Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells.

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated in vitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded in vivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Staphylococcus aureus Isolated from Skin from Atopic-Dermatitis Patients Produces Staphylococcal Enterotoxin Y, Which Predominantly Induces T-Cell Receptor Vα-Specific Expansion of T Cells.

While investigating the virulence traits of Staphylococcus aureus adhering to the skin of atopic-dermatitis (AD) patients, we identified a novel open reading frame (ORF) with structural similarity to a superantigen from genome sequence data of an isolate from AD skin. Concurrently, the same ORF was identified in a bovine isolate of S. aureus and designated SElY (H. K. Ono, Y. Sato'o, K. Narita, I. Naito, et al., Appl Environ Microbiol 81:7034-7040, 2015, https://doi.org/10.1128/AEM.01873-15). Recombinant SElYbov had superantigen activity in human peripheral blood mononuclear cells. It further demonstrated emetic activity in a primate animal model, and it was proposed that SElY be renamed SEY (H. K. Ono, S. Hirose, K. Narita, M. Sugiyama, et al., PLoS Pathog 15:e1007803, 2019, https://doi.org/10.1371/journal.ppat.1007803). Here, we investigated the prevalence of the sey gene in 270 human clinical isolates of various origins in Japan. Forty-two strains were positive for the sey gene, and the positive isolates were from patients with the skin diseases atopic dermatitis and impetigo/staphylococcal scalded skin syndrome (SSSS), with a detection rate of ∼17 to 22%. There were three variants of SEY (SEY1, SEY2, and SEY3), and isolates producing SEY variants formed three distinct clusters corresponding to clonal complexes (CCs) 121, 59, and 20, respectively. Most sey+ isolates produced SEY in broth culture. Unlike SEYbov, the three recombinant SEY variants exhibited stability against heat treatment. SEY predominantly activated human T cells with a particular T-cell receptor (TCR) Vα profile, a unique observation since most staphylococcal enterotoxins exert their superantigenic activities through activating T cells with specific TCR Vß profiles. SEY may act to induce localized inflammation via skin-resident T-cell activation, facilitating the pathogenesis of S. aureus infection in disrupted epithelial barriers.

Fate Decision Between Group 3 Innate Lymphoid and Conventional NK Cell Lineages by Notch Signaling in Human Circulating Hematopoietic Progenitors.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15-dependent cNK cell generation in CD45RA+Flt-3-c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA-Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor-like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor-like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte-committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.

Overexpression of Rev1 promotes the development of carcinogen-induced intestinal adenomas via accumulation of point mutation and suppression of apoptosis proportionally to the Rev1 expression level.

Cancer development often involves mutagenic replication of damaged DNA by the error-prone translesion synthesis (TLS) pathway. Aberrant activation of this pathway plays a role in tumorigenesis by promoting genetic mutations. Rev1 controls the function of the TLS pathway, and Rev1 expression levels are associated with DNA damage induced cytotoxicity and mutagenicity. However, it remains unclear whether deregulated Rev1 expression triggers or promotes tumorigenesis in vivo. In this study, we generated a novel Rev1-overexpressing transgenic (Tg) mouse and characterized its susceptibility to tumorigenesis. Using a small intestinal tumor model induced by N-methyl-N-nitrosourea (MNU), we found that transgenic expression of Rev1 accelerated intestinal adenoma development in proportion to the Rev1 expression level; however, overexpression of Rev1 alone did not cause spontaneous development of intestinal adenomas. In Rev1 Tg mice, MNU-induced mutagenesis was elevated, whereas apoptosis was suppressed. The effects of hREV1 expression levels on the cytotoxicity and mutagenicity of MNU were confirmed in the human cancer cell line HT1080. These data indicate that dysregulation of cellular Rev1 levels leads to the accumulation of mutations and suppression of cell death, which accelerates the tumorigenic activities of DNA-damaging agents.

Linkage between dendritic and T cell commitments in human circulating hematopoietic progenitors.

