Residual DNA Double-strand Breaks Research Articles

KCa channel targeting impairs DNA repair and invasiveness of patient‐derived glioblastoma stem cells in culture and orthotopic mouse xenografts which only in part is predictable by KCa expression levels

AbstractPrognosis of glioblastoma patients is still poor despite multimodal therapy. The highly brain‐infiltrating growth in concert with a pronounced therapy resistance particularly of mesenchymal glioblastoma stem‐like cells (GSCs) has been proposed to contribute to therapy failure. Recently, we have shown that a mesenchymal‐to‐proneural mRNA signature of patient derived GSC‐enriched (pGSC) cultures associates with in vitro radioresistance and gel invasion. Importantly, this pGSC mRNA signature is prognostic for patients' tumor recurrence pattern and overall survival. Two mesenchymal markers of the mRNA signature encode for IKCa and BKCa Ca2+‐activated K+ channels. Therefore, we analyzed here the effect of IKCa‐ and BKCa‐targeting concomitant to (fractionated) irradiation on radioresistance and glioblastoma spreading in pGSC cultures and in pGSC‐derived orthotopic xenograft glioma mouse models. To this end, in vitro gel invasion, clonogenic survival, in vitro and in vivo residual DNA double strand breaks (DSBs), tumor growth, and brain invasion were assessed in the dependence on tumor irradiation and K+ channel targeting. As a result, the IKCa‐ and BKCa‐blocker TRAM‐34 and paxilline, respectively, increased number of residual DSBs and (numerically) decreased clonogenic survival in some but not in all IKCa‐ and BKCa‐expressing pGSC cultures, respectively. In addition, BKCa‐ but not IKCa‐blockade slowed‐down gel invasion in vitro. Moreover, systemic administration of TRAM‐34 or paxilline concomitant to fractionated tumor irradiation increased in the xenograft model(s) residual number of DSBs and attenuated glioblastoma brain invasion and (numerically) tumor growth. We conclude, that KCa‐blockade concomitant to fractionated radiotherapy might be a promising new strategy in glioblastoma therapy.

Seeding the meiotic DNA break machinery and initiating recombination on chromosome axes.

Programmed DNA double-strand break (DSB) formation is a crucial feature of meiosis in most organisms. DSBs initiate recombination-mediated linking of homologous chromosomes, which enables correct chromosome segregation in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We uncover in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms. Both IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), suggesting that DDK contributes to the control of the axial DSB-machinery. Furthermore, we show that axial IHO1 platforms are based on an interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.

USP7 Deregulation Impairs S Phase Specific DNA Repair after Irradiation in Breast Cancer Cells.

The ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme with numerous substrates. Aberrant expression of USP7 is associated with tumor progression. This study aims to investigate how a deregulated USP7 expression affects chromosomal instability and prognosis of breast cancer patients in silico and radiosensitivity and DNA repair in breast cancer cells in vitro. The investigations in silico were performed using overall survival and USP7 mRNA expression data of breast cancer patients. The results showed that a high USP7 expression was associated with increased chromosomal instability and decreased overall survival. The in vitro experiments were performed in a luminal and a triple-negative breast cancer cell line. Proliferation, DNA repair, DNA replication stress, and survival after USP7 overexpression or inhibition and irradiation were analyzed. Both, USP7 inhibition and overexpression resulted in decreased cellular survival, distinct radiosensitization and an increased number of residual DNA double-strand breaks in the S phase following irradiation. RAD51 recruitment and base incorporation were decreased after USP7 inhibition plus irradiation and more single-stranded DNA was detected. The results show that deregulation of USP7 activity disrupts DNA repair in the S phase by increasing DNA replication stress and presents USP7 as a promising target to overcome the radioresistance of breast tumors.

