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.

Background: Ionizing radiation results in an increase in ROS and can be further enhanced in cells with damaged mitochondria, inducing persistent damage and genomic instability, leading to cancer.DNA damage response (DDR) is activated post radiation, and combined with ROS can result in telomere length changes.Mitochondria play a key role in mediating non-targeted effects post radiation exposure.Mitochondrial DNA (mtDNA), due to its less efficient repair as compared to nuclear DNA, is more severely affected by radiation exposure and mutations in essential mtDNA genes can further exacerbate oxidative stress. Aim:We evaluated the impact of mitochondrial defects on radiation-induced DDR and telomere length changes, biomarkers that can foreshadow cancer development. Methods:To better understand this relationship, we have used lymphocytes containing various mitochondrial mutations and studied the kinetics of DNA damage and telomere length changes over time post radiation exposure.In this study we investigated the kinetics of DNA damage response and telomere length changes in cells in lymphoblastoid cell lines with known mitochondrial mutations following a 0.5 Gy dose of Xray or 1 GeV/um Fe ion. Results:Our studies suggest that cells containing different mitochondrial mutations have unique DNA damage and telomere length effects following radiation exposure.The effects on telomere length also reveal differences dependent upon the radiation quality of exposure.Results revealed cells with a mitochondrial mutation in the ND4 subunit of complex 1 showed a decreased growth rate, higher levels of persistent DNA damage, and telomere instability as compared to wild type.In contrast the ATPase 6 mitochondrial mutant showed more subtle changes. Conclusion:These findings point to the importance of mitochondrial integrity and the role it may play in cellular changes that promote cancer.In total results indicate mitochondrial mutations can influence DNA damage kinetics and telomere length and have long-term consequences in regaining homeostasis following radiation exposure.These results may aid in understanding the rationale for the persistent genomic instability following a low dose of radiation and changes leading to cancer promotion.

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.

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 phosphoproteins. 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 (gamma H2AX, 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, gamma H2AX phosphorylation patterns are uniquely different from phosphoproteins 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.

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.

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.

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.

Nonerythroid α spectrin (αIISp) and the Fanconi anemia (FA) protein, FANCD2, play critical roles in DNA interstrand cross-link (ICL) repair during S phase. Both are needed for recruitment of repair proteins, such as XPF, to sites of damage and repair of ICLs. However, the relationship between them in ICL repair and whether αIISp is involved in FANCD2's function in repair is unclear. The present studies show that, after ICL formation, FANCD2 disassociates from αIISp and localizes, before αIISp, at sites of damage in nuclear foci. αIISp and FANCD2 foci do not co-localize, in contrast to our previous finding that αIISp and the ICL repair protein, XPF, co-localize and follow a similar time course for formation. Knock-down of αIISp has no effect on monoubiquitination of FANCD2 (FANCD2-Ub) or its localization to chromatin or foci, though it leads to decreased ICL repair. Studies using cells from FA patients, defective in ICL repair and αIISp, have elucidated an important role for αIISp in the function of non-Ub FANCD2. In FA complementation group A (FA-A) cells, in which FANCD2 is not monoubiquitinated and does not form damage-induced foci, we demonstrate that restoration of αIISp levels to normal, by knocking down the protease μ-calpain, leads to formation of non-Ub FANCD2 foci after ICL damage. Since restoration of αIISp levels in FA-A cells restores DNA repair and cell survival, we propose that αIISp is critical for recruitment of non-Ub FANCD2 to sites of damage, which has an important role in the repair response and ICL repair.

