MDMX phosphorylation-dependent p53 downregulation contributes to an immunosuppressive tumor microenvironment.

A role of tumor-suppressive activity of p53 in the tumor microenvironment (TME) has been implicated but remains fairly understudied. To address this knowledge gap, we leveraged our MdmxS314A mice as recipients to investigate how implanted tumor cells incapacitate host p53 creating a conducive TME for tumor progression. We found that tumor cell-associated stress induced p53 downregulation in peritumor cells via an MDMX-Ser314 phosphorylation-dependent manner. As a result, an immunosuppressive TME was developed, as reflected by diminished immune cell infiltration into tumors and compromised macrophage M1 polarization. Remarkably, ablation of MDMX-Ser314 phosphorylation attenuated p53 decline in peritumor cells, which was associated with mitigation of immunosuppression and significant tumor growth delay. Our data collectively uncover a novel role of p53 in regulating the tumor immune microenvironment, suggesting that p53 restoration in the TME can be exploited as a potential strategy of anticancer therapy.

The MDM2/MDMX/p53 axis in the adaptive stress response.

Via regulation of cellular stress responses, p53 contributes to the maintenance of homeostasis. Contrary to its well-established pro-death function, p53 is also implicated in promoting cell survival by mediating the adaptive stress response. Emerging data reveal that the adaptive stress response is coupled with p53 decline that is a prerequisite for the induction of pro-survival pathways augmenting cell fitness. However, if the adaptive stress responses persist or become chronic, the sustained p53 downregulation would result in a permanent loss of p53 function and p53-dependent homeostasis. The available information suggests a model in which cells respond to different levels of stress by governing the activity and abundance of p53 that, in turn, determines the cell fate dependent on not only the intensity but also the duration of stress.

Functional interplay between p53 and Δ133p53 in adaptive stress response.

Apart from its well-known prodeath activity, p53 is also implicated in promoting cell survival. How p53 can mediate such seemingly opposing effects is largely unclear. We report here a novel mechanism in which p53-mediated proapoptosis is switched to antiapoptosis via its interaction with a p53 isoform, Δ133p53. We show that the expression of Δ133p53 is induced by mild or a moderate level of stress via an HIF1-dependent mechanism. Increased Δ133p53 levels contribute to the adaptive response by shifting the p53 binding at the Bcl2 promoter from suppressive responsive elements (RE) to activating REs, resulting in induction of Bcl2. In accordance with this mode of action, pretreatment of mice with mild stress induces Δ133p53 and Bcl2, which is associated with protection of animals from toxicity caused by high doses of DNA damage agents. Collectively, our work uncovers a novel functional interplay between p53 and Δ133p53 determining cell fate; survival or death in response to stress.

ZBTB7A governs estrogen receptor alpha expression in breast cancer.

ZBTB7A, a member of the POZ/BTB and Krüppel (POK) family of transcription factors, has been shown to have a context-dependent role in cancer development and progression. The role of ZBTB7A in estrogen receptor alpha (ERα)-positive breast cancer is largely unknown. Approximately 70% of breast cancers are classified as ERα-positive. ERα carries out the biological effects of estrogen and its expression level dictates response to endocrine therapies and prognosis for breast cancer patients. In this study, we find that ZBTB7A transcriptionally regulates ERα expression in ERα-positive breast cancer cell lines by binding to the ESR1 promoter leading to increased transcription of ERα. Inhibition of ZBTB7A in ERα-positive cells results in decreased estrogen responsiveness as demonstrated by diminished estrogen-response element-driven luciferase reporter activity, induction of estrogen target genes, and estrogen-stimulated growth. We also report that ERα potentiates ZBTB7A expression via a post-translational mechanism, suggesting the presence of a positive feedback loop between ZBTB7A and ERα, conferring sensitivity to estrogen in breast cancer. Clinically, we find that ZBTB7A and ERα are often co-expressed in breast cancers and that high ZBTB7A expression correlates with improved overall and relapse-free survival for breast cancer patients. Importantly, high ZBTB7A expression predicts a more favorable outcome for patients treated with endocrine therapies. Together, these findings demonstrate that ZBTB7A contributes to the transcriptional program maintaining ERα expression and potentially an endocrine therapy-responsive phenotype in breast cancer.