The relationships between commitments of dendritic cells (DCs) and T cells in human hematopoietic stem cells are not well understood. In this study, we enumerate and characterize conventional DC and plasmacytoid DC precursors in association with T cell and thymus-derived types of NK cell precursors among CD34(+) hematopoietic progenitor cells (HPCs) circulating in human peripheral blood. By limiting-dilution analyses using coculture with stroma cells expressing Notch1 ligand, the precursor frequencies (PFs) of DCs in HPCs were found to significantly correlate with T cell PFs, but not with NK cell PFs, among healthy donors. Clonal analyses showed that the majority of T/NK dual- and T single-lineage precursors-but only a minority of NK single-lineage precursors-were associated with the generation of DC progenies. All clones producing both DC and T cell progenies were found with monocyte and/or granulocyte progenies, suggesting DC differentiation via myeloid DC pathways. Analyses of peripheral blood HPC subpopulations revealed that the lineage split between DC and T/NK cell progenitor occurs at the stage prior to bifurcation into T and NK cell lineages. The findings suggest a strong linkage between DC and T cell commitments, which may be imprinted in circulating lymphoid-primed multipotent progenitors or in more upstream HPCs.

Age-associated changes in the differentiation potentials of human circulating hematopoietic progenitors to T- or NK-lineage cells.

Age-associated changes of T and NK cell (T/NK) potential of human hematopoietic stem cells are unknown. In this study, we enumerate and characterize T/NK precursors among CD34(+)Lin(-) cell populations circulating in normal human adult peripheral blood (PB) by a limiting-dilution assay using coculture with OP9-DL1 stroma cells expressing Notch 1 ligand, Delta-like 1. The frequency of T cell precursors in CD34(+)Lin(-) cells was found to decrease with donor age, whereas the ratio of NK to T cell precursor frequency (NK/T ratio) increased with age, suggesting that lymphoid differentiation potential of PB progenitors shifts from T to NK cell lineage with aging. Clonal analyses of CD34(+)Lin(-) cells showed that differences in the NK/T ratio were attributable to different distributions of single- and dual-lineage T/NK precursor clones. Because nearly all of the clones retained monocyte and/or granulocyte differentiation potentials in coculture with OP9-DL1 cells, T/NK precursors in PB are considered to be contained in the pool of T/NK/myeloid multipotent progenitors. The age-associated increase in NK over T cell commitment might occur in precursor cells with T/NK/myeloid potential.

Genomic analysis and identification of a novel superantigen, SargEY, in Staphylococcus argenteus isolated from atopic dermatitis lesions.

During surveillance of Staphylococcus aureus in lesions from patients with atopic dermatitis (AD), we isolated Staphylococcus argenteus, a species registered in 2011 as a new member of the genus Staphylococcus and previously considered a lineage of S. aureus. Genome sequence comparisons between S. argenteus isolates and representative S. aureus clinical isolates from various origins revealed that the S. argenteus genome from AD patients closely resembles that of S. aureus causing skin infections. We previously reported that 17%-22% of S. aureus isolated from skin infections produce staphylococcal enterotoxin Y (SEY), which predominantly induces T-cell proliferation via the T-cell receptor (TCR) Vα pathway. Complete genome sequencing of S. argenteus isolates revealed a gene encoding a protein similar to superantigen SEY, designated as SargEY, on its chromosome. Population structure analysis of S. argenteus revealed that these isolates are ST2250 lineage, which was the only lineage positive for the SEY-like gene among S. argenteus. Recombinant SargEY demonstrated immunological cross-reactivity with anti-SEY serum. SargEY could induce proliferation of human CD4+ and CD8+ T cells, as well as production of TNF-α and IFN-γ. SargEY showed emetic activity in a marmoset monkey model. SargEY and SET (a phylogenetically close but uncharacterized SE) revealed their dependency on TCR Vα in inducing human T-cell proliferation. Additionally, TCR sequencing revealed other previously undescribed Vα repertoires induced by SEH. SargEY and SEY may play roles in exacerbating the respective toxin-producing strains in AD. IMPORTANCE: Staphylococcus aureus is frequently isolated from active lesions of atopic dermatitis (AD) patients. We reported that 17%-22% of S. aureus isolated from AD patients produced a novel superantigen staphylococcal enterotoxin Y (SEY). Unlike many S. aureus superantigens that activate T cells via T-cell receptor (TCR) Vß, SEY activates T cells via TCR Vα and stimulates cytokine secretion. Staphylococcus argenteus was isolated from AD patients during the surveillance for S. aureus. Phylogenetic comparison of the genome indicated that the isolate was very similar to S. aureus causing skin infections. The isolate encoded a SEY-like protein, designated SargEY, which, like SEY, activated T cells via the TCR Vα. ST2250 is the only lineage positive for SargEY gene. ST2250 S. argenteus harboring a superantigen SargEY gene may be a novel staphylococcal clone that infects human skin and is involved in the exacerbation of AD.

