Search

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.

While whole-body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early-life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non-lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub-lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV-infected animals experiencing 4Gy, but not lower, dose of sub-lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV-infected, but not irradiated, mice were both protected to the same high level as the old non-irradiated, uninfected controls. Analysis of the quality and quantity of anti-WNV immunity showed that higher mortality in MCMV-positive WBI mice correlated with increased levels of MCMV-specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late-life mortality following early-life irradiation and late-life acute infection.

The percutaneous absorption of a fentanyl (FEN)-patch is affected by various external factors including the volume of sebum secretion, which causes changes in the skin surface environment. In this study, we prepared a lard-based sebum-like secretion (SLS), and applied it to investigate the effect of different skin surface conditions on the drug penetration of a FEN-patch. In vitro work to test drug release using the Franz diffusion cell indicated that drug release was significantly suppressed by treatment with 5% SLS, which is equivalent to the amount of daily human sebum secretion. Conversely, in ex vivo experiments using rat skin, the amount of FEN that accumulated in the skin tissue of the 5% SLS-treated rats was higher in comparison with the non-SLS treated group. Furthermore, in vivo experiments indicated that the plasma FEN concentration in rats treated with the FEN-patch was significantly increased by treatment with 5% SLS. These results suggest that the sebum affected the release, accumulation, and absorption of FEN from the FEN-patch, and the FEN concentration in the blood was reflected by the balance of the suppression of drug release and the enhancement of drug accumulation in the skin with SLS.

A popular “DNA-damage theory” of aging posits that unrepaired DNA damage leads to cellular (and organismal) senescence. Indeed, some hallmarks of immune aging are more prevalent in individuals exposed to Whole-Body Irradiation (WBI). To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent Murine Cytomegalovirus (MCMV) and early-life WBI (i) over the course of the lifespan; (ii) in response to a West Nile virus (WNV) live attenuated vaccine; and (iii) following lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non-lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system despite causing widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub-lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV-infected animals experiencing 4Gy, but not lower, dose of sub-lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV-infected, but not irradiated, mice were both protected to the same high level as the old non-irradiated, uninfected controls. Analysis of the quality and quantity of anti-WNV immunity showed that higher mortality in MCMV-positive WBI mice correlated with increased levels of MCMV-specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late-life mortality following early-life irradiation and late-life acute infection.

To identify risk factors for the prognosis of prostate cancer (PC), we retrospectively analyzed the impact of lifestyle-related disorders as well as PC characteristics at initial diagnosis on the progression to castration-resistant PC (CRPC) in PC patients undergoing hormone therapy. Of 648 PC patients, 230 who underwent hormone therapy and met inclusion criteria were enrolled in this study. CRPC developed in 48 patients (20.9%). Univariate analysis using Cox proportional hazard model indicated that newly developed diabetes mellitus (DM) following hormone therapy (postDM), but not preexisting DM, as well as PC characteristics at initial diagnosis including prostate-specific antigen (PSA) ≥ 18 were significantly associated with the progression to CRPC. A similar tendency was also observed in the relationship between newly developed hypertension following hormone therapy and CRPC progression. On the other hand, neither dyslipidemia nor hyperuricemia, regardless the onset timing, exhibited any association with CRPC progression. In multivariate analysis, postDM and PSA ≥ 18 were extracted as independent risk factors for CRPC progression (adjusted hazard ratios, 3.38 and 2.34; p values, 0.016 and 0.019, respectively). Kaplan–Meier analysis and log-rank test clearly indicated earlier progression to CRPC in PC patients who developed postDM or had relatively advanced initial PC characteristics including PSA ≥ 18. Together, the development of lifestyle-related disorders, particularly DM, following hormone therapy, as well as advanced PC characteristics at initial diagnosis is considered to predict earlier progression to CRPC and poor prognosis in PC patients undergoing hormone therapy.

The objective of this study was to evaluate effects of whole body radiation exposure early in life on influenza vaccination immune responses much later in life. A total of 292 volunteers recruited from the cohort members of ongoing Adult Health Study (AHS) of Japanese atomic bomb (A-bomb) survivors completed this observational study spanning two influenza seasons (2011-2012 and 2012-2013). Peripheral blood samples were collected prior to and three weeks after vaccination. Serum hemagglutination inhibition (HAI) antibody titers were measured as well as concentrations of 25 cytokines and chemokines in culture supernatant from peripheral blood mononuclear cells, with and without in vitro stimulation with influenza vaccine. We found that influenza vaccination modestly enhanced serum HAI titers in this unique cohort of elderly subjects, with seroprotection ranging from 18 to 48% for specific antigen/season combinations. Twelve percent of subjects were seroprotected against all three vaccine antigens post-vaccination. Males were generally more likely to be seroprotected for one or more antigens post-vaccination, with no differences in vaccine responses based on age at vaccination or radiation exposure in early life. These results show that early life exposure to ionizing radiation does not prevent responses of elderly A-bomb survivors to seasonal influenza vaccine.

