A minimally invasive assay for individual assessment of the ATM/CHEK2/p53 pathway activity

Abstract

Ionizing radiation induces DNA Double-Strand Breaks (DSBs) which activate the ATM/CHEK2/p53 pathway leading to cell cycle arrest and apoptosis through transcription of genes including CDKN1A (p21) and BBC3 (PUMA). This pathway prevents genomic instability and tumorigenesis as demonstrated in heritable syndromes [e.g. Ataxia Telangiectasia (AT); Li-Fraumeni syndrome (LFS)]. Here, a simple assay based on gene expression in peripheral blood to measure accurately ATM/CHEK2/p53 pathway activity is described. The expression of p21, Puma and Sesn2 was determined in blood from mice with different gene copy numbers of Atm, Trp53 (p53), Chek2 or Arf and in human blood and mitogen stimulated T-lymphocyte (MSTL) cultures from AT, AT carriers, LFS patients, and controls, both before and after ex vivo ionizing irradiation. Mouse Atm/Chek2/p53 activity was highly dependent on the copy number of each gene except Arf. In human MSTL, an AT case, AT carriers and LFS patients showed responses distinct from healthy donors. The relationship between gene copy number and transcriptional induction upon radiation was linear for p21 and Puma and correlated well with cancer incidence in p53 variant mice. This reliable blood test provides an assay to determine ATM/CHEK2/p53 pathway activity and demonstrates the feasibility of assessing the activity of this essential cancer protection pathway in simple assays. These findings may have implications for the individualized prediction of cancer susceptibility.

Figures

Figure 1

Expression level of p21 (A), Puma (B) and Sesn2 (C) 2 h following ex vivo exposure of blood from individual mice to a 2 Gy dose [IR (2 Gy)+] and sham irradiation [IR(2 Gy)-] analyzed by MQRT-PCR. Data are shown for individual mouse samples (each point represents a different mouse with bars representing the mean in each group). 25 wild type [14 males (M), 11 females (F)], 12 p53 KO (6 M, 6 F), 6 p53 heterozygotes (3 M, 3 F), 15 p53-tg (7 M, 8 F), 7 p53-tgb (4 M, 3 F) mice. Data were analyzed with the ANOVA general linear model. Differences between means where significant are shown for irradiated samples only for clarity. *p value < 0.05, **p value < 0.01 and ***p value < 0.001.

Figure 2

Upregulation of p21 (A), Puma (B) and Sesn2 (C) gene expression following ex vivo blood exposure dependant on p53 gene copy number. The data from Figure 1 are represented as fold change (ratio of level of expression after irradiation divided by the un-irradiated expression levels) obtained with p53 KO (no p53 gene), p53 heterozygotes mice (1 gene copy), wild type (2 copies), p53-tg (3 copies) and p53-tgb (4 copies) mice. Values were standardized using Hprt data as control of cDNA quantity. The means of independent mouse samples (numbers as in Fig. 1 legend) are presented with error bars representing the standard error of the mean (SE).

Figure 3

Upregulation of p21, PUMA and Sesn2 transcription following ex-vivo exposure of blood is directly dependant on Atm gene copy number, partially on Chek2 but not on Arf. (A) 5 Atm heterozygote (3 M, 2 F) and 5 Atm KO (3 M, 2 F) mice, (B) 5 Chek2 heterozygote (all M) and 2 Chek2 KO (both M) mice, (C) 6 Arf KO (4 M, 2 F) mice. Each point represents a different mouse and bars the mean in each sample group. Data have been analyzed with the ANOVA general linear model. Differences where statistically significant are shown for irradiated samples only for clarity. *p value < 0.05, **p value < 0.01 and ***p value < 0.001 based on comparison of mean values.

Figure 4

p53 copy number dependence of cancer incidence. (A) Cancer incidences for mice carrying different copy numbers of p53. Data presented here were obtained from previous publications (refs. , and 21). (B) Relationship between the p21 radiation response levels (obtained in mice carrying different copy numbers of p53 (see Fig. 2) and cancer incidence. (C) Same as (B) for Puma.

Figure 5

Expression level of PUMA following radiation exposure as a method for determining the strength of the ATM/CHEK2/p53 pathway in human samples. (A) Quantitative real-time PCR analysis of PUMA gene expression in human MSTL. Fold changes in expression compared to sham-treated cells (relative to HPRT expression) in response to 2 Gy irradiation. Data represent the mean (open circles) ±SD of the fold change and individual values (filled circles) for one AT patient, three LFS patients, four AT heterozygotes and nine healthy donors. The mean value of two independent experiments done in duplicate (RT-PCR done in triplicate each time) is presented. Differences from the healthy donor average are shown where significant **p value <0.01. (B) MQRT-PCR analysis of PUMA gene expression 2 h following 2 Gy irradiation in human blood from seven healthy donors (3 men, 4 women). Data represent the mean of fold change of three independent experiments (filled circle, error bar represent 95% confidence intervals). The straight line is from mouse data from Figure 2B, see text for further details.