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. 2011 Apr 1;10(7):1152–1161. doi: 10.4161/cc.10.7.15231

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

Sylwia Kabacik 1, Ana Ortega-Molina 2, Alejo Efeyan 2, Paul Finnon 1, Simon Bouffler 1, Manuel Serrano 2, Christophe Badie 1,✉
PMCID: PMC3100888  PMID: 21389785

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.

Key words: lymphocyte, gene expression, ATM/CHEK2/Tp53 pathway, blood, cancer, susceptibility, radiation, ATM, p53

Introduction

Mammalian cell DNA is subject to damage caused either spontaneously by oxygen radicals or during DNA replication, or by external agents such as ultraviolet or ionizing irradiation (IR). IR is a physiologically important stress inducing a large range of DNA lesions1 to which cells respond by the activation of multiple signaling pathways. A complex network called the DNA damage response (DDR) that regulates cell proliferation, cell death and coordinates repair is activated to ensure that the lesions are repaired efficiently and accurately with minimal impact on genome stability.2 This is particularly true for DNA double-strand breaks (DSBs) which are often cytotoxic but can also promote chromosomal rearrangement and genomic instability, thus contribute to cancer. DSBs rapidly activate the chief transducer of the DSB damage signal, the nuclear Ataxia Telangiectasia mutated (ATM) gene through its direct activator, the sensor of DSBs, the Mre11-Rad50-Nbs1 (MRN) complex.3 ATM is activated through autophosphorylation at Ser1981 and the conversion of ATM dimers to monomers.4 Monomeric ATM is a nuclear serine-threonine kinase which is essential for rapid activation of cell cycle checkpoints and DNA repair in response to IR.5 It acts upstream of p53 (encoded by TP53) by activating p53 protein through phosphorylation6 or through another protein kinase CHEK2, leading to p53 stabilization and activation of transcription factor functions which collectively provide a cell fate determination point for abnormal or stress-exposed cells leading to apoptosis or survival with DNA damage which is repaired or converted to mutations.7

Following DNA damage and ATM/CHEK2/p53 pathway activation, cells enter either cell cycle arrest or apoptosis, dependent on which downstream pathways are predominant in the specific cell type and environment. The fate determination depends on the ability of p53 to induce the transcription of genes such as the cyclin-dependent kinase inhibitor CDKN1A (p21),8 and the pro-apoptotic BBC3 (PUMA).9 Genome maintenance is crucial to prevent DNA damage that can contribute to cancer and ageing.10 Mutations or polymorphisms in any of the pathway signaling components influence p53 function and tumor susceptibility; loss of this checkpoint function is associated with genomic instability and tumor predisposition11 in heritable syndromes like Ataxia Telangiectasia (AT, ATM mutants) and Li-Fraumeni (LFS, TP53 and CHEK2 mutants12).

Germline mutations of TP53 usually reduce its tumor suppression function but the site of the mutation and the biochemical properties of the amino acid substitution lead to a gradient of tumor suppressor function, ranging from hypomorphic mutations which retain near normal structure and function (i.e., R337H) to those that are essentially inactive (R175H). The degree of p53 function correlates with cancer risk.13 Similarly, other TP53 polymorphisms have been found in association with an increased risk of breast, ovarian and colon cancer.14 Alterations in other components of the p53 signaling pathway also influence tumor susceptibility. For example, the MDM2 promoter polymorphism SNP309 results in elevated levels of MDM2,15 leading to the weakening of the p53 pathway. The tumor suppressor activity of p53 and associated cancer risk depend on the net effect of all genetic and epigenetic alterations within its signaling pathway.

It is important that molecular diagnostics are developed to improve the identification of individuals in the population with an abnormally low activity of ATM/CHEK2/p53 pathway considering its crucial role in the DDR and its cancer risk implications. The combined genetic variation in components of the ATM/CHEK2/p53 pathway as well as radiation-induced post-transcriptional alterations and epigenetic modifications of pathway components all affect p53 activity. The effect of genetic variants or epigenetic modifications can be determined but this brings partial information on the pathway activity as a whole.

