 (23.1%) in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3%) as compared to CEA/CA19-9 (25%).ConclusionWe have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients.

Highlights

Cytosine DNA methylation of the promoter region of tumor suppressor genes is a common cancer phenomenon [1, 2], and could provide good candidate biomarkers for detection of minimal residual disease using methylation specific polymerase chain reaction (PCR) amplification [3, 4]
cysteine dioxygenase 1 (CDO1) is clearly hypermethylated in cancer cell lines [9], the degree of CDO1 promoter hypermethylation of cloned CDO1 promoter sequences has not been determined
Using bisulfite treatment of DNA followed by sequence analysis, we analyzed the methylation level of the CDO1 promoter sequence cloned from PCR amplified genomic DNA of DLD1 and HepG2 cells

Summary

Introduction

Cytosine DNA methylation of the promoter region of tumor suppressor genes is a common cancer phenomenon [1, 2], and could provide good candidate biomarkers for detection of minimal residual disease using methylation specific PCR amplification [3, 4]. We have identified N-methyl-Daspartate receptor type 2A (NMDAR2A) [6], deafness, autosomal dominant 5 (DFNA5) [7], Oncostatin M receptor-b (OSMR) [8], and cysteine dioxygenase 1 (CDO1) [9] as cancer-prone frequently methylated genes in colorectal cancer (CRC) by using pharmacological unmasking microarrays [1, 2]. We describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP) and extensive analysis of the PCR reaction. Methods: DNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from ‘‘positive control’’ DLD1 cells. CDO1 methylation was not detected as frequently as PLOS ONE | DOI:10.1371/journal.pone.0113546 December 3, 2014

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