Cellular Radiosensitivity Research Articles

Abstract 6180: The role of radiation dose-dependent enhancement of fatty acid oxidation on radiation surviving/resistant lung cancer cells

Abstract Objective: Radiotherapy plays a critical role in the integrated management of lung cancer. However, radioresistance limits the long-term control. Exploring the dynamic changes of metabolic reprogramming in radiation surviving/resistance lung cancer cells is helpful to clarify the metabolic mechanism of radiation resistance and to develop new targets for intervention and early detection. Methods: Multiple lung cancer cell lines were irradiated with different doses (2Gy × 20F, 2Gy × 30F, 2Gy × 40F) in conventional dose fractionation. The cellular radiosensitivity was verified by colony formation assay and neutral comet assay. Cell proliferation ability was determined by EdU assay. Metabonomic analysis was used to identify the differentially expressed metabolites between high-dose radiation-resistant cells and their parent cells. Lipid droplet content was detected by Oil Red O staining. Cell oxygen consumption rate (OCR) was measured by Seahorse XF24e analyzer. Western blot was used to detect the expression of metabolic enzymes. The growth of xenograft tumors from these cell lines in BALB/c nude mice were measured after the treatment of radiation (2Gy × 5F), Etomoxir, or radiation combined with Etomoxir. Results: Compared with parent cells, the radioresitance of surviving/resistant lung cancer cells after different doses of radiation was significantly increased with the increase of radiation exposure. Oil Red O staining showed that fatty deposition of radiation surviving/resistant cells was obviously higher than their parent cells, and more fatty deposition in cells received higher dose of radiation. The ketone body metabolism-related substances, including acetoacetic acid, a metabolite of FAO, were significantly enriched in high-dose radiation-resistant lung cancer cells. The expression of carnitine palmitoyltransferase1 (CPT1) and the OCR in radiation surviving/resistant lung cancer cells were also radiation-dose dependently increased. Etomoxir, an inhibitor of fatty acid oxidation, significantly enhanced the radiosensitivity and decreased the OCR and DNA repair ability of various surviving/resistant lung cancer cells exposed to radiation. We further confirmed that Etomoxir could significantly inhibit proliferation of radiation surviving/resistant cells in vivo, which also presented with radiation-dose dependent model. Conclusions: The establishment of equidifferent dose radiation surviving/resistance lung cancer cell lines in this study provides a preclinical research model for exploring the mechanism of dose-dependent radiation resistance. The enhancement of radiation dose-dependent FAO promotes radiation surviving/resistance of lung cancer cells. CPT1A, a key metabolic enzyme mediating FAO, may be a potential target for treatment of radiation resistant lung cancer. Funding: 81972853, 81572279, 2016J004, LC2019ZD009, 2018CR033. Citation Format: Xiaoxia Zhu, Shangbiao Li, Lijuan Wang, Zhihao Zheng. The role of radiation dose-dependent enhancement of fatty acid oxidation on radiation surviving/resistant lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6180.

Abstract 6507: Inhibition of the translation initiation factor eIF4A1 enhances tumor cell radiosensitivity

