Modeling Radiation-Induced Epithelial Cell Injury in Murine Three-Dimensional Esophageal Organoids.

Esophageal squamous cell carcinoma (ESCC) is a deadly consequence of radiation exposure to the esophagus. ESCC arises from esophageal epithelial cells that undergo malignant transformation and features a perturbed squamous cell differentiation program. Understanding the dose- and radiation quality-dependence of the esophageal epithelium response to radiation may provide insights into the ability of radiation to promote ESCC. We have explored factors that may play a role in esophageal epithelial radiosensitivity and their potential relationship to ESCC risk. We have utilized a murine three-dimensional (3D) organoid model that recapitulates the morphology and functions of the stratified squamous epithelium of the esophagus to study persistent dose- and radiation quality-dependent changes. Interestingly, although high-linear energy transfer (LET) Fe ion exposure induced a more intense and persistent alteration of squamous differentiation and 53BP1 DNA damage foci levels as compared to Cs, the MAPK/SAPK stress pathway signaling showed similar altered levels for most phospho-proteins with both radiation qualities. In addition, the lower dose of high-LET exposure also revealed nearly the same degree of morphological changes, even though only ~36% of the cells were predicted to be hit at the lower 0.1 Gy dose, suggesting that a bystander effect may be induced. Although p38 and ERK/MAPK revealed the highest levels following high-LET exposure, the findings reveal that even a low dose (0.1 Gy) of both radiation qualities can elicit a persistent stress signaling response that may critically impact the differentiation gradient of the esophageal epithelium, providing novel insights into the pathogenesis of radiation-induced esophageal injury and early stage esophageal carcinogenesis.

Dose Fractionation During Puberty Is More Detrimental to Mammary Gland Development Than an Equivalent Acute Dose of Radiation Exposure.

PURPOSE: Computed tomographic (CT) scans in adolescents have increased dramatically in recent years. However, the effects of cumulative low-dose exposures on the development of radiation sensitive organs, such as the mammary gland, is unknown. The purpose of this work was to define the effects of dose rate on mammary organ formation during puberty, an especially sensitive window in mammary development. We used a fractionated low-dose x-ray exposure to mimic multiple higher dose CT scans, and we hypothesized that fractionated exposure would have less of an effect on the number of mammary gland defects compared with an acute exposure. METHODS AND MATERIALS: Female mice were subjected to fractionated low-dose x-ray exposure (10 cGy/d for 5 days), acute x-ray exposure (1 × 50 cGy), or sham exposure. As the wide genetic diversity in humans can play a role in a person's response to irradiation, 2 genetically diverse mouse strains differing in radiation sensitivity (BALB/c-sensitive; C57BL/6-resistant) were used to investigate the role of genetic background on the magnitude of the effect. RESULTS: Unexpectedly, our data reveal that multiple low-dose exposures produce greater immune and mammary defects for weeks after exposure compared with controls. The most pronounced defects being increased ductal branching in both strains and a greater percentage of terminal end buds in the BALB/c strain of mice exposed to fractionated radiation compared with sham. Radiation-induced defects near the terminal end bud were also increased in both strains. CONCLUSIONS: The findings suggest that fractionated low-dose exposures are potentially more damaging to organ development compared with an equivalent, single acute exposure and that genetic background is an important parameter modifying the severity of these effects.

Comparison of signaling profiles in the low dose range following low and high LET radiation.