As most of the functions of p53 are attributable to its role as a transcriptional activator in response to damage DNA, the level of activity of the ATM/CHEK2/p53 pathway will affect the expression levels of p53 transcriptional targets following exposure to DNA damage agents such as IR. Very recently, Slee et al.16 demonstrated that mice carrying a p53 Ser312 mutation are predisposed to tumorigenesis following IR exposure, at least partially due to the inability to fully induce p53 target genes like p21. Therefore, we have carefully monitored the messenger RNA transcript abundances of specific p53 downstream target genes in response to radiation and use this as a measure of ATM/CHEK2/p53 pathway activity. Gene expression can be an important indicator of genetic and environmental effects on cellular state. It has been successfully used to identify susceptibility loci for complex diseases such as for example diabetes and allergic asthma.17

We developed a highly sensitive and reproducible multiplex quantitative RT-PCR (MQRT-PCR) method to investigate the variation in response to x-rays of genes transcriptionally activated through p53; the method has successfully identified individuals with severe normal tissue reactions to radiotherapy as well as a case of Ataxia Telangiectasia (AT).18 We report here an extension of this work that has led to the development of a minimally invasive assay to assess the activity of the ATM/CHEK2/p53 pathway in mouse and human blood samples. These results may provide a route to develop simple tests for the detection of individuals with higher cancer susceptibility.

Results

The effect of p53 gene copy number on expression of three p53 transcriptional target genes (p21, Puma and Sesn2) was assessed in 2 Gy irradiated or sham irradiated mouse blood by MQ-RTPCR. Blood samples from mice carrying between zero and four p53 copies were exposed (or sham exposed) to 2 Gy γ-radiation and transcriptional responses were assessed two hours later. Results are shown in Figure 1. Basal expression of p21, Puma and Sesn2 assessed in the sham exposed samples was not greatly dependent on p53 copy number. Marginally significant differences were observed for Puma expression in blood between mice with zero and one p53 copies (p = 0.04) and with zero and two p53 copies (p = 0.05); otherwise basal expression levels were independent of p53 copy number (Fig. 1A–C). By contrast, the transcriptional activation of all three genes following irradiation is clearly and significantly dependent on p53 copy number (Fig. 1). Plotting the data in terms of radiation-induced fold change in gene expression against p53 copy number (Fig. 2A–C) illustrates the strong dependence of the response to p53 copy number. Linear regression fits to the data for all three genes and are excellent with R2 values of 0.96–0.98. The slope of the regression lines for p21 and Puma are very close to unity (1.07 and 0.95 respectively) indicating a simple quantitative relationship between p53 copy number and activity of the Atm/Chek2/p53 pathway as assessed in these assays. Therefore the transcriptional response of p21, Puma and Sesn2 in blood to ionizing radiation provides an accurate measure of p53 copy number.

Figure 1.

Figure 1

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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.

Figure 2

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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).

The Atm and Chek2 dependence of the transcriptional responses was also investigated (Fig. 3A and B). As with p53 copy number dependence, again basal expression of the three genes was not greatly affected by Atm or Chek2 copy number. Radiation responses show a greater and generally significant dependence on Atm and Chek2 copy number (Fig. 3A and B). In blood from Chek2 knockout mice, p21 upregulation following irradiation was somewhat greater than observed in samples from p53 and Atm knockouts, this may relate to the fact that Atm also phosphorylates p53 directly to activate checkpoints. Overall, the response of p21 and Puma to radiation provides reliable indications of Atm/Chek2/p53 pathway function. The fact that irradiation is required to identify the differences between strains with differing p53, Atm and Chek2 copy numbers indicates that the pathway is activated primarily in response to DNA damage. Arf knockout mice show similar basal and post-irradiation expression of p21, Puma and Sesn2 (Fig. 3C) no significant Arf copy number dependent differences were observed. This confirms that p53 is not activated through Arf in the context of DNA damage.

Figure 3.

Figure 3

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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.