The present study investigated the association between single nucleotide polymorphisms (SNPs) in immune- or DNA repair-related genes and the integration pattern of human papillomavirus (HPV), a promising prognostic marker in cervical cancer. The HPV integration patterns of cervical cancer patients were determined by polymerase chain reaction and in situ hybridization, and categorized as episomal (group A), single-copy or multi-copy tandem repetition integrated (group B), and undetectable HPV types (group C). After sample and SNP quality control, 166,505 SNPs in 161 samples (38, 111, and 12 patients in groups A, B, and C, respectively) were examined. None of the SNPs reached genome-wide significance, and several candidate SNPs for future study were selected, including rs10999435 on chromosome 10q22, rs1322054 on chromosome 9q32-33, and rs10902171 on chromosome 11p15. Luciferase assay identified rs1322054 as the primary functional variant to regulate gene expression in immune cell. Further studies are needed to determine the genetic background of different integration patterns of HPV in cervical cancer patients.

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.

PURPOSE Defects in the DNA damage repair process can cause genomic instability and play an important role in cervical carcinogenesis. The purpose of this study was to analyze the association of 29 candidate single nucleotide polymorphisms (SNPs) in genes in the DNA repair pathway, TP53, and TP53BP1 with the risk of cervical cancer. MATERIALS AND METHODS Twenty-nine SNPs in four genes in the DNA repair pathway (ERCC2, ERCC5, NBS1, and XRCC1), TP53, and TP53BP1 were genotyped for 478 cervical cancer patients and 922 healthy control subjects, and their effects on cervical carcinogenesis were analyzed. RESULTS The most significant association was found for rs17655 in ERCC5, with an age-adjusted p-value < 0.0001, for which a strong additive effect of the risk allele C was observed (odds ratio, 2.01 for CC to GG). On the other hand, another significant polymorphism rs454421 in ERCC2 showed a dominant effect (odds ratio, 1.68 for GA+AA to GG) with an age-adjusted p-value of 0.0009. The association of these polymorphisms remained significant regardless of the age of onset. The significant result for rs17655 was also consistent for subgroups of patients defined by histology and human papillomavirus (HPV) types. However, for rs454421, the association was observed only in patients with squamous cell carcinoma and non-HPV 18 type. CONCLUSION The results of this study show a novel association of cervical cancer and the genes involved in the nucleotide excision pathway in the Korean population.

Summary Aging‐related decline in immunity is believed to be the main driver behind decreased vaccine efficacy and reduced resistance to infections in older adults. Unrepaired DNA damage is known to precipitate cellular senescence, which was hypothesized to be the underlying cause of certain age‐related phenotypes. Consistent with this, some hallmarks of immune aging were more prevalent in individuals exposed to whole‐body irradiation (WBI), which leaves no anatomical repository of undamaged hematopoietic cells. To decisively test whether and to what extent WBI in youth will leave a mark on the immune system as it ages, we exposed young male C57BL/6 mice to sublethal WBI (0.5–4 Gy), mimicking human survivor exposure during nuclear catastrophe. We followed lymphocyte homeostasis thorough the lifespan, response to vaccination, and ability to resist lethal viral challenge in the old age. None of the irradiated groups showed significant differences compared with mock‐irradiated (0 Gy) animals for the parameters measured. Even the mice that received the highest dose of sublethal WBI in youth (4 Gy) exhibited equilibrated lymphocyte homeostasis, robust T‐ and B‐cell responses to live attenuated West Nile virus (WNV) vaccine and full survival following vaccination upon lethal WNV challenge. Therefore, a single dose of nonlethal WBI in youth, resulting in widespread DNA damage and repopulation stress in hematopoietic cells, leaves no significant trace of increased immune aging in a lethal vaccine challenge model.

The findings from previously published studies have suggested that radiation exposure is associated with increased mortality and incidence of gastric cancer. However, few cohort studies have incorporated risk factors such as Helicobacter pylori (H. pylori) infection or chronic atrophic gastritis (CAG). The current study is aimed at evaluating the modifying effect of CAG on radiation risk of noncardia gastric cancer by histological type, by reanalyzing data from a nested case-control study conducted within the longitudinal clinical cohort of atomic bomb survivors. The analysis was restricted to 297 intestinal- or diffuse-type noncardia cases and 873 controls rematched to the cases on gender, age, city, and time and type of serum storage, and countermatched on radiation dose. Multivariable-adjusted relative risks [95% confidence interval (CI)] of noncardia gastric cancer were 3.9 (2.1-7.2) for H. pylori IgG seropositivity with cytotoxin-associated gene A (CagA) IgG low titer, 2.6 (1.9-3.6) for CAG, 1.9 (1.3-2.8) for current smoking, and 1.4 (1.1-1.9) for 1 Gy irradiation. Among subjects without CAG, the relative risk (95% CI) of noncardia gastric cancer at 1 Gy was 2.3 (1.4-3.7), whereas relative risk (95% CI) at 1 Gy was 1.1 (0.8-1.5) among subjects with CAG (for the overall interaction, P = 0.012). By histological type, the risk at 1 Gy was high for diffuse type without CAG, with adjusted relative risk (95% CI) of 3.8 (2.0-7.6), but was not high for diffuse type with CAG or for intestinal-type irrespective of CAG status. The results indicate that radiation exposure is associated with increased risk of diffuse-type noncardia gastric cancer without CAG, and this association exists despite adjustment for H. pylori infection and smoking habit.

