Number Of Double-strand Breaks Research Articles

Dose and DNA damage modelling of diffusing alpha-emitters radiation therapy using Geant4

Purpose:Diffusing alpha-emitters radiation therapy (DaRT) is a brachytherapy technique using α-particles to treat solid tumours. The high linear energy transfer (LET) and short range of α-particles make them good candidates for the targeted treatment of cancer. Treatment planning of DaRT requires a good understanding of the dose from α-particles and the other particles released in the 224Ra decay chain. Methods:The Geant4 Monte Carlo toolkit has been used to simulate a DaRT seed to better understand the dose contribution from all particles and simulate the DNA damage due to this treatment. Results:Close to the seed α-particles deliver the majority of dose, however at radial distances greater than 4 mm, the contribution of β-particles is greater. The RBE has been estimated as a function of number of double strand breaks (DSBs) and complex DSBs. A maximum seed spacing of 5.5 mm and 6.5 mm was found to deliver at least 20 Gy RBE weighted dose between the seeds for RBEDSB and RBEcDSB respectively. Conclusions:The DNA damage changes with radial distance from the seed and has been found to become less complex with distance, which is potentially easier for the cell to repair. Close to the seed α-particles contribute the majority of dose, however the contribution from other particles cannot be neglected and may influence the choice of seed spacing.

Abstract LB397: Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation

Abstract CRISPR-Cas9, a highly effective gene editing tool, is a molecular scissor that induces double strand breaks (DSBs) at its targeted genomic locations. Ionizing radiation (IR), in contrast, generates DSBs and other DNA lesions, making it potentially more toxic than CRISPR-Cas9 induced DSBs. Alternatively, CRISPR-Cas9 might be more toxic as the Cas9 activity would continue if the DSB were repaired to wild type, resulting in more scissions per target. We designed multitarget sgRNAs targeting 0-16 sites in the human genome and performed similar dose-response experiments with IR (0-10 Gy) over 21 days on two pancreatic cancer (PC) cell lines. Growth inhibition was measured through clonogenicity and alamarBlue assays, and γ-H2AX staining performed to calculate DSBs. Surviving colonies from sgRNA transduction underwent whole genome sequencing (WGS) and targeted deep sequencing to quantify mutations. Cell growth was assessed through cell counting and sgRNA tag survival over time. Chromosomal changes were elucidated through chromosome breakage assays and XY FISH. We found that cytotoxicity increased as a function of both IR dose and number of sgRNA target site. At a comparable number of DSBs, CRISPR-Cas9 demonstrated higher cytotoxicity (~44% growth inhibition from 5-target sgRNA, ~15 γ-H2AX foci in the PC cells) compared to IR (9% inhibition from 0.5 Gy, ~25 foci). WGS of 5-cutter treated cells also revealed ~13 CRISPR-Cas9 induced DSBs in the PC cells. >99% cytotoxicity was achieved with 12-target sgRNA and 8 Gy. Moreover, sequencing of surviving colonies from sgRNA-treated cells confirmed lack of widespread off-target events. Notably, no surviving colonies could be obtained from the 12- and 16-cutter treated Panc10.05 cells and 8-14 cutters treated TS0111 cells.Both CRISPR-Cas9 and IR assays demonstrated delayed cell death, and cell death began 7 days after transduction of multitarget sgRNAs. Cytogenetic analyses on cells transduced with a 14-cutter sgRNA over 21 days revealed accumulation of karyotypic abnormalities peaking at day 14, and polyploidization peaking at day 10 and decreasing by day 21. Structural variant (SV) analyses from WGS data showed that the number of SVs per colony increased and peaked in 6-cutter colonies (average = 42 SVs), then decreased in 8- and 14-cutter colonies. Finally, surviving colonies that were resistant to 5-, 8-, and 16-cutter sgRNAs were re-challenged with their original multitarget sgRNA, 12-, and 14-cutter sgRNAs. Re-treating with original sgRNA showed no growth inhibition, while >95% cytotoxicity was observed by re-challenging them with 12- or 14-cutter sgRNAs.In conclusion, CRISPR-Cas9 kills pancreatic cancers by inducing chromosomal catastrophe, and resistance to original set of CRISPR-Cas9 induced DSBs can be easily overcome by targeting different sites. For the same number of DSBs, CRISPR-Cas9 was surprisingly more toxic than IR. Citation Format: Selina Shiqing K. Teh, Akhil Kotwal, Eitan Halper-Stromberg, Laura Morsberger, Kirsten Bowland, Alexis Bennett, Michael Goldstein, Ying S. Zou, James R. Eshleman. Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB397.

MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner

DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by forming a vital intermediate known as the TOP2-DNA cleavage complex (TOP2cc). Although the TOP2cc is often transient, stabilization can be achieved by TOP2 poisons, a family of anti-cancer chemotherapeutic agents targeting TOP2, such as etoposide (VP-16), and then induce double-strand breaks (DSBs) in cellular DNA. TOP2cc first needs to be proteolyzed before it can be processed by TDP2 for the removal of these protein adducts and to produce clean DNA ends necessary for proper repair. However, the mechanism by which TOP2βcc is proteolyzed has not been thoroughly studied. In this study, we report that after exposure to VP-16, MDM2, a RING-type E3 ubiquitin ligase, attaches to TOP2β and initiates polyubiquitination and proteasomal degradation. Mechanistically, during exposure to VP-16, TOP2β binds to DNA to form TOP2βcc, which promotes MDM2 binding and subsequent TOP2β ubiquitination and degradation, and results in a decrease in TOP2βcc levels. Biologically, MDM2 inactivation abrogates TOP2β degradation, stabilizes TOP2βcc, and subsequently increases the number of TOP2β-concealed DSBs, resulting in the rapid death of cancer cells via the apoptotic process. Furthermore, we demonstrate the combination activity of VP-16 and RG7112, an MDM2 inhibitor, in the xenograft tumor model and in situ lung cancer mouse model. Taken together, the results of our research reveal an underlying mechanism by which MDM2 promotes cancer cell survival in the presence of TOP2 poisons by activating proteolysis of TOP2βcc in a p53-independent manner, and provides a rationale for the combination of MDM2 inhibitors with TOP2 poisons for cancer therapy.

Redox-Active Cerium Fluoride Nanoparticles Selectively Modulate Cellular Response against X-ray Irradiation In Vitro.

Ionizing radiation-induced damage in cancer and normal cells leads to apoptosis and cell death, through the intracellular oxidative stress, DNA damage and disorders of their metabolism. Irradiation doses that do not lead to the death of tumor cells can result in the emergence of radioresistant clones of these cells due to the rearrangement of metabolism and the emergence of new mutations, including those in the genes responsible for DNA repair. The search for the substances capable of modulating the functioning of the tumor cell repair system is an urgent task. Here we analyzed the effect of cerium(III) fluoride nanoparticles (CeF3 NPs) on normal (human mesenchymal stem cells-hMSC) and cancer (MCF-7 line) human cells after X-ray radiation. CeF3 NPs effectively prevent the formation of hydrogen peroxide and hydroxyl radicals in an irradiated aqueous solution, showing pronounced antioxidant properties. CeF3 NPs are able to protect hMSC from radiation-induced proliferation arrest, increasing their viability and mitochondrial membrane potential, and, conversely, inducing the cell death of MCF-7 cancer cells, causing radiation-induced mitochondrial hyperpolarization. CeF3 NPs provided a significant decrease in the number of double-strand breaks (DSBs) in hMSC, while in MCF-7 cells the number of γ-H2AX foci dramatically increased in the presence of CeF3 4 h after irradiation. In the presence of CeF3 NPs, there was a tendency to modulate the expression of most analyzed genes associated with the development of intracellular oxidative stress, cell redox status and the DNA-repair system after X-ray irradiation. Cerium-containing nanoparticles are capable of providing selective protection of hMSC from radiation-induced injuries and are considered as a platform for the development of promising clinical radioprotectors.

Modeling of DNA Damage Repair and Cell Response in Relation to p53 System Exposed to Ionizing Radiation.