To summarize these data, the upregulation of p21 and Puma in response to radiation provides a robust readout of Atm/Chek2/p53 pathway activity in mouse blood showing high dependence on p53, Atm and Chek2 copy number (Figs. 1 and 3). p53 copy number also affects cancer risk in mice. Data available in the literature19–21 on cancer incidence in mouse strains is plotted in Figure 4A to demonstrate the p53 copy number dependence of cancer incidence (R2 = 0.98). Using data presented in Figure 2A and 2B to express p53 copy number in terms of the response of p21 or Puma to irradiation, the relationship between the p21 or Puma radiation response and cancer incidence can be obtained (Fig. 4B and 4C). It demonstrates that Atm/Chek2/p53 pathway activity as assessed by the Puma response to radiation correlates well with cancer incidence in these mice with differing p53 copy number (R2 = 0.98).

Figure 4.

Figure 4

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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. 19, 20 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.

To validate these findings in humans the PUMA response in cells from healthy donors, an AT case, AT carriers and LFS patients was investigated. Obtaining fresh blood from the patients was not possible; therefore, although recognized as not ideal as culture conditions may affect gene expression responses, mitogen stimulated lymphocyte cultures established from frozen materials were used. A range of PUMA upregulation in response to irradiation was observed amongst healthy donors (Fig. 5A). The range reflects the activity of the Atm/Chek2/p53 pathway in the sample group. The one AT case examined had a very weak response while the AT heterozygous carrier samples showed an intermediate response (Fig. 5A). The AT heterozygote response is significantly different to that in normal donors (p = 0.007); the ATM dependence is similar to that in mice (R2 = 0.999). The upregulation of PUMA is also reduced in the LFS samples compared to normal donor samples although it does not reach statistical significance with only three samples (p = 0.06). The heterozygous mutations in the AT carrier and LFS samples are unknown. The overlap of AT heterozygote and LFS patient ranges with that of normal donors may be in part due to the presence of partially active mutations being present.

Figure 5.

Figure 5

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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.

Fresh blood was available from seven normal donors (distinct from those used to establish lymphocyte cultures) and these showed a mean of 2.52-fold (95% confidence intervals: 1.45–3.59) upregulation of PUMA following irradiation (Fig. 5B). This is quantitatively very similar to the upregulation seen in wild-type mice (3.33-fold, see Fig. 2). However, it should be noted that the upregulation of PUMA was greater in dividing lymphocytes (mean 15.63) than fresh blood (mean 2.52). A similar difference in response in lymphocytes and blood has been observed for other genes.22

Discussion

DDR pathways help maintain genome stability following external genotoxic insult. A prime example is the cellular response to DNA double-strand breaks, which activates the ATM/CHEK2/p53 pathway. Mutations in ATM and TP53 lead to insufficient DNA damage surveillance allowing damaged cells to proceed into mitosis, which eventually results in increased cancer susceptibility.2 Recently, large epidemiological and molecular studies have provided conclusive evidence that ATM mutations that cause Ataxia-Telangiectasia are breast cancer susceptibility alleles.23,24 It has also been recently demonstrated that a rare ATM missense variant plays a clinically significant role in radiation-induced contralateral breast cancer in women treated by radiotherapy.25 Even without external stress, cellular DNA is exposed to endogenous reactive oxygen species (ROS), p53 also protects the genome from oxidation by endogeneous ROS, a major cause of DNA damage and genetic instability.26 Two main groups of signals activate TP53, DNA damage (signaled through the ATM and CHEK kinases) and oncogenic stress. Data presented in this study demonstrate the strict dependence of the transcriptional response of three p53 regulated genes to ionizing radiation on p53 gene copy number in mouse blood (Fig. 1). By contrast basal expression of the genes (p21, Puma and Sesn2) was little affected by p53 copy number. The radiation responses were also dependent on Atm and Chek2 copy number. Again, basal levels of expression were relatively insensitive to Atm or Chek2 copy number. The transcriptional response of the three genes to ionizing radiation is not however dependent on Arf gene copy number (Fig. 3C). This confirms that the tumor suppressor Arf does not regulate p53 in response to DNA damage, but rather by oncogenic disruption of the cell cycle27,28 thus validating the specificity of this assay to the Atm/Chek2/p53 pathway.

