Establishment of a human cell line for the detection of demethylating agents.

To detect xenobiotics affecting the DNA methylation pattern we have established a human clonal cell line (A4/4) derived from the 293 epithelial cells. This cell line is stable after more than 1 year in culture and we report here its characteristics. The A4/4 cells carry the Escherichia coli lacI gene and the symmetric lacO sequence upstream of the beta-galactosidase coding gene (lacZ). Both sequences have been independently integrated into the genomic DNA. The lacZ gene is under the control of the metal-inducible mouse metallothionein-I (mMT-I) promoter. Vector integration followed by cell ageing in culture gave rise to the methylation of the 5'CpG3' sites in the mMT-I promoter and the 5' part of the lacZ gene. The reactivation of the lacZ gene by 5-azacytidine (5-Aza-CR) treatment allowed a tremendous lacZ expression which is correlated with demethylation in the mMT-I promoter and its neighboring regions. This enhanced transcription is easily quantified by measuring the beta-galactosidase activity in cell extracts. 5-Aza-CR, the specific isopropyl beta-D-thiogalactoside inducer, and heavy metal ions added together trigger an increase of the beta-galactosidase activity up to 600-fold over the basal level. These cells can be used for a rapid assessment of the demethylating potential of environmental chemicals.