DETECTION OF 5-BROMO-2-DEOXYURIDINE INCORPORATED INTO DNA BY LABELING STRAND BREAKS INDUCED BY PHOTOLYSIS (SBIP)

A novel method to identify DNA replicating cells is described. In this method DNA strand breaks at sites of incorporation of 5-bromo-2-deoxyuridine (BrdUrd) are induced by photolysis and labeled with digoxygenin- or biotin conjugated dUTP. The reaction is catalyzed by exogenous terminal deoxynucleotidyl transferase. This approach, in conjunction with DNA content analysis by flow cytometry, was applied to studies of the proliferation kinetics of human leukemic HL-60 or MOLT-4 cells during pulse and pulse-chase incubation with BrdUrd. A 30-60 min incubation with BrdUrd led to selective labeling of S phase cells and the progression of a cohort of labeled cells through late S, G2 and G1 was revealed by pulse (30 min) - chase (8 h) labeling with the precursor. The presence of apoptotic cells did not interfere with identification of DNA replicating cells, as the subpopulation of apoptotic cells could be distinguished by low DNA content resulting from extraction of DNA during the procedure. The technique could be applied as well to human breast cancer tissue incubated in vitro with BrdUrd. There is no need for DNA denaturation as is required for the conventional immunocytochemical detection of BrdUrd, which can often impair analysis of the cell phenotype. The SBIP methodology may be uniquely advantageous when there is a need for characterization of the phenotype of proliferating cells, or preservation of some features that would otherwise be destroyed during the step of DNA denaturation.