Nonerythroid nuclear A spectrin (ASpII) is critical for repair of DNA interstrand crosslinks (ICLs) and for genomic stability. We have previously shown that there is a deficiency in ASpII in the inherited chromosomal instability disorder, Fanconi anemia (FA), which has a defect in ability to repair DNA ICLs and a predisposition to cancer. Eight FA proteins, FANC -A, B, C, E, F, G, L, and M, form a core complex essential for monoubiquitination of FANCD2 (FANCD2-Ub), a process which is critical for ICL repair. However, whether any of these FA core proteins play additional roles in ICL repair is not clearly known. The present study was undertaken to address this question and to examine whether one of these proteins, FANCF, is involved in steps in the ICL repair process in which ASpII also plays a role. Immunofluorescence microscopy was used to determine whether FANCF co-localizes with ASpII in nuclear foci in normal human cells after they are damaged with an ICL agent, 8-methoxypsoralen plus UVA light (8-MOP). Time course measurements showed that FANCF co-localized in nuclear foci with ASpII and followed a similar time course for formation. This time course was similar to that of the ICL repair protein, XPF, which produces incisions at sites of ICLs and acts downstream of FANCD2-Ub. FANCF foci, like ASpII and XPF foci, were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. This association of FANCF with ASpII was corroborated by co-immunoprecipitation studies which demonstrated that FANCF has enhanced binding to ASpII after ICL damage. These studies indicate that FANCF associates with ASpII and is involved with ASpII in the repair process. Since we have demonstrated that ASpII, like XPF, acts downstream of FANCD2-Ub and that FANCF co-localizes with ASpII after ICL damage, this suggests that FANCF interacts with ASpII in repair events downstream of FANCD2-Ub. In FA-A cells, FANCF is present as in normal cells, but does not form nuclear foci after ICL damage. Transfection of FA-A cells with a cDNA expressing FANCA, however, led to restoration of ASpII levels to normal and to formation of FANCF nuclear foci, which co-localized with ASpII foci. This indicates that ASpII is needed in localization of FANCF to sites of damage. These studies support a model we have proposed in which ASpII acts as a scaffold in the recruitment of proteins involved in the repair process to sites of ICL damage. They also show that FANCF has an additional function in ICL repair besides monoubiquitination of FANCD2. We propose that after DNA damage and monoubiquitination of FANCD2, ASpII and FANCF act downstream of FANCD2, along with XPF, and that the interaction between these proteins at sites of damage is critical for the repair process and maintenance of genomic stability. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. FANCF, a Fanconi anemia core complex protein involved in monoubiquitination of FANCD2, also has a role with nuclear alpha spectrin in DNA interstrand crosslink repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2758.

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.

The NASA Space Radiation Risk project is responsible for integrating new experimental and computational results into models to predict risk of cancer and acute radiation syndrome (ARS) for use in mission planning and systems design, as well as current space operations. The project has several parallel efforts focused on proving NASA's radiation risk projection capability in both the near and long term. This presentation will give an overview, with select results from these efforts including the following topics: verification, validation, and streamlining the transition of models to use in decision making; relative biological effectiveness and dose rate effect estimation using a combination of stochastic track structure simulations, DNA damage model calculations and experimental data; ARS model improvements; pathway analysis from gene expression data sets; solar particle event probabilistic exposure calculation including correlated uncertainties for use in design optimization.