A fast Monte Carlo cell-by-cell simulation for radiobiological effects in targeted radionuclide therapy using pre-calculated single-particle track standard DNA damage data

IntroductionWe developed a new method that drastically speeds up radiobiological Monte Carlo radiation-track-structure (MC-RTS) calculations on a cell-by-cell basis.MethodsThe technique is based on random sampling and superposition of single-particle track (SPT) standard DNA damage (SDD) files from a “pre-calculated” data library, constructed using the RTS code TOPAS-nBio, with “time stamps” manually added to incorporate dose-rate effects. This time-stamped SDD file can then be input into MEDRAS, a mechanistic kinetic model that calculates various radiation-induced biological endpoints, such as DNA double-strand breaks (DSBs), misrepairs and chromosomal aberrations, and cell death. As a benchmark validation of the approach, we calculated the predicted energy-dependent DSB yield and the ratio of direct-to-total DNA damage, both of which agreed with published in vitro experimental data. We subsequently applied the method to perform a superfast cell-by-cell simulation of an experimental in vitro system consisting of neuroendocrine tumor cells uniformly incubated with 177Lu.Results and discussionThe results for residual DSBs, both at 24 and 48 h post-irradiation, are in line with the published literature values. Our work serves as a proof-of-concept demonstration of the feasibility of a cost-effective “in silico clonogenic cell survival assay” for the computational design and development of radiopharmaceuticals and novel radiotherapy treatments more generally.

Fisetin overcomes non-targetability of mutated KRAS induced YB-1 signaling in colorectal cancer cells and improves radiosensitivity by blocking repair of radiation-induced DNA double-strand breaks

Background and purposeKRAS is frequently mutated, and the Y-box binding protein 1 (YB-1) is overexpressed in colorectal cancer (CRC). Mutant KRAS (KRASmut) stimulates YB-1 through MAPK/RSK and PI3K/AKT, independent of epidermal growth factor receptor (EGFR). The p21-activated kinase (PAK) family is a switch-site upstream of AKT and RSK. The flavonoid compound fisetin inhibits RSK-mediated YB-1 signaling. We sought the most effective molecular targeting approach that interferes with DNA double strand break (DSB) repair and induces radiosensitivity of CRC cells, independent of KRAS mutation status. Materials and MethodsKRAS activity and KRAS mutation were analyzed by Ras-GTP assay and NGS. Effect of dual targeting of RSK and AKT (DT), the effect of fisetin as well as targeting PAK by FRAX486 and EGFR by erlotinib on YB-1 activity was tested by Western blotting after irradiation in vitro and ex vivo. Additionally, the effect of DT and FRAX486 on DSB repair pathways was tested in cells expressing reporter constructs for the DSB repair pathways by flow cytometry analysis. Residual DSBs and clonogenicity were examined by γH2AX- and clonogenic assays, respectively. ResultsErlotinib neither blocked DSB repair nor inhibited YB-1 phosphorylation under KRAS mutation condition in vitro and ex vivo. DT and FRAX486 effectively inhibited YB-1 phosphorylation independent of KRAS mutation status and diminished homologous recombination (HR) and alternative non-homologous end joining (NHEJ) repair. DT and FRAX486 inhibited DSB repair in CaCo2 but not in isogenic KRASG12V cells. Fisetin inhibited YB-1 phosphorylation, blocked DSB repair and increased radiosensitivity, independent of KRAS mutation status. ConclusionCombination of fisetin with radiotherapy may improve CRC radiation response, regardless of KRASmut status.

The Effect of Xevinapant Combined with Ionizing Radiation on HNSCC and Normal Tissue Cells and the Impact of Xevinapant on Its Targeted Proteins cIAP1 and XIAP

The poor prognosis of HNSCC is partly due to treatment resistance. The SMAC mimetic Xevinapant is a promising new approach to targeted cancer therapy. Xevinapant inhibits cIAP1/2 and XIAP, leading to apoptosis, necroptosis and inhibition of prosurvival signaling. Combining Xevinapant with IR could improve therapeutic potential. The effect of Xevinapant in combination with IR on HNSCC and healthy tissue cells was investigated. Cell growth, cell death, clonogenic survival and DNA double-strand breaks (DSBs) were studied, and intracellular cIAP1 and XIAP levels were evaluated. Xevinapant had cytostatic and cytotoxic, as well as radiosensitizing, effects on the malignant cells, while healthy tissue cells were less affected. Apoptotic and necrotic cell death was particularly affected, but the increase in residual DSBs and the reduced survival implied an additional effect of Xevinapant on DNA damage repair and other cell inactivation mechanisms. cIAP1 and XIAP levels varied for each cell line and were affected by Xevinapant and IR treatment. There was an association between higher IAP levels and increased cell death. Xevinapant appears to be a potent new drug for HNSCC therapy, especially in combination with IR. IAP levels could be an indicator for impaired DNA damage repair and increased susceptibility to cellular stress.