Following DNA damage, p53 regulates the transcription of many genes involved in cell cycle arrest, senescence apoptosis and DNA repair, in order to suppress cancer and loss of its function is associated with genomic instability and tumor predisposition.29 Several recent studies demonstrate that the DDR network is activated early in tumorigenesis11,30 and serves as a barrier to cancer progression. For example, heterogeneity in the severity of specific TP53 germinal mutations affects residual trans-activation potential and seems to be a predictor of disease expression in terms of age of onset and number of tumors.31,32 It has also been shown that the TP53 polymorphism with arginine at codon 72 leads to differential levels of transcription and favors apoptosis influencing the incidence of some cancers and the longevity of some populations.33 Similarly, the CHEK2*1100delC carriers have a higher risk of developing breast cancer.34 There is also a clear genotype-phenotype relationship for AT patients as the severity of the phenotype depends on the amount of residual kinase activity as determined by the genotype.35 ATM mutations that cause AT in bi-allelic carriers are breast cancer susceptibility alleles in mono-allelic carriers.23 Therefore there is good evidence for Atm, p53 and Chek2 acting as tumor suppressor genes in both mouse and human. The strong role of p53 in tumor suppression in the mouse is revealed by plotting available cancer incidence data for mouse strains with differing p53 copy number (Fig. 4A). Our data indicate that there is a strict linear quantitative relationship between p53 copy number and the transcriptional response of p21, Puma and Sesn2 to ionizing radiation (Fig. 2). The transcriptional assays therefore provide a quantitative assay of the strength of the Atm/Chek2/p53 pathway activity. The most robust response was observed for Puma which was also affected quantitatively by Atm and Chek2 copy number. As illustrated in Figure 4A and B the p21 and Puma transcriptional response correlates directly to cancer risk in the mouse strains with differing p53 copy number. This suggests that the transcriptional response of genes such as p21 and Puma could be used to predict cancer risk.

Although we focused on three genes (ATM, CHEK2 and TP53), the ATM/CHEK2/p53 pathway is complex and many other genes influence its activity. For example the chromosomal instability disorder Nijmegen Breakage Syndrome (NBS) is caused by germ line mutations in the NBS1 gene which act upstream of ATM in the MRE11-RAD50-NBS1 (MRN) complex. NBS disorder shares clinical and cellular features with AT. Functional NBS1 is necessary for the pathway's activation and there is an increased risk of developing cancer in clinically asymptomatic heterozygotes.36,37 Also, Ataxia Telangiectasia-like disorder (ATLD) patients have been identified, carrying mutations in the MRE11 gene.38 Polymorphisms in the MDM2 gene affect p53 degradation15 and have an impact on the age of tumor onset in LFS.39 So, not only mutations in the ATM, CHEK2 and TP53 genes affect the functional activity of the pathway leading to apoptosis and senescence, but also mutational changes in genes associated with the pathway. MicroRNAs also play a role as mir-34a, b and c, for example, are transcriptionally transactivated by p53,40 whereas others like miR-125b and miR-33, negatively regulate p53 expression.41,42 ATM expression can also be modified by microRNAs, for example miR-421.43 In addition, p53 isoform variants can also influence the pathway by interacting with wild-type p53 and alter its function.44

As there are many genetic and epigenetic mechanisms underlying variation in gene expression following radiation exposure, it is a complex task to obtain a full understanding of the impact of individual variations on the activity of the ATM/CHEK2/p53 pathway.45 The efficiency of the p53 response to IR has been found to decline significantly in various tissues of aging mice, as demonstrated by reduced function of the Atm kinase, lower p53 transcriptional activity and lower p53-dependent apoptosis.46 The time of onset of this decreased p53 response correlated well with the life span of mice suggesting a correlation between tumorigenesis and aging process. The transcriptional response of genes such as Puma to radiation will therefore reflect all the influences on p53 activity contributed by many cancer relevant genes and pathways.

