Increase In Radioresistance Research Articles

Исследование геропротекторных и радиопротекторных эффектов берберина и трихостатина А на модели Drosophila melanogaster

The search for drugs that slow down the rate of aging and stimulate radioresistance is an urgent task of biology, ecology, and medicine. In this work we have studied the effects of trichostatin A and berberine on the lifespan, resistance to the paraquat prooxidant and acute gamma irradiation of the Drosophila melanogaster fruit fly. Trichostatin A demonstrated a better geroprotective effect, increasing the Drosophila lifespan by 3-9 %. However, an increase in radioresistance was observed only after the application of berberine in males. Their median survival after gamma irradiation increased by 25 %. Trichostatin A, on the contrary, increased the sensitivity of flies to gamma irradiation genotoxic effects, reducing the survival by 7-17 %.

488 SNAIL TRANSFECTION INDUCES EPITHELIAL-MESENCHYMAL TRANSITION WITH PROPERTIES OF CANCER STEM-LIKE CELL IN ESOPHAGEAL SQUAMOUS CELL CANCER

Abstract Cancer stem-like cell (CSC) is an important player in tumorigenesis and tumor progression. Snail has been demonstrated as a key driver for epithelial-mesenchymal transition (EMT) that is closely linked with generation of stem-like cell in human cancer. We aim to investigate if Snail transfection induces EMT and properties of stem-like cell in esophageal squamous cell carcinoma (ESCC) cell lines. Methods A lentivirus system was used to transfect human Snail via a plasmid pLV-EF1a-hSnail-Hyg into KYSE-170 and KYSE-510 ESCC cell lines. Immunoblotting was used to determine expression of EMT associated markers including Vimentin, E-cadherin, Fibronectin and N-cadherin. Assays for cell migration and invasion were conducted in Snail-transfected cells and its vector control. Cytotoxicity (MTT) and cell proliferation assay was used to determine cell growth. Sphere formation assay and flow cytometry (FCM) were employed to characterize stem cell properties while examining expression of stemness genes by using real-time polymerase chain reaction (PCR). Results After Snail transfection, mesenchymal markers, Vimentin, N-cadherin increased, whereas epithelial marker E-cadherin decreased. Snail-transfected. ESCC cells presented a significant increase in RNA expression of stemness genes such as Nanog, Oct4, ABCG2 and Sox2 with an induction in sphere formation. Moreover, ability of cell migration and invasion increased after Snail-transfection in ESCC with increased chemoresistance to cisplatin, taxol, and 5-Fluorouracil(5-FU) and an increase in radioresistance as well. Conclusion Our results demonstrated that Snail transfection induced EMT with properties of CSC in ESCC cell lines.

Multicellular Spheroids as In Vitro Models of Oxygen Depletion During FLASH Irradiation

The differential response of normal and tumor tissues to ultrahigh-dose-rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study was to investigate these effects within hypoxic multicellular tumor spheroids through simulations and experiments. Physicobiological equations were derived to model (1) the diffusion and metabolism of oxygen within spheroids; (2) its depletion through reactions involving radiation-induced radicals; and (3) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored postradiation. Clonogenic survival of 2 other cell lines was also investigated. The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroids compared with conventional irradiation, but no significant difference was observed for well-oxygenated 2-dimensional cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy. Tumor spheroids can be used as a model to study FLASH irradiation invitro. The improved survival of tumor spheroids receiving FLASH radiation confirms that ultrafast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect.