Abstract The cellular radioresponse is regulated by constitutively expressed proteins and by new gene expression. We have previously shown that radiation induces changes in gene expression primarily via translational control. Regulation of gene translation largely occurs at the initiation stage, and many factors that regulate initiation impact the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4F is comprised of the cap-binding protein eIF4E, the RNA helicase eIF4A, and the scaffold protein eIF4G. Because eIF4A is considered to regulate the translation of mRNAs involved in cell survival and proliferation, we investigated it as a target for tumor cell radiosensitization. Knockdown of eIF4A1 in the U251 glioblastoma and the HeLa cervical carcinoma cell lines resulted in a decrease in translational efficiency as determined by polysome profiling and an increase in cellular radiosensitivity as determined by clonogenic survival analysis. There was also an increase in γH2AX foci remaining at 24h after irradiation, consistent with an inhibition of DNA double strand break repair. These results suggest that eIF4A serves as a determinant of cellular radiosensitivity. As an additional approach to inhibiting eIF4A, we utilized the rocaglate hydroxamate (-)-SDS-1-021, which prevents eIF4A inclusion into eIF4F. Treatment of U251, HeLa and PSN1 pancreatic carcinoma cells with 10 nM (-)-SDS-1-021 resulted in decreased translational efficiency, which was accompanied by a decrease in protein synthesis. Treatment of the tumor cell lines with (-)-SDS-1-021 immediately before radiation resulted in an increase in radiosensitivity as well as an increase in γH2AX foci remaining at 24h, similar to eIF4A1 knockdown. Importantly, whereas treatment of the normal human fibroblast cell line MRC9 with (-)-SDS-1-021 also inhibited translation and protein synthesis, there was no effect on radiosensitivity. To gain a better understanding of the mechanism of radiosensitization by (-)-SDS-1-021, RNAseq was performed using polysome-bound mRNAs isolated from U251 and MRC9 cells. Cells were treated with 10 nM (-)-SDS-1-021, immediately irradiated (2Gy), and then harvested six hours post-irradiation. In U251 cells treated with 2Gy alone, 192 genes were translationally upregulated. The upregulation of 160 of these genes was inhibited by (-)-SDS-1-021 in the combination treatment group. Similarly, in MRC9 cells, 322 genes were translationally upregulated in response to radiation, and the upregulation of 173 of these was inhibited in the combination treatment group. By Ingenuity Pathway Analysis, the radiation-induced, (-)-SDS-1-021-sensitive genes in U251 were significantly enriched for proteins involved in DNA replication, recombination, and repair, which is in contrast to the MRC9 (-)-SDS-1-021 genes. Altogether, these findings suggest that eIF4A1 is a viable target for tumor radiosensitization. Citation Format: Stacey L. Lehman, Theresa Wechsler, Kayla R. Schwartz, Amy Wahba, Lauren E. Brown, John A. Porco, Jerry Pelletier, Kevin Camphausen, Philip J. Tofilon. Inhibition of the translation initiation factor eIF4A1 enhances tumor cell radiosensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6507.

Non-Coding RNAs in Cancer Radiosensitivity: MicroRNAs and lncRNAs as Regulators of Radiation-Induced Signaling Pathways.

Radiotherapy is a cancer treatment that applies high doses of ionizing radiation to induce cell death, mainly by triggering DNA double-strand breaks. The outcome of radiotherapy greatly depends on radiosensitivity of cancer cells, which is determined by multiple proteins and cellular processes. In this review, we summarize current knowledge on the role of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in determining the response to radiation. Non-coding RNAs modulate ionizing radiation response by targeting key signaling pathways, including DNA damage repair, apoptosis, glycolysis, cell cycle arrest, and autophagy. Additionally, we indicate miRNAs and lncRNAs that upon overexpression or inhibition alter cellular radiosensitivity. Current data indicate the potential of using specific non-coding RNAs as modulators of cellular radiosensitivity to improve outcome of radiotherapy.

The Unfolded Protein Response: Neutron-Induced Therapy Autophagy as a Promising Treatment Option for Osteosarcoma.

Radiotherapy using high linear energy transfer (LET) radiation results in effectively killing tumor cells while minimizing dose (biological effective) to normal tissues to block toxicity. It is well known that high LET radiation leads to lower cell survival per absorbed dose than low LET radiation. High-linear energy transfer (LET) neutron treatment induces autophagy in tumor cells, but its precise mechanisms in osteosarcoma are unknown. Here, we investigated this mechanism and the underlying signaling pathways. Autophagy induction was examined in gamma-ray-treated KHOS/NP and MG63 osteosarcoma cells along with exposure to high-LET neutrons. The relationship between radiosensitivity and autophagy was assessed by plotting the cell surviving fractions against autophagy levels. Neutron treatment increased autophagy rates in irradiated KHOS/NP and MG63 cells; neutrons with high-LETs showed more effective inhibition than those with lower LET gamma-rays. To determine whether the unfolded protein response and Akt-mTOR pathways triggered autophagy, phosphorylated eIF2α and JNK levels, and phospho-Akt, phosphor-mTOR, and phospho-p70S6 levels were, respectively, investigated. High-LET neutron exposure inhibited Akt phosphorylation and increased Beclin 1 expression during the unfolded protein response, thereby enhancing autophagy. The therapeutic efficacy of high-LET neutron radiation was also assessed in vivo using an orthotopic mouse model. Neutron-irradiated mice showed reduced tumor growth without toxicity relative to gamma-ray-treated mice. The effect of high-LET neutron exposure on the expression of signaling proteins LC3, p-elF2a, and p-JNK was investigated by immunohistochemistry. Tumors in high-LET-neutron radiation-treated mice showed higher apoptosis rates, and neutron exposure significantly elevated LC3 expression, and increased p-elF2a and p-JNK expression levels. Overall, these results demonstrate that autophagy is important in radiosensitivity, cell survival, and cellular resistance against high-LET neutron radiation. This correlation between cellular radiosensitivity and autophagy may be used to predict radiosensitivity in osteosarcoma.

