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  • Allele specific methylation has been


    Allele-specific methylation has been observed in CpG islands of imprinted genes.27, 32 In the present study, however, methylation of CpG1 was specific to ALL src inhibitors and occurred in the leukemia cells of 28% of children with ALL, but it was not found in normal leukocytes from the same patients. Methylation in CpG1 was also not detected in leukemia cells from children with primary AML, a form of leukemia that is less sensitive to MTX therapy. Furthermore, we identified four “H.sapiens CpG island DNA genomic Mse1 fragments” in GenBank that are homologous to GGH CpG1. These CpG island genomic DNA MseI fragments are GC-rich chromosomal fragments that are not methylated in primary, normal blood cells but are methylated after in vitro treatment with methylase. A library of CpG island genomic DNA MseI fragments has been used to identify hypermethylated CpG islands in cancer cells, but this has not been reported for GGH. Consistent with our data is that no evidence exists that GGH is an imprinted gene,27, 32 and no imprinted genes have been reported in the locus around the human GGH coding region. Although GGH promoter methylation was restricted to the G allele for SNP −649G→A in Nalm6 cells, methylation of the A allele was observed in primary ALL cells from two of five patients (fig. 3B). The GGH promoter contains multiple transcriptional start sites, all in the CpG2 region. However, we found that methylation in GGH CpG1 has a more pronounced effect on GGH expression (down-regulation) than does methylation in CpG2. Methylation in CpG2 was common in leukemia cells and normal leukocytes from patients with ALL, but it occurred at a somewhat higher frequency in the leukemia cells. However, there was no relationship between CpG2 methylation status and GGH expression in ALL cells, and an absence of CpG2 methylation in normal cells was not associated with higher GGH expression. This finding is consistent with earlier reports that a high level of methylation around a transcriptional start site does not always block transcription, especially for genes with multiple transcriptional start sites. Hypermethylation of CpG islands in cancer exhibits heterogeneity both within the same tumor types and among different tumor types.36, 37 Methylated CpG islands are often, but not always, associated with transcriptional silencing.38, 39 A recent study suggested that aberrant methylation of CpG islands in malignancy might be less frequent than expected, because only a small set of promoters was differentially methylated in normal and transformed human cells. Our study has revealed that aberrant methylation in cancer cells can occur in specific CpG islands (e.g., GGH CpG1) but not in other CpG islands (e.g., GGH CpG2) within the same gene promoter region. Furthermore, the present findings indicate that cancer-specific CpG-island promoter methylation can preferentially target CpG islands that have an effect on gene expression (i.e., CpG1 was methylated only in cancer cells, whereas CpG2 was methylated in cancer and normal cells). Therefore, a global assessment of methylation profile—for example, by use of restriction landmark genomic scanning or an oligonucleotide microarray-based methylation analysis—would need to be performed in a manner that permits one to determine whether methylation in specific CpG islands (certain promoter regions) of a given gene is associated with altered gene transcription or cancer-cell phenotype (e.g., enzyme activity). An src inhibitors array based on a CpG island library created by isolation of genomic fragments that contain CpG dinucleotide regions that are poorly methylated in normal tissue has been used as a genomewide method of detecting cancer-specific CpG island methylation.33, 42 Consistent with our findings, this CpG island library contains the CpG1 region but not the CpG2 region from the GGH promoter region. Mechanisms by which CpG islands are hypermethylated in cancer have not been fully elucidated. CpG island methylation in cancer cells may be blocked by the binding of the transcription factor Sp1,43, 44 and it has been suggested that a boundary sequence separating methylated and unmethylated promoter regions exists.45, 46 Recent studies have indicated that DNA methylation in cancer cells can be induced by micro-RNAs.47, 48 De novo methylation in cancer cells has been suggested to start from a methylation “hotspot” consisting of several nonadjacent CpG sites. These hotspots are methylated in both normal and cancer cells, and they serve as a starting point for CpG island hypermethylation in cancer cells. Because we found that methylation frequently occurs in GGH CpG2 in both leukemia cells and normal leukocytes and that both CpG1 and CpG2 are methylated on a single allele, it is conceivable that methylation in CpG2 serves as the seeding point from which de novo methylation of the entire GGH promoter occurs in leukemia cells. However, the finding that the leukemia cells of several patients had GGH promoter methylation of only CpG1 suggests that leukemia cell–specific methylation of CpG1 arises by another mechanism. Further study is needed to determine whether methylation of a particular allele is a selective process. Our findings that none of the GGH promoter SNPs examined was associated with altered GGH mRNA expression and that either GGH allele (G or A at nt −649) can be methylated in primary ALL cells indicate that GGH promoter SNPs are not the driving force for single-allele methylation.