gamma-H2AX Foci Research Articles

Modulation of ATM enhances DNA repair in G2/M phase of cell cycle and averts senescence in Fuchs endothelial corneal dystrophy

Fuchs Endothelial Corneal Dystrophy (FECD) is an aging disorder characterized by expedited loss of corneal endothelial cells (CEnCs) and heightened DNA damage compared to normal CEnCs. We previously established that ultraviolet-A (UVA) light causes DNA damage and leads to FECD phenotype in a non-genetic mouse model. Here, we demonstrate that acute treatment with chemical stressor, menadione, or physiological stressors, UVA, and catechol estrogen (4-OHE2), results in an early and increased activation of ATM-mediated DNA damage response in FECD compared to normal CEnCs. Acute stress with UVA and 4OHE2 causes (i) greater cell-cycle arrest and DNA repair in G2/M phase, and (ii) greater cytoprotective senescence in NQO1−/− compared to NQO1+/+ cells, which was reversed upon ATM inhibition. Chronic stress with UVA and 4OHE2 results in ATM-driven cell-cycle arrest in G0/G1 phase, reduced DNA repair, and cytotoxic senescence, due to sustained damage. Likewise, UVA-induced cell-cycle reentry, gamma-H2AX foci, and senescence-associated heterochromatin were reduced in Atm-null mice. Remarkably, inhibiting ATM activation with KU-55933 restored DNA repair in G2/M phase and attenuated senescence in chronic cellular model of FECD lacking NQO1. This study provides insights into understanding the pivotal role of ATM in regulating cell-cycle, DNA repair, and senescence, in oxidative-stress disorders like FECD.

Severe Combined Immunodeficiency from a Homozygous DNA Ligase 1 Mutant with Reduced Catalytic Activity but Increased Ligation Fidelity

A cell’s ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3’ end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.

Nicaraven attenuates the acquired radioresistance of established tumors in mouse models via PARP inhibition.

Nicaraven has been reported to inhibit the activity of poly (ADP-ribose) polymerase (PARP). In this study, we investigated the probable ability of nicaraven to attenuate cancer radioresistance during fractionated radiotherapy. Tumor models were established in C57BL/6 mice and BALB/c nude mice by subcutaneous injection of Lewis mouse lung carcinoma cancer cells and A549 human lung cancer cells, respectively. When the tumors had grown to approximately 100 mm3, we initiated fractionated radiotherapy. Nicaraven or saline was administered immediately after each irradiation exposure. Compared to saline treatment, nicaraven administration significantly induced gamma-H2AX foci formation and cell apoptosis in tumors at 1 or 3days after an additional challenge exposure to 10Gy and inhibited tumor growth during the short-term follow-up period, suggesting increased radiosensitivity of cancer cells. Moreover, the expression of PARP in tumor tissue was decreased by nicaraven administration. Our data suggest that nicaraven likely attenuates the acquired radioresistance of cancers through PARP inhibition.

Biodosimetry Based on Gamma-H2AX Quantification in Human Peripheral Blood Lymphocytes after Partial-body Irradiation.

Quantification of gamma-H2AX foci can estimate exposure to ionizing radiation. Most nuclear and radiation accidents are partial-body irradiation, and the doses estimated using the total-body irradiation dose estimation formula are often lower than the actual dose. To evaluate the dose-response relation of gamma-H2AX foci in human peripheral blood lymphocytes after partial-body irradiation and establish a simple and high throughput model to estimate partial-body irradiation dose, we collected human peripheral blood and irradiated with 0-, 0.5-, 1-, 2-, 3-, 4-, 5-, 6-, and 8-Gy gamma rays to simulate total-body irradiation in vitro. Gamma-H2AX foci were quantitated by flow cytometry at 1 h after irradiation, and a dose-response curve was established for total-body irradiation dose estimation. Then, a partial-body irradiation dose-response calibration curve was established by adding calibration coefficients based on the Dolphin method. To reflect the data distribution of all doses more realistically, the partial-body irradiation dose-response calibration curve was divided into two sections. In addition, partial-body irradiation was simulated in vitro, and the PBI data were substituted into curves to verify the accuracy of the two partial-body irradiation calibration curves. Results showed that the dose estimation variations were all less than 30% except the 25% partial-body irradiation group at 1 Gy, and the partial-body irradiation calibration dose-response curves were YF 1 = - 3.444 x 2 + 18.532 x + 3.109, R 2 = 0.92 (YF ≤ 27.95); YF 2 = - 2.704 x 2 + 37.97 x - 56.45, R 2 = 0.86 (YF > 27.95). Results also suggested that the partial-body irradiation dose-response calibration curve based on the gamma-H2AX foci quantification in human peripheral blood lymphocytes is a simple and high throughput model to assess partial-body irradiation dose.