The role of radiation dose-dependent enhancement of fatty acid oxidation in radiation surviving/resistant lung cancer cells.

e21724 Background: Radiotherapy plays a critical role in the integrated management of lung cancer. However, radioresistance limits the long-term control. Exploring the dynamic changes of metabolic reprogramming in radiation surviving/resistant (S/R) lung cancer cells is helpful to clarify the metabolic mechanism of radiation resistance and to develop new targets for intervention and early detection. Methods: Cell lines were irradiated with different doses (2Gy × 20F, 2Gy × 30F, 2Gy × 40F) in conventional dose fractionation. The cellular radiosensitivity was verified by colony formation assay and neutral comet assay. Cell proliferation ability was determined by EdU assay. Metabonomic analysis was used to identify the differentially expressed metabolites between high-dose radiation-resistant cells and their parent cells. Lipid droplet content was detected by Oil Red O (ORO) staining. Cell oxygen consumption rate (OCR) was measured by Seahorse XF24e analyzer. Western blot was used to detect the expression of metabolic enzymes. The growth of xenograft tumors from these cell lines in BALB/c nude mice were measured after the treatment of radiation (2Gy×5F), Etomoxir, or radiation combined with Etomoxir. Results: Compared with parent cells, the radioresitance of S/R lung cancer cells after different doses of radiationwas significantly increased with the increase of radiation exposure. ORO staining showed that fatty deposition of radiation S/R cells was obviously higher than their parent cells, and more fatty deposition in cells received higher dose of radiation. The ketone body metabolism-related substances, including acetoacetic acid, a metabolite of FAO, were significantly enriched in high-dose radiation-resistant cells. The expression of carnitine palmitoyltransferase1 (CPT1) and the OCR in radiation S/R cells were also radiation-dose dependently increased. Etomoxir, an inhibitor of fatty acid oxidation, significantly enhanced the radiosensitivity and decreased the OCR and DNA repair ability of various S/R cells exposed to radiation. We further confirmed that Etomoxir could significantly inhibit proliferation of radiation S/R cells in vivo, which also presented with radiation-dose dependent model. Conclusions: The enhancement of radiation dose-dependent FAO promotes radiation surviving/resistance of lung cancer cells. CPT1A, a key metabolic enzyme mediating FAO, may be a potential target for treatment of radiation resistant lung cancer. Funding: 81972853, 81572279, 2016J004, LC2019ZD009, 2018CR033.

Diagnosis of radiosensitive severe combined immunodeficiency disease (RS-SCID) by Comet Assay, management of bone marrow transplantation.

Severe combined immunodeficiency disease (SCID) is a rare inherited severe immunodeficiency, in which functions of T cells and B cells are impaired. SCID is inherited either in X-linked recessive, or autosomal recessive forms, and is either radiosensitive or radioresistant. Artemis (DCLRE1C gene), DNA ligase IV, DNA-PKC, and Cernunnos/XLF proteins are regarded as NHEJ (Non-Homologous End-Joining) proteins that are involved in the repair process of double-strand DNA breaks and their mutations would lead to cellular radiosensitivity. Diagnostic radiosensitivity assays are important for the management of clinical BMT (Bone Marrow Transplantation) conditions, such as what conditioning agents and doses should be used. In this study, five SCID patients and healthy controls were examined. Skin fibroblasts were cultured. After X-irradiation, cells either underwent clonogenic assay or incubated to allow DNA repair and examined by the alkaline comet assay. Finally, DCLRE1C, RAG-1, and RAG-2 genes sequenced. By clonogenic assay, three patients were detected as radiosensitive with possible mutations in NHEJ genes such as DCLRE1C gene. The percentage of DNA in the tail measured by comet assay, in all three patients, was significantly different from the two other patients and the control group (p-value < 0.05). By using Sanger sequencing, a mutation in DCLRE1C gene was detected in one of the radiosensitive patients and two mutations in RAG-1, and RAG-2 genes were detected in the two radioresistant patients. Our findings suggest that comet assay is a fast technique for the diagnosis of the radiosensitive form of SCID and is very suitable for the timely diagnosis of RS-SCID before BMT.