P2.03-59 The Role of Radiation Dose-Dependent Lipid Metabolism Reprogramming on Radiation Survival/Resistance in Lung Cancer Cells

Radiotherapy plays a more and more important role in the management of lung cancer. However, radioresistance still limits the long-term control of tumors. Exploration of the dynamic changes of metabolic reprogramming in surviving/resistant lung cancer cells exposed to increased doses of radiation is helpful to elucidate the metabolic mechanism of radiation resistance in lung cancer cells, to develop new targets for the clinical intervention and the early detection of radiation resistance. Lung cancer cells A549, H520 and H460 were treated in vitro with fractionated radiation (2 Gy) to a cumulative total dose of 40Gy, 60Gy or 80Gy. Cellular radiation sensitivity was verified by colony survival assay and comet experiments. Cell proliferation was determined by EdU assay. Transcriptome sequencing and metabolomic analysis were performed to identify differentially expressed genes and metabolites in radiation-resistant cells and their parental cells. Oil Red O staining was used to detect lipid droplet content. RT-PCR and WB were used to detect the expression of genes related to lipid metabolism. Lung cancer cell sublines that had been exposed to 20, 30 or 40 fractions of 2Gy had a significant increase in radioresistance as compared with their parental cells. Furthermore, this radioresistance of cells increased with the increase of previous radiation dose exposure. We found that lipid droplet deposition and the expression of lipid metabolism genes, such as fatty acid oxidation-related genes and glycerol metabolism-related genes, showed an increasing trend with previous radiation dose in surviving/resistant lung cancer cells exposed to radiation. Metabolomic analysis revealed that lipid metabolites such as glycerol and oxaloacetic acid also increased significantly. Inhibition of carnitine palmitoyltransferase 1A (CPT1A) by Etomoxir, a fatty acid oxidation inhibitor, significantly enhanced the radiosensitivity and decreased the DNA repair ability of various surviving/resistant lung cancer cells exposed to radiation. The surviving/resistant lung cancer cells generated by sequential irradiation provide models for future investigations of the dose-dependent mechanisms of radioresistance in a preclinical setting. Radiation dose-dependent lipid metabolism reprogramming may contribute to radiation survival/resistance in lung cancer cells. Support: 81572279, 2016J004, LC2016PY016, 2018CR033.

The CD98 Heavy Chain Is a Marker and Regulator of Head and Neck Squamous Cell Carcinoma Radiosensitivity.

The heavy chain of the CD98 protein (CD98hc) is encoded by the SLC3A2 gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High SLC3A2 mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein-mediated molecular mechanisms of tumor radioresistance. CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD50) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC). High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC in vitro and in vivo models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx. We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.

Rationale for Combining Radiotherapy and Immune Checkpoint Inhibition for Patients With Hypoxic Tumors.

In order to compensate for the increased oxygen consumption in growing tumors, tumors need angiogenesis and vasculogenesis to increase the supply. Insufficiency in this process or in the microcirculation leads to hypoxic tumor areas with a significantly reduced pO2, which in turn leads to alterations in the biology of cancer cells as well as in the tumor microenvironment. Cancer cells develop more aggressive phenotypes, stem cell features and are more prone to metastasis formation and migration. In addition, intratumoral hypoxia confers therapy resistance, specifically radioresistance. Reactive oxygen species are crucial in fixing DNA breaks after ionizing radiation. Thus, hypoxic tumor cells show a two- to threefold increase in radioresistance. The microenvironment is enriched with chemokines (e.g., SDF-1) and growth factors (e.g., TGFβ) additionally reducing radiosensitivity. During recent years hypoxia has also been identified as a major factor for immune suppression in the tumor microenvironment. Hypoxic tumors show increased numbers of myeloid derived suppressor cells (MDSCs) as well as regulatory T cells (Tregs) and decreased infiltration and activation of cytotoxic T cells. The combination of radiotherapy with immune checkpoint inhibition is on the rise in the treatment of metastatic cancer patients, but is also tested in multiple curative treatment settings. There is a strong rationale for synergistic effects, such as increased T cell infiltration in irradiated tumors and mitigation of radiation-induced immunosuppressive mechanisms such as PD-L1 upregulation by immune checkpoint inhibition. Given the worse prognosis of patients with hypoxic tumors due to local therapy resistance but also increased rate of distant metastases and the strong immune suppression induced by hypoxia, we hypothesize that the subgroup of patients with hypoxic tumors might be of special interest for combining immune checkpoint inhibition with radiotherapy.

