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Research examining the potential for circulating miRNA to serve as markers for preneoplastic lesions or early-stage hepatocellular carcinoma (HCC) is hindered by the difficulties of obtaining samples from asymptomatic individuals. As a surrogate for human samples, we identified hub miRNAs in gene co-expression networks using HCC-bearing C3H mice. We confirmed 38 hub miRNAs as associated with HCC in F2 hybrid mice derived from radiogenic HCC susceptible and resistant founders. When compared to a panel of 12 circulating miRNAs associated with human HCC, two had no mouse ortholog and 7 of the remaining 10 miRNAs overlapped with the 38 mouse HCC hub miRNAs. Using small RNA sequencing data generated from serially collected plasma samples in F2 mice, we examined the temporal levels of these 7 circulating miRNAs and found that the levels of 4 human circulating markers, miR-122-5p, miR-100-5p, miR-34a-5p and miR-365-3p increased linearly as the time approaching HCC detection neared, suggesting a correlation of miRNA levels with oncogenic progression. Estimation of change points in the kinetics of the 4 circulating miRNAs suggested the changes started 17.5 to 6.8 months prior to HCC detection. These data establish these 4 circulating miRNAs as potential sentinels for preneoplastic lesions or early-stage HCC., (© 2022. The Author(s).)

Galactic cosmic rays are primarily composed of protons (85%), helium (14%), and high charge/high energy ions (HZEs) such as 56 Fe, 28 Si, and 16 O. HZE exposure is a major risk factor for astronauts during deep-space travel due to the possibility of HZE-induced cancer. A systems biology integrated omics approach encompassing transcriptomics, proteomics, lipidomics, and functional biochemical assays was used to identify microenvironmental changes induced by HZE exposure. C57BL/6 mice were placed into six treatment groups and received the following irradiation treatments: 600 MeV/n 56 Fe (0.2 Gy), 1 GeV/n 16 O (0.2 Gy), 350 MeV/n 28 Si (0.2 Gy), 137 Cs (1.0 Gy) gamma rays, 137 Cs (3.0 Gy) gamma rays, and sham irradiation. Left liver lobes were collected at 30, 60, 120, 270, and 360 days post-irradiation. Analysis of transcriptomic and proteomic data utilizing ingenuity pathway analysis identified multiple pathways involved in mitochondrial function that were altered after HZE irradiation. Lipids also exhibited changes that were linked to mitochondrial function. Molecular assays for mitochondrial Complex I activity showed significant decreases in activity after HZE exposure. HZE-induced mitochondrial dysfunction suggests an increased risk for deep space travel. Microenvironmental and pathway analysis as performed in this research identified possible targets for countermeasures to mitigate risk.

High-charge, high-energy ion particle (HZE) radiations are extraterrestrial in origin and characterized by high linear energy transfer (high-LET), which causes more severe cell damage than low-LET radiations like γ-rays or photons. High-LET radiation poses potential cancer risks for astronauts on deep space missions, but the studies of its carcinogenic effects have relied heavily on animal models. It remains uncertain whether such data are applicable to human disease. Here, we used genomics approaches to directly compare high-LET radiation-induced, low-LET radiation-induced and spontaneous hepatocellular carcinoma (HCC) in mice with a human HCC cohort from The Cancer Genome Atlas (TCGA). We identified common molecular pathways between mouse and human HCC and discovered a subset of orthologous genes (mR-HCC) that associated high-LET radiation-induced mouse HCC with a subgroup (mrHCC2) of the TCGA cohort. The mrHCC2 TCGA cohort was more enriched with tumor-suppressing immune cells and showed a better prognostic outcome than other patient subgroups.

Future space missions will include a return to the Moon and long duration deep space roundtrip missions to Mars. Leaving the protection that Low Earth Orbit provides will unavoidably expose astronauts to higher cumulative doses of space radiation, in addition to other stressors, e.g., microgravity. Immune regulation is known to be impacted by both radiation and spaceflight and it remains to be seen whether prolonged effects that will be encountered in deep space can have an adverse impact on health. In this study, we investigated the effects in the overall metabolism of three different low dose radiation exposures (γ-rays, 16 O, and 56 Fe) in spleens from male C57BL/6 mice at 1, 2, and 4 months after exposure. Forty metabolites were identified with significant enrichment in purine metabolism, tricarboxylic acid cycle, fatty acids, acylcarnitines, and amino acids. Early perturbations were more prominent in the γ irradiated samples, while later responses shifted towards more prominent responses in groups with high energy particle irradiations. Regression analysis showed a positive correlation of the abundance of identified fatty acids with time and a negative association with γ-rays, while the degradation pathway of purines was positively associated with time. Taken together, there is a strong suggestion of mitochondrial implication and the possibility of long-term effects on DNA repair and nucleotide pools following radiation exposure.

Characterization of lipids by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is of great interest because not only are lipids important structural molecules in both the cell and internal organelle membranes, but they are also important signaling molecules. MALDI-MSI combined with spatial image segmentation has been previously used to identify tumor heterogeneities within tissues with distinct anatomical regions such as the brain. However, there has been no systematic study utilizing MALDI-MSI combined with spatial image segmentation to assess the tumor microenvironment in the liver. Here, we present that image segmentation can be used to evaluate the tumor microenvironment in the liver. In particular, to better understand the molecular mechanisms of irradiation-induced hepatic carcinogenesis, we used MALDI-MSI in the negative ion mode to identify lipid changes 12 months post exposure to low dose 28 Si and 137 Cs γ ray irradiation. We report here the changes in the lipid profiles of male C3H/HeNCrl mice liver tissues after exposure to irradiation and analyzed using the spatial shrunken centroid clustering algorithm. These findings provide valuable information as astronauts will be exposed to high-charge high-energy (HZE) particles and low-energy γ-ray irradiation during deep space travel. Even at low doses, exposure to these irradiations can lead to cancer. Previous studies infer that irradiation of mice with low-dose HZE particles induces oxidative damage and microenvironmental changes that are thought to play roles in the pathophysiology of hepatocellular carcinoma., Competing Interests: The authors declare no competing financial interest.