Prediction of late adverse events in pelvic cancer patients receiving definitive radiotherapy using radiation-induced gamma-H2AX foci assay.

Radiation can induce DNA double-stranded breaks, which are typically detected by the fluorescence of phosphorylated histone H2AX. In this study, we examined the usefulness of the dynamics of radiation-induced gamma-H2AX foci of peripheral blood lymphocytes (PBLs), as a marker of DNA repair ability, in predicting late adverse events from radiotherapy. A total of 46 patients with cervical, vaginal and anal canal cancers treated with radical radiotherapy between 2014 and 2019 were included in this analysis. Concurrent chemotherapy was administered in 36 cases (78.3%). Peripheral blood was obtained before treatment, and then irradiated ex vivo with 1Gy X-ray. The ratio of radiation-induced gamma-H2AX foci in PBLs measured at 30min and at 4h was defined as the foci decay ratio (FDR). With a median follow-up of 54months, 9 patients (19.6%) were observed to have late genitourinary or gastrointestinal (GU/GI) toxicity. The FDR ranged from 0.51 to 0.74 (median 0.59), with a significantly higher incidence of Grade 1 or higher late adverse events in the FDR ≥ 0.59 group. In multivariate analysis, FDR ≥ 0.59 and hypertension also emerged as significant factors associated with the development of late toxicities. Overall, our results suggest that measurement of radiation-induced gamma-H2AX foci in PBLs may predict the risk of late GU/GI toxicities from chemoradiotherapy, which can enable tailoring the radiation dose to minimize adverse effects.

Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing

BackgroundEarly-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined.MethodsHere, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes.ResultsAnalysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of gammaH2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased gammaH2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities.ConclusionsTogether, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.

RENEB Inter-Laboratory Comparison 2021: The Gamma-H2AX Foci Assay.

The Running the European Network of biological and retrospective dosimetry (RENEB) network of laboratories has a range of biological and physical dosimetry assays that can be deployed in the event of a radiation incident to provide exposure assessment. To maintain operational capability and provide training, RENEB runs regular inter-laboratory comparison (ILC) exercises. The RENEB ILC2021 was carried out with all the biological and physical dosimetry assays employed in the network. The focus of this paper is to evaluate the results from 6 laboratories that took part using the gamma-H2AX radiation-induced foci assay. For two laboratories this was their first RENEB ILC. Blood samples were homogenously exposed to 240 kVp X rays (1 Gy/min) to provide calibration data, (0-4 Gy), and a few weeks later three blind coded test samples, (0, 1.2 and 3.5 Gy) were prepared. All samples were allowed a 2 h repair time at 37°C before being transported, on ice packs, to the participating laboratories. On arrival, the samples were processed, scored either manually or automatically for gamma-H2AX foci and dose estimates for the 3 blind coded samples sent to the organizing laboratory. The temperature of samples during transit and the time taken to report the dose estimates were recorded. Subsequent examination of the data from each laboratory used the doses estimates to assign triage categories to the samples. After receipt of the samples, the quickest report of dose estimates was 4.6 h. Analysis of variance revealed that the laboratory carrying out the assay had a significant effect on the foci yield (P < 0.001) for the calibration data, but not on the dose estimates of the blind coded samples (P = 0.101). All laboratories correctly identified the unirradiated and irradiated samples, although the dose estimates for the latter tended to under-estimate the dose. Two participants seriously under-estimated the dose for the highly exposed sample, which resulted in the sample being placed in the lowest triage category not the highest. However, this under-estimation resulted from the samples not remaining cold during shipment, due to a delay in transit and was not related to the experience of the participating laboratory. Overall, the RENEB network laboratories have demonstrated it is possible to quickly identify a recent whole-body acute exposure using the gamma-H2AX assay within the conditions of the ILC. In addition, an ILC provides a useful training and harmonization exercise for laboratories.

RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays.

Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.

Abstract 6613: Label-free multimodal multiphoton microscopy for predicting DNA damage response in patient derived non-small cell lung cancer organoids

Abstract Background: DNA damage response (DDR) is a fundamental mechanism for evaluating efficacy of cancer therapeutics, many of which target DNA or associated processes (e.g., etoposide: inhibits DNA synthesis, cisplatin: crosslinks DNA). Current techniques to evaluate DDR rely on immunostaining for gamma H2AX foci (γH2AX), which is an indicator of DNA double-strand break formation. While γH2AX immunostaining can provide a snapshot of DDR in fixed cellular and tissue samples, this method is technically cumbersome as: 1) temporal monitoring of DDR requires timepoint replicates; 2) extensive assay development efforts for 3D samples such as organoids, and 3) protocols for γH2AX immunostaining and its evaluation are time-consuming. The goal of this project is to reduce overall burden on assay duration and development in NSCLC organoids by leveraging label-free multiphoton imaging. Method: To predict DDR in 3D organoid samples, label-free multimodal multiphoton techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) imaging can provide intracellular information based on endogenous contrast mechanisms. SLAM makes use of a single laser to excite autofluorescence in multiple cellular components including FAD and NAD(P)H, enabling the calculation of the optical redox ratio, which estimates the equilibrium of electron donating versus accepting species in cells. Moreover, label-free, multiphoton systems enable imaging of live samples without the need to add labels or generate sacrificial samples and have improved image acquisition in 3D space over conventional confocal microscopy. Using SLAM, we propose to predict DDR in non-small cell lung cancer (NSCLC) organoids. NSCLC organoids recapitulate histological and genetic features of their derived patient tissue, retaining tumor variability. Results: Predictive modeling via gradient boost regressor between handcrafted SLAM image features and γH2AX immunostained images on weakly-paired images confirmed strong correlation between SLAM image features and γH2AX speckles/nuclei. Across multiple patient derived NSCLC organoid lines and multiple DNA targeting chemotherapeutics, the optical redox ratio was demonstrated to robustly predict DDR. Conclusion: Label-free imaging via SLAM can be used to predict DDR in live 3D NSCLC organoids with minimal sample handling and without the need to add labelled reporters, enabling quicker and less technically involved DDR assaying. Citation Format: Terrence T. Roh, Aneesh Alex, Janet E. Sorrells, Prasanna Chandramouleeswaran, Marina Marjanovic, Steve R. Hood, BanuPriya Sridharan, Stephen A. Boppart. Label-free multimodal multiphoton microscopy for predicting DNA damage response in patient derived non-small cell lung cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6613.

Anticancer activity of sinapic acid by inducing apoptosis in HT-29 human colon cancer cell line.

Colorectal cancer is the third most lethal and fourth most commonly diagnosed cancer worldwide. Sinapic acid, a derivative of hydroxycinnamic acid, is a promising phytochemical exhibiting numerous pharmacological activities in various systems. It is a substantial chain-breaking antioxidant that operates as a radical scavenger. The aim of this research was to investigate the antiproliferative effect of sinapic acid on the HT-29 cell line besides the mechanisms underlying this activity. The effect of sinapic acid on the viability of HT-29 cell line was investigated using XTT assay. The levels of BCL-2, cleaved caspase 3, BAX, cleaved PARP, and 8-oxo-dG were measured using ELISA. Gamma-H2AX and cytochrome c expressions were assessed semiquantitatively using immunofluorescence staining. Sinapic acid at 200 µm and higher doses produced a significant antiproliferative effect on HT-29 cells. The IC50 value was found to be 317.5 µm for 24 h. Sinapic acid (317.5µm) significantly elevated cleaved caspase 3, BAX, cleaved PARP, and 8-oxo-dG levels. The levels of gamma-H2AX foci are significantly higher, while the levels of cytochrome c are lower in sinapic acid-treated HT-29 cells. These results indicate that sinapic acid has antiproliferative, apoptotic, and genotoxic effects on colon cancer cells.

