p53 and cell cycle effects after DNA damage

Abstract

Flow cytometry, a valuable technique that employs the principles of light scattering, light excitation, and emission of fluorochrome molecules, can be used to assess the cell cycle position of individual cells based on DNA content. After the permeabilization of cells, the DNA can be stained with a fluorescent dye. Cells which have a 2N amount of DNA can be distinguished from cells with a 4N amount of DNA, making flow cytometry a very useful tool for the analysis of cell cycle checkpoints following DNA damage. A critical feature of the cellular response to DNA damage is the ability to pause and repair the damage so that consequential mutations are not passed along to daughter generations of cells. If cells arrest prior to DNA replication, they will contain a 2N amount of DNA, whereas arrest after replication but before mitosis will result in a 4N amount of DNA. Using this technique, the role that p53 plays in cell cycle checkpoints -following DNA damage can be evaluated based on changes in the profile of the G1, S, and G2/M phases of the cell cycle.

Fig. 1

Wild-type p53 cells maintain a G1 and G2/M arrest after treatment with doxorubicin, whereas p53−/− cells primarily arrest in G2/M. (a) HCT 116 WT and p53−/− colorectal carcinoma cells were treated with 0.05 and 0.1 μg/ml doxorubicin for 24 h before harvesting for flow analysis. (b) RKO (wild-type p53) and RKO E6 (p53 is targeted for degradation by HPV E6) colorectal carcinoma cells were treated with 0.01 and 0.05 μg/ml doxorubicin for 24 h before harvesting for flow analysis. The analysis cell profiles are shown for untreated and treated cells. The percentage of cells in G1, S, and G2/M are indicated in the upper right hand corner of each histogram.

Fig. 2

Treatment with doxorubicin in WT p53 mouse embryonic fibroblasts (MEFs) results in a significant reduction of S phase cells. Wild-type and p53−/− MEFs were treated with 0.1 and 0.2 μg/ml doxorubicin for 24 h prior to being pulsed with 10 μM BrdU for 4 h. Following antibody treatment and propidium iodide staining the cells were analyzed by dual parameter flow cytometry. Both single and dual parameter histograms are shown for each condition. The percentage of cells in G1, S, and G2/M are indicated in the upper right hand corner of each histogram.

Fig. 3

Phosphorylation of histone H3 is diminished after DNA damage with doxorubicin in WT p53 cells. HCT 116 p53 WT and −/− cells were treated with 0.1 and 0.25 μg/ml doxorubicin or 0.5 μg/ml nocodozole for 24 h and stained for Ser10-phosphorylated histone H3 and DNA content (propidium iodide). The dual parameter flow analysis is shown. Phosphorylated histone H3 positive cells are boxed and denoted as R2, whereas cells in G1 and G2 are grouped in R1.