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.
Biard
D S
DS
Laboratory of Molecular Genetics, UPR 42, CNRS, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
Cordier
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Sarasin
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D002460:Cell Line; D002999:Clone Cells; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004261:DNA Replication; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D007763:Lac Operon; D008668:Metallothionein / Q000235:genetics; D008745:Methylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping
D004274:DNA, Recombinant; D008668:Metallothionein; D004248:DNA (Cytosine-5-)-Methyltransferases