Background The telomeric 3′-overhang (G-tail) length is essential for the biological effects of telomere dysfunction in vitro, but the association of length with aging and cardiovascular risk is unclear in humans. We investigated the association between the telomere G-tail length of leukocytes and cardiovascular risk, age-related white matter changes (ARWMCs), and endothelial function. Methods Patients with a history of cerebrovascular disease and comorbidity were enrolled (n = 102; 69 males and 33 females, 70.1 ± 9.2 years). Total telomere and telomere G-tail lengths were measured using a hybridization protection assay. Endothelial function was evaluated by ultrasound assessment of brachial flow-mediated dilation (FMD). Findings Shortened telomere G-tail length was associated with age and Framingham risk score (P = 0.018 and P = 0.012). In addition, telomere G-tail length was positively correlated with FMD values (P = 0.031) and negatively with the severity of ARWMCs (P = 0.002). On multivariate regression analysis, telomere G-tail length was independently associated with FMD values (P = 0.022) and the severity of ARWMCs (P = 0.033), whereas total telomere length was not associated with these indicators. Interpretation Telomere G-tail length is associated with age and vascular risk factors, and might be superior to total telomere length as a marker of endothelial dysfunction and ARWMC severity., Graphical abstract, Highlights • Telomere G-tail length was measured using a hybridization protection assay in patients with vascular risk factors. • Telomere G-tail length was independently related to endothelial function and age-related white matter changes. • Telomere G-tail length might be a promising biomarker of vascular damage.

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.

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.

Predictors of survival in patients with stage II/III gastric cancer (GC) who received tegafur/gimeracil/oteracil (S-1) adjuvant chemotherapy (ACT) subsequent to gastrectomy were examined. Additionally, the association between dose intensity of S-1 and survival rate was investigated. A total of 62 patients with stage II/III gastric cancer were retrospectively evaluated, each of whom had received a curative D2 gastrectomy and S-1 ACT. The relative performance (RP; administered/planned S-1 doses ×100%), body mass index (BMI), prognostic nutritional index (PNI) and body weight (BW) were calculated, and the association of survival with these factors and other clinicopathological parameters was examined. The 1-year treatment continuation rate for S-1 was 94.2%, excluding patients who experienced cancer recurrences during their ACT year. The initial S-1 reduction rate was 38.7%. Patients with stage II/IIIA disease exhibited significantly improved 5-year overall survival rates compared with patients with stage IIIB GC, 81.6/73.7 vs. 33.8% (P7 lymph node metastases were significantly associated with outcome (P10.6% and pathological stages were independent prognostic factors for survival. Continuing S-1 ACT for the full year exhibited a greater effect on survival compared with dosage. Early postoperative nutritional deterioration may decrease the survival rates in these patients.

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.

Inflammatory markers have been associated with increased risk of several cancers, including colon, lung, breast and liver, but the evidence is inconsistent. We conducted a nested case-control study in the longitudinal cohort of atomic-bomb survivors. The study included 224 hepatocellular carcinoma (HCC) cases and 644 controls individually matched to cases on gender, age, city and time and method of serum storage, and countermatched on radiation dose. We measured C-reactive protein (CRP) and interleukin (IL)-6 using stored sera obtained within 6 years before HCC diagnosis from 188 HCC cases and 605 controls with adequate volumes of donated blood. Analyses with adjustment for hepatitis virus infection, alcohol consumption, smoking habit, body mass index (BMI) and radiation dose showed that relative risk (RR) of HCC [95% confidence interval (CI)] in the highest tertile of CRP levels was 1.94 (0.72-5.51) compared to the lowest tertile (p = 0.20). RR of HCC (95% CI) in the highest tertile of IL-6 levels was 5.12 (1.54-20.1) compared to the lowest tertile (p = 0.007). Among subjects with BMI > 25.0 kg/m(2) , a stronger association was found between a 1-standard deviation (SD) increase in log IL-6 and HCC risk compared to subjects in the middle quintile of BMI (21.3-22.9 kg/m(2) ), resulting in adjusted RR (95% CI) of 3.09 (1.78-5.81; p = 0.015). The results indicate that higher serum levels of IL-6 are associated with increased HCC risk, independently of hepatitis virus infection, lifestyle-related factors and radiation exposure. The association is especially pronounced among subjects with obesity.

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.

