Flow cytometric detection of drugs altering the DNA methylation pattern.
We have developed a model system for assessing the demethylating potential of external agents. Disruption in the DNA methylation pattern was evaluated at the translational level of the Escherichia coli beta-galactosidase coding gene (lacZ). We have constructed a clonal cell line (A4/4 cells) derived from the adenovirus-transformed human embryonic kidney 293 strain. The A4/4 cells contain the E. coli lacZ gene under the control of the mouse metallothionein 1 promoter which is down-regulated by a natural DNA methylation pattern. Furthermore, the lacZ transcription is also regulated by the E. coli lac operator/repressor system and by mouse metallothionein 1 metal responsiveness offering a wide range in lacZ expression. In this system, the beta-galactosidase activity was only recovered in the presence of a demethylating agent such as 5-azacytidine. The demethylating potential of 5-azacytidine, 5-aza 2'-deoxycytidine and sodium butyrate was rapidly assessed by a flow cytometric method using fluorescein di-beta-D galactopyranoside as a fluorescent probe. A tremendous induction of lacZ expression was triggered by these drugs. Analysis of cell cycles showed little disruptions with 5-azacytidine and sodium butyrate, but an important blockage in the S-phase following 5-aza 2'-deoxycytidine treatment was observed. This approach allows a rapid identification and study of environmental demethylating agents.
Biard
D S
DS
Laboratory of Molecular Genetics (UPR42), CNRS, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
Maratrat
M
M
Thybaud
V
V
Melcion
C
C
Sarasin
A
A
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000494:pharmacology; D001483:Base Sequence; D002087:Butyrates / Q000032:analysis* / Q000494:pharmacology; D020148:Butyric Acid; D002453:Cell Cycle / Q000187:drug effects; D002472:Cell Transformation, Viral / Q000502:physiology; D002478:Cells, Cultured; D004269:DNA, Bacterial / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D000077209:Decitabine; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005434:Flow Cytometry; D005470:Fluorometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000166:cytology; D007763:Lac Operon / Q000187:drug effects / Q000235:genetics; D008745:Methylation; D008969:Molecular Sequence Data; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D001616:beta-Galactosidase / Q000187:drug effects / Q000235:genetics
D002087:Butyrates; D004269:DNA, Bacterial; D020148:Butyric Acid; D000077209:Decitabine; D001616:beta-Galactosidase; D001374:Azacitidine