Abstract Genomic instability is a hallmark of the genetic disorder, Fanconi anemia (FA), which is characterized by bone marrow failure, an increased incidence of cancer, congenital abnormalities and a defect in ability to repair DNA interstrand cross-links (ICLs). We have previously shown that FA cells have a deficiency in the structural protein, nonerythroid A spectrin (ASpII), which is critical for repair of DNA ICLs and binds to cross-linked DNA. Eight FA proteins, FANC -A, B, C, E, F, G, L, and M, form a core complex which is essential for monoubiquitination of FANCD2, an important step in ICL repair. However, whether any of these core proteins play additional roles in the ICL repair process is not clearly known. The present study was undertaken to address this question and to examine whether one of these proteins, FANCF, is involved in the ICL repair process with ASpII. Immunofluorescence microscopy was used to determine whether these two proteins co-localize in nuclear foci after normal human cells are damaged with an ICL agent, 8-methylpsoralen plus UVA light (8-MOP). Time course measurements showed that, in normal human cells, FANCF nuclear foci formed over a similar time course as did those of ASpII. This time course was also similar to that of FANCA and the ICL repair protein, XPF. FANCF foci, as well as ASpII, FANCA and XPF foci, were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. FANCF foci, over this time course, co-localized with ASpII foci, indicating that FANCF is associated with ASpII during ICL repair. This association was corroborated by co-immunoprecipitation studies which demonstrated that FANCF has enhanced binding to ASpII after ICL damage. This indicates that FANCF is involved in the same steps in the repair process as ASpII. In FA-A cells, FANCF is present as in normal cells, but does not form nuclear foci after ICL damage. Transfection of FA-A cells with a cDNA expressing FANCA, however, led to restoration of ASpII levels to normal and to formation of FANCF nuclear foci, which colocalized with ASpII foci. Our studies indicate that this is due to restoration of ASpII levels to normal. These studies support a model we have proposed in which ASpII acts as a scaffold in the recruitment of proteins (i.e., FANCF, FANCA, and XPF) to sites of ICL damage and that FA proteins, such as FANCA, are needed for maintenance of ASpII stability in the cell. In the transfected FA-A cells, expression of FANCA leads to enhanced stability of ASpII, which then participates in the ICL repair process. These studies indicate that the FA core complex protein, FANCF, has an additional function in ICL repair, besides monoubiquitination of FANCD2, and is involved with ASpII in its role in the repair process and in maintaining genomic stability after DNA ICL damage. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. FANCF, a Fanconi anemia core protein, functions outside of monoubiquitinating FANCD2 in DNA interstrand crosslink repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3018. doi:10.1158/1538-7445.AM2015-3018

Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, an increased incidence of cancer, genomic instability, congenital abnormalities and a defect in ability to repair DNA interstrand crosslinks (ICLs). We have previously shown that FA cells have a deficiency in the structural protein, nonerythroid alpha spectrin (SpII), which is critical for repair of DNA ICLs and binds to crosslinked DNA. The FA protein, FANCD2, after monoubiquitination (FANCD2-Ub), has also been shown to be critical for ICL repair. However, the relationship between SpII and FANCD2 and whether they are involved in the same steps or events in this process is not known. The present study was undertaken to address these questions and to examine the role of SpII in the function of non-ubiquitinated FANCD2 (non-Ub FANCD2) and FANCD2-Ub in the FA pathway after ICL damage. Immunofluorescence microscopy was used to determine whether these proteins co-localize in nuclear foci after cells are damaged with an ICL agent, 8-methylpsoralen plus UVA light or mitomycin C. Time course measurements showed that formation of FANCD2 foci in normal human cells is different from those of SpII, FANCA and the ICL repair protein, XPF. FANCD2 foci were visible 2 hours after damage, plateaued at 16 hours and were still present at 72 hours. In contrast, SpII, FANCA and XPF foci were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. SpII foci, over this time course, did not co-localize with FANCD2 foci after ICL damage and this is corroborated by co-immunoprecipitation studies which show that non-Ub FANCD2 and FANCD2-Ub dissociate from SpII after ICL damage. Studies knocking down expression of SpII by siRNA show that SpII is not needed for monoubiquitination of FANCD2 or its localization to chromatin and nuclear foci after ICL damage. This indicates that SpII and FANCD2-Ub are involved in different steps in the repair process, functioning sequentially, independently and/or in parallel to each other within the FA network. However, in FA-A cells, where FANCD2 is present but is not monoubiquitinated and does not form nuclear foci after ICL damage, restoration of SpII levels to normal by knocking down µ-calpain, a protease which cleaves SpII, leads to formation of FANCD2 foci. However, FANCD2 is not monoubiquitinated. This indicates that SpII is needed for formation of non-Ub FANCD2 nuclear foci after ICL damage and suggests that this may be an important process in the ICL repair response, particularly since we have previously shown that restoration of SpII levels in FA-A cells to normal leads to reversal of a number of the phenotypic deficiencies in these cells. Thus both FANCD2-Ub and non-Ub FANCD2 may be involved in distinct but important steps in the repair process; SpII appears to be of critical importance in the role of non-Ub FANCD2 in the FA pathway and in maintenance of genomic stability after ICL damage. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. Separate but important roles of αSpII and FANCD2 in the FA pathway after DNA interstrand crosslink damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2014-2380

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 (TGFβ1)-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 TGFβ1 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 TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/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 TGFβ1, but also in the absence of TGFβ1.