Dual PI3K/mTOR Inhibitor NVP-BEZ235 Enhances Radiosensitivity of Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Due to Suppressed Double-Strand Break (DSB) Repair by Non-Homologous End Joining

The PI3K/Akt/mTOR pathway is frequently altered in human papillomavirus (HPV)-positive and negative squamous cell carcinoma of the head and neck (HNSCC) and overstimulation is associated with poor prognosis. PI3K drives Akt activation and constitutive signaling acts pro-proliferative, supports cell survival, DNA repair, and contributes to radioresistance. Since the small molecule NVP-BEZ235 (BEZ235) is a potent dual inhibitor of this pathway, we were interested whether BEZ235 could be an efficient radiosensitizer. The 50 nM BEZ235 was found to abrogate endogenous and irradiation-induced phosphorylation of Akt (Ser473). The anti-proliferative capacity of the drug resulted in an increase in G1-phase cells. Repair of radiation-induced DNA double-strand breaks (DSBs) was strongly suppressed. Reduction in DSB repair was only apparent in G1- but not in G2-phase cells, suggesting that BEZ235 primarily affects non-homologous end joining. This finding was confirmed using a DSB repair reporter gene assay and could be attributed to an impaired phosphorylation of DNA-PKcs (S2056). Cellular radiosensitivity increased strongly after BEZ235 addition in all HNSCC cell lines used, especially when irradiated in the G0 or G1 phase. Our data indicate that targeting the PI3K/Akt/mTOR pathway by BEZ235 with concurrent radiotherapy may be considered an effective strategy for the treatment of HNSCC, regardless of the HPV and Akt status.

Impact of accumulated alterations in driver and passenger genes on response to radiation therapy.

Although various single genetic factors have been shown to affect radiosensitivity, high-throughput DNA sequencing analyses have revealed complex genomic landscapes in many cancer types. The aim of this study is to elucidate the association between accumulated alterations in driver and passenger genes and radiation therapy response. We used 59 human solid cancer cell lines derived from 11 organ sites. Radiation-induced cell death was measured using a standard colony-forming assay delivered as a single dose ranging from 0 to 12 Gy. Comprehensive genomic data for the cell lines were acquired from the Catalogue Of Somatic Mutations In Cancer v. 80. Random forest classifiers were constructed to predict radioresistant phenotypes using genomic features. The Cancer Genome Atlas data sets were used to evaluate the clinical impact of the genomic feature following radiotherapy. The 59 cancer cell lines harbored either nucleotide variations or copy number variations in a median of 157 genes per cell. Radiosensitivity of the cancer cells was correlated with neither the number of driver gene mutations nor the number of passenger gene mutations. However, the proportion of driver gene alterations to total gene alterations in gene sets selected from the Kyoto Encyclopedia Genes and Genomes predicted radioresistant cells with sensitivity of 85% and specificity of 73%. High probability of radioresistance predicted by the model was associated with worse overall survival following definitive radiotherapy in patients of The Cancer Genome Atlas data sets. Cellular radiosensitivity was associated with the proportion of driver to total gene alterations in the selected oncogenic pathways, which may be a biomarker candidate for response to radiation therapy. These findings suggest that accumulated alterations in not only driver genes but also passenger genes affect radiosensitivity.

MiR-450a-5p inhibits autophagy and enhances radiosensitivity by targeting dual-specificity phosphatase 10 in esophageal squamous cell carcinoma.