In a series of studies of atomic bomb survivors, radiation-dose-dependent alterations in peripheral T-cell populations have been reported. For example, reduced size in naïve T-cell pools and impaired proliferation ability of T cells were observed. Because these alterations are also generally observed with human aging, we hypothesized that radiation exposure may accelerate the aging process of the T-cell immune system. To further test this hypothesis, we conducted cross-sectional analyses of telomere length, a hallmark of cellular aging, of naïve and memory CD4 T cells and total CD8 T cells in the peripheral blood of 620 atomic bomb survivors as it relates to age and radiation dose, using fluorescence in situ hybridization with flow cytometry. Since telomere shortening has been recently demonstrated in obesity-related metabolic abnormalities and diseases, the modifying effects of metabolic status were also examined. Our results indicated nonlinear relationships between T-cell telomere length and prior radiation exposure, i.e., longer telomeres with lower dose exposure and a decreasing trend of telomere length with individuals exposed to doses higher than 0.5 Gy. There were associations between shorter T-cell telomeres and higher hemoglobin Alc levels or fatty liver development. In naïve and memory CD4 T cells, radiation dose and high-density lipoprotein (HDL) cholesterol were found to positively interact with telomere length, suggesting that the decreasing trend of telomere length from a higher radiation dose was less conspicuous in individuals with a higher HDL cholesterol. It is therefore likely that radiation exposure perturbs T-cell homeostasis involving telomere length maintenance by multiple biological mechanisms, depending on dose, and that long-term-radiation-induced effects on the maintenance of T-cell telomeres may be modified by the subsequent metabolic conditions of individuals.

Immune system impairments reflected by the composition and function of circulating lymphocytes are still observed in atomic bomb survivors, and metabolic abnormalities including altered blood triglyceride and cholesterol levels have also been detected in such survivors. Based on closely related features of immune and metabolic profiles of individuals, we investigated the hypothesis that long-term effects of radiation exposure on lymphocyte subsets might be modified by metabolic profiles in 3,113 atomic bomb survivors who participated in health examinations at the Radiation Effect Research Foundation, Hiroshima and Nagasaki, in 2000-2002. The lymphocyte subsets analyzed involved T-, B- and NK-cell subsets, and their percentages in the lymphocyte fraction were assessed using flow cytometry. Health examinations included metabolic indicators, body mass index, serum levels of total cholesterol, high-density lipoprotein cholesterol, C-reactive protein and hemoglobin A1c, as well as diabetes and fatty liver diagnoses. Standard regression analyses indicated that several metabolic indicators of obesity/related disease, particularly high-density lipoprotein cholesterol levels, were positively associated with type-1 helper T- and B-cell percentages but were inversely associated with naive CD4 T and NK cells. A regression analysis adjusted for high-density lipoprotein cholesterol revealed a radiation dose relationship with increasing NK-cell percentage. Additionally, an interaction effect was suggested between radiation dose and C-reactive protein on B-cell percentage with a negative coefficient of the interaction term. Collectively, these findings suggest that radiation exposure and subsequent metabolic profile changes, potentially in relationship to obesity-related inflammation, lead to such long-term alterations in lymphocyte subset composition. Because this study is based on cross-sectional and exploratory analyses, the implications regarding radiation exposure, metabolic profiles and circulating lymphocytes warrant future longitudinal and molecular mechanistic studies.

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.

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

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.—Hayashi, T., Morishita, Y., Khattree, R., Misumi, M., Sasaki, K., Hayashi, I., Yoshida, K., Kajimura, J., Kyoizumi, S., Imai, K., Kusunoki, Y., Nakachi, K. Evaluation of systemic markers of inflammation in atomic-bomb survivors with special reference to radiation and age effects.

Background The case-cohort study design has received significant methodological attention in the statistical and epidemiological literature but has not been used as widely as other cohort-based sampling designs, such as the nested case-control design. Despite its efficiency and practicality for a wide range of epidemiological study purposes, researchers may not yet be aware of the fact that the design can be analysed using standard software with only minor adjustments. Furthermore, although the large number of options for design and analysis of case-cohort studies may be daunting, they can be reduced to a few simple recommendations. Methods We review conventional methods for the design and analysis of case-cohort studies and describe empirical comparisons based on a study of radiation, gene polymorphisms and cancer in the Japanese atomic bomb survivor cohort. Results Stratified, as opposed to simple, random subcohort selection is recommended, especially for studies of gene-environment interaction, which are notorious for lacking statistical power. Methods based on the score-unbiased exact pseudo-likelihood (or its analogue with stratified case-cohort data) are recommended for use in conjunction with the asymptotic variance estimator. Conclusions We present an example of how to implement case-cohort analysis methods using SPSS, a popular statistical package that lacks some of the features necessary to directly adapt and implement published methods based on other software platforms. We also illustrate case-control analysis using Epicure, which provides greater risk-modelling flexibility than other software. Our conclusions and recommendations should help investigators to better understand and apply the case-cohort design in epidemiological research.

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.

This study aims to deepen understanding of lymphocyte phenotypes related to the course of hepatitis C virus (HCV) infection and progression of liver fibrosis, in a cohort of atomic-bomb survivors. The study subjects comprise three groups: 162 HCV persistently infected, 145 spontaneously cleared, and 3511 uninfected individuals. We found increased percentages of peripheral blood TH1 and total CD8 T cells and decreased percentages of NK cells in the HCV persistence group, compared with the other two groups, after adjustment for age, gender, and radiation exposure dose. Subsequently, we found that increased TH1 cell percentages in the HCV persistence group were significantly associated with an accelerated time-course reduction in platelet counts―accelerated progression of liver fibrosis―while TC1 and NK cell percentages were inversely associated with the progression. This study suggests that TH1 immunity is enhanced by persistent HCV infection, and that percentages of peripheral TH1, TC1, and NK cells may help predict progression of liver fibrosis., This research was based on RERF Research Protocols 3-09, 4-02, 2-00, 9-92, and was supported in part by the U.S. National Institute of Allergy and Infectious Diseases (NIAID Contract HHSN272200900059C).