Characterization of Intrinsic Radiation Sensitivity in a Diverse Panel of Normal, Cancerous and CRISPR-Modified Cell Lines.

Intrinsic radiosensitivity is a major determinant of radiation response. Despite the extensive amount of radiobiological data available, variability among different studies makes it very difficult to produce high-quality radiosensitivity biomarkers or predictive models. Here, we characterize a panel of 27 human cell lines, including those derived from lung cancer, prostate cancer, and normal tissues. In addition, we used CRISPR-Cas9 to generate a panel of lines with known DNA repair defects. These cells were characterised by measuring a range of biological features, including the induction and repair of DNA double-strand breaks (DSBs), cell cycle distribution, ploidy, and clonogenic survival following X-ray irradiation. These results offer a robust dataset without inter-experimental variabilities for model development. In addition, we used these results to explore correlations between potential determinants of radiosensitivity. There was a wide variation in the intrinsic radiosensitivity of cell lines, with cell line Mean Inactivation Doses (MID) ranging from 1.3 to 3.4 Gy for cell lines, and as low as 0.65 Gy in Lig4-/- cells. Similar substantial variability was seen in the other parameters, including baseline DNA damage, plating efficiency, and ploidy. In the CRISPR-modified cell lines, residual DSBs were good predictors of cell survival (R2 = 0.78, p = 0.009), as were induced levels of DSBs (R2 = 0.61, p = 0.01). However, amongst the normal and cancerous cells, none of the measured parameters correlated strongly with MID (R2 < 0.45), and the only metrics with statistically significant associations are plating efficiency (R2 = 0.31, p = 0.01) and percentage of cell in S phase (R2 = 0.37, p = 0.005). While these data provide a valuable dataset for the modelling of radiobiological responses, the differences in the predictive power of residual DSBs between CRISPR-modified and other subgroups suggest that genetic alterations in other pathways, such as proliferation and metabolism, may have a greater impact on cellular radiation response. These pathways are often neglected in response modelling and should be considered in the future.

Modelling oxygen effects on the in- and out-of-field radiosensitivity of cells exposed to intensity-modulated radiation fields

Objective. The delivery of intensity-modulated radiation fields has improved the conformity of dose to tumour targets during radiotherapy (RT). Previously, it has been shown that intercellular communication between cells positioned in- and outside of the radiation field impacts cellular radiosensitivity under hypoxic and normoxic conditions. However, the mechanism of intercellular communication in hypoxia remains to be fully understood. In this study, the cell-killing effects of intercellular communication in hypoxia were modelled in an effort to better understand the underlying mechanisms of response. Approach. By irradiating a 50% area of the culture dish (half-field exposure), experimental dose-response curves for cell survival and residual DNA double-strand breaks (DSBs) were generated in prostate (DU145) and non-small cell lung cancer (H1299) cells. The oxygen enhancement ratio (OER) was determined from early DSB yields (corresponding to relative direct damage) and used to model the in- and out-of-field radiosensitivity. Main results. The developed integrated microdosimetric-kinetic (IMK) model successfully predicted the experimental dose responses for survival and lethal lesions, and provides a mechanistic interpretation that the probability of hits for releasing cell-killing signals is dependent on oxygen. This experimental and modelling study also suggests that residual DSBs correspond to logarithmic survival fraction (meaning lethal lesions) for in- and out-of-field cells. Our data suggest that the OER value determined using uniform-field exposure can be applied to predict the in- and out-of-field radiosensitivity of cells following exposure to intensity modulated beams. Significance. The developed IMK model facilitates a more precise understanding of intercellular signalling following exposure to intensity-modulated radiation fields.

Optimal timing of a γH2AX analysis to predict cellular lethal damage in cultured tumor cell lines after exposure to diagnostic and therapeutic radiation doses.

Phosphorylated H2AX (γH2AX) is a sensitive biomarker of DNA double-strand breaks (DSBs). To assess the adverse effects of low-dose radiation (<50mGy), γH2AX levels have typically been measured in human lymphocytes within 30min of computed tomography (CT) examinations. However, in the presence of DSB repair, it remains unclear whether γH2AX levels within 30min of irradiation completely reflect biological effects. Therefore, we investigated the optimal timing of a γH2AX analysis to predict the cell-surviving fraction (SF). Three tumor cell lines were irradiated at different X-ray doses (10-4000mGy), and the relationships between SF and relative γH2AX levels were investigated 15min and 2, 6, 12 and 24h after irradiation. Data were analyzed for high-dose (0-4000mGy) and low-dose (0-500mGy) ranges. Correlations were observed between SF and the relative number of γH2AX foci/nucleus at 12h only (R2 = 0.68, P = 0.001 after high doses; R2 = 0.37, P = 0.016 after low doses). The relative intensity of γH2AX correlated with SF 15min to 12h after high doses and 2 to 12h after low doses, with the maximum R2 values being observed 2h after high doses (R2 = 0.89, P < 0.001) and 12h after low doses (R2 = 0.65, P < 0.001). Collectively, cellular lethal damage in tumor cells was more accurately estimated with residual DSBs 12h after low-dose (10-500mGy) irradiation. These results may contribute to determination of the optimal timing of biodosimetric analyses using γH2AX in future studies.

Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase.

During meiosis, recombination between homologous chromosomes (homologs) generates crossovers that promote proper segregation at the first meiotic division. Recombination is initiated by Spo11-catalyzed DNA double strand breaks (DSBs). 5' end resection of the DSBs creates 3' single strand tails that two recombinases, Rad51 and Dmc1, bind to form presynaptic filaments that search for homology, mediate strand invasion and generate displacement loops (D-loops). D-loop processing then forms crossover and non-crossover recombinants. Meiotic recombination occurs in two temporally distinct phases. During Phase 1, Rad51 is inhibited and Dmc1 mediates the interhomolog recombination that promotes homolog synapsis. In Phase 2, Rad51 becomes active and functions with Rad54 to repair residual DSBs, making increasing use of sister chromatids. The transition from Phase 1 to Phase 2 is controlled by the meiotic recombination checkpoint through the meiosis-specific effector kinase Mek1. This work shows that constitutive activation of Rad51 in Phase 1 results in a subset of DSBs being repaired by a Rad51-mediated interhomolog recombination pathway that is distinct from that of Dmc1. Strand invasion intermediates generated by Rad51 require more time to be processed into recombinants, resulting in a meiotic recombination checkpoint delay in prophase I. Without the checkpoint, Rad51-generated intermediates are more likely to involve a sister chromatid, thereby increasing Meiosis I chromosome nondisjunction. This Rad51 interhomolog recombination pathway is specifically promoted by the conserved 5'-3' helicase PIF1 and its paralog, RRM3 and requires Pif1 helicase activity and its interaction with PCNA. This work demonstrates that (1) inhibition of Rad51 during Phase 1 is important to prevent competition with Dmc1 for DSB repair, (2) Rad51-mediated meiotic recombination intermediates are initially processed differently than those made by Dmc1, and (3) the meiotic recombination checkpoint provides time during prophase 1 for processing of Rad51-generated recombination intermediates.

Patient derived ex vivo tissue slice cultures demonstrate a profound DNA double-strand break repair defect in HPV-positive oropharyngeal head and neck cancer