Relationship between radioadaptive response and individual radiosensitivity to low doses of gamma radiation: an extended study of chromosome damage in blood lymphocytes of three donors

Purpose: Our study aimed at evaluating: 1) whether well-established variability in radioadaptive response (AR) in various donor blood lymphocytes might be attributed to inter-individual differences in radiosensitivity to different low dose levels; 2) whether AR is reproducibly present over time in the lymphocytes of AR-positive individuals.Experimental procedure: Whole blood samples of three donors were exposed to low doses (2–30 cGy) of γ-radiation alone (G0 phase) or followed by a 1 Gy challenge dose (late S/early G2 phase), and chromosome aberration were scored to assess the dose-response relationship and adaptive response, correspondingly. Three experiments were performed on blood samples of the same donors at six month intervals.Results: Significant differences in dose response relationship for blood lymphocytes were found among individuals. In most cases, the donors exhibited initial low-dose hypersensitivity (HRS) followed by an increase in radioresistance (IRR). AR could be successfully induced by some particular priming doses in the lymphocytes of each donor; however, the doses resulting in a protective response were quite different for all three donors. These protective doses could equally belong to either HRS or IRR region on the individual dose-response curves. In most cases, no clear AR outcome dependence on the priming dose was found at all. Moreover, pre-exposure to the same low dose could result in opposite effects in the lymphocytes of the same donor in different experiments.Conclusions: AR variability in human lymphocytes is not attributed to variation in radiosensitivity among individuals and is more drastic than was believed. It seems doubtful that AR is a universal phenomenon which has a consistent impact on the effects of radiation exposure on humans.

Abstract LB-210: IL-6 promotes stemness of cancer stem cells and may contribute to radiation resistance of non-small cell lung cancer

Abstract Background: Lung cancer is the leading cause of cancer-related deaths among both men and women. Long-term survival of lung cancer patients remains low despite aggressive therapies including surgery, radiation, and chemotherapy. Ionizing radiation (IR) is the standard therapy for patients diagnosed with localized unresectable non-small cell lung cancer (NSCLC). However local tumor control by standard fractionated radiotherapy (RT) remains poor due to tumor resistance to IR. Accumulating evidences have indicated that cancer stem cells (CSCs, also known as cancer initiating cells) exist as a very minor population in NSCLC tumors, which are composed of heterogeneous cell populations. Our laboratory and others found that CSC population increases upon irradiation, which may contribute to radiation resistance. The role of IL-6 in NSCLC progression has been suggested since high IL-6 levels were detected in sera of lung cancer patients, but not detectable in most patients with benign lung diseases. However, the exact mechanism by which IL-6 influences lung cancer progression and its role in CSCs mediated radioresistance remain unresolved. Methods: We isolated CD133+ CSC population from 3 NSCLC cell lines (NSCLC-CSCs) and studied the role of IL-6 in CD133+ CSCs growth by treating cells with IL-6. In addition, we manipulated IL-6 expression levels in the IL-6 expressing A549 and H157 cells by the knockdown of IL-6 using lentiviral infection system carrying IL-6siRNA and isolated CD133+ cells for further analyses. The growth and stem cell marker expressions of these CSCs, IL-6 treated or non-treated, or IL-6 expressing or knocked down cells upon irradiation were then investigated. The differences in the activation of signaling pathways and neuroendocrine differentiation were also studied. Results and Conclusions: We found that IL-6 increased self-renewal of NSCLC-CSCs significantly and thereby increased NSCLC-CSC population. We also found that IL-6 increased stemness of the isolated CD133+ CSCs when CD133+ cells were treated with IL-6. More importantly, when we irradiated the CD133+ CSCs isolated from the IL-6 knocked down and scramble control A549 cells, we found that the CSCs isolated from the IL-6 knocked down cells exhibited lower survival than the CSCs of control cells. Therefore, we suggest that IL-6 may contribute to NSCLC-CSCs mediated-radioresistance via (i) direct triggering radioresistance, and (ii) increasing self-renewal and stemness of the survived CSCs. Mechanistic dissection studies revealed that the increased self-renewal of CSCs by IL-6 is due to the activation of the hedgehog signaling and the increased expression of bcl-2, while the IL-6 mediated increase of radioresistance is due to the activation of neuroendocrine differentiation, Stat 3 signaling pathway, and increased expression of bcl-2. Citation Format: Yuhchyau Chen, Ying Tsai, Jong-Wei Hsu, Shan-Zhou Duan, Peter Keng, Soo Ok Lee. IL-6 promotes stemness of cancer stem cells and may contribute to radiation resistance of non-small cell lung cancer. [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 LB-210. doi:10.1158/1538-7445.AM2014-LB-210