Lung cancer is a leading cause of cancer death worldwide. Prevention could be improved by identifying susceptible individuals as well as improving understanding of interactions between genes and etiological environmental agents, including radiation exposure. The epidermal growth factor receptor (EGFR)-signaling pathway, regulating cellular radiation sensitivity, is an oncogenic cascade involved in lung cancer, especially adenocarcinoma. The cytosine adenine (CA) repeat number polymorphism in the first intron of EGFR has been shown to be inversely correlated with EGFR production. It is hypothesized that CA repeat number may modulate individual susceptibility to lung cancer. Thus, we carried out a case-cohort study within the Japanese atomic bomb (A-bomb) survivor cohort to evaluate a possible association of CA repeat polymorphism with lung cancer risk in radiation-exposed or negligibly exposed (5 mGy) A-bomb survivors. First, by dividing study subjects into Short and Long genotypes, defined as the summed CA repeat number of two allelesor = 37 andor = 38, respectively, we found that the Short genotype was significantly associated with an increased risk of lung cancer, specifically adenocarcinoma, among negligibly exposed subjects. Next, we found that prior radiation exposure significantly enhanced lung cancer risk of survivors with the Long genotype, whereas the risk for the Short genotype did not show any significant increase with radiation dose, resulting in indistinguishable risks between these genotypes at a high radiation dose. Our findings imply that the EGFR pathway plays a crucial role in assessing individual susceptibility to lung adenocarcinoma in relation to radiation exposure.

Cytolethal distending toxin (CDT) induces apoptosis using the caspase-dependent classical pathway in the majority of human leukemic T cells (MOLT-4). However, we found the process to cell death is only partially inhibited by pretreatment of the cells with a general caspase inhibitor, z-VAD-fmk. Flow cytometric analysis using annexin V and propidium iodide showed that a 48-h CDT treatment decreased the living cell population by 35% even in the presence of z-VAD-fmk. z-VAD-fmk completely inhibited caspase activity in 24 h CDT-intoxicated cells. Further, CDT with z-VAD-fmk treatment clearly increased the cell population that had a low level of intracellular reactive oxygen. This is a characteristic opposite to that of caspase-dependent apoptosis. Overexpression of bcl2 almost completely inhibited cell death using CDT treatment in the presence of z-VAD-fmk. The data suggest there are at least two different pathways used in CDT-induced cell death: conventional caspase-dependent (early) apoptotic cell death and caspase-independent (late) death. Both occur via the mitochondrial membrane disruption pathway.

Using flow cytometry, we quantified the number of micronucleated reticulocytes in peripheral blood of whole-body X-irradiated mice in order to evaluate the radiation sensitivity and the induced genomic instability of the hematopoietic system. An acute effect of radiation dose as small as 0.1 Gy was detectable 2 days after irradiation, and the radiation dose effect was significantly greater in BALB/c mice than in C57BL/6 mice, that is, 3.0- and 2.3-fold increases in frequencies of micronuclei were noted in the two groups of mice, respectively. Even 1 year after irradiation, mice irradiated with 2.5 Gy of X-rays showed significantly increased frequencies of micronucleated reticulocytes, that is, 1.6- and 1.3-fold increases in BALB/c and C57BL/6 mice, respectively. However, this delayed effect was not apparent when the same mice were analyzed for T-cell receptor mutant frequencies in splenocytes. A significant mouse strain difference in the delayed radiation effect on micronucleated reticulocyte frequencies was noted as well. The results indicate that delayed genomic effects of irradiation on the murine hematopoietic system can persist in vivo for prolonged periods, and that there are mouse strain differences in sensitivity to radiation-induced genomic instability.

Background and Purpose: Telomere length of leukocytes is associated with cardiovascular risk. Recently, telomere G-tails, which are extensions of a guanine-rich single-stranded 3’-overhang, are thought to be key structures that protect telomere DNA from DNA damage. The aim of this study was to investigate the associations between telomere G-tail lengths, total telomere lengths and clinical factors, including biological inflammation and oxidative stress markers. Methods: Patients with history of cerebrovascular diseases and comorbidities were enrolled in this study (n=102; 69 males, 70.1±9.2 years). Total telomere lengths and telomere G-tail lengths were measured by hybridization protection assay (Tahara H et al. Nat Methods. 2005). Serum reactive oxygen species (ROS) levels were measured by using the total ROS assay system (Hayashi I et al. Mutat Res. 2007). Endothelial function was evaluated by ultrasound assessment of brachial flow-mediated dilation (FMD) as an index of endothelium-dependent vasodilation. Results: Telomere G-tail length (13653.0±2787.4 RLU/μg DNA) was positively correlated with total telomere length (176698.3±20308.0 RLU/μg DNA; R2=0.156, P Conclusion: Telomere G-tail lengths were independently related to the endothelial function after adjustment for vascular risk factors, chronic inflammation and oxidative stress. Telomere G-tail lengths might be more useful marker for the endothelial function which was evaluated by FMD than total telomere lengths.