An EZH2-mediated epigenetic mechanism behind p53-dependent tissue sensitivity to DNA damage.

Renewable tissues exhibit heightened sensitivity to DNA damage, which is thought to result from a high level of p53. However, cell proliferation in renewable tissues requires p53 down-regulation, creating an apparent discrepancy between the p53 level and elevated sensitivity to DNA damage. Using a combination of genetic mouse models and pharmacologic inhibitors, we demonstrate that it is p53-regulated MDM2 that functions together with MDMX to regulate DNA damage sensitivity by targeting EZH2 (enhancer of zeste homolog 2) for ubiquitination/degradation. As a methyltransferase, EZH2 promotes H3K27me3, and therefore chromatin compaction, to determine sensitivity to DNA damage. We demonstrate that genetic and pharmacologic interference of the association between MDM2 and MDMX stabilizes EZH2, resulting in protection of renewable tissues from radio-/chemotherapy-induced acute injury. In cells with p53 mutation, there are diminished MDM2 levels, and thus accumulation of EZH2, underpinning the resistant phenotype. Our work uncovers an epigenetic mechanism behind tissue sensitivity to DNA damage, carrying important translation implications.

A functional interplay between Δ133p53 and ΔNp63 in promoting glycolytic metabolism to fuel cancer cell proliferation.

Although ΔNp63 is known to promote cancer cell proliferation, the underlying mechanism behind its oncogenic function remains elusive. We report here a functional interplay between ΔNp63 and Δ133p53. These two proteins are co-overexpressed in a subset of human cancers and cooperate to promote cell proliferation. Mechanistically, Δ133p53 binds to ΔNp63 and utilizes its transactivation domain to upregulate GLUT1, GLUT4, and PGM expression driving glycolysis. While increased glycolysis provides cancer cells with anabolic metabolism critical for proliferation and survival, it can be harnessed for selective cancer cell killing. Indeed, we show that tumors overexpressing both ΔNp63 and Δ133p53 exhibit heightened sensitivity to vitamin C that accumulate to a lethal level due to accelerated uptake via overexpressed GLUT1. These observations offer a new therapeutic avenue that could be exploited for clinical applications.

Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX-MDM2 complex.

Deregulation of the tyrosine kinase signalling is often associated with tumour progression and drug resistance, but its underlying mechanisms are only partly understood. In this study, we investigated the effects of the receptor tyrosine kinase AXL on the stability of the MDMX-MDM2 heterocomplex and the activity of p53 in melanoma cells. Our data demonstrated that AXL overexpression or activation through growth arrest-specific 6 (Gas6) ligand stimulation increases MDMX and MDM2 protein levels and decreases p53 activity. Upon activation, AXL stabilizes MDMX through a post-translational modification that involves phosphorylation of MDMX on the phosphosite Ser314, leading to increased affinity between MDMX and MDM2 and favouring MDMX nuclear translocation. Ser314 phosphorylation can also protect MDMX from MDM2-mediated degradation, leading to stabilization of the MDMX-MDM2 complex. We identified CDK4/6 and p38 MAPK as the two kinases mediating AXL-induced modulation of the MDMX-MDM2 complex, and demonstrated that suppression of AXL, either through siRNA silencing or pharmacological inhibition, increases expression levels of p53 target genes P21, MDM2, and PUMA, improves p53 pathway response to chemotherapy, and sensitizes cells to both Cisplatin and Vemurafenib. Our findings offer an insight into a novel signalling axis linking AXL to p53 and provide a potentially druggable pathway to restore p53 function in melanoma.

Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex: IMPLICATION OF A NOVEL MDMX SER-314 PHOSPHOSITE.