Radioresistance reduces the success of therapy for patients with ESCC. Enhancing our understanding of the cardinal principles of radioresistance may improve the response of patients to irradiation. MicroRNAs perform a key role in posttranscriptional regulation, which is linked with the response of tumors to irradiation. Here, we successfully constructed a radioresistant cell line model, ECA109R, from parental esophageal cancer cell line ECA109. We used RNA-Seq analysis and qRT-PCR to compare the miRNA expression profiles of the ECA109 and ECA109R cell lines. The results revealed that miR-450a-5p was downregulated in the radioresistant cells. Functional analysis indicated that miR-450a-5p increases cellular radiosensitivity and suppresses autophagy in ESCC cells. We utilized a luciferase reporter assay to identify the target gene, DUSP10, as an indispensable regulator of the p38 and SAPK/JNK signaling pathways. Upregulation or downregulation of DUSP10 expression could reverse the effects of miR-450a-5p overexpression or inhibition. Tumor xenograft experiments verified that miR-450a-5p overexpression could increase sensitivity to radiation therapy in vivo. In general, our findings indicate that miR-450a-5p is a latent radiosensitizer and may represent a potential novel therapeutic target for radioresistance in ESCC.

Electron Scattering in Conventional Cell Flask Experiments and Dose Distribution Dependency

Electron beam therapy (EBT) is commonly used for treating superficial and subdermal tumors. Previous cellular radiosensitivity research using EBT may be underestimating the contribution from flask wall scattering and the corresponding dose distribution. Single cell suspensions of Chinese hamster ovary (CHO) cells were plated on flasks and irradiated with 3, 4, 7, 9, and 18 MeV energy electron beams from two different institutions, and the spatial locations of surviving colonies were recorded. Gafchromic film dosimetry and Monte Carlo simulations were carried out to determine the spatial electron scattering contribution from the flask walls. Low electron irradiation resulted in an uneven surviving colony distribution concentrated near the periphery of the flasks, while spatial colony formation was statistically uniform at energies above 7 MeV. Our data demonstrates that without proper dosimetric corrections, studies using low energy electrons can lead to misinterpretations of energy dependent cellular radiosensitivity in culture vessels, and radiotherapeutic applications.

5-HT2A and 5-HT3 receptors contribute to the exacerbation of targeted and non-targeted effects of ionizing radiation-induced cell death in human colon carcinoma cells

Purpose: Serotonin (5-HT) is implicated in the underlying mechanisms which mediate cell death following ionizing radiation exposure, however, effects appear to be cell type-dependent. We sought to further characterize the role of 5-HT and 5-HT receptors (5-HTRs) in the exacerbation of cell death following ionizing radiation exposure in human colon carcinoma cells.Materials and methods: We examined the clonogenic survival of colon carcinoma HCT116 cells treated with 5-HT and the selective 5-HTR antagonists ketanserin (5-HT2A) and ondansetron (5-HT3), following exposure to direct ionizing radiation and irradiated cell-conditioned medium (ICCM). The relative expression of these target receptors was measured using western blotting.Results: Western blotting results revealed that relative protein levels of the 5-HT2A and 5-HT3 receptors were similar. 5-HT concentration-dependent increases in cell death that occurred following direct ionizing radiation exposure were abolished by both 5-HTR antagonists. Death of nonirradiated cells recipient of ICCM was increased in a concentration-dependent manner by 5-HT when present during donor cell irradiation. Both 5-HTR antagonists completely abolished the increases in bystander-induced cell death generated by 5-HT. Finally, we show that exposure of cells to 5-HT prior to receipt of ICCM can also dictate the degree of bystander-induced cell death.Conclusions: Our findings demonstrate a definitive role for 5-HT in the exacerbation of cell death following ionizing radiation exposure in colon carcinoma cells and highlight 5-HTRs as potential markers for predicting cellular radiosensitivity.

Erastin, a ferroptosis-inducing agent, sensitized cancer cells to X-ray irradiation via glutathione starvation in vitro and in vivo.