BackgroundHPV-positive head and neck squamous cell carcinoma of the oropharynx (OPSCC) are more sensitive towards radiation than HPV-negative OPSCC. Two main theories exist regarding the underlying mechanism. Stronger lymphocyte infiltration points to an enhanced immunogenicity, whereas data from HPV-positive HNSCC cell lines suggest an enhanced cellular radiosensitivity based on a defect in DNA double-strand break (DSB) repair. The critical limitation of the latter theory is that the evidence was largely derived from a small number of established HPV-positive HNSCC cell lines. Methods and materialsFresh patient-derived OPSCC samples were cut in 400 µm sections and cultured on cell culture inserts. Slice cultures were irradiated, in part combined with ATM inhibition, and fixed and frozen after 2 and 24 h. DSBs were analyzed by quantification of 53BP1 foci in nuclei co-stained with the SCC marker p63 via immunofluorescence microscopy. ResultsEx vivo OPSCC tumor slice cultures maintained stable oxygenation and proliferation characteristics for at least 3 days. Areas of p63-positivity in immunofluorescence microscopy matched histologically confirmed tumor cell areas in serial sections, indicating the suitability of p63 as a tumor cell marker. p63-positive nuclei in HPV-positive OPSCC tissues (n = 14) showed profoundly elevated numbers of residual radiation-induced DSBs as compared to those from HPV-negative OPSCC (n = 12) (3 Gy: on average 4.9 vs. 1.2 foci per nucleus; p < 0.0001). Within the HPV-positive subgroup, samples derived from patients with a smoking history of less than 10 pack years demonstrated higher residual DSBs as compared to those derived from patients with 10 or more pack years (3 Gy: on average 6.5 vs. 3.2 foci per nucleus; p = 0.0105). Additional ATM inhibition resulted in a substantial increase in residual foci in all 4 HPV-negative samples tested but strikingly only in 2 out of 11 HPV-positive samples. ConclusionsIn summary, our data provide robust, cell line-independent experimental evidence for an intrinsic DSB repair deficiency in HPV-positive OPSCC, strongly suggesting a meaningful contribution to the enhanced clinical radiosensitivity. The reduced effectiveness of ATM inhibition indicates a defect in the ATM-orchestrated DNA damage response. Lower numbers of residual 53BP1 nuclear foci in the ex vivo assay may identify HPV-positive patients with effective DSB repair who should potentially be excluded from de-intensification approaches.

Characterization of radiation-induced micronuclei associated with premature senescence, and their selective removal by senolytic drug, ABT-263

Radiotherapy is well-recognized as an efficient non-invasive remedy for cancer treatment. Since 10 Gy, a weekly total dose for conventional radiotherapy, was proven to create unreparable and residual DNA double-strand breaks (DSBs), they were found to give rise to mitotic failure, such as mitotic catastrophe, which resulted in multiple micronuclei associated with premature senescence. We demonstrated that pulverization of micronuclear DNA was caspase-dependent and triggered not ATM-dependent but DNA-PK-dependent DNA damage response, including phosphorylation of histone H2AX. Pulverization of micronuclear DNA and senescence-associated secretory phenotype (SASP) worsen tumor microenvironment after radiotherapy, so that senolytic drug was applied to eliminate senescent cancer cells. Prematurely senescent cancer cells with micronuclei caused by 10 Gy of γ-irradiation were subjected to 5 μM of ABT-263, a Bcl-2 family inhibitor, and selective cancer cell death by apoptosis was observed, while ABT-263 had little effect on growing cancer cells. Western blot analysis showed augmented expression of both apoptotic and anti-apoptotic proteins in senescent cells, indicating that increased apoptotic factors are essential for selective apoptotic cell death in combination with ABT-263. Our results suggested that selective elimination of senescent cells alleviates SASP and micronuclei-mediated the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) activation, both of which lead to unfavorable adverse effects caused by radiotherapy.

Molecular Response to Combined Molecular- and External Radiotherapy in Head and Neck Squamous Cell Carcinoma (HNSCC).

Simple SummaryOur previous preclinical trial in a head and neck squamous cell carcinoma (HNSCC) xenograft model showed a high potential for the improvement of curative treatment outcome upon the combination treatment of a radiolabeled (Yttrium-90) anti-EGFR antibody (Cetuximab) and external radiotherapy. We aim to elucidate the molecular response of HNSCC tumors upon this combination. Here, we show that the combination treatment leads to an increasing number and complexity of DNA double strand breaks. The upregulation of p21cip1/waf1 expression and cleaved caspase-3 suggest a blockage of cell cycle transition and an induction of programmed cell death. Collectively, a complex interplay between molecular mechanisms involved in cell death induction, cell cycle arrest, and DNA double strand break repair accounts for the beneficial potential using Yttrium-90-Cetuximab in combination with external radiotherapy. Combination treatment of molecular targeted and external radiotherapy is a promising strategy and was shown to improve local tumor control in a HNSCC xenograft model. To enhance the therapeutic value of this approach, this study investigated the underlying molecular response. Subcutaneous HNSCC FaDuDD xenografts were treated with single or combination therapy (X-ray: 0, 2, 4 Gy; anti-EGFR antibody (Cetuximab) (un-)labeled with Yttrium-90 (90Y)). Tumors were excised 24 h post respective treatment. Residual DNA double strand breaks (DSB), mRNA expression of DNA damage response related genes, immunoblotting, tumor histology, and immunohistological staining were analyzed. An increase in number and complexity of residual DNA DSB was observed in FaDuDD tumors exposed to the combination treatment of external irradiation and 90Y-Cetuximab relative to controls. The increase was observed in a low oxygenated area, suggesting the expansion of DNA DSB damages. Upregulation of genes encoding p21cip1/waf1 (CDKN1A) and GADD45α (GADD45A) was determined in the combination treatment group, and immunoblotting as well as immunohistochemistry confirmed the upregulation of p21cip1/waf1. The increase in residual γH2AX foci leads to the blockage of cell cycle transition and subsequently to cell death, which could be observed in the upregulation of p21cip1/waf1 expression and an elevated number of cleaved caspase-3 positive cells. Overall, a complex interplay between DNA damage repair and programmed cell death accounts for the potential benefit of the combination therapy using 90Y-Cetuximab and external radiotherapy.

