Anti-MAGEA1 polyclonal antibody

Genome-wide reduction in 5-methylcytosine is an epigenetic hallmark of human tumorigenesis. Experimentally induced hypomethylation in mice promotes genomic instability and is sufficient to initiate tumorigenesis. Here, we report that global hypomethylation is common in primary human glioblastomas [glioblastoma multiforme (GBM)] and can affect up to an estimated 10 million CpG dinucleotides per haploid tumor genome. Demethylation involves satellite 2 (Sat2) pericentromeric DNA at chromosomes 1 and 16, the subtelomeric repeat sequence D4Z4 at chromosomes 4q and 10q, and interspersed AIu elements. Severe hypornethylation of Sat2 sequences is associated with copy number alterations of the adjacent euchromatin, suggesting that hypornethylation may be one factor predisposing to specific genetic alterations commonly occurring in GBMs. An additional apparent consequence of global hypomethylation is reactivation of the cancer-testis antigen MAGEA1 via promoter demethylation, but only in GBMs and GBM cell lines exhibiting a 5-methylcytosine content below a threshold of similar to 50%. Primary GBMs with significant hypomethylation tended to be heterozygous or homozygous for the low-functioning Val allele of the rate-limiting methyl group metabolism gene methylenetetrahydrofolate reductase (MTHFR), or had a deletion encompassing this gene at 1p36. Tumors with severe genomic hypomethylation also had an elevated proliferation index and deletion of the MTHFR gene. These data suggest a model whereby either excessive cell proliferation in the context of inadequate methyl donor production from MTHFR deficiency promotes genomic hypornethylation and further genomic instability, or that MTHFR deficiency-associated demethylation leads to increased proliferative activity in GBM.

Melanoma-associated antigen A1 (MAGEA1) and BORIS (also known as CTCFL) are members of the cancer testis antigen (CTA) family. Their functions and expression-regulation mechanisms are not fully understood. In this study, we reveal new functions and regulatory mechanisms of MAGEA1 and BORIS in breast cancer cells, which we investigated in parental and genetically manipulated breast cancer cells via gene overexpression or siRNA-mediated downregulation. We identified the interaction between MAGEA1 and CTCF, which is required for the binding of MAGEA1 to the BORIS promoter and is critical for the recruitment of DNMT3a. A protein complex containing MAGEA1, CTCF and DNMT3a was formed before or after conjunction with the BORIS promoter. The binding of this complex to the BORIS promoter accounts for the hypermethylation and repression of BORIS expression, which results in cell death in the breast cancer cell lines tested. Multiple approaches were employed, including co-immunoprecipitation, glutathione S-transferase pull-down assay, co-localization and cell death analyses using annexin V-FITC/propidium iodide double-staining and caspase 3 activation assays, chromatin immunoprecipitation and bisulfite sequencing PCR assays for methylation. Our results have implications for the development of strategies in CTA-based immune therapeutics.