A range of transcriptional response of PUMA was observed in lymphocyte cultures and fresh blood from healthy human donors. Similar to the findings in mice, in human cells ATM variants affected the response as did mutations carried by three LFS patients (Fig. 5A). The mean upregulation of PUMA in the healthy donor blood samples (2.52-fold) was quantitatively similar to the upregulation of Puma in wild type mice (3.33-fold). The range of response in healthy human donor bloods reflects the range of activity of the ATM/CHEK2/p53 pathway and will therefore relate in some way to cancer risk in humans as it does in mice (Fig. 4B). Insufficient human data relating p53 copy number or function to cancer risk quantitatively are available. Nonetheless, the mean and range of PUMA response in human blood can be compared to that in blood of mice with differing p53 copy number (Fig. 5B). The human healthy donor samples will all carry two functional p53 copies but information on mutations or polymorphisms affecting the ATM/CHEK2/p53 pathway activity is not available. As shown in Figure 5B the range in human response can be translated into an implied ‘virtual’ p53 copy number. While the relationship of this virtual copy number to cancer risk is unknown it is reasonable to assume that low virtual copy number will be associated with higher cancer risk.

Although new technologies will soon allow the rapid sequencing of the whole genome of individuals, it is extremely difficult to take in account all individual specific genetic and epigenomic differences influencing any pathway to assess its activity. Moreover, despite the identification of many genes involved in any one pathway, it is likely that there will be always more unidentified genes or allelic variants that affect it. Therefore, a more integrative biological approach is required to provide a simple readout of the activity of any individual pathway. The key finding of our study is that it is possible to measure accurately the activity of the Atm/Chek2/p53 pathway in mouse blood samples using simple transcriptional assays. Furthermore, this may extend to assessment of the pathway activity in humans; therefore it may be a useful biomarker that can identify individuals who are more or less likely to develop cancer. Humans are exposed to ionizing radiation (IR) through the natural environment, medical practices and industrial applications and tests assessing the activity of the ATM/CHEK2/p53 pathway could be relevant in terms of individual risk assessment of those exposed.

To summarize, we have described here a minimally invasive method to assess the activity of the ATM/CHEK2/p53 pathway in mice and humans. Although confirmation on a larger number of human samples will be needed in a large prospective study looking at cancer incidence, it might be possible in the future to assess the activity of this pathway in individuals in order to identify those who may have a higher risk of developing cancer using reliable tests.

Materials and Methods

Mouse blood collection.

Blood samples were collected from 10-week-old C57BL/6 mice either wild-type (wt), p53-tg (containing 3 p53 gene copies; two genomic copies plus one copy of a p53 transgene), p53-tgb (4 p53 copies; two genomic copies plus two copies of a p53 transgene),19 p53 knock-out (KO, no p53 copies), p53 heterozygotes (het, one p53 copies),47 Atm heterozygotes, Atm KO,48 Arf KO,49 Chek2 heterozygotes or Chek2 KO.50

A maximum of 1 ml of blood was obtained by cardiac puncture of mice euthanized by CO2 asphyxiation; blood samples were collected into anticoagulant EDTA tubes and kept at 4°C for a maximum of two hours. Mice were treated in accordance with Spanish and European Union Laws and the Guidelines for Humane Endpoints for Animals Used in Biomedical Research.

Human blood collection and mitogen stimulated T-lymphocyte culture.

Fresh blood from healthy donors was used to investigate human responses. However, when fresh blood was not available (i.e., AT patient, AT carriers and LFS patients), mitogen stimulated T-lymphocyte (MSTL) cultures were prepared from thawed vials of unstimulated leukocyte samples purified by separation on Histopaque-1077 (Sigma-Aldrich, Poole UK) that has been stored frozen in liquid nitrogen prior to use.

Blood.

