Application of a bromodeoxyuridine-Hoechst/ethidium bromide technique for the analysis of radiation-induced cell cycle delays in asynchronous cell populations

A flow cytometric technique utilizing the continuous incorporation of bromodeoxyuridine (BrdU) into asynchronous cells to measure radiation-induced cell cycle delay is described. Following the incorporation of the BrdU label the cells are stained with ethidium bromide and the bis-benzimidazole Hoechst 33258. These fluorochromes have differential staining patterns. Hoechst 33258 fluoresces blue and is quenched by BrdU incorporated into cellular DNA during S phase. Ethidium bromide fluoresces red and is not quenched by BrdU. Therefore in cells that are cycling and synthesizing DNA new G(1) and G(2) compartments are created and this can be used to measure cell cycle delays following ionizing radiation to asynchronous cells. We have used this technique to evaluate two cell lines: a normal diploid human embryo fibroblast cell line MRC 5, which has inducible p53 and shows delays at both G(1) and G(2) checkpoints, and the human cervix carcinoma cell line HX 156. This cell line has been infected with human papilloma virus (HPV) 16, and therefore has inactivated p53 function and is blocked only at the G(2) checkpoint. Using this method, cell cycle-dependent effects relating to the G(2) block can be observed. The radiation-induced G(2) block differs from that induced by drugs or heating in that cells are blocked in G(2) irrespective of the phase of the cell cycle they are treated in. This method allows these different types of G(2) block to be quantified.