Radiation tolerance of Bacillus cereus pre-treated with carvacrol alone or in combination with nisin after exposure to single and multiple sub-lethal radiation treatment

Currently little information exists on the response of Bacillus cereus after repetitive exposure to combined mild treatments involving antimicrobials and gamma irradiation. Therefore, the aim of this present study was to evaluate the radiation stress on growth and physiology of B. cereus LSPQ 2872 vegetative cells at stationary phase, following exposure to single and repetitive sub-lethal γ-radiation treatment at 1 kGy simultaneously with carvacrol alone or combined with nisin at sub-inhibitory concentrations. Results indicated that the combination of carvacrol and nisin significantly enhance the radiation sensitivity of B. cereus since lower D10 values were recorded after both single and repetitive irradiation treatments. Flow cytometric analysis of radiation-stressed B. cereus cells following repetitive treatment revealed the heterogenic behaviour of the bacterium leading to the induction of a radiation tolerance response. When compared to carvacrol alone, the combination of carvacrol and nisin developed also increased radioresistance if repetitively processed with γ-radiation at 1 kGy, since the decrease percentage of dead cells was accompanied by an increase in the number of injured cells. However, good agreement was not found between classical plate counting (log cfu reductions) and flow cytometry method. For both antimicrobials, the increase of radioresistance after repetitive mild treatment was not accompanied by augmentations of lag phase or growth rate. The structural changes of the outer membranes were assessed by TEM analysis and results revealed that radioresistance might be related to changes in the cell wall.

HELIUM-NEON LASER PROVIDE A CHANGE TO THE RADIATION RESISTANCE OF BIOLOGICAL CELLS

Розглянуто публікації, присвячені підвищенню радіаційної стійкості клітинних культур шляхом їх опромінення лазером. Показано, що реалізуються протилежні результати в залежності від того, чи використовуються інтенсивност, менші ніж “дотеплова” величина, або навпаки.

Abstract 2132: Characterization of galectin 3 as a novel BARD1 interaction partner

Abstract BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid (Y2H) screen as a binding partner of BRCA1. The functional heterodimer BRCA1/BARD1 is required for several of the cellular and tumor-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair pathway, transcription, cell cycle control, and ubiquitination. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a Y2H screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for galectin 3 C-terminal region, residues 170-250 (OMIM 153619). Galectin 3 is a galactoside binding protein involved in several cellular processes including apoptosis control and RNA splicing. Moreover there are evidences that galectin 3 variants are correlated with tumor development and progression in breast and thyroid cancer. We confirmed galectin 3/BARD1 interaction conducting co-immunoprecipitation analysis of ectopically expressed BARD1 and galectin-3 in HEK293T cells. The interaction between endogenous galectin 3 and BARD1 were validated in HeLa nuclear extracts, and by confocal microscopy. Using ectopically expressed galectin 3 fragments we determined the interaction region (aa 100-215). Moreover, galectin 3 fragment comprising aminoacids 100-165 is able to disrupt the endogenous interaction with BARD1. Co-immunoprecipitation assays suggested the presence of BRCA1, BARD1 and galectin-3 in the same complex. Interestingly the galectin 3 observed in this complex corresponds to a mono-ubiquitinated form. This ubiquitination reaction occurs mainly in the nucleus but is not BRCA1 dependent. The role of galectin 3 in DNA damage pathway was investigated using galectin 3 silenced HeLa cells exposed to increasing doses of ionizing radiation. These cells exhibited a slightly increase in radioresistance, but no effect was observed in cell cycle control. In addition the phosphorylation of histone H2AX seems to be delayed after damage in cells lacking galectin 3. Other DNA damage agents, such as carboplatin and etoposide did not affected differently the cell viability of these cells. We are now evaluating the protein interaction network of galectin 3 using a tandem affinity purification approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2132. doi:1538-7445.AM2012-2132