BRCA1 interactors, RAD50 and BRIP1, as prognostic markers for triple-negative breast cancer severity.

Introduction: BRIP1 (BRCA1-interacting protein 1) is one of the major interacting partners of BRCA1, which plays an important role in repair by homologous recombination (HR). This gene is mutated in around 4% of cases of breast cancer; however, its mechanism of action is unclear. In this study, we presented the fundamental role of BRCA1 interactors BRIP1 and RAD50 in the development of differential severity in triple-negative breast cancer (TNBC) among various affected individuals. Methods: We have analyzed the expression of DNA repair-related genes in different BC cells using Real-time PCR and western blotting analysis and assessed changes in stemness property and proliferation through Immunophenotyping. We have performed cell cycle analysis to see the defect in checkpoints and also immunofluorescence assay to confirm the accumulation of gamma-H2AX and BRCA1 foci and subsequent incidence. We have performed a severity analysis using TCGA data sets for comparing the expression in MDA-MB-468 MDA-MB-231 and MCF7 cell line. Results: We showed that in some TNBC cell lines such as MDA-MB-231, the functioning of both BRCA1/TP53 is compromised. Furthermore, the sensing of DNA damage is affected. Due to less damage-sensing capability and low availability of BRCA1 at the damage sites, the repair by HR becomes inefficient, leading to more damage. Accumulation of damage sends a signal for over activation of NHEJ repair pathways. Over expressed NHEJ molecules with compromised HR and checkpoint conditions lead to higher proliferation and error-prone repair, which increases the mutation rate and corresponding tumour severity. The in-silico analysis of the TCGA datasets with gene expression in the deceased population showed a significant correlation of BRCA1 expression with overall survival (OS) in TNBCs (0.0272). The association of BRCA1 with OS became stronger with the addition of BRIP1 expression (0.000876**). Conclusion: The severity phenotypes were more in cells having compromised BRCA1-BRIP1 functioning. Since the OS is directly proportional to the extent of severity, the data analysis hints at the role of BRIP1 in controlling the severity of TNBC.

Bystander Response Following High-Dose X-irradiation; Time-dependent Nature of GammaH2AX Foci and Cell Death Consequences.

The paradigm shifts in target theory could be defined as the radiation-triggered bystander response in which the radiation deleterious effects occurred in the adjacent cells. This study aims to assess bystander response in terms of DNA damage and their possible cell death consequences following high-dose radiotherapy. Temporal characteristics of gH2AX foci as a manifestation of DNA damage were also evaluated. In this experimental study, bystander response was investigated in human carcinoma cells of HeLa and HN5, neighboring those that received high doses. Medium transfer was performed from 10 Gy-irradiated donors to 1.5 Gy-irradiated recipients. GammaH2AX foci, clonogenic and apoptosis assays were investigated. The gH2AX foci time-point study was implemented 1, 4, and 24 h after the medium exchange. DNA damage was enhanced in HeLa and HN5 bystander cells with the ratio of 1.27 and 1.72, respectively, which terminated in more than two-fold clonogenic survival decrease, along with gradual apoptosis increase. GammH2AX foci temporal characterization revealed maximum foci scoring at the 1 h time-point in HeLa, and also 4 h in HN5, which remained even 24 h after the medium sharing in higher level than the control group. The time-dependent nature of bystander-induced gH2AX foci as a DNA damage surrogate marker was highlighted with the persistent foci at 24 h. considering an outcome of bystander-induced DNA damage, predominant role of clonogenic cell death was also elicited compared to apoptosis. Moreover, the role of high-dose bystander response observed in the current work clarified bystander potential implications in radiotherapy.

Targeting PLK1 As a Novel Strategy for Acute Myeloid Leukemias with Fanconi Anemia Pathway Mutations

The Fanconi anemia (FA) signaling network is critical to maintaining genomic integrity during interphase and mitosis. Biallelic germline mutation of any of the 22 genes that constitute this pathway (FANCA-FANCW) results in the disorder FA, which is characterized by congenital malformations, a predisposition to bone marrow failure, and pediatric acute myeloid leukemias (AMLs). Among the general population, acquired genetic disruptions of the FA pathway are present in 18% of sporadic pediatric AMLs, underscoring the importance of this pathway in the prevention of hematological malignancy. FANCA is the gene most commonly mutated in FA and is one of the most frequently disrupted FA pathway genes in sporadic AML. Therefore, the identification of precision therapies for FANCA-deficient AML is a high clinical priority. To identify synthetic lethal targets of FANCA, we previously identified mitotic phosphosignaling pathways required for the survival of FANCA-/- patient-derived fibroblasts through a kinome-wide shRNA screen. This screen identified the mitotic kinases CHEK1, PLK1, and BUB1 as hits. These findings corroborate work by others who have identified synthetic lethal interactions between PLK1 and the FA pathway components FANCG and BRCA1, suggesting inactivation of the FA pathway may sensitize cancers to PLK1 inhibition. The clinical success of PLK1 inhibitors, such as volasertib and onvansertib, has been limited by the absence of predictive biomarkers of response and adverse side effects. To test our hypothesis that FA gene mutations may predict sensitivity to PLK1 inhibition we generated an isogenic panel of human AML cells with a stable knockout of the FANCA gene. Utilizing in vitro cell viability assays, we have demonstrated the selective killing of FA-deficient AMLs by pharmacological PLK1 inhibition through low doses of volasertib and onvansertib. We also found that PLK1-inhibition impairs the hematopoietic potential of Fanca-/- murine hematopoietic stem and progenitor cells by performing in vitro bone marrow colony formation assays. Similarly, we demonstrated that primary human AMLs with multiple disruptions within the FA-pathway were hypersensitive to low doses of volasertib. To investigate the mechanism of synthetic lethality, we performed quantitative proteomic analysis of FANCA immunoprecipitates, which identified PLK1 as a novel FANCA-binding partner during mitosis. We validated this finding through high-resolution immunofluorescence microscopy and proximity ligation assays, which revealed a direct interaction between FANCA and PLK1 at kinetochores. Additionally, we demonstrated that volasertib treatment induces monoubiquitination of FANCD2 and colocalization of FANCA with FANCD2 at gamma-H2AX foci. Together, these findings indicate that PLK1 inhibition causes damage to mitotic chromosomes and subsequent activation of the FA pathway. Our work identifies a mitosis-specific vulnerability of FA-deficient cells and suggests that genetic disruptions of the FA pathway may be predictive of sensitivity to PLK1 inhibition, providing a preclinical rationale for the development of precision therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Upregulation of Long Noncoding RNA MALAT1 in Colorectal Cancer Promotes Radioresistance and Aggressive Malignance.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a conserved transcript with 8000 nt, is highly associated with malignancy of numerous cancer types. However, the function of MALAT1 plays in regulating the response to radiotherapy in colorectal cancer (CRC) remains unclear. Thus, the object of this study is to investigate the functions of MALAT1 in CRC radioresistance. First, the expression of MALAT1 in colon adenocarcinoma (COAD) was analyzed through the Cancer Genome Atlas (TCGA) database. Then, we detected the expression level of MALAT1 in tumor tissues and CRC cell lines and analyzed the relevance of MALAT1 and clinicopathological parameters. In the end, the effect of silencing MALAT1 on the radiosensitivity of CRC cells was investigated, and its potential mechanism was preliminarily illustrated. The analysis of TCGA data showed that MALAT1 was closely related to the type of tumor, and high expression of MALAT1 was remarkably relevant to poor outcome. MALAT1 was highly expressed in CRC tissues and cell lines and related to tumor stages. Knockdown of MALAT1 could significantly suppress colony survival, proliferation, and migration and increase apoptosis, G2/M phase arrest, and formation of gamma-H2AX foci in HCT116, whether in combination with X-rays or not. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that the regulated proteins were principally enriched in the glycosaminoglycan degradation pathway after silencing MALAT1. Our results implied that MALAT1 was highly expressed in CRC and associated with tumor stage and prognosis. Silencing MALAT1 can increase HCT116 cell radiosensitivity, which may be potentially influenced by glycosaminoglycan degradation pathway.