Deregulated receptor tyrosine kinase (RTK) signaling is frequently associated with tumorigenesis and therapy resistance, but its underlying mechanisms still need to be elucidated. In this study, we have shown that the RTK human epidermal growth factor receptor 4 (Her4, also known as Erbb4) can inhibit the tumor suppressor p53 by regulating MDMX-mouse double minute 2 homolog (MDM2) complex stability. Upon activation by either overexpression of a constitutively active vector or ligand binding (Neuregulin-1), Her4 was able to stabilize the MDMX-MDM2 complex, resulting in suppression of p53 transcriptional activity, as shown by p53-responsive element-driven luciferase assay and mRNA levels of p53 target genes. Using a phospho-proteomics approach, we functionally identified a novel Her4-induced posttranslational modification on MDMX at Ser-314, a putative phosphorylation site for the CDK4/6 kinase. Remarkably, inhibition of Ser-314 phosphorylation either with Ser-to-Ala substitution or with a specific inhibitor of CDK4/6 kinase blocked Her4-induced stabilization of MDMX-MDM2 and rescued p53 activity. Our study offers insights into the mechanisms of deregulated RTK-induced carcinogenesis and provides the basis for the use of inhibitors targeting RTK-mediated signals for p53 restoration.

MDMX under stress: the MDMX-MDM2 complex as stress signals hub.

The tumor suppressor p53 plays a central role in safeguarding cellular homeostasis. Upon various types of stress signals such as DNA damage or oncogenic stress, p53 is promptly activated to prevent and repair damages that can threaten the genome stability. The two major negative regulators of p53 are MDM2 and MDMX, two homolog proteins that control p53 activity and turnover, hence keeping it in check during normal cell cycling. In the event of cellular stress, they have to be inhibited in order to relieve p53 from their suppression and allow its activation. As the essential upstream modulator of p53, the MDMX-MDM2 complex integrates multiple signaling pathways regulating p53 response to perturbations of cellular homeostasis. Given its predominantly cytoplasmic localization in normal conditions, we hypothesize that MDMX, rather than MDM2, is the first recipient of signaling cues directed towards the MDMX-MDM2 complex and aimed at modulating p53. In this review we give a synthetic overview of the phosphorylation sites of MDMX that are known to affect its degradation, ubiquitination, intracellular localization and interaction with MDM2 and p53, ultimately modulating the stability and activity of p53. The role of MDMX in response to the main types of cellular stress is also briefly discussed, along with the potential of the MDMX-MDM2 complex as therapeutic target to restore p53 activity.

UXT, a novel MDMX-binding protein, promotes glycolysis by mitigating p53-mediated restriction of NF-κB activity.

The importance of stress-induced p53 activation has been extensively investigated and well established. How the basal activity of p53 prevents carcinogenesis, however, remains incompletely understood. We report the identification of a novel p53 inhibitor, UXT, which binds to MDMX and suppresses the basal activity of p53. Interestingly, human TCGA database indicates that the UXT gene is frequently amplified in human sarcoma where p53 mutation is rare. We thus used sarcoma as a model to show that UXT acts as an oncogene promoting cell proliferation in vitro and tumor progression in vivo. A screening of 10 major cellular pathways uncovered that UXT-mediated p53 inhibition results in an activation of NF-κB, leading to induction of glycolysis. While elevated glycolytic metabolism provides growth advantage it also renders UXT expressing sarcoma cells heightened sensitivity to glycolysis inhibition. Altogether, our data demonstrate a crucial role for the basal activity of p53 in restriction of NF-κB. By impeding such an activity of p53, UXT unleashes the oncogenic activity of NF-κB resulting in induction of glycolysis fueling carcinogenesis.

Glycolytic metabolism influences global chromatin structure.