During space travel astronauts will be exposed to a very low, mixed field of radiation containing different high LET particles of varying energies, over an extended period. Thus, defining how human cells respond to these complex low dose exposures is important in ascertaining risk. In the current study, we have chosen to investigate how low doses of three different ion's at various energies uniquely change the kinetics of three different phospho-proteins. A normal hTERT immortalized fibroblast cell line, 82-6, was exposed to a range of lower doses (0.05-0.5 Gy) of radiation of different qualities and energies (Si 1000 MeV/u, Si 300 MeV/u, Si 173 MeV/u, Si 93 MeV/u, Fe 1000 MeV/u, Fe 600 MeV/u, Fe 300 MeV/u, Ti 300 MeV/u, Ti 326 MeV/u, Ti 386 MeV/u), covering a wide span of LET's. Exposed samples were analyzed for the average intensity of signal as a fold over the geometric mean level of the sham controls. Three phospho-proteins known to localize to DNA DSBs following radiation (γH2AX, pATF2, pSMC1) were studied. The kinetics of their response was quantified by flow cytometery at 2 and 24 h post exposure. These studies reveal unique kinetic patterns based on the ion, energy, fluence and time following exposure. In addition, γH2AX phosphorylation patterns are uniquely different from phospho-proteins known to be primarily phosphorylated by ATM. This latter finding suggests that the activating kinase(s), or the phosphatases deactivating these proteins, exhibit differences in their response to various radiation qualities and/ or doses of exposure. Further studies will be needed to better define what the differing kinetics for the kinases activated by the unique radiation qualities plays in the biological effectiveness of the particle.

Genetic variation and radiation quality impact cancer promoting cellular phenotypes in response to HZE exposure.

There exists a wide degree of genetic variation within the normal human population which includes disease free individuals with heterozygote defects in major DNA repair genes. A lack of understanding of how this genetic variation impacts cellular phenotypes that inform cancer risk post heavy ion exposure poses a major limitation in developing personalized cancer risk assessment astronauts. We initiated a pilot study with Human Mammary Epithelial Cell strains (HMEC) derived from wild type, a p16 silenced derivative of wild type, and various genetic variants that were heterozygote for DNA repair genes; BRCA1, BRCA2 and ATM. Cells strains were exposed to different high and low LET radiation qualities to generate both simple and complex lesions and centrosome aberrations were examined as a surrogate marker of genomic instability and cancer susceptibility post different exposures. Our results indicate that centrosome aberration frequency is higher in the genetic variants under study. The aberration frequency increases with dose, complexity of the lesion generated by different radiation qualities and age of the individual. This increase in genomic instability correlates with elevated check-point activation post radiation exposure. These studies suggest that the influence of individual genetics on cell cycle regulation could modify the degree of early genomic instability in response to complex lesions and potentially define cancer predisposition in response to HZE exposure. These results will have significant implications in estimating cancer susceptibility in genetically variant individuals exposed to HZE particles.

Lesion complexity drives age related cancer susceptibility in human mammary epithelial cells.

Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations and changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population.

Evaluating biomarkers to model cancer risk post cosmic ray exposure.

Robust predictive models are essential to manage the risk of radiation-induced carcinogenesis. Chronic exposure to cosmic rays in the context of the complex deep space environment may place astronauts at high cancer risk. To estimate this risk, it is critical to understand how radiation-induced cellular stress impacts cell fate decisions and how this in turn alters the risk of carcinogenesis. Exposure to the heavy ion component of cosmic rays triggers a multitude of cellular changes, depending on the rate of exposure, the type of damage incurred and individual susceptibility. Heterogeneity in dose, dose rate, radiation quality, energy and particle flux contribute to the complexity of risk assessment. To unravel the impact of each of these factors, it is critical to identify sensitive biomarkers that can serve as inputs for robust modeling of individual risk of cancer or other long-term health consequences of exposure. Limitations in sensitivity of biomarkers to dose and dose rate, and the complexity of longitudinal monitoring, are some of the factors that increase uncertainties in the output from risk prediction models. Here, we critically evaluate candidate early and late biomarkers of radiation exposure and discuss their usefulness in predicting cell fate decisions. Some of the biomarkers we have reviewed include complex clustered DNA damage, persistent DNA repair foci, reactive oxygen species, chromosome aberrations and inflammation. Other biomarkers discussed, often assayed for at longer points post exposure, include mutations, chromosome aberrations, reactive oxygen species and telomere length changes. We discuss the relationship of biomarkers to different potential cell fates, including proliferation, apoptosis, senescence, and loss of stemness, which can propagate genomic instability and alter tissue composition and the underlying mRNA signatures that contribute to cell fate decisions. Our goal is to highlight factors that are important in choosing biomarkers and to evaluate the potential for biomarkers to inform models of post exposure cancer risk. Because cellular stress response pathways to space radiation and environmental carcinogens share common nodes, biomarker-driven risk models may be broadly applicable for estimating risks for other carcinogens.