Impact of Epidermal Growth Factor Receptor Expression on Disease-Free Survival and Rate of Pelvic Relapse in Patients With Advanced Cancer of the Cervix Treated With Chemoradiotherapy

To determine the impact of the expression of epidermal growth factor receptor (EGFR) on disease-free survival (DFS) and on pelvic relapse in patients with advanced cancer of the cervix receiving concurrent chemoradiotherapy. In 112 consecutive patients with advanced cancer of the cervix (11 stage IB2-IIA, 25 IIB, 63 IIIB, 13 IVA) treated with chemoradiotherapy between December 1994 and September 2004, the expression of EGFR using histoimmunochemistry was measured and used in univariate and multivariate analysis, along with variables such as age, International Federation of Gynecology and Obstetrics Staging System for Epithelial Ovarian Cancer (FIGO) stage, histology, Eastern Cooperative Oncology Group (ECOG), tumor size, and ganglia involvement diagnosed with computerized axial tomography, treatment with cisplatin to evaluate its impact on DFS and pelvic relapse. Of the 112 biopsies, 32 (28.6%) were negative or slightly positive (EGFR±) and 80 (71.4%) were moderate or intensely positive (EGFR++/+++). The overexpression of EGFR (++/+++) was significantly associated with an epidermoid histology (P < 0.0001), with a higher rate of pelvis relapse and a decreased DFS (hazard ratio [HR]: 2.31 [1.08-4.96]; P = 0.03). Overall, treatment with cisplatin increased DFS (HR: 0.51 [0.26-0.97]; P = 0.04). Patients with tumors of the cervix and overexpression of the EGFR++/+++ show a higher probability of pelvic relapses and a decreased disease-free survival. The poor prognosis of these tumors may be a consequence of an increase in radio-resistance.

Abstract 4276: In-vivo imaging of glioma cancer stem cells

Abstract Recent data support that glioblastoma (GB) is organized hierarchically with a small population of cancer stem cells (CSCs), able to repopulate an entire tumor. The cell of origin in GB is currently unknown, but animal data suggest that CSCs derive from the normal SC population and that the normal stem cell niche in the brain may harbor CSCs in patients with GB. Because therapies targeting CSCs directly may potentially improve patient outcome, novel means are needed to track CSCs and their interaction with the tumor microenvironment to develop and test novel therapeutic approaches. Using limiting dilution assays we studied if glioma CSCs (GCSCs) can be serially transplanted in Rag2−/− γc −/− KO mice. Radiation responses of the population of glioma GCSCs were investigated using FACS. Radiation sensitivity assessed in sphere forming capacity assays. To studying proteasome activity in normal neuronal stem cells a transgenic animal model was generated that reports activities of the proteasome. Finally, the role of the normal tissue SC niche for radiation therapy outcome was investigated in a retrospective analysis of patient data. GCSCs showed increased tumorigenicity over non-tumorigenic cells in the Rag2−/− γc −/− mouse model supporting a hierarchical CSC model in GB. GCSCs could be serially transplanted over at least 5 generations and reassembled the typical histopathology of GB. When unselected glioma cells were subjected to fractionated irradiation, cells were highly enriched for GSCSs. We did not detect differences in H2AX phosphorylation directly after irradiation, which was consistent with previous studies reporting comparable amounts of initial DNA damage in GCSCs and non-tumorigenic cells. Single cell sphere formation after irradiation indicated a 1.3-fold increase in radioresistance for GCSCs (measured at 10% survival) supporting previously reported increased DNA repair in CSCs. Using sections of brains from transgenic animals, which report lack of proteasome function by accumulation of ZsGreen, we identified a rare cell population in the subventricular zone of the brain that lacks proteasome function, consistent with normal stem cell origin of GCSCs. Clinically, radiation dose of 43Gy and higher delivered to the periventricular region of the brain of patients with high-grade glioma was correlated with improved progression-free survival and (n=55, 7.2 vs. 15 months, p=0.028, log-rank) and a decreased risk ratio in a Cox proportional Hazard model (p=0.034). We conclude that lack of proteasome activity is a valuable marker for tracking GCSCs and their treatment responses. Low proteasome activity seems to be shared by malignant and normal stem cells in the brain, supporting the hypothesis that normal tissue stem cells in the brain are the cells of origin in GB. Our clinical data indicate that the normal tissue stem cell niche in the brain may harbor GCSCs and that targeted treatment of this region could improve patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4276.

