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  • Cancer has been linked to both genetic and epigenetic change

    2019-11-16

    Cancer has been linked to both genetic and epigenetic changes. Epigenetic modifications, mainly DNA methylation at CpG dinucleotides, affect the regulation of gene transcription without altering the gene\'s sequence. Short sequences rich in CpG dinucleotides (i.e., CpG islands) are often found in the 5′ promoter region of genes and can extend into the first exon and sometimes into intron 1., Aberrant methylation of CpG islands is a common feature of cancer cells,, and methylated CpG islands have been consistently detected in primary human acute leukemia samples, sometimes with consequent silencing of gene expression. In the current study, we examined epigenetic modifications of in leukemia benzbromarone australia and normal leukocytes from pediatric patients with ALL and identified two CpG islands in the promoter region of . CpG island 1 (CpG1) was methylated in leukemia cells but not in normal leukocytes, whereas CpG island 2 (CpG2) was methylated in both normal leukocytes and leukemia cells. Methylation of CpG1 alone in leukemia cells was associated with modest down-regulation of expression, whereas methylation in both CpG1 and CpG2 was associated with significantly reduced mRNA expression and GGH catalytic activity. Our findings establish that epigenetic mechanisms alter GGH activity and MTXPG accumulation in human leukemia cells via methylation of CpG islands in the human promoter region. Subjects and Methods
    Results
    Discussion Because MTXPG accumulation in leukemia cells is a determinant of the in vivo antileukemic effects of MTX,4, 5, 23 we and others have focused on elucidating mechanisms underlying the highly variable accumulation of MTXPG in leukemia cells.7, 24, 25 We have already documented significantly differential expression of the genes encoding reduced folate carrier (SLC19A1 [MIM 600424]); breast cancer resistance protein (ABCG2 [MIM 603756]); ATP-binding cassette, subfamily C, member 1 (ABCC1 [MIM 158343]); and folylpolyglutamate synthetase (FPGS [MIM 136510]) as a mechanism underlying differences in MTXPG accumulation among some subtypes of ALL. We have also shown that the catalytic activity of GGH in ALL cells exhibits substantial interindividual differences that are influenced by the number of copies of chromosome 8 containing a wild-type GGH allele and that GGH activity is inversely related to accumulation of long-chain MTXPG in BNHD-ALL cells. However, before now, we did not know what mechanism is responsible for differences in GGH mRNA expression and catalytic activity in ALL cells of patients with a diploid wild-type GGH genotype. Here, we report epigenetic mechanisms controlling GGH expression and catalytic activity in B-lineage ALL cells containing two wild-type GGH alleles. We found significant concordance between GGH activity and GGH mRNA expression in leukemia cells from these patients. Furthermore, we observed methylation spanning the entire GGH promoter region in leukemia cells of ∼15% of pediatric patients with ALL and its concordance with significantly reduced GGH mRNA expression and catalytic activity. Moreover, we showed that this was associated with higher intracellular MTXPG concentrations after in vivo treatment with MTX. DNA methylation is an important mechanism regulating gene expression, and it is required for normal mammalian development.26, 27, 28 Alteration in DNA methylation is a hallmark of cancer10, 29, 30 and may be observed as a loss of global methylation or as a gain of methylation of selected CpG islands, either of which can result in alteration of the expression of hundreds of genes.12, 13, 31 We identified two CpG islands in the region extending from the GGH promoter through the first exon and into intron 1. To perform population screening for GGH promoter methylation status, we designed an NMSP that requires as little as 40 ng of genomic DNA from each patient sample. We confirmed GGH promoter methylation status in primary leukemia cells and a BNHD-ALL cell line by sequencing bisulfite-treated samples. We found that methylation of CpG1 has a much greater effect on GGH expression than does methylation of CpG2 and that methylation of both CpG islands has the most pronounced effect on GGH expression.