Evaluation of systemic markers of inflammation in atomic-bomb survivors with special reference to radiation and age effects.

Past exposure to atomic bomb (A-bomb) radiation has exerted various long-lasting deleterious effects on the health of survivors. Some of these effects are seen even after >60 yr. In this study, we evaluated the subclinical inflammatory status of 442 A-bomb survivors, in terms of 8 inflammation-related cytokines or markers, comprised of plasma levels of reactive oxygen species (ROS), interleukin (IL)-6, tumor necrosis factor α (TNF-α), C-reactive protein (CRP), IL-4, IL-10, and immunoglobulins, and erythrocyte sedimentation rate (ESR). The effects of past radiation exposure and natural aging on these markers were individually assessed and compared. Next, to assess the biologically significant relationship between inflammation and radiation exposure or aging, which was masked by the interrelationship of those cytokines/markers, we used multivariate statistical analyses and evaluated the systemic markers of inflammation as scores being calculated by linear combinations of selected cytokines and markers. Our results indicate that a linear combination of ROS, IL-6, CRP, and ESR generated a score that was the most indicative of inflammation and revealed clear dependences on radiation dose and aging that were found to be statistically significant. The results suggest that collectively, radiation exposure, in conjunction with natural aging, may enhance the persistent inflammatory status of A-bomb survivors.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

TORC-SIK cascade regulates CREB activity through the basic leucine zipper domain.

The transcription factor cAMP response element-binding protein (CREB) plays important roles in gene expression induced by cAMP signaling and is believed to be activated when its Ser133 is phosphorylated. However, the discovery of Ser133-independent activation by the activation of transducer of regulated CREB activity coactivators (TORC) and repression by salt inducible kinase cascades suggests that Ser133-independent regulation of CREB is also important. The activation and repression are mediated by the basic leucine zipper domain of CREB. In this review, we focus on the basic leucine zipper domain in the regulation of transcriptional activity of CREB and describe the functions of TORC and salt inducible kinase.

Dephosphorylation of TORC initiates expression of the StAR gene.

Cyclic AMP responsive element (CRE) binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. However, transducer of regulated CREB activity (TORC), a CREB specific co-activator, upregulates CREB activity in a phospho-Ser133-independent manner. Interestingly, TORC is also regulated by phosphorylation; the phospho-form is inactive, and the dephospho-form active. When PKA phosphorylates CREB, it inhibits TORC kinases simultaneously and accelerates dephosphorylation of TORC. We show in this report that staurosporine, a kinase inhibitor, induces the expression of the StAR gene in Y1 adrenocortical cells, possibly a result of an increase in the population of dephospho-TORC. The expression of the StAR gene is known to be regulated by SF-1 and CREB, and the co-activators CBP/p300 may mediate the actions of both factors. Our experiments using KG501, a disruptor of the interaction between phospho-CREB and CBP/p300, also support the importance of TORC in the regulation of StAR gene expression.

Tumor induction by monoenergetic neutrons in B6C3F1 mice.

This study was undertaken to investigate induction of tumors by monoenergetic neutrons in B6C3F1 mice. Individual groups of 6 week-old animals of both sexes (about 30 mice/group) were exposed to 0.5 Gy of various monoenergetic neutrons (dose rate 0.5 cGy/min) and then observed for 13 months. The incidences of tumors (mainly liver neoplasms) in non-irradiated male and female controls were 11% and 0%, respectively. In the irradiated animals, the incidences were 53%, 50%, 60% and 43% in males, and 75%, 81%, 71%, and 85% in females, after 0.18, 0.32, 0.6 and 1.0 MeV neutron exposure, respectively. There were no significant differences in the tumor induction rate among the different energy groups.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

Late Effects of Exposure to Ionizing Radiation and Age on Human Thymus Morphology and Function.