10 ml peripheral blood samples were obtained anonymously from seven healthy donors (HD) (three males, age range 41–47 years old and four females, age range 37–53 years old) with informed consent and ethical approval from Oxford Research Ethics Committee. Peripheral blood was collected via venipuncture into anticoagulant EDTA vacutainer tubes (Becton Dickinson, Oxford, UK) and kept on ice until 30 minutes prior to irradiation.

MSTL.

MSTL cultures were prepared from nine HD unrelated to the seven blood HD (age range 43 to 61; five males and four females), four obligate carriers of ATM mutations (range 25–58 years; four males) provided by Prof. A.M.R. Taylor, University of Birmingham, UK (C7183, D2537, C5967, C5537), one AT patient (AT58) obtained from Dr. C. Arlett, University of Sussex, UK and three Li-Fraumeni patients (BD2284, GR0001, DD3508, age range 7 to 50; one man, two women; unknown mutations) obtained from the Health Protection Agency Culture Collection (ECACC, UK).

Briefly, after thawing, leukocytes were seeded at 3 × 105 cells/ml in stimulating growth medium (SR10-RPMI 1640 (Dutch modification) supplemented with 10% heat inactivated FBS, 1 mM sodium pyruvate, 2 mM L-Glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol (all from Invitrogen Ltd., Paisley, UK), 250 IU/ml recombinant interleukin-2 (Novartis Pharmaceuticals UK Ltd., Camberley, UK) and 0.4 µg/ml phytohemagglutinin (PHA) [Remel Ltd., Lenexa, USA)]. Cultures were also supplemented with 1.5 × 105 cells/ml GM1899A lethally-irradiated feeder cells as described in references 51 and 52. Cells were left undisturbed for 4 days and thereafter they were disaggregated and counted daily. When the cells reached a density of 1 × 106 cells/ml they were diluted 1:2 with growth medium (GR10, SR10 without PHA). After a week of culture sufficient cells were available for each experiment; at this stage no feeder cells could be detected by microscopy.

Irradiation.

Mouse blood (1 ml), human blood (10 ml) or MSTL (10 ml of culture, 4 × 106 cells) samples were split into two aliquots of respectively 500 µl (mouse) or 5 ml (human) each. All pairs of samples were incubated for 30 min at 37°C before irradiation, and then, one of the aliquot was irradiated with 2 Gy using an X-ray set (Siemens AG) (output 14 mA, 250 kV peak, 1.2 mm Cu HVL, 0.7 Gy min−1) at the Medical Research Council (MRC), Harwell (human samples) or a 137Cs irradiator (MDS Nordion) as a source of gamma rays (Gammacell 1000 Research Irradiator source Cs-137, activity: 541 Ci, 3.46 Gy min−1) (mouse samples). Irradiations were conducted at room temperature and samples were maintained for 2 h after irradiation at 37°C. Human blood leukocytes were captured on LeukoLOCK™ filters (Applied Biosystems/Ambion, Warrington, UK); MSTL were pelleted by centrifugation and all cells were then preserved in 1 ml of RNAlater (Ambion, Applied Biosystems, Foster City, CA USA) was added and samples were stored at −80°C until being processed for RNA extraction. Mouse blood was also preserved in RNAlater (1.3 ml to each 500 µl sample).

RNA purification from blood samples and MSTL.

Total RNA from mouse blood was prepared by using Mouse RiboPure™-Blood RNA Isolation Kit (Applied Biosystems/Ambion). Total RNA from human blood was prepared by using LeukoLOCK™ Total RNA Isolation Kit (Applied Biosystems/Ambion, Warrington, UK). DNA contamination was removed by using DNase, DNA-free™ (Ambion). Total RNA from MSTL was prepared by using RNAqueous®-4PCR (Applied Biosystems/Ambion) kit. Again, DNA contamination was removed by using DNase, DNA-free™ (Applied Biosystems/Ambion). RNA quantity was assessed by spectrophotometry (Nanodrop ND1000) and RNA quality was assessed on 1.3% agarose gel. For multiplex PCR, reverse transcriptase reactions were performed using High Capacity cDNA Reverse transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol with 700 ng of total RNA in a 50 µl reaction volume.

