Comprehensive Analysis of Methylation of CpG Islands on the Long Arm of Chromosome 7.

Abstract

Deletion of the long arm of chromosome 7 is frequently observed in hematological malignancy and constitutes one of the most important prognostic factors. So, it is supposed that a tumor suppressing gene is located in this region, and several attempts with classical analysis methods have been executed. However, none of the genes has been identified thus far. On the other hand, methylation of the promoter region of genes is now recognized as a common mechanism of gene silencing in malignancy and frequently observed in hematological malignancy. Especially in myelodysplastic syndrome, demethylating agents such as 5-aza-2′ deoxycytidine are shown to have clinical usefulness. In order to find a gene inactivated in hematological malignancy, we comprehensively analyzed the CpG island methylation on the long arm of chromosome 7 of 5 normal blood, 13 primary leukemic bone marrow, and cell lines established from various hematological malignancies. We treated the sample genome with sodium bisulfite and directly sequenced the PCR-amplified CpG islands. Degrees of CpG island hypermethylation are widely varying in different tumors samples with much higher frequencies observed in cultured tumor cell lines. The levels of methylation are considerably varying among different samples, with higher methylation levels in cultured tumor cell lines, intermediate for primary tumor samples, and lowest in normal tissues. In contrast, however, patterns of methylation are largely similar among different samples with varying methylation levels, which creates a unique clustering pattern of methylated CpG islands, indicating some regions of the chromosome arm are more prone to methylation than others. We also examined the expression levels of DNA methyltransferases (DNMT) 1, 3a, and 3b by real-time PCR. However, there is no correlation between the expression of these genes and overall methylation level of CpG islands. We identified 25 genes that show malignancy-specific methylation. We consider that these genes are candidates of tumor suppressor genes and inquiring into deletion, methylation, and methylation analysis. We also show that methylation level of CpG islands is inversely correlated with the density of SP1 binding sequence in normal cells, which is obscured in case of primary tumors and no more observed in cultured cell lines. We also show that CpG islands that are on 5′-end of genes show lower methylation levels than those not on 5′ of genes in normal samples. Again, this trend tends to be a little lost in primary leukemia samples and further collapsed in cultured tumor cell lines, though the relation is still in a significant level. Together with previous observations, we support the hypothesis that SP1-binding sequence or active transcription protects normal CpG islands from methylation. Our observation that these phenomena disappear or diminish shows that the mechanism of aberrant methylation in malignancy is the loss of these protection mechanisms.