We have previously shown that natural cytotoxic activity of peripheral blood lymphocytes was inversely related to cancer development based on a prospective cohort study. The genetic fraction of cytotoxic activity needs to be clarified, identifying individuals immunogenetically susceptible to cancer. A case-control study within the cohort members was designed: 102 cancer cases with peripheral lymphocyte DNA available and three control groups, each of which consisted of 204 subjects with each tertile level of cytotoxic activity. We first compared two control groups with high and low cytotoxic activity in terms of the single nucleotide polymorphisms in the natural killer complex gene region on chromosome 12p, identifying the haplotype alleles that were associated with the activity. Next, cancer risks were assessed for these haplotypes. We found two haplotype blocks, each of which generated two major haplotype alleles: low-activity-related LNK1 (frequency 0.478 and 0.615 in groups with high and low activity, respectively; P < 0.00008) and high-activity-related HNK1 (0.480 and 0.348; P < 0.0001), LNK2 (0.711 and 0.821; P < 0.0002), and HNK2 (0.272 and 0.174; P < 0.0008). These NKG2D haplotype alleles showed a significant difference between cases (0.632 for LNK1 and 0.333 for HNK1) and controls (0.554 for LNK1 and 0.406 for HNK1). The haplotype HNK1/HNK1 revealed a decreased risk of cancer (odds ratio, 0.471; 95% confidence interval, 0.233-0.952) compared with LNK1/LNK1. Individuals who are genetically predisposed to have low or high natural cytotoxic activity can in part be determined by NKG2D haplotyping, which in turn reveals an increased or decreased risk of cancer development. (Cancer Res 2006; 66(1): 563-70)

It has previously been reported that hemizygous mutant fraction (Mf) at the glycophorin A (GPA) locus in erythrocytes increased with radiation dose in heterozygotes among Hiroshima and Nagasaki atomic bomb survivors. In the present study, we analyzed the relationship between GPA Mf and cancer risk using newly developed cancers among previously cancer-free subjects whose GPA Mf had been measured between 1988 and 1996. Among 1,723 survivors (1,117 in Hiroshima and 606 in Nagasaki), we identified 186 subjects who developed a first cancer by the end of 2000. We compared the radiation dose responses of GPA Mf between cancer and cancer-free groups using a linear-quadratic model fit by multiple regression analysis in combination with age, sex, and city. The slope of the GPA Mf dose-response curve was significantly higher in the cancer group than in the cancer-free group among Hiroshima subjects. Moreover, no significant difference of GPA Mf between cancer and cancer-free groups was found in unexposed controls in the two cities. The same conclusions were obtained using a linear dose-response model and by further analysis using Cox regression of cancer incidence. These findings suggest that there might be interindividual variation in mutability of somatic genes and that Hiroshima survivors who have higher mutability in response to radiation exposure would be expected to have a higher probability of suffering radiation-related cancer.

Estimating human cancer risk based on host-environment interaction is one task of epidemiology, and it has provided indispensable knowledge for prevention of cancer. The recent develop-ment of gene-engineered mice has also provided solid evidence about the relationship between cancer development and immunity. The aim of this review is to discuss the possible contribution of epidemiology to understanding the role of immunity in host defense against cancer, and also to assess the involvement of inflammation in the occurrence of selected cancers. Here we look at the concepts of cancer immunosurveillance and infection-inflammation-cancer, and include a brief introduction to recent studies in humans and experimental animal models. It has been postulated for many years that the immune system has the ability to recognize and eliminate nascent transformed cells in the body (so-called cancer immunosurveillance hypothesis), and this idea has recently obtained strong support from animal experiments. In humans, follow-up studies among immunosuppressed transplant recipients revealed a remarkably increased risk of not only selected malignancies, but also cancers with no known viral etiology. On the other hand, a prospective cohort study among the general population revealed that individuals with low natural cytotoxic activity of peripheral blood lymphocytes had an increased risk of cancer development. More studies are warranted to allow the construction of a model for the interaction between host immunity, aging, and the environment. The host immune system is also involved in inflammatory responses to pathogen infection: insufficient immune function of the host, or repeated infection, may result in persistent inflammation, where growth/survival factors continuously act on initiated cells. The combined use of biomarkers will be necessary to define low-grade persistent inflammation in future cohort studies; and, in addition to these phenotype marker-based cohort studies, one plausible future direction will be a genomic approach that can be undertaken within cohort studies, looking at the genetic background underlying individual variations in phenotype markers.

Suzuki, G., Shimada, Y., Hayashi, T., Akashi, M., Hirama, T. and Kusunoki, Y. An Association between Oxidative Stress and Radiation-Induced Lymphomagenesis. Radiat. Res. 161, 642–647 (2004). It is generally thought that reactive oxygen species (ROS) play an important role in carcinogenesis. However, direct evidence supporting this idea is still lacking. In the present study, we measured ROS in thymocytes at the thymic prelymphoma stage in C57BL/6 mice. Mice (n = 20) were irradiated at 1.6 Gy/week for 4 consecutive weeks and the levels of ROS were measured 8 to 11 weeks later by dehydrorhodamine 123, which accumulated in mitochondria and became fluorescent dye upon oxidation. Unirradiated littermates (n = 17) served as controls. Thymic prelymphoma cells were diagnosed by the aberrant CD4/CD8 staining profile and monoclonal or oligoclonal T-cell receptor gene rearrangement. A significant fraction of mice (11/13) bearing thymic prelymphoma cells exhibited elevated levels of ROS in thymocytes (P < 0....