Defining the Biological Effectiveness of Components of High-LET Track Structure.

During space travel, astronauts are exposed to a wide array of high-linear energy transfer (LET) particles, with differing energies and resulting biological effects. Risk assessment of these exposures carries a large uncertainty predominantly due to the unique track structure of the particle's energy deposition. The complex damage elicited by high charge and energy (HZE) particles results from both lesions along the track core and from energetic electrons, δ rays, generated as a consequence of particle traversal. To better define how cells respond to this complex radiation exposure, a normal hTERT immortalized skin fibroblast cell line was exposed to a defined panel of particles carefully chosen to tease out track structure effects. Phosphorylation kinetics for several key double-strand break (DSB) response proteins (γ-H2AX, pATF2 and pSMC1) were defined after exposure to ten different high-LET radiation qualities and one low-LET radiation (X ray), at two doses (0.5-2 Gy) and time points (2 and 24 h). The results reveal that the lower energy particles (Fe 300, Si 93 and Ti 300 MeV/u), with a narrower track width and higher number and intensity of δ rays, cause the highest degree of persistent damage response. The persistent γ-H2AX signal at lower energies suggests that damage from these exposures are more difficult to resolve, likely due to the greater complexity of the associated DNA lesions. However, different kinetics were observed for the solely ATM-mediated phosphorylations (pATF2 and pSMC1), revealing a shallow induction at early times and a higher level of residual phosphorylation compared to γ-H2AX. The differing phospho-protein profiles exhibited, compared to γ-H2AX, suggests additional functions for these proteins within the cell. The strong correspondence between the predicted curves for energy deposition per nucleosome for each ion/energy combination and the persistent levels of γ-H2AX indicates that the nature of energy distribution defines residual levels of γ-H2AX, an indicator of unrepaired DSBs. Our results suggest that decreasing the energy of a particle results in more complex damage that may increase genomic instability and increase the risk of carcinogenesis.

Protons sensitize epithelial cells to mesenchymal transition.

Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFß1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFß1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFß1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFß Receptor 1 (TGFßR1) kinase inhibitor, can efficiently block TGFß1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFß1, but also in the absence of TGFß1.

Increased Artemis levels confer radioresistance to both high and low LET radiation exposures.

BACKGROUND: Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation. RESULTS: Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage. CONCLUSIONS: These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.

Knockdown of alphaII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair.

Nonerythroid alpha-spectrin (alphaIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that alphaIISp plays in normal human cells and in the repair defect in FA, alphaIISp was knocked down in normal cells using siRNA. Depletion of alphaIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced alphaIISp and XPF nuclear foci. Thus depletion of alphaIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.

Deficiency in incisions produced by XPF at the site of a DNA interstrand cross-link in Fanconi anemia cells.

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.

alphaII-Spectrin interacts with five groups of functionally important proteins in the nucleus.

Nonerythroid alpha-spectrin (alphaSpIISigma( *)) is a structural protein that has been identified in the nucleus of mammalian cells and shown to be involved in DNA repair. It is also deficient in cells from the clinically diverse genetic disorder Fanconi anemia (FA). In order to get a clearer understanding of the role of alphaSpIISigma( *) in DNA repair, and whether it may have other important functions in the nucleus, studies were undertaken to identify specific alphaSpIISigma( *) protein binding partners in the nucleus. The results demonstrate that multiple proteins co-immunoprecipitate with alphaSpIISigma( *) from nuclear extracts from normal human lymphoblastoid and HeLa cells. These can be grouped into five categories: structural proteins, proteins involved in DNA repair, chromatin remodeling proteins, FA proteins, and transcription and RNA processing factors. These studies indicate that alphaSpIISigma( *) may play a role in a number of diverse and important processes in the nucleus and that a deficiency in this protein, as occurs in FA, could affect a number of critical cellular pathways.