DNA damage induced by radiation exposure from cardiac catheterization – an analysis in patients and operators

Abstract Background The biological effects of low-dose radiation from cardiac imaging or intervention remain largely unknown. This study aimed to evaluate the effects of ionized radiation from cardiac catheterization on genomic DNA integrity and inflammatory cytokines in patients and operators. Methods Peripheral mononuclear cells (MNCs) were isolated from patients (n=52) and operators (n=35) before and after coronary angiography and/or percutaneous coronary intervention. Expression of gammaH2AX, a marker for DNA double-strand breaks, was measured by immunofluorescence. Dicentric chromosomes (DICs), a form of chromosome aberrations, were assayed using a fluorescent in situ hybridization technique. Results In the patient MNCs, the numbers of gammaH2AX foci and DICs increased after cardiac catheterization by 101±75% and 28±99%, respectively (P&amp;lt;0.05 for both). The mRNA expressions of interleukin (IL)-1α, IL-1β, leukemia inhibitory factor (LIF) and caspase-1 were significantly increased by radiation exposure from cardiac catheterization. The increase in IL-1β was significantly correlated with that of gammaH2AX, but not with dose area product. In the operators, neither gammaH2AX foci nor DICs level was changed, but IL-1β mRNA was significantly increased. Protein expression of IkappaBα was significantly decreased in both groups. Conclusions DNA damage was increased in the MNCs of patients, but not of operators, who underwent cardiac catheterization. Inflammatory cytokines were increased both in the patients and operators, presumably through activation of NF-kappaB. Further efforts to reduce radiation exposure from cardiac catheterization are necessary both for patients and operators. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grants-in-Aid for Scientific Research (KAKENHI)

Abstract 1630: Development of a clinically relevant in vitro model of chemo-radioresistant cervical cancer cells for insights into treatment resistance

Abstract Resistance to therapeutic treatment is a common phenomenon that contributes to relapse in several cancers including cervical that is the fourth most common cancer in women across the world. Acquired resistance to chemotherapy, radiotherapy or their combination contributes to a majority of relapse cases which suggests the need to develop models that can be used for preliminary screening of drugs or decipher the associated signaling mechanisms contributing to this resistance. Various chemo-resistant, as well as radio-resistant models, have been established as in vitro models. In cervical cancer treatment, often, radiation therapy is accompanied by the addition of radio-sensitizers such as cisplatin that increase its treatment efficiency. Hence, a combined chemo-radio-resistant in vitro model seems more relevant clinically in cervical cancer. Therefore, we designed our study to establish a cellular model that is resistant to a combined treatment of chemotherapy and radiotherapy. A chemo-radio resistant cervical cancer cell line (HeLa) was developed in vitro by a fractionated exposure of a combination of cisplatin and megavoltage X-rays. Briefly, HeLa cells were sensitized with cisplatin (0.1µM increase at every fraction) prior to radiation exposure (2Gy). The cells were treated once in 5 days and eventually after 25 fractions, delivered in 20 weeks, the cells were made resistant up to a total dose of 2.5µM cisplatin + 50Gy. These cells were further characterized and compared with radioresistant HeLa cells as well as parental cells. For this purpose, proliferation assay, clonogenic survival assay, cell cycle analysis and apoptosis assay were performed. The resistivity was confirmed by comet assay and Gamma-H2AX staining. Interestingly, an increase in cell proliferation and clonogenic survival rates was observed in chemo-radio-resistant cells. FACS analysis revealed that the proportion of cells in G2/M phase increased and apoptotic rates declined in chemo-radio-resistant cells. Further, changes in comet tails, olive moment and Gamma-H2AX foci formation in chemo-radio-resistant groups confirmed resistivity. These cells were also analyzed for their metastatic properties and results suggest that induced chemo-radio-resistant cells are more tumorigenic as compared to only radioresistant cells as well as the parental ones. In conclusion, the establishment of a chemo-radio-resistant cell line may serve as a model to study signalling mechanisms, drug screening and develop novel drugs. Further studies are underway to induce the chemo-radio resistance in other cell lines by a similar method. Citation Format: Sanchita Khurana, Surbhi Singla, Reena Sharma, Bhavana Rai, Shalmoli Bhattacharyya. Development of a clinically relevant in vitro model of chemo-radioresistant cervical cancer cells for insights into treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1630.

Evaluation of Capability and Relationship of Different Radiobiological Endpoints for Radiosensitivity Prediction in Human Tumor Cell Lines Compared with Clonogenic Survival.

Background: Establishing a predictive assay of radiosensitivity (as an appropriate, practical and cost-effective method) has been challenging.Objective: The purpose of this study is to evaluate the capability and relationship of various endpoints, including GammaH2AX, micronuclei; and apoptosis in determining the human tumor cell lines radiosensitivities compared with clonogenic survival.Material and Methods: In an experimental in-vitro study, the response of carcinoma cell lines of HN5 and HeLa to 2 Gy of 6 MV photon beam was investigated via various assays.Results: Survival fraction at 2 Gy (SF2) of HeLa and HN5 was indicated as 0.42 ± 0.06 and 0.5 ± 0.03 respectively, proposing more radioresistance of HN5. This finding was confirmed with “2 Gy apoptosis enhancement ratio” which was 1.77 and 1.42 in HeLa and HN5. The increased levels of DNA DSBs were observed after irradiation; significant in HeLa with enhancement rate of 19.24. The micronuclei formation followed an ascending trend post irradiation; but with the least difference between two cells. Although the relationship between micronuclei and clonogenic survival was moderate (R2 = 0.35), a good correlation was observed between apoptosis and clonogenic survival (R2 = 0.71).Conclusion: The results of studied endpoints agreed with the SF2, highlighting their capabilities in radiosensitivity prediction. In terms of the enhancement ratio, gammaH2AX foci scoring could be a valid indicator of radiosensitivity but not the exact surrogate marker of survival because no correlation was observed. Moreover, considering the chief determents comprising lack of time and money, the apoptotic induction might be an appropriate indicator with the best correlation coefficient.

Biopsy-derived oral keratinocytes – A model to potentially test for oral mucosa radiation sensitivity

PurposeTo establish stable in vitro growth of keratinocytes from very small biopsy specimens and successfully apply new test systems to determine their radiosensitivity. Materials and MethodsOral mucosa biopsies (diameter: 1.7 mm) from 15 subjects were immobilized with custom-made cups onto culture plates. Outgrowing cells were tested for cytokeratin 5/14 and Ki67, expanded, radiated at different doses, and seeded onto circumscribed areas before being allowed to spread centrifugally. In this newly developed spreading assay, cell-covered areas were measured by image analysis. For statistical analysis, a linear mixed regression model was used; additionally, results were correlated to the radiation dose applied. Colony forming efficiency (CFE) was used to validate the results. DNA damage repair was analysed by gammaH2AX and 53BP1 foci quantification using immunofluorescence microscopy 24 h and 96 h after irradiation. ResultsStable keratinocyte growth continued for up to 7 weeks in 14 biopsies. Cells spread reliably from an initial 16.6 mm2 up to a median of 119.2 mm2 (range: 54.4–290). Radiated cells spread to only 100.7 mm2 (2 Gy; range: 55.3–266.7); 73.2 mm2 (4 Gy; 15–240.4); 47 mm2 (6 Gy; 2–111.9), and 22.7 mm2 (8 Gy; 0–80). Similarly, CFE decreased from 0.223 (0 Gy) to 0.0028 (8 Gy). Using an individual donor as a random factor, cell spread correlated with CFE, where radiation dose was the main driver (decrease by 0.50, adjusted for area). Upon irradiation with 6 Gy, radiation-induced DNA damage was increased after 24 h in all samples, and even after 96 h in 5 out of 7 samples, as detected by a higher number of gammaH2AX/53BP1 foci in irradiated cells (mean 3.7 for 24 h; mean 0.6 for 96 h). ConclusionIn vitro propagation of keratinocytes derived from a small biopsy is feasible. Radiation impairs cellular migration and proliferation, and the newly described spreading assay allows ranking for cellular radioresistance. The keratinocyte model also supports classical functional assays such as clonogenic survival and DNA double strand repair. The clinical relevance awaits upcoming investigations.