Repair of DNA damage induced by ionizing radiation plays an important role in the cell response to ionizing radiation. Radiation-induced DNA damage also activates the p53 system, which determines the fate of cells. The kinetics of repair, which is affected by the cell itself and the complexity of DNA damage, influences the cell response and fate via affecting the p53 system. To mechanistically study the influences of the cell response to different LET radiations, we introduce a new repair module and a p53 system model with NASIC, a Monte Carlo track structure code. The factors determining the kinetics of the double-strand break (DSB) repair are modeled, including the chromosome environment and complexity of DSB. The kinetics of DSB repair is modeled considering the resection-dependent and resection-independent compartments. The p53 system is modeled by simulating the interactions among genes and proteins. With this model, the cell responses to low- and high-LET irradiation are simulated, respectively. It is found that the kinetics of DSB repair greatly affects the cell fate and later biological effects. A large number of DSBs and a slow repair process lead to severe biological consequences. High-LET radiation induces more complex DSBs, which can be repaired by slow processes, subsequently resulting in a longer cycle arrest and, furthermore, apoptosis and more secreting of TGFβ. The Monte Carlo track structure simulation with a more realistic repair module and the p53 system model developed in this study can expand the functions of the NASIC code in simulating mechanical radiobiological effects.

Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity.

In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4PPH-4.1 phosphatase and ATRATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4PPH-4.1, ATRATL-1, and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.

Double-strand breaks quantification by statistical length analysis of DNA fragments imaged with AFM

DNA, a molecule carrying our genome, is constantly exposed to various endogenous and exogenous damaging factors. These factors may lead to the formation of single- or double-strand breaks. Recently, an atomic force microscopy emerged as an attractive tool for determination of the length of DNA fragments deposited at surfaces, allowing quantification of double-strand breaks (DSBs). In spite of this, the length of objects provided via AFM is strictly related to the image spatial resolution, which in consequence leads to underestimation of DSBs quantity. To correct this deficiency, we provide here a novel methodology based on statistics of DNA fragments length. This approach allows for highly accurate determination of the DSBs number. Moreover, we provide the software that enables to automatically calculate the number of DSBs according to the described methodology. Finally, the method is applied for characterization of the DNA plasmid fragmentation by bleomycin - an anticancer chemotherapeutic drug.

Different Mechanisms of DNA Radiosensitization by 8-Bromoadenosine and 2'-Deoxy-2'-fluorocytidine Observed on DNA Origami Nanoframe Supports.

DNA origami nanoframes with two parallel DNA sequences are used to evaluate the effect of nucleoside substituents on radiation-induced DNA damage. Double strand breaks (DSB) of DNA are counted using atomic force microscopy (AFM), and total number of lesions is evaluated using real-time polymerase chain reaction (RT-PCR). Enhanced AT or GC content does not increase the number of DNA strand breaks. Incorporation of 8-bromoadenosine results in the highest enhancement in total number of lesions; however, the highest enhancement in DSB is observed for 2'-deoxy-2'-fluorocytidine, indicating different mechanisms of radiosensitization by nucleoside analogues with the halogen substituent on base or sugar moieties, respectively. "Bystander" effects are observed, when the number of DSB in a sequence is enhanced by a substituent in the parallel DNA sequence. The present approach eliminates limitations of previously developed methods and motivates detailed studies of poorly understood conformation or bystander effects in radiation induced damage to DNA.

Epigenetic Anti-Cancer Treatment With a Stabilized Carbocyclic Decitabine Analogue.

5‐Aza‐2’‐deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2’‐deoxycytidine (dC) to 5‐methyl‐dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM‐13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti‐proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off‐target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

Targeted methylation facilitates DNA double strand breaks and enhances cancer suppression: A DNA intercalating/methylating dual-action chimera Amonafidazene

A DNA intercalating agent Amonafide interferes with topoisomerase 2 (Topo II) activity and prevents re-ligation of DNA strands, leading to double strand breaks (DSB). If DSB repair fails, cells stop dividing and eventually die. In a search of approaches to enhance anti-cancer activities of Topo II inhibitors, we hypothesized that introduction of additional damage in proximity to the DSB may suppress DNA repair and enhance cancer cell killing. Accordingly, chimeric molecules were created that target a DNA alkylating component to the proximity of Topo II-induced DSBs. These chimeras consist of Amonafide or its 4-amino isomer, and DNA methylating methyl triazene moiety Azene protected with a carbamate group, connected via linker. Treatment of cancer cells with the chimeric molecules leads to significantly higher number of DSBs, which were repaired slower compared to Amonafide or monomethyl triazene-treated cells. On the other hand, methyl triazene linked to non-intercalating Amonafide analogs was ineffective. Together, these data strongly support our hypothesis. In line with increased DSBs, the chimeric molecules exhibited significantly higher antiproliferative activity in cancer cell lines compared to Amonafide or monomethyl triazene constituent Azene. We utilized the fluorescent properties of chimera Amonafidazene to develop ''photo-switchable'' reporting system to monitor the prodrug activation. Using this approach, we found that the chimera accumulated and was activated at the tumor sites specifically and demonstrated significantly stronger tumor suppressing activities compared to Amonafide in a xenograft model. Therefore, targeting alkylating groups to the proximity of DSB sites may become an effective approach towards enhancing anti-cancer activities of inhibitors of topoisomerases.