Metabolic rewiring, specifically elevated glycolytic metabolism is a hallmark of cancer. Global chromatin structure regulates gene expression, DNA repair, and also affects cancer progression. But the interrelationship between tumor metabolism and chromatin architecture remain unclear. Here we show that increased glycolysis in cancer cells promotes an open chromatin configuration. Using complementary methods including Micrococcal nuclease (MNase) digestion assay, electron microscope and immunofluorescence staining, we demonstrate that glycolysis inhibition by pharmacological and genetic approaches was associated with induction of compacted chromatin structure. This condensed chromatin status appeared to result chiefly from histone hypoacetylation as restoration of histone acetylation with an HDAC inhibitor reversed the compacted chromatin state. Interestingly, glycolysis inhibition-induced chromatin condensation impeded DNA repair efficiency leading to increased sensitivity of cancer cells to DNA damage drugs, which may represent a novel molecular mechanism that can be exploited for cancer therapy.

Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

A low-dose arsenic-induced p53 protein-mediated metabolic mechanism of radiotherapy protection.

Radiotherapy is the current frontline cancer treatment, but the resulting severe side effects often pose a significant threat to cancer patients, raising a pressing need for the development of effective strategies for radiotherapy protection. We exploited the distinct metabolic characteristics between normal and malignant cells for a metabolic mechanism of normal tissue protection. We showed that low doses of arsenic induce HIF-1α, which activates a metabolic shift from oxidative phosphorylation to glycolysis, resulting in increased cellular resistance to radiation. Of importance is that low-dose arsenic-induced HIF-1α requires functional p53, limiting the glycolytic shift to normal cells. Using tumor-bearing mice, we provide proof of principle for selective normal tissue protection against radiation injury.

Sequentially-induced responses define tumour cell radiosensitivity.

PURPOSE: Our aim was to define dose-dependent and genotype-dependent components of radiosensitivity by resolving patterns of radiation-induced clonal inactivation into specific responses. METHODS: In a set of 10 tumour cells with varying expression of radiosensitivity and genotype, we identified doses at which all tumour cells change in their rate of clonogenic inactivation. We tested intervening dose-segments as to whether inactivation was constant, expressing inactivation as a log-linear function of dose. We compared these segments to components proposed in the Hit-target (HT) model and the Linear-quadratic (LQ) model. Temporal changes in redistribution in cell-cycle prevalence and apoptosis were examined as essential components of cellular radiosensitivity. RESULTS: We identified four distinct responses induced sequentially in all cells independent of genotype. Rates of inactivation within each response varied with expression of genotype and identified: (i) A hypersensitive component H (0.0-0.10 Gy); (ii) a resistant component R (0.1-0.2 Gy); (iii) an induced repair response alpha* (0.2 Gy and higher); and (iv) a more sensitive component omega* (3.0 Gy and higher). The H, alpha* and omega* components were fitted well by log-linear patterns, the R response did not. CONCLUSIONS: Four distinct, sequentially-induced responses comprise cellular radiosensitivity. H and R responses are associated with low dose hyper-radiosensitivity and early apoptosis, while the alpha* and omega* responses share characteristics of the HT and LQ models and are associated with post-repair apoptosis. Radiation induces these four responses at the same doses in all cells, but the rate of inactivation over each response depends on genotype.

The role of gap junction communication and oxidative stress in the propagation of toxic effects among high-dose α-particle-irradiated human cells.

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.

Differential role of DNA-PKcs phosphorylations and kinase activity in radiosensitivity and chromosomal instability.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G(1) cells to (137)Cs γ rays. DNA-PKcs mutant cells defective in phosphorylation at multiple sites within the T2609 cluster or within the PI3K domain displayed extreme radiosensitivity. Cells defective at the S2056 cluster or T2609 single site alone were only mildly radiosensitive, but cells defective at even one site in both the S2056 and T2609 clusters were maximally radiosensitive. Thus a synergism between the capacity for phosphorylation at the S2056 and T2609 clusters was found to be critical for induction of radiosensitivity.

Tumor response to radiotherapy is dependent on genotype-associated mechanisms in vitro and in vivo.