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.

A simple, fast, and new ion pair chromatographic method was developed and validated, for the simultaneous estimation of Tramadol hydrochloride and paracetamol in pharmaceutical formulations. The developed method uses acetonitrile: 5% sodium lauryl sulphate: methanol in the ratio of 45:15:40 v/v with flow rate of 1mL/minute. The optimum separation was achieved in less than 5 minutes using WATERS Symmetry C18 column (250mm X 4.6mm, 5μm, i.d.) detection was carried out using UV detector, measuring the response at 217nm. Beer's law was obeyed in the concentration range of 20–187.5μg/mL for tramadol and 20–1625 μg/mL for paracetamol, with a detection limit of 4ng/mL for tramadol and 5ng/mL for paracetamol, and a quantitation limit of 15ng/mL for tramadol and 16ng/mL for paracetamol. Intra-day and Inter-day precision and accuracy of the methods have been established according to the current ICH guidelines. The regression co-efficient (r2) value for tramadol and paracetamol was found to be 0.9998 and 0.9996 respectively. The average recovery for tramadol was 100.03% and 100.28% for paracetamol. No interferences were observed from the excipients. The proposed method was found to be accurate, precise and rapid for the simultaneous estimation of tramadol and paracetamol.

We have previously shown that there is a deficiency in the structural protein, nonerythroid alpha spectrin (alphaIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of alphaIISp and correlates with a decreased level of repair of DNA interstrand cross-links and chromosomal instability in FA cells. An important factor in the stability of alphaIISp is its susceptibility to cleavage by the protease, mu-calpain. We hypothesized that an increased level of mu-calpain cleavage of alphaIISp in FA cells leads to an increased level of breakdown of alphaIISp and that knocking down expression of mu-calpain in FA cells should restore levels of alphaIISp and correct a number of the phenotypic defects observed. The results showed that there is increased mu-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in alphaIISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to mu-calpain. We hypothesize that this binding may lead to inhibition of mu-calpain activity in normal cells. Knocking down mu-calpain by siRNA in FA-A cells restored levels of alphaIISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the chromosomal instability observed after exposure to a DNA interstrand cross-linking agent. These studies indicate that FA proteins may play an important role in maintaining the stability of alphaIISp in the cell by regulating its cleavage by mu-calpain. Thus, by reducing the level of breakdown of alphaIISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.

The structural protein nonerythroid alpha spectrin (alpha IISp) plays a role in the repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), in which there is a defect in ability to repair such cross-links. We have proposed a model in which alpha IISp, whose stability is dependent on FA proteins, acts as a scaffold to aid in recruitment of repair proteins to sites of damage. In order to get a clearer understanding of the proposed. role of FA proteins in maintaining stability of alpha IISp, yeast two-hybrid analysis was carried out to determine whether FA proteins directly interact with alpha IISp and, if so, to map the sites of interaction. Four overlapping regions of wwwctJlSp were constructed. FANCG interacted with one of these regions and specifically with the SH3 domain in this region of alpha IISp. The site of interaction in FANCG was mapped to a motif that binds to SH3 domains and contains a consensus sequence with preference for the SH3 domain of wwwallSp. This site of interaction was confirmed using site-directed mutagenesis. Two FA proteins that did not contain motifs that bind to SH3 domains, FANCC and FANCF, did not interact with the SH3 domain of wwwallSp. These results demonstrate that one of the FA proteins, FANCG, contains a motif that interacts directly with the SH3 domain of (wwwxIISp. We propose that this binding of FANCG to alpha IISp may be important for the stability of alpha IISp in cells and the role wwwwallSp plays in the DNA repair process.

Abstract Abstract 3196 Poster Board III-133 The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . Disclosures No relevant conflicts of interest to declare.