Ultraviolet-induced detection of halogenated pyrimidines: simultaneous analysis of DNA replication and cellular markers.
BACKGROUND
We describe a new nonenzymatic methodology that allows the simultaneous detection of DNA replication and other cellular markers such as immunophenotyping. DNA replicating cells are identified by their incorporation of halogenated thymidine analogs, e.g., 5-bromo-deoxyuridine (BrdUrd).


METHODS
Irradiation with ultraviolet (UV)-B or UV-A light in the presence of Hoechst 33258 and subsequent treatment with a hypotonic buffer makes BrdUrd accessible to monoclonal antibodies (mAb), thus allowing its sensitive detection.


RESULTS
The photolysis of BrdUrd in DNA with UV light is sequence dependent and results in DNA damage, allowing the detection of remaining BrdUrd using hypotonic conditions. However, treatment with other inducers of single or double- strand breaks of DNA such as gamma irradiation or hydrogen peroxide did not allow BrdUrd detection. The new methodology is compatible with both mild crosslinking fixation, i.e., aldehydes, or coagulative fixation, i.e., alcohols. The successful identification of CD34+, CD138+, or CD19+ cells out of heterogeneous cell suspensions and their cell-cycle analysis are described. Results correlated very well with acid denaturation (r = 0.972). The average coefficient of variation (CV) of G(1) in the DNA histogram was smaller than 5%, resulting in good preservation of DNA distribution. Also, the signal-to-noise ratio was almost twice as high as for 2N acid denaturation, facilitating convenient discrimination of BrdUrd-positive cells.


CONCLUSIONS
In contrast to previous approaches, this methodology eliminates the need for any additional enzymatic treatment such as DNA digestion or strand-break labeling after UV irradiation. The method is fast, convenient, and inexpensive and should be able to promote the use of halogenated pyrimidines in basic and clinical research of cancer, immunology, and pharmacology.
Institute of Immunology and Transfusion Medicine, University of Luebeck School of Medicine, Germany. hj-hammers@yahoo.com
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D018941:Antigens, CD19 / Q000032:analysis*; D018952:Antigens, CD34 / Q000032:analysis*; D006690:Bisbenzimidazole / Q000032:analysis; D001973:Bromodeoxyuridine / Q000032:analysis*; D002453:Cell Cycle; D004247:DNA / Q000528:radiation effects*; D004261:DNA Replication / Q000528:radiation effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D016130:Immunophenotyping; D008562:Membrane Glycoproteins / Q000032:analysis*; D011509:Proteoglycans / Q000032:analysis*; D053668:Syndecan-1; D053667:Syndecans; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D018941:Antigens, CD19; D018952:Antigens, CD34; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D011509:Proteoglycans; C508241:SDC1 protein, human; D053668:Syndecan-1; D053667:Syndecans; D004247:DNA; D001973:Bromodeoxyuridine; D006690:Bisbenzimidazole