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy.

The quantitative detection of radiation caused DNA double-strand breaks (DSB) by immunostained γ-H2AX foci using direct stochastic optical reconstruction microscopy (dSTORM) provides a deeper insight into the DNA repair process at nanoscale in a time-dependent manner. Glioblastoma (U251) cells were irradiated with 250 keV X-ray at 0, 2, 5, 8 Gy dose levels. Cell cycle phase distribution and apoptosis of U251 cells upon irradiation was assayed by flow cytometry. We studied the density, topology and volume of the γ-H2AX foci with 3D confocal microscopy and the dSTORM superresolution method. A pronounced increase in γ-H2AX foci and cluster density was detected by 3D confocal microscopy after 2 Gy, at 30 min postirradiation, but both returned to the control level at 24 h. Meanwhile, at 24 h a considerable amount of residual foci could be measured from 5 Gy, which returned to the normal level 48 h later. The dSTORM based γ-H2AX analysis revealed that the micron-sized γ-H2AX foci are composed of distinct smaller units with a few tens of nanometers. The density of these clusters, the epitope number and the dynamics of γ-H2AX foci loss could be analyzed. Our findings suggest a discrete level of repair enzyme capacity and the restart of the repair process for the residual DSBs, even beyond 24 h. The dSTORM superresolution technique provides a higher precision over 3D confocal microscopy to study radiation induced γ-H2AX foci and molecular rearrangements during the repair process, opening a novel perspective for radiation research.

In vitro cellular and proteome assays identify Wnt pathway and CDKN2A-regulated senescence affected in mesenchymal stem cells from mice after a chronic LD gamma irradiation in utero

Reliable data on the effects of chronic prenatal exposure to low dose (LD) of ionizing radiation in humans are missing. There are concerns about adverse long-term effects that may persist throughout postnatal life of the offspring. Due to their slow cell cycle kinetics and life-long residence time in the organism, mesenchymal stem cells (MSCs) are more susceptible to low level genotoxic stress caused by extrinsic multiple LD events. The aim of this study was to investigate the effect of chronic, prenatal LD gamma irradiation to the biology of MSCs later in life. C3H mice were exposed in utero to chronic prenatal irradiation of 10 mGy/day over a period of 3 weeks. Two years later, MSCs were isolated from the bone marrow and analyzed in vitro for their radiosensitivity, for cellular senescence and for DNA double-strand break recognition after a second acute gamma-irradiation. In addition to these cellular assays, changes in protein expression were measured using HPLC–MS/MS and dysregulated molecular signaling pathways identified using bioinformatics. We observed radiation-induced proteomic changes in MSCs from the offspring of in utero irradiated mice (leading to ~ 9.4% of all detected proteins being either up- or downregulated) as compared to non-irradiated controls. The proteomic changes map to regulation pathways involved in the extracellular matrix, the response to oxidative stress, and the Wnt signaling pathway. In addition, chronic prenatal LD irradiation lead to an increased rate of in vitro radiation-induced senescence later in life and to an increased number of residual DNA double-strand breaks after 4 Gy irradiation, indicating a remarkable interaction of in vivo radiation in combination with a second acute dose of in vitro radiation. This study provides the first insight into a molecular mechanism of persistent MSC damage response by ionizing radiation exposure during prenatal time and will help to predict therapeutic safety and efficacy with respect to a clinical application of stem cells.

