Late health effects and changes in lifestyle factors after cancer in childhood with and without subsequent second primary cancers - the KiKme case-control study.

Background: Improved treatments for childhood cancer result in a growing number of long-term childhood cancer survivors (CCS). The diagnosis and the prevalence of comorbidities may, however, influence their lifestyle later in life. Nonetheless, little is known about differences in late effects between CCS of a first primary neoplasm (FPN) in childhood and subsequent second primary neoplasms (SPN) and their impact on lifestyle. Therefore, we aim to investigate associations between the occurrence of FPN or SPN and various diseases and lifestyle in the later life of CCS. Methods: CCS of SPN (n=101) or FPN (n=340) and cancer-free controls (n=150) were matched by age and sex, and CCS additionally by year and entity of FPN. All participants completed a self-administered questionnaire on anthropometric and socio-economic factors, medical history, health status, and lifestyle. Mean time between FPN diagnosis and interview was 27.3 years for SPN and 26.2 years for FPN CCS. To confirm results from others and to generate new hypotheses on late effects of childhood cancer as well as CCS´ lifestyles, generalized linear mixed models were applied. Results: CCS were found to suffer more likely from diseases compared to cancer-free controls. In detail, associations with cancer status were observed for hypercholesterinemia and thyroid diseases. Moreover, CCS were more likely to take regular medication compared to controls. A similar association was observed for CCS of SPN compared to CCS of FPN. In contrast to controls, CCS rarely exercise more than 5 hours per week, consumed fewer soft and alcoholic drinks, and were less likely to be current, former, or passive smokers. Additionally, they were less likely overweight or obese. All other exploratory analyses performed on cardiovascular, chronic lung, inflammatory bone, allergic, and infectious diseases, as well as on a calculated health-score revealed no association with tumor status. Conclusion: CCS were more affected by pathologic conditions and may consequently take more medication, particularly among CCS of SPN. The observed higher disease burden is likely related to the received cancer therapy. To reduce the burden of long-term adverse health effects in CCS, improving cancer therapies should therefore be in focus of research in this area.

POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis.

BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions.

ATRX and RECQ5 define distinct homologous recombination subpathways.

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

Turning end-joining upside down in mitosis.

How cells deal with DNA breaks during mitosis is not well understood. While canonical non-homologous end-joining predominates in interphase, it is inhibited in mitosis to avoid telomere fusions. DNA polymerase θ mediated end-joining appears to be repressed in interphase, but promotes break repair in mitosis. The nature and induction time of breaks might determine their fate during mitosis.

Spontaneous and Radiation-Induced Chromosome Aberrations in Primary Fibroblasts of Patients With Pediatric First and Second Neoplasms.

The purpose of the present study was to investigate whether former childhood cancer patients who developed a subsequent secondary primary neoplasm (SPN) are characterized by elevated spontaneous chromosomal instability or cellular and chromosomal radiation sensitivity as surrogate markers of compromised DNA repair compared to childhood cancer patients with a first primary neoplasm (FPN) only or tumor-free controls. Primary skin fibroblasts were obtained in a nested case-control study including 23 patients with a pediatric FPN, 22 matched patients with a pediatric FPN and an SPN, and 22 matched tumor-free donors. Clonogenic cell survival and cytogenetic aberrations in Giemsa-stained first metaphases were assessed after X-irradiation in G1 or on prematurely condensed chromosomes of cells irradiated and analyzed in G2. Fluorescence in situ hybridization was applied to investigate spontaneous transmissible aberrations in selected donors. No significant difference in clonogenic survival or the average yield of spontaneous or radiation-induced aberrations was found between the study populations. However, two donors with an SPN showed striking spontaneous chromosomal instability occurring as high rates of numerical and structural aberrations or non-clonal and clonal translocations. No correlation was found between radiation sensitivity and a susceptibility to a pediatric FPN or a treatment-associated SPN. Together, the results of this unique case-control study show genomic stability and normal radiation sensitivity in normal somatic cells of donors with an early and high intrinsic or therapy-associated tumor risk. These findings provide valuable information for future studies on the etiology of sporadic childhood cancer and therapy-related SPN as well as for the establishment of predictive biomarkers based on altered DNA repair processes.

An Assessment of Radiation Doses From Radon Exposures Using a Mouse Model System.