Our previous studies have revealed a clear dose-dependent decrease in the percentage of naïve CD4 T cells that are phenotypically CD45RA+ in PBL among A-bomb survivors. However, whether there is a similar radiation effect on CD8 T cells has remained undetermined because of the unreliability of CD45 isoforms as markers of naïve and memory subsets among the CD8 T-cell population. In the present study, we used double labeling with CD45RO and CD62L for reliable identification of naïve and memory cell subsets in both CD4 and CD8 T-cell populations among 533 Hiroshima A-bomb survivors. Statistically significant dose-dependent decreases in the percentages of CD45RO-/CD62L+ naïve cells were found in the CD8 T-cell population as well as in the CD4 T-cell population. Furthermore, the percentages of CD45RO+/CD62L+ and CD45RO+/CD62L- memory T cells were found to increase significantly with increasing radiation dose in the CD8 T-cell population but not in the CD4 T-cell population. These results suggest that the prior A-bomb exposure has induced long-lasting deficits in both naïve CD4 and CD8 T- cell populations along with increased proportions of these particular subsets of the memory CD8 T-cell population.

To study the long-term effects of radiation-induced T-cell depletion on the T-cell receptor (TCR) Vbeta repertoires of human peripheral CD4 T-cell populations, we measured the percentages of CD4 T cells representing each of the full range of possible TCR Vbeta families in a cohort of atomic bomb survivors. We then estimated the extent to which the expression levels for individual TCR Vbeta families differed from the average expression level for that particular TCR Vbeta family across the entire cohort. We found no evidence of a systematic change in the TCR Vbeta repertoires of the naive CD4 T-cell populations, but memory CD4 T-cell TCR Vbeta family expression levels diverged significantly from the population average for counterpart families, especially in individuals who had been exposed to higher doses and were at least 20 years of age at the time of the bombing. Comparisons of the TCR Vbeta family expression profiles in the naive and memory CD4 T-cell pools of the same group of adult survivors revealed that differences in the TCR Vbeta repertoires of these two types of CD4 T-cell pool were larger in more heavily exposed survivors than in unexposed controls. These findings suggest that the memory CD4 T-cell pools of individuals who received significant radiation doses in adulthood may well have become (and could still be) dependent upon a much less diverse complement of TCR Vbeta families than would otherwise have been the case.

We propose a classification of human CD4+CD45RO+ memory T cells into three new subsets based on cell surface expression levels of CD43. The first subset consists of cells whose CD43 expression is relatively high; this subset also contains the highest proportion of recall Ag-reactive precursors, and its constituent cells respond far more strongly than cells in either of the other subsets to immobilized CD3 Ab in addition to secreting substantially more IFN-γ and IL-4. Cells of the second subset express similar levels of CD43 to naive cells, and they also respond weakly to TCR-mediated stimuli as judged by either their ability to proliferate or capacity for cytokine production. The third subsets consists of cells whose CD43 expression levels are clearly down-regulated; its cells appear to be anergic to TCR-mediated stimuli, and when examined ex vivo many of them appear to be undergoing either spontaneous apoptosis via a caspase-independent pathway or Fas-mediated apoptosis via a caspase-dependent pathway, even in the resting state. An analysis of telomere lengths revealed that the typical telomere of a cell in the second subset was significantly longer than the typical telomere in the first or third subset. Taken together, these results appear to indicate that CD4+CD45RO+ T cells fall into three functionally differing subsets, one being a subset of cells with fully matured memory phenotype, a second being a less mature subset of cells that retain longer telomeres and whose memory functionality is marginal, and a third consisting of anergic cells that give every appearance of being death-prone and/or in the process of dying.

Background and Purpose: Pathogenesis of white matter lesions is thought to be associated with endothelial dysfunction and inflammation. Ultrasound assessment of brachial artery flow mediated dilation (FMD) as a noninvasive and useful method has been recently used as an index of endothelium-dependent vasodilation. The aim of this study was to investigate the associations between endothelial function which was evaluated by FMD and the severity of white matter lesions in patients with chronic cerebrovascular disease and comorbidity. Additionally, we evaluated association between FMD and the number of circulating progenitor cells. Methods: Patients with cerebrovascular diseases history and other untypical neurological problems were enrolled in this study (n=63; 39 male, 69.8±10.0 years). Patients were classified according to periventricular hyperintensities (PVH) and deep white matter hyperintensities (DWMH) scores by fluid-attenuate inversion recovery image of magnetic resonance images (Fazekas gradings). Endothelial dysfunction was defined as a FMD≤4.5%. The numbers of CD34+/CD133+ cells defined as circulating progenitor cells were evaluated by flow cytometry. Results: The patients with endothelial dysfunction (n=39, 61.9%) were older (72.4±7.6 vs. 65.5±11.8, P=0.007), more frequently male (74.4% vs. 41.7%, P=0.016) and more frequently had hypertension (89.7% vs. 37.5%, P2 =0.120, p=0.006, R 2 =0.280, p2 =0.234, p2 =0.078, p=0.034). After multivariate analysis with age, sex, presence of stroke and comorbidities, endothelial dysfunction was independently associated with the severity of PVH (standardized partial regression coefficient [β] 0.703, p=0.002), as well as DWMH (β 0.589, p=0.042). Conclusion: Endothelial dysfunction which was evaluated by FMD might be predictive for the grade of cerebral small vessel disease.

