Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color.
Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated nucleotides has been recently proposed as a means of analyzing DNA replication and detecting proliferating cells. The presence of numerous in situ DNA strand breaks is also an inherent feature of apoptotic cells. The aim of the present study was to differentially label DNA strand breaks in apoptotic cells vs photolysis-induced breaks in BrdUrd incorporating cells. This would allow one, by multicolor staining, to identify these respective cells in the same sample preparation. Toward this end, exponentially growing HL-60 cells were pulse labeled with BrdUrd and then were subjected to hyperthermia or treated with DNA topoisomerase I inhibitor camptothecin to induce apoptosis. DNA strand breaks in apoptotic cells were first labeled directly with fluorochrome-conjugated dUTP or dCTP, followed by dideoxynucleotide (to terminate chain elongation), in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The cells were subsequently exposed to UV light to photolyze DNA containing the incorporated BrdUrd. The photolysis-induced DNA strand breaks were, in turn, labeled with digoxygenin- or biotin-conjugated dUTP followed by digoxygenin antibody or avidin, respectively, conjugated with fluorochrome of another color. Alternatively, DNA strand breaks were labeled with BrdUTP which was then detected by FITC-conjugated anti-BrdUrd MoAb. Following counterstaining of cellular DNA with a fluorochrome of a third color it was possible to identify apoptotic cells, cells incorporating BrdUrd, and cells having no DNA strand breaks. Cell fluorescence was measured either by flow cytometry or with the microscope-based laser scanning cytometer. The SBIP approach also offers a possibility to study a colocalization of the immunocytochemically detectable cell constituents at the DNA replication points by microscopy. Using this approach the presence of the proliferating cell nuclear antigen at the DNA replication sites was revealed in MCF-7 breast carcinoma cells.
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Cancer Research Institute, New York Medical College, Valhalla 10595, USA.
Melamed
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Darzynkiewicz
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D000911:Antibodies, Monoclonal; D017209:Apoptosis; D001973:Bromodeoxyuridine; D002166:Camptothecin / Q000494:pharmacology; D003080:Cold Temperature; D003592:Cytophotometry / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004261:DNA Replication; D003845:Deoxycytosine Nucleotides; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018922:HL-60 Cells; D006801:Humans; D007834:Lasers; D010782:Photolysis; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate
D000911:Antibodies, Monoclonal; D003845:Deoxycytosine Nucleotides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D018809:Proliferating Cell Nuclear Antigen; D059004:Topoisomerase I Inhibitors; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D001973:Bromodeoxyuridine; D014544:Uridine Triphosphate; D002166:Camptothecin