Background: One of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z > 13), which can lead to the induction of hepatocellular carcinoma (HCC). However, little is known on the molecular mechanisms of HZE irradiation-induced HCC., Results: We performed comparative RNA-Seq transcriptomic analyses to assess the carcinogenic effects of 600 MeV/n 56 Fe (0.2 Gy), 1 GeV/n 16 O (0.2 Gy), and 350 MeV/n 28 Si (0.2 Gy) ions in a mouse model for irradiation-induced HCC. C3H/HeNCrl mice were subjected to total body irradiation to simulate space environment HZE-irradiation, and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the biological effects of these HZE ions, particularly immunological, such as Acute Phase Response Signaling, B Cell Receptor Signaling, IL-8 Signaling, and ROS Production in Macrophages. Also seen in this study were novel unannotated transcripts that were significantly affected by HZE. To investigate the biological functions of these novel transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples (45 HZE-irradiated, 15 non-irradiated control) from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes (e.g., Acute Phase Response Signaling, B Cell Receptor Signaling, IL-3 Signaling), and RNA Transcription/Expression., Conclusions: A large number of transcripts were found differentially expressed post-HZE irradiation. These results provide valuable information for uncovering the differences in molecular mechanisms underlying HZE specific induced HCC carcinogenesis. Additionally, a handful of novel differentially expressed unannotated transcripts were discovered for each HZE ion. Taken together, these findings may provide a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation and may also have important implications for the discovery of potential countermeasures against and identification of biomarkers for HZE-induced HCC.

Background: mRNA interaction with other mRNAs and other signaling molecules determine different biological pathways and functions. Gene co-expression network analysis methods have been widely used to identify correlation patterns between genes in various biological contexts (e.g., cancer, mouse genetics, yeast genetics). A challenge remains to identify an optimal partition of the networks where the individual modules (clusters) are neither too small to make any general inferences, nor too large to be biologically interpretable. Clustering thresholds for identification of modules are not systematically determined and depend on user-settable parameters requiring optimization. The absence of systematic threshold determination may result in suboptimal module identification and a large number of unassigned features., Results: In this study, we propose a new pipeline to perform gene co-expression network analysis. The proposed pipeline employs WGCNA, a software widely used to perform different aspects of gene co-expression network analysis, and Modularity Maximization algorithm, to analyze novel RNA-Seq data to understand the effects of low-dose 56 Fe ion irradiation on the formation of hepatocellular carcinoma in mice. The network results, along with experimental validation, show that using WGCNA combined with Modularity Maximization, provides a more biologically interpretable network in our dataset, than that obtainable using WGCNA alone. The proposed pipeline showed better performance than the existing clustering algorithm in WGCNA, and identified a module that was biologically validated by a mitochondrial complex I assay., Conclusions: We present a pipeline that can reduce the problem of parameter selection that occurs with the existing algorithm in WGCNA, for applicable RNA-Seq datasets. This may assist in the future discovery of novel mRNA interactions, and elucidation of their potential downstream molecular effects.

In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis, and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin) 24 h after lethal radiation exposure (9 Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post exposure prevents the disintegration of GI crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also upregulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24 h post exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment.

Despite considerable progress in identifying health risks to crewmembers related to exposure to galactic/cosmic rays and solar particle events (SPE) during space travel, its long-term effects on the pulmonary system are unknown. We used a murine risk projection model to investigate the impact of exposure to space-relevant radiation (SR) on the lung. C3H mice were exposed to (137)Cs gamma rays, protons (acute, low-dose exposure mimicking the 1972 SPE), 600 MeV/u (56)Fe ions, or 350 MeV/u (28)Si ions at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. Animals were irradiated at the age of 2.5 mo and evaluated 23.5 mo postirradiation, at 26 mo of age. Compared with age-matched nonirradiated mice, SR exposures led to significant air space enlargement and dose-dependent decreased systemic oxygenation levels. These were associated with late mild lung inflammation and prominent cellular injury, with significant oxidative stress and apoptosis (caspase-3 activation) in the lung parenchyma. SR, especially high-energy (56)Fe or (28)Si ions markedly decreased sphingosine-1-phosphate levels and Akt- and p38 MAPK phosphorylation, depleted anti-senescence sirtuin-1 and increased biochemical markers of autophagy. Exposure to SR caused dose-dependent, pronounced late lung pathological sequelae consistent with alveolar simplification and cellular signaling of increased injury and decreased repair. The associated systemic hypoxemia suggested that this previously uncharacterized space radiation-associated lung injury was functionally significant, indicating that further studies are needed to define the risk and to develop appropriate lung-protective countermeasures for manned deep space missions., (Copyright © 2015 the American Physiological Society.)

