Ultraviolet-induced detection of halogenated pyrimidines: Simultaneous analysis of DNA replication and cellular markers

Background: We describe a new nonenzymatic method ology that allows the sim ultaneous detection of DNA replication and other cellular markers such as i mmunophenotyping. DNA replicating cells are identified by their incorporati on 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 Brd Urd accessible to monoclonal antibodies (mAb), thus allowing its sensitive detection. Results: The photolysis of BrdUrd in DNA with UV light is sequence dependen t and results in DNA damage, allowing the detection of remaining BrdUrd usi ng hypotonic conditions. However, treatment with other inducers of single o r double- strand breaks of DNA such as gamma irradiation or hydrogen peroxi de did not allow BrdUrd detection. The new methodology is compatible with b oth mild crosslinking fixation, i.e., aldehydes, or coagulative fixation, i .e., alcohols. The successful identification of CD34+, CD138+, or CD19+ cel ls 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 denatura tion, facilitating convenient discrimination of BrdUrd-positive cells. Conclusions: In contrast to previous approaches, this methodology eliminate s the need for anp 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 pyrim idines in basic and clinical research of cancer, immunology, and pharmacolo gy. (C) 2000 Wiley Liss, Inc.