Flow cytometric detection of drugs altering the DNA methylation pattern

• Published in: Cancer research (Chicago, Ill.) Vol. 52; no. 19; pp. 5213 - 5218
• Main Authors: BIARD, D. S. F, MARATRAT, M, THYBAUD, V, MELCION, C, SARASIN, A
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 01.10.1992
• Subjects: Antineoplastic agents; Azacitidine - analogs & derivatives; Azacitidine - analysis; Azacitidine - pharmacology; Base Sequence; beta-Galactosidase - drug effects; beta-Galactosidase - genetics; Biological and medical sciences; Butyrates - analysis; Butyrates - pharmacology; Butyric Acid; Cell Cycle - drug effects; Cell Transformation, Viral - physiology; Cells, Cultured; DNA, Bacterial - drug effects; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Flow Cytometry; Fluorometry; Gene Expression Regulation, Enzymologic - drug effects; General aspects; Humans; Kidney - cytology; Lac Operon - drug effects; Lac Operon - genetics; Medical sciences; Methylation; Molecular Sequence Data; Pharmacology. Drug treatments; Transcription, Genetic - drug effects; Transcription, Genetic - genetics; Antineoplastic agent; Human; Flow cytometry; Embryo; Adenoviridae; Enzyme; Toxicity; Transformed cell; Chemical compound; Escherichia coli; β-D-Galactosidase; Kidney; Virus; Plasmid; Demethylation; Gene; Investigation method; Human adenovirus; DNA; Bacteria; Mastadenovirus; Mechanism of action; Nucleic acid; Enterobacteriaceae
• ISSN: 0008-5472; 1538-7445

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.