Amyloid precursor protein gene expression in neural cell lines: influence of DNA cytosine methylation

Transcription of the gene encoding amyloid precursor protein (APP) varies in a cell-specific and developmentally regulated manner. The 5' region of this gene possesses a high frequency of CpG dinucleotides as well as copies of a GC-rich sequence, a potential trans factor binding element. These findings raise the possibility that DNA cytosine methylation could participate in the regulation of APP gene expression. We examined APP mRNA/18S rRNA ratio in three neural cell lines (N18TG2, SN6, SN17) cultured in 5-azacytidine (5-AZA), an inhibitor of maintenance methylase which results in loss of cytosine methylation in proliferating cells. Culture in 5-AZA globally reduced methylation in genomic DNA as assessed by an increase in HpaII restriction sites, reduced cytosine methylation in the APP gene as assessed by Southern blotting of HpaII digests, and increased APP mRNA steady state abundance in all studied cell lines. Cell lines re-acquired APP gene methylation 48 h after removal of 5-AZA from media. These results indicate that in vitro alteration of DNA methylation can affect APP gene expression, and suggest that the APP gene in neuronal cell lines may be rapidly inactivated in vitro, perhaps to neutralize its potential toxicity.