The thymus is essential for proper development and maintenance of a T-cell repertoire that can respond to newly encountered antigens, but its function can be adversely affected by internal factors such as pregnancy and normal aging or by external stimuli such as stress, infection, chemotherapy and ionizing radiation. We have utilized a unique archive of thymus tissues, obtained from 165 individuals, exposed to the 1945 atomic bomb blast in Hiroshima, to study the long-term effects of receiving up to ∼3 Gy dose of ionizing radiation on human thymus function. A detailed morphometric analysis of thymus activity and architecture in these subjects at the time of their natural deaths was performed using bright-field immunohistochemistry and dual-color immunofluorescence and compared to a separate cohort of nonexposed control subjects. After adjusting for age-related effects, increased hallmarks of thymic involution were observed histologically in individuals exposed to either low (5-200 mGy) or moderate-to-high (>200 mGy) doses of ionizing radiation compared to unirradiated individuals (<5 mGy). Sex-related differences were seen when the analysis was restricted to individuals under 60 years of attained age at sample collection, but were not observed when comparing across the entire age range. This indicates that while females undergo slower involution than males, they ultimately attain similar phenotypes. These findings suggest that even low-dose-radiation exposure can accelerate thymic aging, with decreased thymopoiesis relative to nonexposed controls evident years after exposure. These data were used to develop a model that can predict thymic function during normal aging or in individuals therapeutically or accidentally exposed to radiation.

A miso (Japanese soybean paste) diet conferred greater protection against hypertension than a sodium chloride diet in Dahl salt-sensitive rats.

The purpose of this study was to compare the effects of miso and sodium chloride (NaCl) on blood pressure in both sexes of Dahl and SD rats. The systolic and diastolic blood pressures (SBP/DBP) were measured at 2, 4, 8 and 12 weeks of treatment with a miso diet including 2.3% NaCl, a high-sodium diet including 2.3% or 1.9% NaCl, or a normal diet including 0.3% NaCl (MF diet; Oriental Yeast Co., Tokyo, Japan). The rats were autopsied after 12 weeks on a diet. DBP in male Dahl rats was significantly increased by the 2.3% NaCl diet as compared with that in the MF group (p < 0.01) or miso group (p < 0.05) from 4 weeks of treatment. Thereafter, SBP and DBP in both the high NaCl groups were significantly increased when compared with the MF or miso group. SBP in female Dahl rats on 2.3% NaCI was significantly increased from 8 weeks after treatment. Nephropathy was observed in both sexes of Dahl rats but not SD rats. These results show that blood pressure was not increased by the miso diet.

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.

Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors.

It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34(+)Lin(-)) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34(+)Lin(-) cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34(+)Lin(-) cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34(+)Lin(-) cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels.

Long term effects of radiation exposure on telomere lengths of leukocytes and its associated biomarkers among atomic-bomb survivors.

Ionizing radiation (IR) is a major source of cellular damage and the immediate cellular response to IR has been well characterized. But the long-term impact of IR on cell function and its relationship with aging are not known. Here, we examined the IR effects on telomere length and other biomarkers 50 to 68 years post-exposure (two time points per person) in survivors of the atomic bombing at Hiroshima during WWII. We found that telomere length of leukocytes was inversely correlated with the dose of IR (p=0.008), and this effect was primarily found in survivors who were exposed at younger ages; specifically those <12 years old (p=0.0004). Although a dose-related retardation of telomere shortening with age was observed in the cross-sectional data, longitudinal follow-up after 11 years did not show IR exposure-related alteration of the rate of telomere shortening with age. In addition, IR diminished the associations between telomere length and selected aging biomarkers that were observed in survivors with no dose. These included uric acid metabolism, cytokines, and blood T cell counts. These findings showed long-lasting detrimental effects of IR on telomere length of leukocytes in both dose- and age-at-exposure dependent manner, and on alterations of biomarkers with aging.

Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure.