Fully automated counting of DNA damage foci in tumor cell culture: A matter of cell separation.

Analysis and quantification of residual, unrepaired DNA double-strand breaks by detecting damage-associated γH2AX or 53BP1 foci is a promising approach to evaluate radiosensitivity or radiosensitization in tumor cells. Manual foci quantification by eye is well-established but unsatisfactory due to inconsistent foci numbers between different observers, lack of information about foci size and intensity and the time-consuming scoring process. Therefore, automated foci counting is an important goal. Several software solutions for automated foci counting in separately acquired fluorescence microscopy images have been established. The AKLIDES NUK technology by Medipan combines automated microscopy and image processing/ counting, enabling affordable high throughput foci analysis as a routine application. Using this machine, automated foci counting is well established for lymphocytes but has not yet been reported for adherent tumor cells with their irregularly shaped nuclei and heterogeneous foci textures. Here we aimed to use the AKLIDES NUK system for adherent tumor cells growing in clusters. We identified cell separation as a critical step to ensure fast and reliable automated nuclei detection. We validated our protocol for the fully automated quantification of (i) the IR-dose dependent increase and (ii) the ATM as well as PARP inhibitor-induced radiosensitization. Collectively, with this protocol the AKLIDES NUK system facilitates cost effective, fast and high throughput quantitative fluorescence microscopic analysis of DNA damage induced foci such as γH2AX and 53BP1 in adherent tumor cells.

The role of DNA damage response in chemo- and radio-resistance of cancer cells: Can DDR inhibitors sole the problem?

Up to now, many improvements have been made in providing more therapeutic strategies for cancer patients. The lack of susceptibility to common therapies like chemo- and radio-therapy is one of the reasons why we need more methods in the field of cancer therapy. DNA damage response (DDR) is a set of mechanisms which identifies DNA lesions and triggers the repair process for restoring DNA after causing an arrest in the cell cycle. The ability of DDR in maintaining the genome stability and integrity can be favorable to cancerous cells which are exposed to radiation therapy or are treated with chemotherapeutic agents. When DDR mechanisms are error-free in cancer cells, they can escape the expected cellular death and display resistance to treatment. In this regard, targeting different components of DDR can help to increase the susceptibility of advanced tumors to chemo- and radio-therapy.

Variable interplay of UV-induced DNA damage and repair at transcription factor binding sites.

An abnormally high rate of UV-light related mutations appears at transcription factor binding sites (TFBS) across melanomas. The binding of transcription factors (TFs) to the DNA impairs the repair of UV-induced lesions and certain TFs have been shown to increase the rate of generation of these lesions at their binding sites. However, the precise contribution of these two elements to the increase in mutation rate at TFBS in these malignant cells is not understood. Here, exploiting nucleotide-resolution data, we computed the rate of formation and repair of UV-lesions within the binding sites of TFs of different families. We observed, at certain dipyrimidine positions within the binding site of TFs in the Tryptophan Cluster family, an increased rate of formation of UV-induced lesions, corroborating previous studies. Nevertheless, across most families of TFs, the observed increased mutation rate within the entire DNA region covered by the protein results from the decreased repair efficiency. While the rate of mutations across all TFBS does not agree with the amount of UV-induced lesions observed immediately after UV exposure, it strongly agrees with that observed after 48 h. This corroborates the determinant role of the impaired repair in the observed increase of mutation rate.

Combined EGFR1 and PARP1 Inhibition Enhances the Effect of Radiation in Head and Neck Squamous Cell Carcinoma Models.