Hexamethylene bisacetamide protects peritoneal mesothelial cells from glucose. Background Peritoneal dialysis causes damage to peritoneal mesothelial cells primarily because dialysis fluids have a high glucose concentration. This study examined the abnormalities of gap junctional intercellular communication (GJIC) in human peritoneal mesothelial cells (HPMCs) exposed to relatively high levels of glucose. Also, ability of hexamethylene bisacetamide (HMBA) to up-regulate GJIC in HPMCs exposed to high levels of glucose was measured. Methods An assay that monitors the recovery of fluorescence after photobleaching was used to measure GJIC in primary cultured HPMCs. The cells were exposed to a low (10 mmol/L) or high (50 or 90 mmol/L) glucose level for a total of six days, and some cells were also incubated with or without HMBA (1 or 6 mmol/L) from day 4. The effects of incubation in these various environments on expression of the connexin 43 (Cx43) gene were investigated by the reverse transcription-polymerase chain reaction (to detect Cx43 mRNA) or by immunofluorescence and Western blotting (to detect Cx43 protein). To evaluate the influence of protein kinase C (PKC) or mitogen-activated protein kinase (MAPK) on GJIC, specific inhibitors were added to cultures in a high glucose medium. Results Gap junctional intercellular communication was inhibited in a concentration- and time-dependent manner when cells were exposed to high glucose. The addition of 6 mmol/L HMBA to cultures significantly enhanced GJIC despite the presence of a high glucose concentration. High glucose also down-regulated Cx43 mRNA and protein expression, with the dose-dependent decrease of Cx43 protein at gap junctions paralleled by a decrease in the phosphorylation of this protein. As expected, treatment of cells with 6 mmol/L HMBA increased both Cx43 mRNA and protein levels despite exposure to high glucose. The addition of PKC or MAPK inhibitors to high glucose cultures did not restore GJIC, and there was no significant change of Cx43 phosphorylation in the presence of these inhibitors. Conclusions High glucose down-regulates GJIC in human peritoneal mesothelial cells. It also decreases the levels of both Cx43 mRNA and Cx43 protein, with the latter becoming hypophosphorylated. HMBA appears to reverse all of these changes. These results are consistent with our hypothesis that HMBA protects HPMCs from the adverse effects of high glucose by reversing various processes that would otherwise lead to harmful loss of GJIC.

Ataxia-telangiectasia (AT) is an autosomal recessive disease. The relevant gene has been cloned and designated ATM. We studied the expression of both ATM mRNA and the ATM protein in unirradiated and X-irradiated EBV (Epstein-Barr virus)-transformed lymphoblastoid cell lines (LCLs) derived from donors who were normal (ATM + / + ), AT heterozygotes (ATM + / - ), or AT homozygotes (ATM - / - ), respectively. In ATM + / + LCLs, the levels of ATM mRNA were found to have increased by approximately 1.5-fold within 1 h of exposure to 10 Gy of X-rays, while the ATM protein levels had increased by 1.5- to 2.0-fold within 2 to 3 h of irradiation. The wild-type mRNA and protein levels both returned to their basal values fairly quickly after this time. The results obtained with the ATM + / - LCLs were quite different, however: neither the mRNA nor protein levels were found to have increased as a consequence of X-irradiation in any ATM + / - LCL. Twelve of the mutations in the ATM - / - LCLs we used were truncating mutations, and we suspected that the corresponding truncated ATM proteins would be too labile to be detected by western blot analysis. However, five of the ATM - / - LCLs produced mutant ATM proteins that were identical in molecular weight to the wild-type ATM protein. When cells from three of these five clones were exposed to X-rays, transcription of the mutant ATM genes appeared to reduce somewhat, as were the levels of protein being produced. These results suggest that the normal ATM gene responds to ionizing radiation by up-regulating its activity, whereas none of the mutant ATM genes we studied were able to respond in this way.

Previous studies indicated that transforming RNA, derived from the 3′ half of the U5 small nuclear RNA first stem structure, suppressed the secretory protein translation in vitro. Gap junctions facilitate homeostatic control of cell growth and differentiation and their dysfunction has been correlated with carcinogenesis. Here, we reported that transforming RNA directly suppressed the gap junction protein, connexin 43, translation and thereby inhibited functional gap junction function in rat epithelial cells. Together with previous data, this implies that altered expression of transforming RNA itself is a potential mechanism in inhibiting gap junction function during carcinogenesis.