Homologous Recombination Contributes to the Repair of DNA Double-Strand Breaks Induced by High-Energy Iron Ions

To test the contribution of homologous recombinational repair (HRR) in repairing DNA damage sites induced by high-energy iron ions, we used (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We found that in response to exposure to iron ions, HRR contributed to cell survival in rodent cells and that HRR deficiency abrogated RAD51 focus formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 focus formation. For human cells irradiated with iron ions, cell survival was decreased, and in p53 mutant cells, the levels of mutagenesis were increased when HRR was impaired. Human cells synchronized in S phase exhibited a more pronounced resistance to iron ions compared with cells in G(1) phase, and this increase in radioresistance was diminished by RAD51 knockdown. These results indicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged-particle radiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival after exposure to high-energy high-LET radiation.

Increased Radiation Resistance of Breast Cancer Cells by Endocrine Disrupting Chemical Bisphenol A.

Abstract Purpose: Bisphenol A (BPA) is used in the manufacture of plastics and belongs to a family of compounds called endocrine disrupting chemicals (EDCs). Because of extensive use of BPA in the manufacture of consumer goods and products, humans are exposed to levels of BPA which may cause adverse effects on endocrine-sensitive organs. Here, we evaluated the effects of BPA on the proliferation of breast cancer cells and the effect of BPA on tumor cell radioresponse.Materials/Methods: Proliferation was evaluated in estrogen-sensitive (MCF-7) and estrogen-insensitive (MDA-MB-231) human breast cancer lines that were treated for 72 hour with BPA (0.1µM; environmentally relevant concentration). The effect on radiation sensitivity was evaluated using clonogenic survival assays after exposure to various dose of BPA (0.1, 1, and 10 µM-) followed by irradiation (IR). DNA damage was evaluated using phosphorylated histone H2AX (γH2AX) and the neutral comet assay. BPA induced cell cycle changes were evaluated by staining of phosphor-H3 and flow cytomety. To investigate the mechanism of BPA±IR induced cell growth, we explored the activation of Akt and mitogen-activated protein kinase (MAPK) after treatments.Results: Exposure of BPA at low-dose (0.1µM) for 72 hour resulted in an increase survival rate in MCF-7 cancer cell (150 ± 19% of control) with more limited effects on MDA-MB-231 cancer cell (123 ± 17% of control). Exposure of MCF-7 to BPA for 72 hour before radiation resulted in an increase in radioresistance with dose-reduction factors at a surviving fraction of 0.1 of 1.23 and 1.31 with BPA 0.1µM, 10µM, respectively. BPA had no effect on the number of cells in each phase of the cell cycle nor on the activation of the G2 cell cycle checkpoint after IR. As a measure of DNA double strand breaks, γH2AX were determined as a function of time after the BPA±IR treatments. BPA also significantly decreased γH2AX expression at 24 hours after IR. Expression of phosphorylated Akt and phosphorylated mitogen-activated protein kinase (MAPK) increased with BPA± IR but decreased with IR alone at 24 hr.Conclusions: These results suggest that BPA can enhance phosphorylation of Akt and MAPK possibly leading to a resistance to radiation-induced cell death after IR. Further studies are needed to determine the clinical relevance of these findings. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6073.