High concentrations of antioxidants in cancer cells are huge obstacle in cancer radiotherapy. Erastin was first discovered as an inducer of iron-dependent cell death called ferroptosis accompanied by antioxidant depletion caused by cystine glutamate antiporter inhibition. Therefore, treatment with erastin is expected to potentially enhance cellular radiosensitivity. In this study, we investigated the influence of treatment with erastin on the radiation efficiency against cancers. The clonogenic ability, glutathione peroxidase 4 (GPX4) expression, and glutathione concentration were evaluated using HeLa and NCI-H1975 adenocarcinoma cell lines treated with erastin and/or X-ray irradiation. For in vivo studies, NCI-H1975 cells were transplanted in the left shoulder of nude mice, and then radiosensitizing effect of erastin and glutathione concentration in the cancer were evaluated. Treatment with erastin induced ferroptosis and decreased the concentration of glutathione and GPX4 protein expression levels in the two tumor cell lines. Moreover, erastin enhanced X-ray irradiation-induced cell death in both human tumor cell lines. Furthermore, erastin treatment of a tumor-transplanted mouse model similarly demonstrated the radiosensitizing effect and decrease in intratumoral glutathione concentration in the in vitro study. In conclusion, our study demonstrated the radiosensitizing effect of erastin on two adenocarcinoma cell lines and the tumor xenograft model accompanied by glutathione depletion, indicating that ferroptosis inducers that reduce glutathione concentration could be applied as a novel cancer therapy in combination with radiotherapy.

Some mutations in the xeroderma pigmentosum D gene may lead to moderate but significant radiosensitivity associated with a delayed radiation-induced ATM nuclear localization

Purpose: Xeroderma Pigmentosum (XP) is a rare, recessive genetic disease associated with photosensitivity, skin cancer proneness, neurological abnormalities and impaired nucleotide excision repair of the UV-induced DNA damage. Less frequently, XP can be associated with sensitivity to ionizing radiation (IR). Here, a complete radiobiological characterization was performed on a panel of fibroblasts derived from XP-group D patients (XPD).Materials and methods: Cellular radiosensitivity and the functionality of the recognition and repair of chromosome breaks and DNA double-strand breaks (DSB) was evaluated by different techniques including clonogenic cell survival, micronuclei, premature chromosome condensation, pulsed-field gel electrophoresis, chromatin decondensation and immunofluorescence assays. Quantitative correlations between each endpoint were analyzed systematically.Results: Among the seven fibroblast cell lines tested, those derived from three non-relative patients holding the p.[Arg683Trp];[Arg616Pro] XPD mutations showed significant cellular radiosensitivity, high yield of residual micronuclei, incomplete DSB recognition, DSB and chromosome repair defects, impaired ATM, MRE11 relocalization, significant chromatin decondensation. Interestingly, XPD transduction and treatment with statins and bisphosphonates known to accelerate the radiation-induced ATM nucleoshuttling led to significant complementation of these impairments.Conclusions: Our findings suggest that some subsets of XPD patients may be at risk of radiosensitivity reactions and treatment with statins and bisphosphonates may be an interesting approach of radioprotection countermeasure. Different mechanistic models were discussed to better understand the potential specificity of the p.[Arg683Trp];[Arg616Pro] XPD mutations.

Mitochondrial fission promotes radiation-induced increase in intracellular Ca2+ level leading to mitotic catastrophe in mouse breast cancer EMT6 cells

Mitochondrial dynamics are crucial for cellular survival in response to various stresses. Previously, we reported that Drp1 promoted mitochondrial fission after x-irradiation and its inhibition resulted in reduced cellular radiosensitivity and mitotic catastrophe. However, the mechanisms of radiation-induced mitotic catastrophe related to mitochondrial fission remain unclear. In this study, we investigated the involvement of cellular ATP production, ROS generation, and Ca2+ levels in mitotic catastrophe in EMT6 cells. Knockdown of Drp1 and Fis1, which are mitochondrial fission regulators, resulted in elongated mitochondria and significantly attenuated cellular radiosensitivity. Reduced mitochondrial fission mainly decreased mitotic catastrophe rather than necrosis and apoptosis after irradiation. Cellular ATP contents in Drp1 and Fis1 knockdown cells were similar to those in control cells. N-acetylcysteine and 2-glucopyranoside ascorbic acid have no effect on mitotic catastrophe after irradiation. The cellular [Ca2+]i level increased after irradiation, which was completely suppressed by Drp1 and Fis1 inhibition. Furthermore, BAPTA-AM significantly reduced radiation-induced mitotic catastrophe, indicating that cellular Ca2+ is a key mediator of mitotic catastrophe induction after irradiation. These results suggest that mitochondrial fission is associated with radiation-induced mitotic catastrophe via cytosolic Ca2+ regulation.

