Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation.
In situ presence of numerous DNA strand breaks is a typical feature of apoptotic cells. Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated DNA precursors has recently been proposed as a method of analysing DNA replication. Detection of DNA strand breaks, thus, enables one to identify apoptotic and/or DNA replicating cells. The current methods for DNA strand break labelling rely on the use of exogenous terminal deoxynucleotidyl transferase which either directly attaches the fluorochrome conjugated triphosphodeoxynucleotides to 3'OH ends in the breaks, or indirectly labels 3'OH ends with digoxygenin or biotin conjugated triphosphodeoxynucleotides. A limitation of these methodologies, especially restricting their routine application in the clinic, is high cost of reagents. In the present study we have tested whether relatively simple compound BrdUTP, which is approximately three orders of magnitude less expensive than dUTP conjugated to digoxygenin, can be used as marker of DNA strand breaks. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin. The incorporated BrdUTP was detected by fluoresceinated anti-BrdUrd MoAb. Cellular fluorescence was measured by flow cytometry as well as by Laser Scanning Cytometer (LSC). The data show that intensity of DNA strand break labelling with BrdUTP was nearly four- and two-fold higher than that obtained with the indirect labelling using biotin- or digoxygenin-conjugated dUTP, respectively, and over eight-fold higher than in the case of direct labelling with the fluorochrome (fluorescein or BODIPY)-conjugated deoxynucleotides. The increased labelling of DNA strand breaks with BrdUTP may reflect more efficient incorporation of this precursor by terminal transferase, compared to the nucleotides with bulky fluorochrome conjugates. DNA strand break labelling with BrdUTP, thus, offers a possibility of more sensitive (and at lower cost) detection of apoptotic or DNA replicating cells, compared to the alternative methods of DNA strand break labelling.
Li
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Cancer Research Institute, New York Medical College, Valhalla 10523, USA.
Darzynkiewicz
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D017209:Apoptosis / Q000502:physiology*; D015415:Biomarkers; D001896:Boron Compounds; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D004249:DNA Damage / Q000502:physiology*; D003856:Deoxyuracil Nucleotides; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D018922:HL-60 Cells / Q000166:cytology*; D006801:Humans; D012680:Sensitivity and Specificity; D059004:Topoisomerase I Inhibitors
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D015415:Biomarkers; D001896:Boron Compounds; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D059004:Topoisomerase I Inhibitors; C032549:bromodeoxyuridine triphosphate; D016650:Fluorescein-5-isothiocyanate; D002166:Camptothecin