Quantitative proteomic analysis of radioresistant breast cancer cell lines.

Abstract Abstract #5074 Background: Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. We aimed to develop a novel in vitro model of radioresistance using a breast cancer cell line and to subsequently identify molecular biomarkers which may be associated with the radioresistant phenotype. We utilised a quantitative proteomics technique (iTRAQ) based on MALDI-TOF/TOF mass spectrometry (MS) to identify differentially expressed proteins.&amp;#x2028; Material and Methods: We established 3 novel breast cancer cell sublines which were significantly resistant to radiotherapy when compared with the parental cells. The radioresistant sublines were created by irradiating cells in fractionated doses of 2Gy up to a total dose of 40Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for each novel cell subline when compared with parental cells. One radioresistant/parental cell pair was first analysed using in-solution digestion and liquid chromatographic separation with protein identification by MALDI-TOF/TOF (LC-MALDI analysis) on an Applied Biosystems 4800 Plus instrument. Quantitative iTRAQ (Applied Biosystems) was then performed on the same instrument for all 3 radioresistant/parental cell pairs.&amp;#x2028; Results: A total of 586 and 652 proteins were identified in T47D and T47DRR cells respectively by LC-MALDI. Those proteins identified in both cell lines and any redundant entries were removed to reveal those proteins which were unique to each cell line. In total 244 unique proteins were identified in T47D cells and 311 unique proteins were identified in T47DRR cells.&amp;#x2028; Comparison of the 3 pairs of radioresistant/parental cell samples by iTRAQ revealed a number of differentially expressed proteins. Using a standard ≥2-fold change in expression, these iTRAQ analyses revealed significant changes in the expression of 51 proteins in one or more of the radio-resistant derivatives.&amp;#x2028; Further confirmation by immunoblotting is underway. Currently the decrease in expression of 26S proteasome associated subunits has been confirmed by this method.&amp;#x2028; Conclusion: LC-MALDI and iTRAQ analysis has revealed a large number of candidate proteins which may be associated with a radioresistant phenotype. These now require further confirmatory studies. These MS-based techniques offer a powerful proteomic approach to identify candidate biomarkers which may be involved in radioresistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5074.

Antibody microarray analysis identifies biomarkers associated with radioresistant breast cancer cell lines.

Abstract Abstract #5072 Background: Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. In addition, if patients with radioresistant breast cancers can be identified, harmful side effects from exposure to unnecessary ionizing radiation could be prevented. We aimed to develop a novel in vitro model of radioresistance using breast cancer cell lines and to subsequently identify molecular biomarkers which may be associated with the radioresistant phenotype. Antibody microarrays offer a complementary approach for proteomic analysis in conjunction with standard screening methods such as two dimensional gel electrophoresis/ mass spectrometry and other quantitative proteomic techniques. We previously utilised the Panorama Cell Signalling Antibody Microarray Kit (Sigma-Aldrich) consisting of 224 antibodies (Smith et al Mol Cancer Ther 5:2115-20, 2006). In this study we utilised a novel high density 725 antibody microarray to screen for proteins associated with radioresistance.&amp;#x2028; Material and Methods: We established novel breast cancer cell sublines which were significantly resistant to radiotherapy when compared with the parental cells (T47D; MCF-7). The radioresistant sublines were created by irradiating cells in fractionated doses of 2Gy up to a total dose of 40Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for the novel cell subline when compared with parental cells. The radioresistant/parental cell pairs were analysed using the Panorama Antibody Microarray XPRESS Profiler725 Kit (Sigma-Aldrich). The microarray comprised 725 different antibodies on nitrocellulose coated microscope slides. The antibodies were selected from a wide variety of pathways, including apoptotic and cell signalling pathways.&amp;#x2028; Results: Utilising a Cy3/Cy5 labelling strategy the antibody microarray approach yielded a number of a total of 28 targets for further study. Of these, three proteins were identified independently from both of the radioresistant cell lines. These were GFI1 (Growth Factor Independence-1), DR4 (Death Receptor 4) and Importin a1. Immunoblotting and other proteomic approaches have confirmed the identities and differential expression of some candidate protein targets.&amp;#x2028; Conclusion: High density antibody microarrays potentially offer a powerful new proteomic technique to allow the global analysis of many proteins simultaneously. This analysis has produced both complementary and confirmatory data in our proteomic screening for putative biomarkers associated with radiotherapy resistance. We successfully identified a number of protein targets which may be associated with a radioresistant phenotype. Further confirmatory and validation studies are ongoing. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5072.