Dosimetric Evaluation of the Effect of Receptor Heterogeneity on the Therapeutic Efficacy of Peptide Receptor Radionuclide Therapy: Correlation with DNA Damage Induction and InVivo Survival.

Our rationale was to build a refined dosimetry model for 177Lu-DOTATATE invivo experiments enabling the correlation of absorbed dose with double-strand break (DSB) induction and cell death. Methods: Somatostatin receptor type 2 expression of NCI-H69 xenografted mice, injected with 177Lu-DOTATATE, was imaged at 0, 2, 5, and 11 d. This expression was used as input to reconstruct realistic 3-dimensional heterogeneous activity distributions and tissue geometries of both cancer and heathy cells. The resulting volumetric absorbed dose rate distributions were calculated using the GATE (Geant4 Application for Tomographic Emission) Monte Carlo code and compared with homogeneous dose rate distributions. The absorbed dose (0-2 d) on micrometer-scale sections was correlated with DSB induction, measured by γH2AX foci. Moreover, the absorbed dose on larger millimeter-scale sections delivered over the whole treatment (0-14 d) was correlated to the modeled invivo survival to determine the radiosensitivity parameters α and β for comparison with experimental data (cell death assay, volume response) and external-beam radiotherapy. The DNA-damage repair half-life Tμ and proliferation doubling time TD were obtained by fitting the DSB and tumor volume data over time. Results: A linear correlation with a slope of 0.0223 DSB/cell mGy-1 between the absorbed dose and the number of DSBs per cell has been established. The heterogeneous dose distributions differed significantly from the homogeneous dose distributions, with their corresponding average S values diverging at 11 d by up to 58%. No significant difference between modeled invivo survival was observed in the first 5 d when using heterogeneous and uniform dose distributions. The radiosensitivity parameter analysis for the invivo survival correlation indicated that the minimal effective dose rates for cell kill was 13.72 and 7.40 mGy/h, with an α of 0.14 and 0.264 Gy-1, respectively, and an α/β of 100 Gy; decreasing the α/β led to a decrease in the minimal effective dose rate for cell kill. Within the linear quadratic model, the best matching invivo survival correlation (α = 0.1 Gy-1, α/β = 100 Gy, Tμ = 60 h, TD = 14.5 d) indicated a relative biological effectiveness of 0.4 in comparison to external-beam radiotherapy. Conclusion: Our results demonstrated that accurate dosimetric modeling is crucial to establishing dose-response correlations enabling optimization of treatment protocols.

Using Machine Learning Techniques for Asserting Cellular Damage Induced by High-LET Particle Radiation

This is a study concerning the use of Machine Learning (ML) techniques to ascertain the impacts of particle ionizing radiation (IR) on cell survival and DNA damage. Current empirical models do not always take into account intrinsic complexities and stochastic effects of the interactions of IR and cell populations. Furthermore, these models often lack in biophysical interpretations of the irradiation outcomes. The linear quadratic (LQ) model is a common way to associate the biological response of a cell population with the radiation dose. The parameters of the LQ model are used to extrapolate the relation between the dosage and the survival fraction of a cell population. The goal was to create a ML-based model that predicts the α and β parameters of the well known and established LQ model, along with the key metrics of DNA damage induction. The main target of this effort was, on the one hand, the development of a computational framework that will be able to assess key radiobiophysical quantities, and on the other hand, to provide meaningful interpretations of the outputs. Based on our results, as some metrics of the adaptability and training efficiency, our ML models exhibited 0.18 median error (relative root mean squared error (RRMSE)) in the prediction of the α parameter and errors of less than 0.01 for various DNA damage quantities; the prediction for β exhibited a rather large error of 0.75. Our study is based on experimental data from a publicly available dataset of irradiation studies. All types of complex DNA damage (all clusters), and the number of double-stranded breaks (DSBs), which are widely accepted to be closely related to cell survival and the detrimental biological effects of IR, were calculated using the fast Monte Carlo Damage Simulation software (MCDS). We critically discussed the varying importance of physical parameters such as charge and linear energy transfer (LET); we also discussed the uncertainties of our predictions and future directions, and the dynamics of our approach.