Cancer stem cells (CSCs) are believed to be resistant to currently available therapies and may be responsible for relapse of cancer in patients. Measuring circulating tumor cells (CTCs) in the blood of patients has emerged as a non-invasive diagnostic procedure for screening patients who may be at high risk for developing metastatic cancers or relapse of the cancer disease. However, accurate detection of CTCs has remained a problem, as epithelial-cell markers used to date are not always reliable for detecting CTCs, especially during epithelial-mesenchymal transition. As CSCs are required to initiate metastatic tumors, our goal was to optimize and standardize a method for identifying circulating CSCs (CCSCs) in patients, using established CSC markers. Here, we report for the first time the detection of CCSCs in the blood of athymic nude mice, bearing metastatic tumors, and in the blood of patients positive for colonic adenocarcinomas. Using a simple and non-expensive method, we isolated a relatively pure population of CSCs (CD45-/CK19+), free of red blood cells and largely free of contaminating CD45+ white blood cells. Enriched CCSCs from patients with colon adenocarcinomas had a malignant phenotype and co-expressed CSC markers (DCLK1/LGR5) with CD44/Annexin A2. CSCs were not found in the blood of non-cancer patients, free of colonic growths. Enriched CCSCs from colon cancer patients grew primary spheroids, suggesting the presence of tumor-initiating cells in the blood of these patients. In conclusion, we have developed a novel diagnostic assay for detecting CSCs in circulation, which may more accurately predict the risk of relapse or metastatic disease in patients. As CSCs can potentially initiate metastatic growths, patients positive for CCSCs can be treated with inhibitory agents that selectively target CSCs, besides conventional treatments, to reduce the risk of relapse/metastatic disease for improving clinical outcomes.

Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the carcinogenic effects of 300 MeV/n 28Si or 600 MeV/n 56Fe ions in a mouse model for radiation-induced acute myeloid leukemia and hepatocellular carcinoma. C3H/HeNCrl mice were irradiated with 0.1, 0.2, 0.4, or 1 Gy of 300 MeV/n 28Si ions, 600 MeV/n 56Fe ions or 1 or 2 Gy of protons simulating the 1972 solar particle event (1972SPE) at the NASA Space Radiation Laboratory. Additional mice were irradiated with 137Cs gamma rays at doses of 1, 2, or 3 Gy. All groups were followed until they were moribund or reached 800 days of age. We found that 28Si or 56Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia. However, 28Si or 56Fe ion irradiated mice had a much higher incidence of hepatocellular carcinoma than gamma ray irradiated or proton irradiated mice. These data demonstrate a clear difference in the effects of these HZE ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis. Also seen in this study was an increase in metastatic hepatocellular carcinoma in the 28Si and 56Fe ion irradiated mice compared with those exposed to gamma rays or 1972SPE protons, a finding with important implications for setting radiation exposure limits for space-flight crew members.

Exposure to sparsely ionising gamma- or X-ray irradiation is known to increase the risk of leukaemia in humans. However, heavy ion radiotherapy and extended space exploration will expose humans to densely ionising high linear energy transfer (LET) radiation for which there is currently no understanding of leukaemia risk. Murine models have implicated chromosomal deletion that includes the hematopoietic transcription factor gene, PU.1 (Sfpi1), and point mutation of the second PU.1 allele as the primary cause of low-LET radiation-induced murine acute myeloid leukaemia (rAML). Using array comparative genomic hybridisation, fluorescence in situ hybridisation and high resolution melt analysis, we have confirmed that biallelic PU.1 mutations are common in low-LET rAML, occurring in 88% of samples. Biallelic PU.1 mutations were also detected in the majority of high-LET rAML samples. Microsatellite instability was identified in 42% of all rAML samples, and 89% of samples carried increased microsatellite mutant frequencies at the single-cell level, indicative of ongoing instability. Instability was also observed cytogenetically as a 2-fold increase in chromatid-type aberrations. These data highlight the similarities in molecular characteristics of high-LET and low-LET rAML and confirm the presence of ongoing chromosomal and microsatellite instability in murine rAML.

Polymorphic variants of DNA repair genes can increase the carcinogenic potential of exposure to ionizing radiation. Two single nucleotide polymorphisms (SNPs) in Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), have been identified in BALB/c mice and linked to reduced DNA-PKcs activity and mammary cancer susceptibility. We examined three additional mouse strains to better define the roles of the BALB/c Prkdc SNPs (R2140C and M3844V). One is a congenic strain (C.B6) that has the C57BL/6 Prkdc allele on a BALB/c background, and the other is a congenic strain (B6.C) that has the BALB/c variant Prkdc allele on a C57BL/6 background. We also examined the LEWES mouse strain, which possesses only one of the BALB/c Prkdc SNPs (M3844V). Our results demonstrate that both Prkdc SNPs are responsible for deficient DNA-PKcs protein expression, DNA repair and telomere function, while the LEWES SNP affects only DNA-PKcs expression and repair capacity. These studies provide insight into the separation of function between the two BALB/c SNPs as well as direct evidence that SNPs positioned within Prkdc can significantly influence DNA-PKcs function involving DNA repair capacity, telomere end-capping, and potentially cancer susceptibility., (© 2011 by Radiation Research Society)

Different types of ionizing radiation produce different dependences of cancer risk on radiation dose/dose rate. Sparsely ionizing radiation (e.g. γ-rays) generally produces linear or upwardly curving dose responses at low doses, and the risk decreases when the dose rate is reduced (direct dose rate effect). Densely ionizing radiation (e.g. neutrons) often produces downwardly curving dose responses, where the risk initially grows with dose, but eventually stabilizes or decreases. When the dose rate is reduced, the risk increases (inverse dose rate effect). These qualitative differences suggest qualitative differences in carcinogenesis mechanisms. We hypothesize that the dominant mechanism for induction of many solid cancers by sparsely ionizing radiation is initiation of stem cells to a pre-malignant state, but for densely ionizing radiation the dominant mechanism is radiation-bystander-effect mediated promotion of already pre-malignant cell clone growth. Here we present a mathematical model based on these assumptions and test it using data on the incidence of dysplastic growths and tumors in the mammary glands of mice exposed to high or low dose rates of γ-rays and neutrons, either with or without pre-treatment with the chemical carcinogen 7,12-dimethylbenz-alpha-anthracene (DMBA). The model provides a mechanistic and quantitative explanation which is consistent with the data and may provide useful insight into human carcinogenesis.