Down-regulation of radioresistance by LexA2 in Deinococcus radiodurans

The extremely radioresistant bacterium Deinococcus radiodurans contains two LexA homologues (LexA1 and LexA2) that are possible transcriptional regulators associated with the DNA damage response. In this study, resequencing revealed that there was an additional cytosine nucleotide (nucleotide position 612) in the D. radiodurans lexA2 gene. Purified LexA2 possessed proteolytic activity that could be stimulated by RecA. In an effort to gain an insight into the role of LexA2 in the radiation response mechanism, recA, lexA1 and lexA2 disruptant strains were generated and investigated. The intracellular level of RecA increased in lexA1 and lexA2 disruptant strains following gamma-irradiation as in the wild-type strain. These results indicated that the two LexA homologues did not possess functional overlap regarding the induction of RecA. The lexA2 disruptant strains exhibited a much higher resistance to gamma-rays than the wild-type strain. Furthermore, a luciferase assay showed that pprA promoter activation was enhanced in the lexA2 disruptant strain following gamma-irradiation. The pprA gene encoding the novel radiation-inducible protein PprA plays a critical role in the radioresistance of D. radiodurans. The increase in radioresistance of the lexA2 disruptant strain is explained in part by the enhancement of pprA promoter activation.

Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy

BackgroundMulticellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids.ResultsVolume of spheroids, fed daily with fresh medium, increased exponentially during 7–28 days of growth through three population doublings. Proportion of G1-phase cells was higher (~60%) than exponentially growing monolayer cells (~48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2–3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1α was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (~2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors.ConclusionDevelopment of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.

Chromosome aberration dosimetry in cosmonauts after single or multiple space flights

Background and aims: Cosmic radiation is one of the main hazards for manned space exploration. Uncertainty in radiation risk estimates for crews of long-term missions are very high, and direct biological measurements are necessary. We measured chromosomal aberrations in peripheral blood lymphocytes from 33 cosmonauts involved in space missions during the past 11 years. Methods: Blood lymphocytes from the cosmonauts were stimulated to grow in vitro and were harvested at their first mitosis. Slides were either stained with Giemsa stain for dicentrics analysis, or painted with whole-chromosome DNA probes for translocation analysis (FISH). Results: A statistically significant increase in the yield of chromosomal aberrations was measured following long-term space missions in lymphocytes from cosmonauts at their first flight. No significant changes in aberration frequencies were observed for short-term taxi flights. The increase in long-term missions was consistent with the values calculated from physical dosimetry data. However, for cosmonauts involved in two or more space flights, the yield of interchromosomal exchanges was not related to the total duration of space sojourn or integral absorbed dose. Indeed, the yield of aberrations at the end of the last mission was generally in the range of background frequencies measured before the first mission. Conclusions: Chromosome aberration dosimetry can detect radiation damage during space flight, and biological measurements support the current risk estimates for space radiation exposure. However, for cosmonauts involved in multiple space missions the frequency of chromosomal aberrations is lower than expected, suggesting that the effects of repeated space flights on this particular endpoint are not simply additive. Changes in the immune system in microgravity and/or adaptive response to space radiation may explain the apparent increase in radioresistance after multiple space flights.