Multiplex quantitative RT-PCR.

Real-time PCR was performed using iQ5 thermocyclers (Bio-Rad, Hercules, CA USA). All reactions were run in triplicate using PerfeCTa® MultiPlex qPCR SuperMix (Quanta Biosciences, Inc., Gaithersburg, MD, USA), with primer and probe sets for target genes (described in Table 1) at 300 nM concentration each and 2.5 µl of cDNA in 30 µl reaction volume. FAM, HEX Texas Red and CY5 (Eurogentec Ltd., Fawley, Hampshire, UK) were used as fluorochrome reporters for the hydrolysis probes analyzed in multiplexed reactions. Cycling parameters were 2 min at 95°C, then 45 cycles of 10 s at 95°C and 60 s at 60°C. Data were collected and analyzed by iQ5 Detection System software. Gene target Ct (cycle threshold) values were normalized to a Hypoxanthine-Guanine phosphoribosyltransferase 1 (HPRT1) internal control. Ct values were converted to transcript quantity using standard curves obtained by serial dilution of PCR-amplified DNA fragments of each gene. The linear dynamic range of the standard curves covering six orders of magnitude (serial dilution from 3.2 × 10−4 to 8.2 × 10−10) gave PCR efficiencies between 93 and 103% for each gene with R2 > 0.998. Relative gene expression levels after irradiation were similarly obtained for comparison with pre-irradiation controls.

Table 1.

Oligonucleotide primers and probes used for MQRT-PCR analysis

Database acc no.
Genes Genebank/dbSNP PCR primers, Fwd, Rev Probes
HPRT1* NM_000194.2 TCA GGC AGT ATA ATC CAA AGA TGG T CGC AAG CTT GCT GGT GAA AAG GAC CC
AGT CTG GCT TAT ATC CAA CAC TT C G
PUMA* NM_014417.3, NM_001127240.1, NM_001127241.1, NM_001127242.1 CGG AGA CAA GAG GAG CAG CCC TCA CCC TGG AGG GTC CTG T
GGA GTC CCA TGA TGA GAT TG
p21* NM_000389.3, NM_078467.1 GCA GAC CAG CAT GAC AG TTT CTA CCA CTC CAA ACG CCG GCT
TAG GGC TT C CTC TT G GA
Hprt NM_013556.2 GGA CAG GAC TGA AAG ACT TG CCC TT G AGC ACA CAG AGG GCC ACA
TAA TCC AGC AGG TCA GCA AA
Puma NM_133234.2 CGG CGG AGA CAA GAA GAG CAT CGA CAC CGA CCC TCA CCC TGG
AGG AGT CCC ATG AAG AGA TTG
Sesn2 NM_144907.1 CGT TTT GAG CTG GAG AAG TCA AGC CTG CTG GTG ACC CCC TCA GC
GTG GAG AAG GCT CCA GGA TA
p21 NM_001111099.1, NM_007669.4 GCA AGA GAA AAC CCT GAA GTG ACG GGA GCC CCG CCC TCT T
CAC ACA GAG TGA GGG CTA AG
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*

Human genes.

Statistical analysis.

The gene expression level means were compared using the General Linear Model (GLM), a generalized statistical model which relates experimental data with model coefficients. GLM analysis of variance (ANOVA) allows the relative significance of the experimental model parameters to be estimated, taking into account the errors associated with each parameter. GLM ANOVA was performed using Minitab statistical software. Linear regression analyses also used Minitab software; R2 values are given.

Acknowledgements

We thank Dr. Liz Ainsbury for advice on statistical analysis, Francois Paillier for oligonucleotide PCR design, Richard Doull (MRC Harwell) for irradiations, Joanna Robbins and Susan Martin for blood supply.

Financial support was provided by the National Institute for Health Research Centre for Research in Public Health Protection at the Health Protection Agency.

Conflicts of Interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

This report is work commissioned by the National Institute for Health Research. The views expressed in this publication are those of the authors and not necessary those of the NHS, the National Institute for Health Research or the department of Health.

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