Head and neck squamous cell carcinoma (HNSCC) is a challenging cancer with little change in five-year overall survival rate of 50-60% over the last two decades. Radiation with or without platinum-based drugs remains the standard of care despite limited benefit and high toxicity. HNSCCs often overexpress epidermal growth factor receptor (EGFR) and inhibition of EGFR signaling enhances radiation sensitivity by interfering with repair of radiation-induced DNA breaks. Poly (adenosine diphosphate-ribose) polymerase-1 (PARP1) also participates in DNA damage repair, but its inhibition provides benefit in cancers that lack DNA repair by homologous recombination (HR) such as BRCA-mutant breast cancer. HNSCCs in contrast are typically BRCA wild-type and proficient in HR repair, making it challenging to apply anti-PARP1 therapy in this model. A recently published study showed that a combination of EGFR and PARP1 inhibition induced more DNA damage and greater growth control than each single agent in HNSCC cells. This led us to hypothesize that a combination of EGFR and PARP1 inhibition would enhance the efficacy of radiation to a greater extent than each single agent, providing a rationale for paradigm-shifting combinatorial approaches to improve the standard of care in HNSCC. Here, we report a proof-of-concept study using Detroit562 HNSCC cells, which are proficient for DNA repair by both HR and non-homologous end joining (NHEJ) mechanisms. We tested the effect of adding cetuximab and/or olaparib (inhibitors of EGFR and PARP1, respectively) to radiation and compared it to that of cisplatin and radiation combination, which is the standard of care. Our results demonstrate that the combination of cetuximab and olaparib with radiation was superior to the combination of any single drug with radiation in terms of induction of unrepaired DNA damage, induction of senescence, apoptosis and clonogenic death, and tumor growth control in mouse xenografts. Combined with our recently published phase I safety data on cetuximab/olaparib/radiation triple combination, the data reported here demonstrate a potential for combining biologically-based therapies that might optimize radiosensitization in HNSCC.

Insulin-like growth factor receptor signaling in tumorigenesis and drug resistance: a challenge for cancer therapy.

Insulin-like growth factors (IGFs) play important roles in mammalian growth, development, aging, and diseases. Aberrant IGFs signaling may lead to malignant transformation and tumor progression, thus providing the rationale for targeting IGF axis in cancer. However, clinical trials of the type I IGF receptor (IGF-IR)-targeted agents have been largely disappointing. Accumulating evidence demonstrates that the IGF axis not only promotes tumorigenesis, but also confers resistance to standard treatments. Furthermore, there are diverse pathways leading to the resistance to IGF-IR-targeted therapy. Recent studies characterizing the complex IGFs signaling in cancer have raised hope to refine the strategies for targeting the IGF axis. This review highlights the biological activities of IGF-IR signaling in cancer and the contribution of IGF-IR to cytotoxic, endocrine, and molecular targeted therapies resistance. Moreover, we update the diverse mechanisms underlying resistance to IGF-IR-targeted agents and discuss the strategies for future development of the IGF axis-targeted agents.

Radio-Susceptibility of Nasopharyngeal Carcinoma: Focus on Epstein- Barr Virus, MicroRNAs, Long Non-Coding RNAs and Circular RNAs.

Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer. As a neoplastic disorder, NPC is a highly malignant squamous cell carcinoma that is derived from the nasopharyngeal epithelium. NPC is radiosensitive; radiotherapy or radiotherapy combining with chemotherapy are the main treatment strategies. However, both modalities are usually accompanied by complications and acquired resistance to radiotherapy is a significant impediment to effective NPC therapy. Therefore, there is an urgent need to discover effective radio-sensitization and radio-resistance biomarkers for NPC. Recent studies have shown that Epstein-Barr virus (EBV)-encoded products, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), which share several common signaling pathways, can function in radio-related NPC cells or tissues. Understanding these interconnected regulatory networks will reveal the details of NPC radiation sensitivity and resistance. In this review, we discuss and summarize the specific molecular mechanisms of NPC radio-sensitization and radio-resistance, focusing on EBV-encoded products, miRNAs, lncRNAs and circRNAs. This will provide a foundation for the discovery of more accurate, effective and specific markers related to NPC radiotherapy. EBVencoded products, miRNAs, lncRNAs and circRNAs have emerged as crucial molecules mediating the radio-susceptibility of NPC. This understanding will improve the clinical application of markers and inform the development of novel therapeutics for NPC.

Regulation of programmed death-ligand 1 expression in response to DNA damage in cancer cells: Implications for precision medicine.

Anti-programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) therapy, which is one of the most promising cancer therapies, is licensed for treating various tumors. Programmed death-ligand 1, which is expressed on the surface of cancer cells, leads to the inhibition of T lymphocyte activation and immune evasion if it binds to the receptor PD-1 on CTLs. Anti-PD-1/PD-L1 Abs inhibit interactions between PD-1 and PD-L1 to restore antitumor immunity. Although certain patients achieve effective responses to anti-PD-1/PD-L1 therapy, the efficacy of treatment is highly variable. Clinical trials of anti-PD-1/PD-L1 therapy combined with radiotherapy/chemotherapy are underway with suggestive evidence of favorable outcome; however, the molecular mechanism is largely unknown. Among several molecular targets that can influence the efficacy of anti-PD-1/PD-L1 therapy, PD-L1 expression in tumors is considered to be a critical biomarker because there is a positive correlation between the efficacy of combined treatment protocols and PD-L1 expression levels. Therefore, understanding the mechanisms underlying the regulation of PD-L1 expression in cancer cells, particularly the mechanism of PD-L1 expression following DNA damage, is important. In this review, we consider recent findings on the regulation of PD-L1 expression in response to DNA damage signaling in cancer cells.