Studies of radiation carcinogenesis in animals allow detailed investigation of how the risk depends on age at exposure and time since exposure and of the mechanisms that determine this risk, e.g., induction of new pre-malignant cells (initiation) and enhanced proliferation of already existing pre-malignant cells (promotion). To assist the interpretation of these patterns, we apply a newly developed biologically based mathematical model to data on several types of solid tumors induced by acute whole-body radiation in mice. The model includes both initiation and promotion and analyzes pre-malignant cell dynamics on two different time scales: comparatively short-term during irradiation and long-term during the entire life span. Our results suggest general mechanistic similarities between radiation carcinogenesis in mice and in human atomic bomb survivors. The excess relative risk (ERR) in mice decreases with age at exposure up to an exposure age of 1 year, which corresponds to mid-adulthood in humans; the pattern for older ages at exposure, for which there is some evidence of increasing ERRs in atomic bomb survivors, cannot be evaluated using the data set analyzed here. Also similar to findings in humans, initiation dominates the ERR at young ages in mice, when there are few background pre-malignant cells, and promotion becomes important at older ages.

Abstract Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the leukemogenic efficacy of one such HZE species, 1 GeV (56)Fe ions, a component of space radiation, in a mouse model for radiation-induced acute myeloid leukemia. CBA/CaJ mice were irradiated with 1 GeV/nucleon (56)Fe ions or (137)Cs gamma rays and followed until they were moribund or to 800 days of age. We found that 1 GeV/nucleon (56)Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia (AML). However, (56)Fe-ion-irradiated mice had a much higher incidence of hepatocellular carcinoma (HCC) than gamma-irradiated mice, with an estimated RBE of approximately 50. These data suggest a difference in the effects of HZE iron ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis.

Since deletion of the PU.1 gene on chromosome 2 is a crucial acute myeloid leukemia (AML) initiating step in the mouse model, we quantified PU.1 deleted cells in the bone marrow of gamma-, X- and 56Fe-ion-irradiated mice at various times postirradiation. Although 56Fe ions were initially some two to three times more effective than X or gamma rays in inducing PU.1 deletions, by 1 month postirradiation, the proportions of cells with PU.1 deletions were similar for the HZE particles and the sparsely ionizing radiations. These results indicate that while 56Fe ions are more effective in inducing PU.1 deletions, they are also more effective in causing collateral damage that removes hit cells from the bone marrow. After X, gamma or 56Fe-ion irradiation, AML-resistant C57BL/6 mice have fewer cells with PU.1 deletions than CBA mice, and those cells do not persist in the bone marrow of the C57B6/6 mice. Our findings suggest that quantification of PU.1 deleted bone marrow cells 1 month postirradiation can be used as surrogate for the incidence of radiation-induced AML measured in large-scale mouse studies. If so, PU.1 loss could be used to systematically assess the potential leukemogenic effects of other ions and energies in the space radiation environment.

Chromosome aberrations in mitotic bone marrow cells of CBA/Ca and C57BL/6 mice were measured 1 day after exposure to 1 Gy of 1 GeV/nucleon 56Fe ions or 3 Gy of gamma rays. The proportion that have lost a region of chromosome 2 containing the PU.1 gene could be explained by a model based on these measurements. The distribution of aberrations among cells was close to the expected Poisson for the gamma-irradiated cells, but for the HZE 56Fe ions the distribution was highly dispersed. The observations were consistent with the results of an analysis similar to that of Edwards and co-workers in 1980 after ex vivo irradiation of human blood with alpha particles. The analysis used to fit the current data was based on a compound Poisson process, also used previously by others, but in addition included the random nature of parameters involved such as cell nuclear diameter, particle traversal lengths through cell nuclei, production of aberrations, and cell cycle arrest per traversal. From the measured numbers of acentric fragments produced, the relative size of chromosome 2 and the region associated with PU.1 deletions, an independent prediction of PU.1 loss agreed well with measurements described in the accompanying paper.

The mechanisms by which cells accurately distinguish between DNA double-strand break (DSB) ends and telomeric DNA ends remain poorly defined. Recent investigations have revealed intriguing interactions between DNA repair and telomeres. We were the first to report a requirement for the nonhomologous end-joining (NHEJ) protein DNA-dependent protein kinase (DNA-PK) in the effective end-capping of mammalian telomeres. Here, we report our continued characterization of uncapped (as opposed to shortened) dysfunctional telomeres in cells deficient for the catalytic subunit of DNA-PK (DNA-PKcs) and shed light on their consequence. We present evidence in support of our model that uncapped telomeres in this repair-deficient background are inappropriately detected and processed as DSBs and thus participate not only in spontaneous telomere-telomere fusion but, importantly, also in ionizing radiation-induced telomere-DSB fusion events. We show that phosphorylation of DNA-PKcs itself (Thr-2609 cluster) is a critical event for proper telomere end-processing and that ligase IV (NHEJ) is required for uncapped telomere fusion. We also find uncapped telomeres in cells from the BALB/c mouse, which harbors two single-nucleotide polymorphisms that result in reduced DNA-PKcs abundance and activity, most markedly in mammary tissue, and are both radiosensitive and susceptible to radiogenic mammary cancer. Our results suggest mechanistic links between uncapped/dysfunctional telomeres in DNA-PKcs-deficient backgrounds, radiation-induced instability, and breast cancer. These studies provide the first direct evidence of genetic susceptibility and environmental insult interactions leading to a unique and ongoing form of genomic instability capable of driving carcinogenesis.