Image processing method for automatic measurement of number of DNA breaks

The number of double-strand breaks can be evaluated from the change of average DNA length. The average DNA length is measured by the single-molecule observation method using fluorescence microscope. The measurement of DNA length in the microscope images is done manually by experienced operators and it is time consuming in many experiments. An image processing method using OpenCV library to measure length of DNA in fluorescence microscope images is developed in this paper. An automation of measurement using deep learning is also proposed.

The molecular control of meiotic double-strand break (DSB) formation and its significance in human infertility.

Meiosis is an essential step in gametogenesis which is the key process in sexually reproducing organisms as meiotic aberrations may result in infertility. In meiosis, programmed DNA double-strand break (DSB) formation is one of the fundamental processes that are essential for maintaining homolog interactions and correcting segregation of chromosomes. Although the number and distribution of meiotic DSBs are tightly regulated, still abnormalities in DSB formation are known to cause meiotic arrest and infertility. This review is a detailed account of molecular bases of meiotic DSB formation, its evolutionary conservation, and variations in different species. We further reviewed the mutations of DSB formation genes in association with human infertility and also proposed the future directions and strategies about the study of meiotic DSB formation.

Biological effect of microwave electromagnetic radiation: methods of registration and protection

The development of modern electronics, communications, high-tech production and other technologies lead to a significant increase in the intensity of microwave electromagnetic radiation. This article discusses the problem of registration (including biological detection) and protection from microwave electromagnetic radiation. To consider the method of integral biological and instrumental recording of the intensity of microwave electromagnetic radiation and to evaluate the method of protection using a passive multifrequency re-emitter generator with a radio holographic antenna (trade mark EQVILUM). A series of experiments were carried out to determine the number of double-strand breaks in DNA (DS DNA) by the foci of γH2AX repair proteins, to assess proliferative activity and clonogenic ability after 3 hours of exposure to microwave electromagnetic radiation with a frequency radiation of 1200 MHz both with shielding and without it on a culture of human lung fibroblasts (MRC5 line). It is shown that no changes in the level of DS DNA were revealed in experimental cells, however, a decrease in the clonogenic ability of irradiated cells without shielding was observed against the background of their high proliferative activity. The mechanisms of the observed cellular effects formation are unclear and we suppose that they can be associated with both a change in the metabolism of irradiated cells and with the triggering of certain intracellular mechanisms initiated by electromagnetic radiation (EMR). This study made it possible to identify effective ways of integral assessment of the biological effects of microwave electromagnetic radiation as well as ways of protection against it. A film microcircuit – an autonomous module of a passive multi-frequency re-emitter generator with a radio holographic antenna (EQVILUM trademark) has shown its high efficiency in protection against man-made microwave electromagnetic radiation and can be recommended as an additional means of effective protection.

Characteristic of the active substance of the Saccharomyces cerevisiae preparation having radioprotective properties

В работе охарактеризованы некоторые биологические особенности радиопротекторного действия препарата двуцепочечной РНК. Обнаружено, что препарат дрожжевой РНК обладает пролонгированным радиопротекторным действием при облучении животных летальной дозой в 9.4 Гр. При облучении через 1 ч и на 4-е сутки после введения 7 мг препарата РНК выживает 100 % животных на 70-е сутки наблюдения, при облучении на 8-е и 12-е сутки – 60 % животных. Были оценены временные параметры процесса репарации двуцепочечных разрывов, индуцированных γ-лучами. Выявлено, что введение препарата РНК в момент максимального количества двуцепо- чечных разрывов, через 1 ч после облучения, снижает эффективность радиопротекторного действия по сравнению с введением за 1 ч до облучения и через 4 ч после облучения. Проведено сравнение эффективности радиозащит- ного действия штатного радиопротектора Б-190 и препарата РНК в одном эксперименте. Установлено, что препарат суммарной РНК не уступает по эффективности препарату Б-190. Выживаемость на 40-е сутки после облучения для группы мышей, получавших препарат РНК, составила 78 %, для Б-190 – 67 % животных. В ходе аналитических иссле- дований препарата суммарной РНК дрожжей обнаружилось, что препарат представляет собой смесь одноцепочеч- ной и двуцепочечной РНК. Радиопротекторными свойствами обладает только двуцепочечная РНК. При введении 160 мкг препарата двуцепочечной РНК выживает 100 % подопытных животных после абсолютно летальной дозы гамма-радиации 9.4 Гр. Установлено, что радиозащитный эффект двуцепочечной РНК зависит не от последователь- ности, а от ее двуцепочечной формы, причем для осуществления радиопротекторного действия двуцепочечная РНК должна иметь «открытые» концы молекулы. Предполагается, что радиозащитное действие препарата двуцепочеч- ной РНК связано с участием молекул РНК в корректном восстановлении поврежденного облучением хроматина в стволовых клетках крови. Сохранившие жизнеспособность стволовые гемопоэтические клетки мигрируют на пери- ферию и достигают селезенки, где активно пролиферируют. Вновь образовавшаяся клеточная популяция восстанав- ливает кроветворную и иммунную системы, что определяет выживание летально облученных животных.