High-dose-rate brachytherapy as monotherapy for prostate cancer: The impact of cellular repair and source decay.

PURPOSE: This work quantifies the influence of intrafraction DNA damage repair and cellular repopulation on biologically effective dose (BED) in Ir-192 high-dose-rate brachytherapy for prostate cancer. In addition, it examines the effect of source-decay-induced BED variation for patients treated at different time points in a source exchange cycle. MATERIALS AND METHODS: Current fractionation schemes are based on simplified-form BED = nd(1 + d/(α/ß)), which assumes that intrafraction repair, interfraction repair, and repopulation are negligible. We took accepted radiobiological parameters of Tk, Tp, and α from the recommendations of the AAPM TG-137, and recalculated the full-form BED. Fraction times were normalized to require 15 min for 20 Gy at 10 Ci. Calculations were carried out for both α/ß = 1.5 and 3 Gy. RESULTS: After accounting for intrafraction repair, interfraction repair, and/or repopulation, full-form BED calculations showed significant values, as compared with simplified-form BED. For 1-fraction 20 Gy fractionation, the full-form BED was only 64-82% of the simplified-form BED. Dose protraction effects were milder for smaller prescriptions (6 Gy/Fx), where full form was 87-94%. With regard to source decay, BED varied >20% for patients treated at the beginning and the end of a source exchange cycle for 20 Gy single-fraction prescription. CONCLUSIONS: Repair and repopulation can be significant in monotherapy high-dose-rate for prostate cancer. As fractionation schemes are established, the simplified BED calculation may not be appropriate. Investigators should consider evaluating BED as a range rather than a discrete value when presenting results unless source activity is explicitly incorporated as well.

DNA damage response of clinical carbon ion versus photon radiation in human glioblastoma cells.

BACKGROUND AND PURPOSE: Carbon ion radiotherapy is a promising therapeutic option for glioblastoma patients due to its high physical dose conformity and greater biological effectiveness than photons. However, the biological effects of carbon ion radiation are still incompletely understood. Here, we systematically compared the biological effects of clinically used carbon ion radiation to photon radiation with emphasis on DNA repair. MATERIALS AND METHODS: Two human glioblastoma cell lines (U87 and LN229) were irradiated with carbon ions or photons and DNA damage response was systematically analyzed, including clonogenic survival, induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and apoptosis or autophagy. γH2AX foci were analyzed by flow cytometry, conventional light microscopy and 3D superresolution microscopy. RESULTS: DSBs were repaired delayed and with slower kinetics after carbon ions versus photons. Carbon ions caused stronger and longer-lasting cell cycle delays, predominantly in G2 phase, and a higher rate of apoptosis. Compared to photons, the effectiveness of carbon ions was less cell cycle-dependent. Homologous recombination (HR) appeared to be more important for DSB repair after carbon ions versus photons in phosphatase and tensin homolog (PTEN)-deficient U87 cells, as opposed to PTEN-proficient LN229 cells. CONCLUSION: Carbon ions induced more severe DSB damage than photons, which was repaired less efficiently in both cell lines. Thus, carbon ion radiotherapy may help to overcome resistance mechanisms of glioblastoma associated with DNA repair for example in combination with repair pathway-specific drugs in the context of personalized radiotherapy.

First experimental proof of Proton Boron Capture Therapy (PBCT) to enhance protontherapy effectiveness.

Protontherapy is hadrontherapy's fastest-growing modality and a pillar in the battle against cancer. Hadrontherapy's superiority lies in its inverted depth-dose profile, hence tumour-confined irradiation. Protons, however, lack distinct radiobiological advantages over photons or electrons. Higher LET (Linear Energy Transfer) 12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in efficient cell killing, i.e. higher Relative Biological Effectiveness (RBE). However, economic and radiobiological issues hamper 12C-ion clinical amenability. Thus, enhancing proton RBE is desirable. To this end, we exploited the p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam. To maximize the reaction rate, we used sodium borocaptate (BSH) with natural boron content. Boron-Neutron Capture Therapy (BNCT) uses 10B-enriched BSH for neutron irradiation-triggered alpha particles. We recorded significantly increased cellular lethality and chromosome aberration complexity. A strategy combining protontherapy's ballistic precision with the higher RBE promised by BNCT and 12C-ion therapy is thus demonstrated.