The phenomenon by which irradiated cells influence non-irradiated neighboring cells, referred to as the bystander effect (BSE), is not well understood in terms of the underlying pathways involved. We sought to enlighten connections between DNA damage repair and the BSE. Utilizing sister chromatid exchange (SCE) frequencies as a marker of the BSE, we performed cell transfer strategies that enabled us to distinguish between generation versus reception of a bystander signal. We find that DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs) and Ataxia Telangectasia Mutated (ATM) are necessary for the generation of such a bystander signal in normal human cells following gamma (gamma)-ray exposure, but are not required for its reception. Importantly, we also show that directly irradiated human cells do not respond to receipt of a bystander signal, helping to explain why the BSE is a low-dose phenomenon. These studies provide the first evidence for a role of the DNA damage response proteins DNA-PKcs and ATM specifically in the generation of a bystander signal and intercellular signaling.

Resveratrol, a polyphenol compound with reported antioxidant and anticarcinogenic effects, a wide range of molecular targets, and toxicity only at extreme doses, has received considerable attention. We evaluated the radioprotective effect of orally administered resveratrol on the frequencies of chromosome aberrations in irradiated mouse bone marrow cells. CBA/CaJ mice were divided into four groups: (1) no treatment, (2) resveratrol only, (3) radiation only, and (4) resveratrol and radiation. Resveratrol treatment (100 mg/kg daily) was initiated 2 days prior to irradiation. Bone marrow was then harvested at 1 and 30 days after a single dose of 3 Gy whole-body gamma radiation. A statistically significant (P < 0.05) reduction in the mean total chromosome aberration frequency per metaphase at both times postirradiation in the resveratrol and radiation group compared to the radiation-only group was observed. This study is the first to demonstrate that resveratrol has radioprotective effects in vivo. These results support the use of resveratrol as a radioprotector with the potential for widespread application.

Interleukin-12 (IL-12), a proinflammatory cytokine, shows anticancer properties. Systemically administered IL-12 causes dose-dependent toxicity. To achieve localized intratumoral gene expression, an adenoviral gene therapy vector with IL-12 controlled by a heat-inducible promoter (heat shock promoter 70B) was developed and tested in a phase I clinical trial in cats with spontaneously arising soft tissue sarcoma. A feasibility study was done in 16 cats with soft tissue sarcoma using murine IL-12 and/or enhanced green fluorescent protein adenoviral vectors under cytomegalovirus or heat shock promoter 70 control. Subsequently, we conducted a phase I clinical trial using an adenoviral feline IL-12 construct in 13 cats with soft tissue sarcoma. The soft tissue sarcomas were irradiated (48 Gy/16 fractions) followed by intratumoral injection of adenovirus. Twenty-four hours postinjection, tumors were heated (41 degrees C, 60 min). Tumor expression of feline IL-12 and IFN-gamma was determined. Cats were monitored for systemic toxicity. For the murine IL-12 construct, an association was noted between viral dose and murine IL-12 levels within tumor, whereas serum levels were minimal. Mild toxicity was noted at 10(11) plaque-forming units (pfu). With the feline IL-12 construct, high levels of feline IL-12 mRNA were detected in tumor biopsies with low or absent IFN-gamma mRNA following gene therapy. Hematologic and hepatic toxicities were noted at the highest viral doses and were associated with detection of IFN-gamma mRNA in tumor. It is possible to localize gene expression and limit systemic toxicity of IL-12 using the hyperthermia-induced gene therapy approach. The maximum tolerated dose of the feline IL-12 adenoviral vector was 10(10) pfu/tumor as dose-limiting toxicities were noted at the 4 x 10(10) pfu dose.

The absence of p53 function increases risk for spontaneous tumorigenesis in the mammary gland. Hormonal stimulation enhances tumor risk in p53-null mammary epithelial cells as well as the incidence of aneuploidy. Aneuploidy appears in normal p53-null mammary epithelial cells within 5 weeks of hormone stimulation. Experiments reported herein assessed a possible mechanism of hormone-induced aneuploidy. Hormones increased DNA synthesis equally between wild-type (WT) and p53-null mammary epithelial cells. There were two distinct responses in p53-null cells to hormone exposure. First, Western blot analysis demonstrated that the levels of two proteins involved in regulating sister chromatid separation and the spindle checkpoint, Mad2 and separase (ESPL1) were increased in null compared with WT cells. In contrast, the levels of securin and Rad21 proteins were not increased in hormone-stimulated p53-null compared with WT cells. ESPL1 RNA was also increased in p53-null mouse mammary cells in vivo by 18 h of hormone stimulation and in human breast MCF7 cells in monolayer culture by 8 h of hormone stimulation. Furthermore, both promoters contained p53 and steroid hormone response elements. Mad2 protein was increased as a consequence of the absence of p53 function. The increase in Mad2 protein was observed also at the cellular level by immunohistochemistry. Second, hormones increased gene amplication in the distal arm of chromosome 2, as shown by comparative genomic hybridization. These results support the hypothesis that hormone stimulation acts to increase aneuploidy by several mechanisms. First, by increasing mitogenesis in the absence of the p53 checkpoint in G2, hormones allow the accumulation of cells that have experienced chromosome missegregation. Second, the absolute rate of chromosome missegregation may be increased by alterations in the levels of two proteins, separase and Mad2, which are important for maintaining chromosomal segregation and the normal spindle checkpoint during mitosis.

