Article

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

Meyerstein Institute of Oncology, The Middlesex Hospital, London, UK.
International Journal of Radiation Biology (impact factor: 2.28). 03/1996; 69(2):251-7. pp.251-7
Source: PubMed

ABSTRACT 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 G1 and G2 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 G1 and G2 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 G2 checkpoint. Using this method, cell cycle-dependent effects relating to the G2 block can be observed. The radiation-induced G2 block differs from that induced by drugs or heating in that cells are blocked in G2 irrespective of the phase of the cell cycle they are treated in. This method allows these different types of G2 block to be quantified.

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Keywords

asynchronous cells
 
bis-benzimidazole Hoechst 33258
 
BrdU label
 
cell cycle-dependent effects
 
cell lines
 
continuous incorporation
 
delays
 
Ethidium bromide fluoresces red
 
flow cytometric technique utilizing
 
G2 checkpoint
 
G2 checkpoints
 
G2 compartments
 
Hoechst 33258 fluoresces blue
 
human cervix carcinoma cell line HX 156
 
human papilloma virus
 
ionizing radiation
 
measure cell cycle delays
 
normal diploid human embryo fibroblast cell line MRC 5
 
radiation-induced G2 block
 
synthesizing DNA new G1