Photochemical Activation of Enediyne Warheads: A Potential Tool for Targeted Antitumor Therapy.

Spatial and temporal control over DNA cleavage by photoactivated enediynes can be complemented by additional factors such as the release of internal strain, chelation, pH changes, intramolecular H-bonds, and substituent effects. This review presents design and reactivity of photoactivated enediynes/enynes and analyses the chemical, biological, and photophysical challenges in their applications.

Glucose starvation impairs DNA repair in tumour cells selectively by blocking histone acetylation.

BACKGROUND AND PURPOSE: Tumour cells are characterized by aerobic glycolysis and thus have high glucose consumption. Because repairing radiation-induced DNA damage is an energy-demanding process, we hypothesized that glucose starvation combined with radiotherapy could be an effective strategy to selectively target tumour cells. MATERIAL AND METHODS: We glucose-starved tumour cells (A549, FaDu) in vitro and analysed their radiation-induced cell responses compared to normal fibroblasts (HSF7). RESULTS: Irradiation depleted intracellular ATP levels preferentially in cancer cells. Consequently, glucose starvation impaired DNA double-strand break (DSB) repair and radiosensitized confluent tumour cells but not normal fibroblasts. In proliferating tumour cells glucose starvation resulted in a reduction of proliferation, but failed to radiosensitize cells. Glucose supply was indispensable during the late DSB repair in confluent tumour cells starting approximately 13 h after irradiation, and glucose starvation inhibited radiation-induced histone acetylation, which is essential for chromatin relaxation. Sirtinol - an inhibitor of histone deacetylases - reverted the effects of glucose depletion on histone acetylation and DNA DSB repair in tumour cells. Furthermore, a glucose concentration of 2.8 mmol/L was sufficient to impair DSB repair in tumour cells and reduced their clonogenic survival under a fractionated irradiation regimen. CONCLUSIONS: In resting tumour cells, glucose starvation combined with irradiation resulted in the impairment of late DSB repair and the reduction of clonogenic survival, which was associated with disrupted radiation-induced histone acetylation. However, in normal cells, DNA repair and radiosensitivity were not affected by glucose depletion.

[Abnormalities of DNA repair and gynecological cancers]. / Anomalies de la réparation de l'ADN et cancers gynécologiques.

The demonstration of frequent defects in the DNA damage response in high grade ovarian cancer has paved the way for a new therapeutic approach aimed at exploiting this unique vulnerability. The efficacy of poly (ADP) ribose polymerase inhibitors (PARPi) in patients with homologous recombination (HR) DNA repair deficient ovarian cancer (OC) resulting from a BRCA1/2 mutation has provided the proof of concept for synthetic lethality. Thus, olaparib is now approved by the EMA as maintenance therapy after response to a platinum regimen for patients with recurrent, platinum-sensitive, high-grade serous, BRCA1/2-mutated ovarian cancer. Furthermore, several recent trials in OC have demonstrated that the benefit of PARPi may not be limited to patients with BRCA mutations. These data, combined with genomic studies suggesting that a significant proportion of OC may harbor somatic and germline alterations in other HR genes open huge perspectives for exploiting DNA repair as a therapeutic strategy. The current priorities are to (i) determine whether new biomarkers of homologous recombination deficiency may identify the BRCA wild-type subset likely to derive benefit from PARPi; (ii) to determine whether the efficacy of PARPi can be improved by combinatorial strategies (with chemotherapy, radiotherapy, immunotherapy, anti-angiogenesis or DNA repair inhibitors) and (iii) to develop new approaches exploiting DNA repair deficiencies in ovarian and other gynecological tumors.

Measuring the response of human head and neck squamous cell carcinoma to irradiation in a microfluidic model allowing customized therapy.