Comparative Therapeutic Exploitability of Acute Adaptation Mechanisms to Photon and Proton Irradiation in 3D Head and Neck Squamous Cell Carcinoma Cell Cultures.

Simple SummaryTreatment resistance is a major obstacle affecting the outcome of patients with head and neck squamous cell carcinomas (HNSCC). Proton beam therapy may be beneficial in the treatment of HNSCC due to optimized dose distribution and consequent sparing of healthy tissue. However, molecular data on tumor cell responses upon proton irradiation appear sparse. The aim of this study was to compare the acute adaptative kinome of HNSCC cell lines to photon and proton irradiation and elucidate their therapeutic potential. Despite pronounced differences in kinome profiles upon photon and proton irradiation, these differences failed to be therapeutically exploitable. Instead, our results reveal radiation type-independent sensitization upon pharmacological inhibition of selected targets.For better tumor control, high-precision proton beam radiation therapy is currently being intensively discussed relative to conventional photon therapy. Here, we assumed that radiation type-specific molecular response profiles in more physiological 3D, matrix-based head and neck squamous cell carcinoma (HNSCC) cell cultures can be identified and therapeutically exploited. While proton irradiation revealed superimposable clonogenic survival and residual DNA double strand breaks (DSB) relative to photon irradiation, kinome profiles showed quantitative differences between both irradiation types. Pharmacological inhibition of a subset of radiation-induced kinases, predominantly belonging to the mitogen-activated protein kinase (MAPK) family, failed to sensitize HNSCC cells to either proton or photon irradiation. Likewise, inhibitors for ATM, DNA-PK and PARP did not discriminate between proton and photon irradiation but generally elicited a radiosensitization. Conclusively, our results suggest marginal cell line-specific differences in the radiosensitivity and DSB repair without a superiority of one radiation type over the other in 3D grown HNSCC cell cultures. Importantly, radiation-induced activity changes of cytoplasmic kinases induced during the first, acute phase of the cellular radiation response could neither be exploited for sensitization of HNSCC cells to photon nor proton irradiation.

Long-term culture of mesenchymal stem cells impairs ATM-dependent recognition of DNA breaks and increases genetic instability

BackgroundMesenchymal stem cells (MSCs) are attracting increasing interest for cell-based therapies, making use of both their immuno-modulating and regenerative potential. For such therapeutic applications, a massive in vitro expansion of donor cells is usually necessary to furnish sufficient material for transplantation. It is not established to what extent the long-term genomic stability and potency of MSCs can be compromised as a result of this rapid ex vivo expansion. In this study, we investigated the DNA damage response and chromosomal stability (indicated by micronuclei induction) after sub-lethal doses of gamma irradiation in murine MSCs at different stages of their in vitro expansion.MethodsBone-marrow-derived tri-potent MSCs were explanted from 3-month-old female FVB/N mice and expanded in vitro for up to 12 weeks. DNA damage response and repair kinetics after gamma irradiation were quantified by the induction of γH2AX/53BP1 DSB repair foci. Micronuclei were counted in post-mitotic, binucleated cells using an automated image analyzer Metafer4. Involvement of DNA damage response pathways was tested using chemical ATM and DNA-PK inhibitors.ResultsMurine bone-marrow-derived MSCs in long-term expansion culture gradually lose their ability to recognize endogenous and radiation-induced DNA double-strand breaks. This impaired DNA damage response, indicated by a decrease in the number of γH2AX/53BP1 DSB repair foci, was associated with reduced ATM dependency of foci formation, a slower DNA repair kinetics, and an increased number of residual DNA double-strand breaks 7 h post irradiation. In parallel with this impaired efficiency of DNA break recognition and repair in older MSCs, chromosomal instability after mitosis increased significantly as shown by a higher number of micronuclei, both spontaneously and induced by γ-irradiation. Multifactorial regression analysis demonstrates that in vitro aging reduced DNA damage recognition in MSCs after irradiation by a multiplicative interaction with dose (p < 0.0001), whereas the increased frequency of micronuclei was caused by an additive interaction between in vitro aging and radiation dose.ConclusionThe detrimental impact of long-term in vitro expansion on DNA damage response of MSCs warrants a regular monitoring of this process during the ex vivo growth of these cells to improve therapeutic safety and efficiency.

