Chk2 down-regulation by promoter hypermethylation in human bulk gliomas

Gliomas account for 80% of malignant brain tumors. DNA damage response and subsequent checkpoint control pathways could maintain the integrity of the genome and thus defend tumorigenesis. Four kinases, ATM, ATR, ChK1 and Chk2 are the damage sensors and the early effectors in DNA damage responses. Given their importance, we investigated the transcriptional regulation of these four genes.

Tissues from ten normal brains and thirty human gliomas were utilized for mRNA analysis via real-time PCR. Another twelve normal brain tissues and forty gliomas were used for confirmation. Methylation-specific PCR (MSP) was used to determine the methylation status of the Chk2 promoter. Quantitative chromatin immunoprecipitation (ChIP) was used to measure the influence of methylation on Sp1 binding.

We found that the expression of ATR, ChK1 and Chk2 in gliomas was significantly down-regulated relative to the normal brain tissues. The most significant reduction of expression was of the Chk2 gene, whose expression was approximately 10-fold decreased in gliomas (P<0.0001). Down-regulation of Chk2 was validated in the second real-time PCR analysis. This reduction in expression was partially due to promoter methylation. The Chk2 proximal promoter recruited Sp1 for transcriptional activation. We found that hypermethylation of the Chk2 promoter undermined the binding of the transcriptional factor Sp1.

Our results indicate that Chk2 methylation could be involved in glioma carcinogenesis and Chk2 expression may potentially be used for the diagnosis of glioma.