Cytometric analysis of DNA replication inhibited by emetine and cyclosporin A.
DNA staining methods based on aspecific interactions with dye molecules have been replaced by an immunofluorescent approach to measure DNA replication. Biotin-11-dUTP was incorporated into permeable thymocytes isolated after emetine or cyclosporin A treatment of mice. Active sites of DNA replication were amplified based on biotin-avidin interaction and verified under fluorescent microscope. Cytometry of fluorescent images allow the direct measurement of replicating DNA without aspecific detection of total cellular DNA. Cytometric analysis of replication revealed that emetine acts at the early S phase, while cyclosporin A blocks in vivo DNA synthesis at mid S phase.
Institute of Biochemistry Department I, Semmelweis Medical University, Budapest, Hungary.
T
T
Schweighoffer
E
E
Schweighoffer
A
A
Apati
F
F
Antoni
G
G
Molnar
K
K
Lapis
G
G
Banfalvi
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D004261:DNA Replication / Q000187:drug effects*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004640:Emetine / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008856:Microscopy, Fluorescence; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology; D014407:Tumor Cells, Cultured
D003856:Deoxyuracil Nucleotides; D001710:Biotin; D016572:Cyclosporine; C045931:biotin-11-dUTP; D004640:Emetine