BACKGROUND: We have previously shown that in vitro radiosensitivity of human tumor cells segregate non-randomly into a limited number of groups. Each group associates with a specific genotype. However we have also shown that abrogation of a single gene (p21) in a human tumor cell unexpectedly sensitized xenograft tumors comprised of these cells to radiotherapy while not affecting in vitro cellular radiosensitivity. Therefore in vitro assays alone cannot predict tumor response to radiotherapy.In the current work, we measure in vitro radiosensitivity and in vivo response of their xenograft tumors in a series of human tumor lines that represent the range of radiosensitivity observed in human tumor cells. We also measure response of their xenograft tumors to different radiotherapy protocols. We reduce these data into a simple analytical structure that defines the relationship between tumor response and total dose based on two coefficients that are specific to tumor cell genotype, fraction size and total dose. METHODS: We assayed in vitro survival patterns in eight tumor cell lines that vary in cellular radiosensitivity and genotype. We also measured response of their xenograft tumors to four radiotherapy protocols: 8 x 2 Gy; 2 x 5 Gy, 1 x 7.5 Gy and 1 x 15 Gy. We analyze these data to derive coefficients that describe both in vitro and in vivo responses. RESULTS: Response of xenografts comprised of human tumor cells to different radiotherapy protocols can be reduced to only two coefficients that represent 1) total cells killed as measured in vitro 2) additional response in vivo not predicted by cell killing. These coefficients segregate with specific genotypes including those most frequently observed in human tumors in the clinic. Coefficients that describe in vitro and in vivo mechanisms can predict tumor response to any radiation protocol based on tumor cell genotype, fraction-size and total dose. CONCLUSIONS: We establish an analytical structure that predicts tumor response to radiotherapy based on coefficients that represent in vitro and in vivo responses. Both coefficients are dependent on tumor cell genotype and fraction-size. We identify a novel previously unreported mechanism that sensitizes tumors in vivo; this sensitization varies with tumor cell genotype and fraction size.

Some unsolved problems and unresolved issues in radiation cytogenetics: a review and new data on roles of homologous recombination and non-homologous end joining.

New data and historical evidence from our own and other laboratories are summarized and discussed bearing on several issues relating to mechanisms and processes involved in the formation of chromosomal aberrations following exposure to ionizing radiations. Specifically addressed are: (1) the lesions and processes affecting the appearance of chromatid-type and/or chromosome-type aberrations after radiation, (2) DNA double strand break rejoining processes and the restitution of breaks vs. the formation of exchanges, (3) the role of homologous recombinational repair in protecting cells from induction of chromatid-type aberrations after irradiation of late S/G2 cells, (4) the role of interphase chromatin structure and nuclear organization in aberration induction, (5) cellular responses for aberration induction in relation to their tissue context, and (6) approaches to the detection of aberrations previously known as "cryptic".

G2-phase chromosomal radiosensitivity of primary fibroblasts from hereditary retinoblastoma family members and some apparently normal controls.

We previously described an enhanced sensitivity for cell killing and gamma-H2AX focus induction after both high-dose-rate and continuous low-dose-rate gamma irradiation in 14 primary fibroblast strains derived from hereditary-type retinoblastoma family members (both affected RB1(+/-) probands and unaffected RB1(+/+) parents). Here we present G(2)-phase chromosomal radiosensitivity assay data for primary fibroblasts derived from these RB family members and five Coriell cell bank controls (four apparently normal individuals and one bilateral RB patient). The RB family members and two normal Coriell strains had significantly higher ( approximately 1.5-fold, P < 0.05) chromatid-type aberration frequencies in the first postirradiation mitosis after doses of 50 cGy and 1 Gy of (137)Cs gamma radiation compared to the remaining Coriell strains. The induction of chromatid-type aberrations by high-dose-rate G(2)-phase gamma irradiation is significantly correlated to the proliferative ability of these cells exposed to continuous low-dose-rate gamma irradiation (reported in Wilson et al., Radiat. Res. 169, 483-494, 2008). Our results suggest that these moderately radiosensitive individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes or may carry epigenetic changes involving genes that more broadly modulate such systems, including G(2)-phase-specific DNA damage responses.