Radiotherapy is the standard treatment for head and neck squamous cell carcinoma (HNSCC), however, radioresistance remains a major clinical problem despite significant improvements in treatment protocols. Therapeutic outcome could potentially be improved if a patient's tumour response to irradiation could be predicted ex vivo before clinical application. The present study employed a bespoke microfluidic device to maintain HNSCC tissue whilst subjecting it to external beam irradiation and measured the responses using a panel of cell death and proliferation markers. HNSCC biopsies from five newly-presenting patients [2 lymph node (LN); 3 primary tumour (PT)] were divided into parallel microfluidic devices and replicates of each tumour were subjected to single-dose irradiation (0, 5, 10, 15 and 20 Gy). Lactate dehydrogenase (LDH) release was measured and tissue sections were stained for cytokeratin (CK), cleaved-CK18 (cCK18), phosphorylated-H2AX (γH2AX) and Ki­67 by immunohistochemistry. In addition, fragmented DNA was detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Compared with non­irradiated controls, higher irradiation doses resulted in elevated CK18-labelling index in two lymph nodes [15 Gy; 34.8% on LN1 and 31.7% on LN2 (p=0.006)] and a single laryngeal primary tumour (20 Gy; 31.5%; p=0.014). Significantly higher levels of DNA fragmentation were also detected in both lymph node samples and one primary tumour but at varying doses of irradiation, i.e., LN1 (20 Gy; 27.6%; p=0.047), LN2 (15 Gy; 15.3%; p=0.038) and PT3 (10 Gy; 35.2%; p=0.01). The γH2AX expression was raised but not significantly in the majority of samples. The percentage of Ki­67 positive nuclei reduced dose-dependently following irradiation. In contrast no significant difference in LDH release was observed between irradiated groups and controls. There is clear inter- and intra-patient variability in response to irradiation when measuring a variety of parameters, which offers the potential for the approach to provide clinically valuable information.

DNA damage and the bystander response in tumor and normal cells exposed to X-rays.

Monolayer and suspension cultures of tumor (BMG-1, CCRF-CEM), normal (AG1522, HADF, lymphocytes) and ATM-mutant (GM4405) human cells were exposed to X-rays at doses used in radiotherapy (high dose and high dose-rate) or radiological imaging (low dose and low dose-rate). Radiation-induced DNA damage, its persistence, and possible bystander effects were evaluated, based on DNA damage markers (γ-H2AX, p53ser15) and cell-cycle-specific cyclins (cyclin B1 and cyclin D1). Dose-dependent DNA damage and a dose-independent bystander response were seen after exposure to high dose and high dose-rate radiation. The level of induced damage (expression of p53ser15, γ-H2AX) depended on ATM status. However, low dose and dose-rate exposures neither increased expression of marker proteins nor induced a bystander response, except in the CCRF-CEM cells. Bystander effects after high-dose irradiation may contribute to stochastic and deterministic effects. Precautions to protect unexposed regions or to inhibit transmission of DNA damage signaling might reduce radiation risks.

NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR.

Exposure of melanocytes to ultraviolet radiation (UVR) induces the formation of UV lesions that can produce deleterious effects in genomic DNA. Encounters of replication forks with unrepaired UV lesions can lead to several complex phenomena, such as the formation of DNA double-strand breaks (DSBs). The NR4A family of nuclear receptors are transcription factors that have been associated with mediating DNA repair functions downstream of the MC1R signaling pathway in melanocytes. In particular, emerging evidence shows that upon DNA damage, the NR4A2 receptor can translocate to sites of UV lesion by mechanisms requiring post-translational modifications within the N-terminal domain and at a serine residue in the DNA-binding domain at position 337. Following this, NR4A2 aids in DNA repair by facilitating chromatin relaxation, allowing accessibility for DNA repair machinery. Using A2058 and HT144 melanoma cells engineered to stably express wild-type or mutant forms of the NR4A2 proteins, we reveal that the expression of functional NR4A2 is associated with elevated cytoprotection against UVR. Conversely, knockdown of NR4A2 expression by siRNA results in a significant loss of cell viability after UV insult. By analyzing the kinetics of the ensuing 53BP1 and RAD51 foci following UV irradiation, we also reveal that the expression of mutant NR4A2 isoforms, lacking the ability to translocate, transactivate, or undergo phosphorylation, display compromised repair capacity.Implications: These data expand the understanding of the mechanism by which the NR4A2 nuclear receptor can facilitate DNA DSB repair. Mol Cancer Res; 15(9); 1184-96. ©2017 AACR.