Simultaneous effect of gamma and Wi-Fi radiation on gamma-H2Ax expression in peripheral blood of rat: A radio-protection note

IntroductionNuclear medicine patients are isolated in a room after the injection of a radiopharmaceutical. They may be active Wi-Fi option of its smartphone mobile or other environmental radiofrequency waves. The hypothesis of this study was the evaluation of increased biological effects of the simultaneous exposure to gamma-ray and the Wi-Fi waves by measuring the level of the increased double strand-breaks DNA in peripheral blood lymphocyte in the rat. Materials and methodsFifty male Wistar rats were exposed for 2, 24, and 72 h only by Wi-Fi, 99m Tc, and simultaneously by Wi-Fi and 99m Tc. The power density levels of Wi-Fi emitter at 15 cm was 4.2nW/ cm2. An activity of 100 μCi of 99m Tc was injected intraperitoneally. Blood samples were taken by cardiac puncture following general anesthesia. Mononuclear cells are extraction by Ficoll-Hypaque density gradient centrifugation. The number of gamma-H2AX foci per nucleus was counted by flow cytometry. The statistical differences between experimental groups at 2, 24, and 72 h were determined with a repeated measure's analysis of variance. The significant difference between groups at the same time was analyzed with the Kruskal-Wallis Test. ResultsThe manner of gamma-H2AX expression was not the same for three groups in time. The number of gamma-H2AX foci between the three groups was a significant difference after 72 h. ConclusionSimultaneous Wi-Fi and gamma-ray exposures can increase the number of double-strand break DNA in peripheral blood lymphocytes to exposure of gamma-ray to 72 h after technetium injection in the rat.

Caffeic acid phenethyl ester induces radiosensitization via inhibition of DNA damage repair in androgen-independent prostate cancer cells.

In the present study, we evaluated the radiomodulatory potential of caffeic acid phenethyl ester (CAPE), an active component of traditional herbal medicine propolis. CAPE has been identified as a potent anticancer agent in multiple cancer types and is reported to have the dual role of radioprotection and radiosensitization. However, the radiomodulatory potential of CAPE in prostate cancer (PCa), which eventually becomes radioresistant is not known. Therefore, we studied the effect of co-treatment of CAPE and gamma radiation on androgen-independent DU145 and PC3 cells. The combination treatment sensitized PCa cells to radiation in a dose-dependent manner. The radiosensitizing effect of CAPE was observed in both cell lines. CAPE enhanced the level of ionizing radiation (IR)-induced gamma H2AX foci and cell death by apoptosis. The combination treatment also decreased the migration potential of PCa cells. This was confirmed by increased expression of E-cadherin and decrease in vimentin expression. CAPE sensitized PCa cells to radiation in vitro and induced apoptosis, augmented phosphorylation of Akt/mTOR, and hampered cell migration. At the mechanistic level, co-treatment of CAPE and IR inhibited cell growth by decreasing RAD50 and RAD51 proteins involved in DNA repair. This resulted in enhanced DNA damage and cell death. CAPE might represent a promising new adjuvant for the treatment of hormone-refractory radioresistant PCa.