Ataxia-telangiectasia (AT) is a genetic syndrome resulting from the inheritance of two defective copies of the ATM gene that includes among its stigmata radiosensitivity and cancer susceptibility. Epidemiological studies have demonstrated that although women with a single defective copy of ATM (AT heterozygotes) appear clinically normal, they may never the less have an increased relative risk of developing breast cancer. Whether they are at increased risk for radiation-induced breast cancer from medical exposures to ionizing radiation is unknown. We have used a murine model of AT to investigate the effect of a single defective Atm allele, the murine homologue of ATM, on the susceptibility of mammary epithelial cells to radiation-induced transformation. Here we report that mammary epithelial cells from irradiated mice with one copy of Atm truncated in the PI-3 kinase domain were susceptible to radiation-induced genomic instability and generated a 10% incidence of dysplastic mammary ducts when transplanted into syngenic recipients, whereas cells from Atm(+/+) mice were stable and formed only normal ducts. Since radiation-induced ductal dysplasia is a precursor to mammary cancer, the results indicate that AT heterozygosity increases susceptibility to radiogenic breast cancer in this murine model system.

Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

We have studied the efficiency of DNA double strand break (DSB) rejoining in primary cells from mouse strains that show large differences in in vivo radiosensitivity and tumor susceptibility. Cells from radiosensitive, cancer-prone BALB/c mice showed inefficient end joining of gamma ray-induced DSBs as compared with cells from all of the other commonly used strains and F1 hybrids of C57BL/6 and BALB/c mice. The BALB/c repair phenotype was accompanied by a significantly reduced expression level of DNA-PKcs protein as well as a lowered DNA-PK activity level as compared with the other strains. In conjunction with published reports, these data suggest that natural genetic variation in nonhomologous end joining processes may have a significant impact on the in vivo radiation response of mice.

Radiation-induced cytogenetic instability has been well documented in a number of laboratories, and we have hypothesized that such instability is the initiating event in the process leading to radiation-induced cancer. To date most studies of radiation-induced instability have used systems in which cells are rapidly dividing. For this phenomenon to have significance for radiation carcinogenesis, it must be established that instability can be induced in vivo in less rapidly dividing fully differentiated tissues known to be at risk. In the present study, we have examined the kinetics of radiation-induced cytogenetic instability in mammary epithelial cells after irradiation in vivo. Having established that instability could arise in vivo in intact mammary tissue, we subsequently demonstrated a dose-response relationship both in vitro and in vivo and demonstrated a lower frequency of instability after fractionated exposures.

A radiosensitive DNA repair-deficient xrs-5 cell line was used to study asbestos cytotoxicity and DNA double strand breaks (DSBs). Although xrs-5 cells did not show any increase in sensitivity to chrysotile fibers in short-term (4-h) treatment when compared with wild-type CHO cells, longer-term exposure (24 h) gave significantly lower cell survival accompanied by a cell growth delay as well as a higher DNA DSB induction in this mutant cell line. These results suggest an important role played by DNA DSBs at the initial stage of asbestos injury.

Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis. The rejoining of DSBs in irradiated cells was significantly inhibited when 20 microM or more of wortmannin was added to the cells. The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration. However, significant sensitization was observed only with a concentration of wortmannin of 20 microM or higher, reflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein. These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs.

There is now substantial evidence that ionizing radiations can induce genomic instability in the form of chromosomal aberrations that appear several cell generations after irradiation. However, questions remain concerning the influence of radiation quality on this phenomenon. In this study, progeny of either gamma- or neutron-irradiated human epithelial MCF-10A cells were examined for chromosomal aberrations between 5 and 40 population doublings postirradiation. Exposure to either type of radiation resulted in an increase in chromatid-type gaps and breaks several doublings after the irradiation; no such effect was observed for chromosome-type aberrations. Neutron-irradiated cells showed consistently elevated frequencies of aberrations compared to nonirradiated controls at all times examined. Aberration frequencies for gamma-irradiated cells were not significantly different from controls until 20 to 35 population doublings postirradiation, where they increased 2-fold above background before returning to near control levels. To our knowledge these data represent the first evidence of chromosomal instability caused by neutron exposure. Results show that while either gamma rays or neutrons are capable of inducing similar types of delayed aberrations, the time course of their appearance can differ markedly.

Genomic instability has been proposed to be the earliest step in radiation-induced tumorigenesis. It follows from this hypothesis that individuals highly susceptible to induction of tumors by radiation should exhibit enhanced radiation-induced instability. BALB/c white mice are considerably more sensitive to radiation-induced mammary cancer than C57BL/6 black mice. In this study, primary mammary epithelial cell cultures from these two strains were examined for the "delayed" appearance of chromosomal aberrations after exposure to 137Cs gamma radiation, as a measure of radiation-induced genomic instability. As expected, actively dividing cultures from both strains showed a rapid decline of initial asymmetrical aberrations with time postirradiation. However, after 16 population doublings, cells from BALB/c mice exhibited a marked increase in the frequency of chromatid-type breaks and gaps which remained elevated throughout the time course of the experiment (28 doublings). No such effect was observed for the cells of C57BL/6 mice; after the rapid clearance of initial aberrations, the frequency of chromatid-type aberrations in the irradiated population remained at or near those of nonirradiated controls. These results demonstrate a correlation between the latent expression of chromosomal damage in vitro and susceptibility for mammary tumors, and provide further support for the central role of radiation-induced instability in the process of tumorigenesis.