Apigenin and Hesperidin Downregulate DNA Repair Genes in MCF-7 Breast Cancer Cells and Augment Doxorubicin Toxicity.

A number of studies have confirmed anti-tumor activity of flavonoids and their ability to enhance the effectiveness of classical anticancer drugs. The mechanism of this phenomenon is difficult to explain because of the ambivalent nature of these compounds. Many therapeutic properties of these compounds are attributed to their antioxidant activity; however, it is known that they can act as oxidants. The aim of this study was to assess the influence of apigenin and hesperidin on MCF-7 breast cancer cells with doxorubicin. The cytotoxic effect was determined using an MTT test and cell cycle analysis. To evaluate the possible interaction mechanism, reduced glutathione levels, as well as the DNA oxidative damage and the double strand breaks, were evaluated. Additionally, mRNA expression of genes related to DNA repair was assessed. It was demonstrated that flavonoids intensified the cytotoxic effect of doxorubicin despite flavonoids reduced oxidative damage caused by the drug. At the same time, the number of double strand breaks significantly increased and expression of tested genes was downregulated. In conclusion, both apigenin and hesperidin enhance the cytotoxic effects of doxorubicin on breast cancer cells, and this phenomenon occurs regardless of oxidative stress but is accompanied by disorders of DNA damage response mechanisms.

Abstract 5812: FOXD1 mediates G2/M transition in clear cell renal cell carcinoma

Abstract The forkhead family of transcription factors are highly conserved and essential during embryonic development. There have been reports showing increased expression of forkheads in various cancers, but little is known about the role of these proteins in the disease context. Analysis of 500 clear cell renal cell carcinoma (ccRCC) transcriptomes in The Cancer Genome Database revealed that FOXD1 is overexpressed in ccRCC and correlates with poor patient outcomes (median survival FOXD1-low=2830 days; FOXD1-high=1913 days). Immunohistochemistry of 46 patient tumors showed that FOXD1 is expressed the majority of tumor cells (95% CI [50.6%, 79.4%]). To understand the biological role of FOXD1, we generated FOXD1-null variants of the 786-O ccRCC cell line. Using an in vivo xenograft model, FOXD1-null cells had smaller initiating tumor sizes (parent=71.05mm3; FOXD1-null=36.39mm3) and stunted tumor growth. In vitro, FOXD1-null lines had significantly reduced growth rates compared to the parental line, and showed increased cell size. Cell cycle analysis revealed that FOXD1-null cells had an increased number of cells in G2 compared to the parental line (parent=21.1%; FOXD1-null=37.1%). After G1 synchronization, all lines progressed through G1, S, and G2 at similar rates (parent=10 hours; FOXD1-null=10 hours). However, FOXD1-null clones spent increased time in G2 compared to the parental line (parent= 4 hours; FOXD1-null= 8 hours). Western blot analysis revealed decreased levels of Cyclin B1 and p-H3, as well as increased levels of p27 10 hours post-synchronization in FOXD1-null cells compared to the parent line. Staining with yH2AX did not indicate any significant change in the number of double strand breaks at any phase. These findings suggest that FOXD1 is a mediator of the transition between G2 and M, and that its loss initializes the G2/M checkpoint. Extrapolating back to our clinical observation, we propose that increased FOXD1 expression in tumor cells accelerates the transition between G2 and M, a point that is normally critical for survey and repair of DNA damage. This could result in greater tolerance for DNA damage, accelerating tumor evolution. Citation Format: Kyle Henry Bond, Ryan Jaikaran, Suizhen Qiu, Leif Oxburgh. FOXD1 mediates G2/M transition in clear cell renal cell carcinoma [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 5812.