RAD51 as a potential surrogate marker for DNA repair capacity in solid malignancies.

Targeting deficient mechanisms of cellular DNA repair still represents the basis for the treatment of the majority of solid tumors, and increased DNA repair capacity is a hallmark mechanism of resistance not only to DNA-damaging treatments such as cytotoxic drugs and radiotherapy, but also to small molecule targeted drugs such as inhibitors of poly-ADP ribose polymerase (PARP). Hence, there is substantial medical need for potent and convenient biomarkers of individual response to DNA-targeted treatment in personalized cancer care. RAD51 is a highly conserved protein that catalyzes DNA repair via homologous recombination, a major DNA repair pathway which directly modulates cellular sensitivity to DNA-damaging treatments. The clinical and biological significance of RAD51 protein expression is still under investigation. Pre-clinical studies consistently show the important role of nuclear RAD51 immunoreactivity in chemo- and radioresistance. Validating data from clinical trials however is limited at present, and some clinical studies show controversial results. This review gives a comprehensive overview on the current knowledge about the prognostic and predictive value of RAD51 protein expression and genetic variability in patients with solid malignancies.

Exploring water radiolysis in proton cancer therapy: Time-dependent, non-adiabatic simulations of H+ + (H2O)1-6.

To elucidate microscopic details of proton cancer therapy (PCT), we apply the simplest-level electron nuclear dynamics (SLEND) method to H+ + (H2O)1-6 at ELab = 100 keV. These systems are computationally tractable prototypes to simulate water radiolysis reactions-i.e. the PCT processes that generate the DNA-damaging species against cancerous cells. To capture incipient bulk-water effects, ten (H2O)1-6 isomers are considered, ranging from quasi-planar/multiplanar (H2O)1-6 to "smallest-drop" prism and cage (H2O)6 structures. SLEND is a time-dependent, variational, non-adiabatic and direct method that adopts a nuclear classical-mechanics description and an electronic single-determinantal wavefunction in the Thouless representation. Short-time SLEND/6-31G* (n = 1-6) and /6-31G** (n = 1-5) simulations render cluster-to-projectile 1-electron-transfer (1-ET) total integral cross sections (ICSs) and 1-ET probabilities. In absolute quantitative terms, SLEND/6-31G* 1-ET ICS compares satisfactorily with alternative experimental and theoretical results only available for n = 1 and exhibits almost the same accuracy of the best alternative theoretical result. SLEND/6-31G** overestimates 1-ET ICS for n = 1, but a comparable overestimation is also observed with another theoretical method. An investigation on H+ + H indicates that electron direct ionization (DI) becomes significant with the large virtual-space quasi-continuum in large basis sets; thus, SLEND/6-31G** 1-ET ICS is overestimated by DI contributions. The solution to this problem is discussed. In relative quantitative terms, both SLEND/6-31* and /6-31G** 1-ET ICSs precisely fit into physically justified scaling formulae as a function of the cluster size; this indicates SLEND's suitability for predicting properties of water clusters with varying size. Long-time SLEND/6-31G* (n = 1-4) simulations predict the formation of the DNA-damaging radicals H, OH, O and H3O. While "smallest-drop" isomers are included, no early manifestations of bulk water PCT properties are observed and simulations with larger water clusters will be needed to capture those effects. This study is the largest SLEND investigation on water radiolysis to date.

Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells.

PURPOSE: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. METHODS AND MATERIALS: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10(6) protons/cm(2). RESULTS: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. CONCLUSIONS: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

Cancer Cell Death-Inducing Radiotherapy: Impact on Local Tumour Control, Tumour Cell Proliferation and Induction of Systemic Anti-tumour Immunity.

Radiotherapy (RT) predominantly is aimed to induce DNA damage in tumour cells that results in reduction of their clonogenicity and finally in tumour cell death. Adaptation of RT with higher single doses has become necessary and led to a more detailed view on what kind of tumour cell death is induced and which immunological consequences result from it. RT is capable of rendering tumour cells immunogenic by modifying the tumour cell phenotype and the microenvironment. Danger signals are released as well as the senescence-associated secretory phenotype. This results in maturation of dendritic cells and priming of cytotoxic T cells as well as in activation of natural killer cells. However, RT on the other hand can also result in immune suppressive events including apoptosis induction and foster tumour cell proliferation. That's why RT is nowadays increasingly combined with selected immunotherapies.