Variations in sensitivity to radiation-induced mammary cancer among different strains of mice are well known. However, the reasons for these variations have not been determined. In the present study, the cell dissociation assay was used to determine the radiation-induced transformation frequencies in sensitive BALB/c mice and resistant C57BL mice as well as the resistant hybrid B6CF1 independent of host environment. The influence of host environment on the progression of transformed cells to the neoplastic phenotype was also examined. Results demonstrated that the variations in sensitivity among these sensitive and resistant mice are a result of inherent differences in the sensitivity of the mammary epithelial cells to radiation-induced transformation. Under the conditions used, host environment played no role in the initiation of transformed cells by radiation or in the progression of these cells to the neoplastic phenotype.

EF42 is a clonally derived preneoplastic cell lineage from irradiated mouse mammary tissue, which becomes neoplastic with time in vitro or in vivo. We now report that multiple mutations in p53 occur before the acquisition of the neoplastic phenotype. The selective expansion of mutant cells is accompanied by loss of heterozygosity at the p53 locus and c-myc amplification. Although p53 mutations represent critical early events, our data argue these mutations were not directly induced by radiation but arose in the progeny of irradiated cells several cell generations later. The data are consistent with a multistep model of carcinogenesis that identifies genomic instability as the earliest step.

These studies have examined alterations in the in vivo growth properties of mammary epithelial cells isolated at 1, 4, and 16 weeks after in vivo irradiation with -137Cs gamma rays or fission-spectrum neutrons. Altered in vitro growth potential was characterized by the proliferation of epithelial foci (EF) from irradiated animals under conditions in which mammary cells from nonexposed animals senesced. These EF were further characterized based on their ability to be subcultured. Both gamma and neutron irradiation resulted in the appearance of cells capable of forming EF. Further, with increased time in situ between irradiation and cell isolation, the frequency of EF which were capable of being subcultured indefinitely (EFs) increased. Reducing the gamma-ray dose rate resulted in fewer EFs while reducing the neutron dose rate resulted in increased frequencies of EFs. These data confirm earlier observations following gamma irradiation and show these cellular changes are also observed following neutron irradiation. In addition, these data indicate that changes in dose rate primarily influence the emergence of immortalized cell populations.

The present paper describes experiments conducted over the last several years which have focused primarily on the development of mammary tumors in BALB/c mice after neutron and gamma irradiation. Time-dose relationships for induction of mammary and lung tumors following irradiation with fission-spectrum neutrons and 137Cs gamma rays are described. Subsequent studies have used in vivo/in vitro approaches to characterize cellular and molecular changes. These studies suggest quantitative and qualitative differences in the effects of neutrons and gamma rays.

To examine the cytotoxic effects of ionizing radiation on mouse mammary epithelial cells, a transplantation assay was used to generate a single-dose survival curve. In these experiments, virgin female BALB/c mice were irradiated with 0, 100, 300, 600, 900, 1200, or 1500 rad whole-body 137Cs radiation. Mammary glands of control and irradiated mice were removed and the mammary epithelial cells were enzymatically dissociated, either immediately or 24 hr following irradiation, serially diluted, and injected into gland-free mammary fat pads of 31/2-week-old virgin female BALB/c host mice. The number of cells required to give ductal growth at 10 weeks in half the fat pads (the mammary cell dose 50%, MCD50) was determined by a maximum likelihood procedure and the ratio of MCD50 for nonirradiated control cells to MCD50 for irradiated cells provided an estimate of cell survival. Fitting the data using a single-hit multitarget model resulted in an estimate for D0 of 250 rad with an n of 2.4. No significant difference in the results was obtained whether the cells were dissociated and transplanted immediately after irradiation or the cells were allowed to remain in situ for 24 hr following irradiation. Thus no "in situ repair" was detected for mouse mammary epithelial cells.

Studies in this laboratory have shown enhancement of the mammary tumorigenic effects of neutron irradiation after low-dose-rate neutron exposures. To investigate possible reasons, a mammary cell system was used which allows quantitation of initiated mammary epithelial cells and examination of the progression of these radiation-altered cells toward the neoplastic phenotype. Female BALB/c mice were irradiated with fission-spectrum neutrons at dose rates of 1 rad/min or 1 rad/day. Twenty-four hours or 16 weeks after irradiation, mammary cells were obtained by enzymatic dissociation. Mammary outgrowths were derived by injection of 10(4) cells into gland-free fat pads of 3-week-old female BALB/c mice. The frequency of ductal dysplasias in outgrowths from cells irradiated at high or low dose rates was similar. Persistence of dysplasias differed markedly. Few of the dysplasias in outgrowths derived from cells irradiated at the high dose rate persisted, while a large fraction of the dysplasias in outgrowths derived from cells irradiated at low dose rate persisted. When cells remained in situ for 16 weeks prior to dissociation a higher frequency of persistent altered cells was also observed in outgrowths derived from cells irradiated at low neutron dose rates. These data suggest that low-dose-rate neutron exposures enhance the probability of progression of carcinogen-altered cells rather than increase the numbers of initiated cells.