Monte Carlo track-structure for the radionuclide Copper-64: characterization of S-values, nanodosimetry and quantification of direct damage to DNA

TOPAS-nBio was used to simulate, collision-to-collision, the complete trajectories of electrons in water generated during the explicit simulation of 64Cu decay. S-values and direct damage to the DNA were calculated representing the cell (C) and the cell nucleus (N) with concentric spheres of 5 μm and 4 μm in radius, respectively. The considered ‘target’←‘source’ configurations, including the cell surface (Cs) and cytoplasm (Cy), were: C←C, C←Cs, N←N, N←Cy and N←Cs. Ionization cluster size distributions were also calculated in a cylinder immersed in water corresponding to a DNA segment of 10 base-pairs in length (diameter 2.3 nm, length 3.4 nm), modeling a radioactive point source moving from the central axis to the edge of the cylinder. For that, the first moment (M1) and cumulative probability of having a cluster size of 2 or more ionizations in the cylindrical volume (F2) were obtained. Finally, the direct damage to the DNA was estimated by quantifying double-strand breaks (DSBs) using the clustering algorithm DBSCAN.The S-values obtained with TOPAS-nBio for 64Cu were 7.879 × 10−4 ± 5 × 10−7, 4.351 × 10−4 ± 6 × 10−7, 1.442 × 10−3 ± 1 × 10−6, 2.596 × 10−4 ± 8 × 10−7, 1.127 × 10−4 ± 4 × 10−7 Gy Bq-s−1 for the configurations C←C, C←Cs, N←N, N←Cy and N←Cs, respectively. The difference of these values, compared with previously reported S-values for 64Cu with the code MNCP and software MIRDCell, ranged from −4% to −25% for the configurations N←N and N←Cs, respectively. On the other hand, F2 was maximum with the source at the center of the cylinder 0.373 ± 0.001, and monotonically decreased until reaching a value of 0.058 ± 0.001 at 2.3 nm. The same behavior was observed for M1 with values ranging from 2.188 ± 0.004 to 0.242 ± 0.002. Finally, the DBSCAN algorithm showed that the mean number of DNA DSBs per decay were 0.187 ± 0.001, 0.0317 ± 0.0005, and 0.0125 ± 0.0002 DSB-(Bq-s)−1 for the configurations N←N, N←Cs, and N←Cy, respectively.In conclusion, the results of the S-values show that the absorbed dose strongly depends on the distribution of the radionuclide in the cell, the dose being higher when 64Cu is internalized in the cell nucleus, which is reinforced by the nanodosimetric study by the presence of DNA DSBs attributable to the Auger electrons emitted during the decay of 64Cu.

The IRI-DICE hypothesis: ionizing radiation-induced DSBs may have a functional role for non-deterministic responses at low doses

Low-dose ionizing radiation (IR) responses remain an unresolved issue in radiation biology and risk assessment. Accurate knowledge of low-dose responses is important for estimation of normal tissue risk in cancer radiotherapy or health risks from occupational or hazard exposure. Cellular responses to low-dose IR appear diverse and stochastic in nature and to date no model has been proposed to explain the underlying mechanisms. Here, we propose a hypothesis on IR-induced double-strand break (DSB)-induced cis effects (IRI-DICE) and introduce DNA sequence functionality as a submicron-scale target site with functional outcome on gene expression: DSB induction in a certain genetic target site such as promotor, regulatory element, or gene core would lead to changes in transcript expression, which may range from suppression to overexpression depending on which functional element was damaged. The DNA damage recognition and repair machinery depicts threshold behavior requiring a certain number of DSBs for induction. Stochastically distributed persistent disruption of gene expression may explain—in part—the diverse nature of low-dose responses until the repair machinery is initiated at increased absorbed dose. Radiation quality and complexity of DSB lesions are also discussed. Currently, there are no technologies available to irradiate specific genetic sites to test the IRI-DICE hypothesis directly. However, supportive evidence may be achieved by developing a computational model that combines radiation transport codes with a genomic DNA model that includes sequence functionality and transcription to simulate expression changes in an irradiated cell population. To the best of our knowledge, IRI-DICE is the first hypothesis that includes sequence functionality of different genetic elements in the radiation response and provides a model for the diversity of radiation responses in the (very) low dose regimen.