What Does the History of Research on the Repair of DNA Double-Strand Breaks Tell Us?-A Comprehensive Review of Human Radiosensitivity.

Our understanding of the molecular and cellular response to ionizing radiation (IR) has progressed considerably. This is notably the case for the repair and signaling of DNA double-strand breaks (DSB) that, if unrepaired, can result in cell lethality, or if misrepaired, can cause cancer. However, through the different protocols, techniques, and cellular models used during the last four decades, the DSB repair kinetics and the relationship between cellular radiosensitivity and unrepaired DSB has varied drastically, moving from all-or-none phenomena to very complex mechanistic models. To date, personalized medicine has required a reliable evaluation of the IR-induced risks that have become a medical, scientific, and societal issue. However, the molecular bases of the individual response to IR are still unclear: there is a gap between the moderate radiosensitivity frequently observed in clinic but poorly investigated in the publications and the hyper-radiosensitivity of rare but well-characterized genetic diseases frequently cited in the mechanistic models. This paper makes a comprehensive review of semantic issues, correlations between cellular radiosensitivity and unrepaired DSB, shapes of DSB repair curves, and DSB repair biomarkers in order to propose a new vision of the individual response to IR that would be more coherent with clinical reality.

The Cell Cycle G2/M Block Is an Indicator of Cellular Radiosensitivity.

Background:Determination of the radiosensitivity of a specific tumor is essential to its precision tumor radiotherapy, but the measurement of cellular radiosensitivity with a routine colony forming assay is both labor- and time-consuming. An alternative option allowing rapid and precise prediction of radiosensitivity is necessary.Methods:In this study, we exposed 4 in vitro cultured cell lines to various doses of X-rays or carbon ions and then measured their radiosensitivities with a routine colony-forming assay, and monitored the kinetics of cell cycle distribution with routine propidium iodine staining and flow cytometry.Results:Based on the results, we correlated cellular radiosensitivity with a dynamic assay of cell cycle distribution, specifically, the negative correlation of cellular radiosensitivity with the accumulated G2/M arrested cells at 48 hours after exposure. The higher the proportion of accumulated G2/M arrested cells at 48 hours after exposure, the lower the radiosensitivity of the cell line, that is, the higher radioresistance of the cell line.Conclusion:These findings provide an optional application of regular cell cycle analysis for the prediction of tumor radiosensitivity.

Evaluation of the effect of three constituent metals of monazita on the radiosensibility of human osteoblasts

Thorium has gained notoriety in recent years, as a potential source of nuclear energy, substituting uranium in power plants. Monazite is an important source of thorium, as well of uranium and rare earths elements. Workers involved in the extraction and manipulation of this mineral are occupationally exposed to a range of metal mixtures containing thorium and to ionizing radiation. As an osteotropic substance, thorium is mostly deposited in bone tissue and may interfere in cellular radiosensitivity. A human osteoblast cell line was used to evaluate the effects of thorium (Th), cerium (Ce) and lanthanum (La) on cell radiosensivity, using proliferation as indicator. Assays were performed using cell cultures exposed to metals alone and metals combined with ionizing radiation. No stimulus of proliferation was observed when samples were exposed to metals or radiation alone. On the other hand, the metals were able to influence cell radiosensivity, in a concentration-dependent manner when metals and radiation were applied simultaneously. Samples irradiated and exposed to metals combinations revealed an interaction between them in all the tested arrangements (Th-Ce, Th-La, Th-Ce-La). All interactions proved to be of the antagonist type relative to the proliferation indicator, with a higher degree seen for the Th-Ce association. Such results showed the possibility that metal mixtures together with radiation may produce combined effects on osteoblasts, through modifications on the degree of radiosensivity. The results indicate the possibility of an enhancement in occupational risk for workers that manipulate monazite byproducts. Thus, the development of risk assessment models that include the evaluation of mixtures and their cytotoxic and radiotoxic effects on tissues and organs must be highlighted.