Nanoscopic analysis of 53BP1, BRCA1 and Rad51 reveals new insights in temporal progression of DNA-repair and pathway choice

The accumulation and spatial distribution of 53BP1, BRCA1 and Rad51, key proteins in DNA double-strand break (DSB) repair, was investigated with high temporal resolution over a time span of 24 h, using STED nanoscopy. DNA lesions were induced by irradiation with high-LET (linear energy transfer) α-particles. We show that 53BP1 IRIF formation occurs quickly in almost all cells and after about 6 h the fraction of 53BP1 IRIF positive cells slowly declines. Against the expectations BRCA1 and Rad51 IRIF formation is only shortly delayed but with the maximum of cells showing foci after 6 and 8 h after irradiation. At this stage, almost all IRIF in a given Rad51-positive cell show Rad51 accumulation, suggesting that repair via homologous recombination is attempted at almost all residual DSB sites. The frequency of BRCA1 IRIF positive cells increases much earlier and remains high after Rad51 positive cells start to decline, supporting models claiming that functional roles of BRCA1 change over time. Correlation analysis showed a high degree of correlation of Rad51 with BRCA1, while the exclusion of 53BP1 from the actual resection-zone is demonstrated by anti-correlation of Rad51 and 53BP1. Interestingly, these correlation and anti-correlation patterns exhibit complementary temporal variation.

Delayed γH2AX foci disappearance in mammary epithelial cells from aged women reveals an age-associated DNA repair defect.

Aging is a degenerative process in which genome instability plays a crucial role. To gain insight into the link between organismal aging and DNA repair capacity, we analyzed DNA double-strand break (DSB) resolution efficiency in human mammary epithelial cells from 12 healthy donors of young and old ages. The frequency of DSBs was measured by quantifying the number of γH2AX foci before and after 1Gy of γ-rays and it was higher in cells from aged donors (ADs) at all times analyzed. At 24 hours after irradiation, ADs retained a significantly higher frequency of residual DSBs than young donors (YDs), which had already reached values close to basal levels. The kinetics of DSB induction and disappearance showed that cells from ADs and YDs repair DSBs with similar speed, although analysis of early times after irradiation indicate that a repair defect may lie within the firing of the DNA repair machinery in AD cells. Indeed, using a mathematical model we calculated a constant factor of delay affecting aged human epithelial cells repair kinetics. This defect manifests with the accumulation of DSBs that might eventually undergo illegitimate repair, thus posing a relevant threat to the maintenance of genome integrity in older individuals.

Residual DNA double strand breaks correlates with excess acute toxicity from radiotherapy

Introduction: A high risk for development of severe side effects after radiotherapy may be correlated with high cellular radiosensitivity. To enhance radiation therapy efficiency a fast and reliable in-vitro test is desirable to identify radiosensitive patients. The aim of present study was to identify the mechanism of radiation induced DNA double-strand breaks (DSBs) and DSB repair kinetics on radiosensitivity and normal tissue complications caused by the radiation therapy. Materials and Methods: The study included 50 breast cancer patients. Blood samples (taken before radiotherapy) were irradiated in vitro with 1 and 2 Gy X-rays and The initial level of double-strand breaks (DSB) and repair kinetics analyzed by flow cytometric method of phosphorylation of histone H2A (γ-H2AX-assay) at 30 minutes, 3 and 24 hours. All the patients received similar tangential irradiation of the whole breast and conventional fractionation and acute normal tissue reactions were assessed by Radiation Therapy Oncology Group criteria. Results: In the in-vitro experiment repair kinetic of DSBs after 3 and 24 hours were strongly correlated with the acute skin toxicity score following irradiation (P=0.0007 and P=0.0005 respectively; Pearson's correlation test). This issue could be subject for treatment adjustment. Conclusion: Our findings strongly suggest that the measurement of DSB by performing γ- H2AX flow cytometric analysis has the potential to be developed into a clinically useful predictive assay for distinguishing the overreactors among breast cancer patients prior to the start of radiotherapy. However, further clinical trials are required to validate these biomarkers.