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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.