Abstract 2921: A dual role of genomic instability on radiosensitivity in cancer cells

Abstract Background: Genomic instability is a hallmark of cancer, which may lead to heterogeneity and provide an advantage on tumor survival and proliferation. However, we previously found that a greater number of genomic mutations were associated with reduced tolerance to radiation. The aim of this study is to elucidate a role of genomic instability on cellular radiosensitivity. Materials and Methods: We used 59 cancer cell lines derived from various types of solid tumors. Radiation-induced cell death was measured by the standard colony-forming assay at the dose range of 0 to 10 Gy. Based on the resulting survival curves, the mean inactivation dose (MID) was calculated as a single value representing cellular radiosensitivity. Somatic gene aberration data for the 59 cell lines were obtained from the Catalogue Of Somatic Mutations In Cancer cell line project v.80. The genomic analysis focused on cancer-related pathways defined by Kyoto Encyclopedia Genes and Genomes. The genomic mutations included nucleotide variations (substitutions, insertions, or deletions) and copy number variations (CNV). Only pathogenic mutations were selected and neutral mutations were disregarded according to the functional analysis through hidden Markov model. Both gain and loss in CNVs were included. Results: There were either nucleotide variations or copy number variations in 598 genes in the 59 cancer cell lines. The mutations were found in 16 caretakers, 51 gatekeepers, 105 oncogenes, and 449 passengers. There were significant positive correlations between the MIDs and the proportions of mutations in the gatekeepers or the oncogenes to the whole mutations. Namely, radioresistant cell lines harbored gatekeeper mutations or oncogene mutations at higher proportions. Conversely, radiosensitive cell lines showed significantly higher proportions of passenger mutations. No correlation was found in between the MIDs and the proportions of caretaker mutations. Conclusions: Genomic instability caused by caretaker mutations may result in not only increased tolerance to radiation through mutations in gatekeeper genes and oncogenes but also reduced tolerance to radiation through accumulating passenger mutations. A balance between the two opposing phenomena is a significant determinant of cancer cell radiosensitivity. Citation Format: Yuji Seo, Keisuke Tamari, Yutaka Takahashi, Kazumasa Minami, Keisuke Otani, Fumiaki Isohashi, Kazuhiko Ogawa. A dual role of genomic instability on radiosensitivity in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2921.

DNA Damage and Cytokine Production in Non-Target Irradiated Lymphocytes.

In advanced radiotherapy, treatment of the tumor with high-intensity modulated fields is balanced with normal tissue sparing. However, the non-target dose delivered to surrounding healthy tissue within the irradiated volume is a potential cause for concern. Whether the effects observed are caused after exposure to out-of-field radiation or bystander effects through neighboring irradiated cells is not fully understood. The goal of this study was to determine the effect of exposure to out-of-field radiation in lymphocyte cell lines and primary blood cells. The role of cellular radiosensitivity in altering bystander responses in out-of-field exposed cells was also investigated. Target cells were positioned in a phantom in the center of the radiation field (in-field dose) and exposed to 2 Gy irradiation. Lymphocyte cell lines (C1, AT3ABR, Jurkat, THP-1, AT2Bi and AT3Bi) and peripheral blood were placed 1 cm away from the radiation field edge (out-of-field dose) and received an average dose of 10.8 ± 4.2 cGy. Double-stranded DNA damage, cell growth and gene expression were measured in the out-of-field cells. Radiosensitive AT3ABR and primary blood cells demonstrated the largest increase in γ-H2AX foci after irradiation. Exposure of normal cells to bystander factors from irradiated radiosensitive cell lines also increased DNA damage. Expression of IL-1, IL-6, TNFα and TGFβ after addition of bystander factors from radiosensitive cells showed differential effects in normally responding cells, with some evidence of an adaptive response observed. Exposure to out-of-field radiation induces DNA damage and reduces growth in radiosensitive cells. Bystander factors produced by directly irradiated cells in combination with out-of-field exposure may upregulate pro- and anti-inflammatory genes in responding cells of different radiosensitivities, with the potential of affecting the tumor microenvironment. A greater understanding of the radio-biological response in normal cells outside the primary treatment field would assist in radiation treatment planning and in reducing early and late toxicities.

EP-2161 miR-454-3p regulates cellular radio-sensitivity by targeting to BTG1 in renal carcinoma cells

EP-2161 miR-454-3p regulates cellular radio-sensitivity by targeting to BTG1 in renal carcinoma cells