Virgin female BALB/c mice were exposed in vivo to whole body gamma-radiation and/or to 7,12-dimethylbenz[a]anthracene (DMBA) p.o. Mammary epithelial cells were isolated and assayed for carcinogen altered cell populations both in vitro by an epithelial focus assay and in vivo by injection into cleared fat pads of syngeneic host mice. Five groups of mice were exposed as follows: (a) sham controls; (b) 50-rad gamma-radiation; (c) 100-rad gamma-radiation; (d) 75 micrograms DMBA; or (e) 50-rad gamma-radiation followed in 1 week by 75 micrograms DMBA. Mammary epithelial cells were isolated and assayed at 24 h and at 1, 4, 16, and 52 weeks after in vivo exposure. Four to 12 mice per treatment per time point were individually assayed. Altered in vitro growth potential was characterized by the proliferation of carcinogen exposed (but not control) cells as epithelial foci which persisted at least 12 weeks in primary culture. Epithelial foci which could then be subcultured at least four times were termed subculturable epithelial foci. Altered in vivo morphogenic potential was characterized by dysplastic or neoplastic growth in host fat pads. With increased time in situ between exposure and assay, cell populations emerged which exhibited both increased in vitro subculturability and enhanced tumorigenic potential including a host response upon injection in vivo. Further, combined radiation and DMBA resulted in higher frequencies of subculturable epithelial foci than either treatment alone. The relevance of these progressive cellular changes to the process of mammary tumor development is discussed.

This study has examined the effect of dose rate or fractionation on the carcinogenic effects of fission neutrons with emphasis on the dose range below 50 rad. The induction of lung adenocarcinomas, mammary adenocarcinomas, and ovarian tumors in female BALB/c mice was examined after whole-body neutron irradiation delivered at a high dose rate as a single exposure, or delivered as two equal fractions separated by intervals of 24 hr or 30 days and compared these effects to those after neutron irradiation at low dose rates. The dose responses for ovarian tumorigenesis after the split-dose fractionation regimen were similar to that observed after single high-dose-rate neutron exposure. However, lowering the dose rate reduced the incidence over the dose range of 0-50 rad. For lung and mammary tumors the results were more complex. These data suggest that fractionation and dose-rate effects are different for different tumor types presumably because of the different mechanisms of tumorigenesis that may be involved.

These studies were undertaken to determine if altered growth potential of mammary epithelial cells could be detected in outgrowths derived from monodispersed mammary cells of virgin female BALB/c mice previously exposed to ionizing radiation or 7, 12-dimethylbenz(a)anthracene (DMBA). Monodispersed mammary epithelial cells were obtained by enzymatic dissociation of mammary tissues of 12-week-old virgin female BALB/c mice. Twenty-four hr prior to cell dissociation, donor animals were exposed to either 100 rads of gamma-ray irradiation, 0.25 mg of DMBA, or 0.075 mg of DMBA. Control donors were untreated. Mammary outgrowths were then derived from these donor cells by injecting either 10(5) or 10(4) cells into the gland-free mammary fat pads of three-week-old virgin female BALB/c mice. Ten weeks after the injection of cells, the outgrowths were examined and classified. Mammary outgrowths were classified either as having a normal ductal architecture or as having ductal dysplasia. Ductal dysplasias were further classified on the basis of an index of severity, which was an arbitrary index based on the number of abnormal ductal structures within each lesion. The data indicated that treatment of donor animals with either gamma-radiation or DMBA increased the frequency of ductal lesions over control levels; however, both the frequency and severity of the lesions depended on the number of cells which were injected into the fat pad. When outgrowths were derived by injection of 10(5) cells into the gland-free fat pads, lesion frequencies in outgrowths from control and treated cells were: 3.3%, control; 15.7%, gamma-rays; 5.3%, 0.25 mg DMBA; in these groups only a few severe lesions were detected. In outgrowths derived from 10(4) cells, less severe lesions (Class I lesions) were common in all groups and occurred in approximately 10 to 15% of the outgrowths. The frequency of severe (Class II and III) ductal dysplasia, however, was increased by treatment in these groups, occurring in 4.5% of control outgrowths in 15.6, 14.9, an 14.3% of the outgrowths derived from donor cells treated with 100 rads gamma-rays, 0.075 mg DMBA, nd 0.25 mg DMBA, respectively. Thus, these data indicated that ductal dysplasias were more common and more severe in outgrowths derived from 10(4) rather than 10(5) cells. The ductal lesions observed in this study resembled both morphologically and histologically ductal abnormalities which have been associated with the pathogenesis of mammary carcinoma in both rats and mice.

The antitumor properties of Corynebacterium parvum have been studied in young (3- to 8-month-old) and aged 18 or more months old) BALB/c mice given s.c., i.m., i.p., or i.v. transplants of the highly malignant, weakly immunogenic line 1 lung carcinoma, and in aged (25- to 33-month-old) BALB/c mice bearing primary mammary tumors. These aged BALB/c mice were shown to be less immunoresponsive than their younger counterparts, and this, in combination with nonimmunological factors, made them more sensitive to the lethal effects of the line 1 carcinoma. Correspondingly, C. parvum proved to have less antitumor activity in aged mice than it did in young mice. In spite of this relatively weaker antitumor activity for C. parvum in aged mice, repeated injections of this agent were able to induce temporary regressions of the primary mammary tumors studied and therby prolong survival time.