BACKGROUND: Radon and its progenies contribute significantly to the natural background radiation and cause several thousands of lung cancer cases per year worldwide. Moreover, patients with chronic inflammatory joint diseases are treated in radon galleries. Due to the complex nature of radon exposure, the doses associated with radon exposures are difficult to assess. Hence, there is a clear need to directly measure dose depositions from radon exposures to provide reliable risk estimates for radiation protection guidelines. OBJECTIVES: We aimed to assess tissue-specific radiation doses associated with radon activity concentrations, that deposit similar dose levels as the annual natural radon exposure or radon gallery visits. METHODS: We exposed mice to defined radon concentrations, quantified the number of 53BP1 foci as a measure of induced DNA damage, and compared it with the number of foci induced by known doses of reference-type radiations. An image-based analysis of the 3-dimensional foci pattern provided information about the radiation type inflicting the DNA damage. RESULTS: A 1-hour exposure to 440 kBq/m3 radon-induced DNA damage corresponding to a dose of ∼10 mGy in the lung and ∼3.3 mGy in the kidney, heart, and liver. A 1-hour exposure to 44 kBq/m3 provided values consistent with a linear relationship between dose and radon concentration. Two-thirds of the dose in the lung was caused by α-particles. The dose in the kidney, heart, and liver and one-third of the dose in the lung likely resulted from ß- and γ-rays. DISCUSSION: We found that radon exposures mainly lead to α-particle-induced DNA damage in the lung, consistent with the lung cancer risk obtained in epidemiologic studies. Our presented biodosimetric approach can be used to benchmark risk model calculations for radiation protection guidelines and can help to understand the therapeutic success of radon gallery treatments.

Fractionation-Dependent Radiosensitization by Molecular Targeting of Nek1.

NIMA (never-in-mitosis gene A)-related kinase 1 (Nek1) is shown to impact on different cellular pathways such as DNA repair, checkpoint activation, and apoptosis. Its role as a molecular target for radiation sensitization of malignant cells, however, remains elusive. Stably transduced doxycycline (Dox)-inducible Nek1 shRNA HeLa cervix and siRNA-transfected HCT-15 colorectal carcinoma cells were irradiated in vitro and 3D clonogenic radiation survival, residual DNA damage, cell cycle distribution, and apoptosis were analyzed. Nek1 knockdown (KD) sensitized both cell lines to ionizing radiation following a single dose irradiation and more pronounced in combination with a 6 h fractionation (3 × 2 Gy) regime. For preclinical analyses we focused on cervical cancer. Nek1 shRNA HeLa cells were grafted into NOD/SCID/IL-2Rγc-/- (NSG) mice and Nek1 KD was induced by Dox-infused drinking water resulting in a significant cytostatic effect if combined with a 6 h fractionation (3 x 2 Gy) regime. In addition, we correlated Nek1 expression in biopsies of patients with cervical cancer with histopathological parameters and clinical follow-up. Our results indicate that elevated levels of Nek1 were associated with an increased rate of local or distant failure, as well as with impaired cancer-specific and overall survival in univariate analyses and for most endpoints in multivariable analyses. Finally, findings from The Cancer Genome Atlas (TCGA) validation cohort confirmed a significant association of high Nek1 expression with a reduced disease-free survival. In conclusion, we consider Nek1 to represent a novel biomarker and potential therapeutic target for drug development in the context of optimized fractionation intervals.

One end to rule them all: Non-homologous end-joining and homologous recombination at DNA double-strand breaks.

Double-strand breaks (DSBs) represent the most severe type of DNA damage since they can lead to genomic rearrangements, events that can initiate and promote tumorigenic processes. DSBs arise from various exogenous agents that induce two single-strand breaks at opposite locations in the DNA double helix. Such two-ended DSBs are repaired in mammalian cells by one of two conceptually different processes, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ has the potential to form rearrangements while HR is believed to be error-free since it uses a homologous template for repair. DSBs can also arise from single-stranded DNA lesions if they lead to replication fork collapse. Such DSBs, however, have only one end and are repaired by HR and not by NHEJ. In fact, the majority of spontaneously arising DSBs are one-ended and HR has likely evolved to repair one-ended DSBs. HR of such DSBs demands the engagement of a second break end that is generated by an approaching replication fork. This HR process can cause rearrangements if a homologous template other than the sister chromatid is used. Thus, both NHEJ and HR have the potential to form rearrangements and the proper choice between them is governed by various factors, including cell cycle phase and genomic location of the lesion. We propose that the specific requirements for repairing one-ended DSBs have shaped HR in a way which makes NHEJ the better choice for the repair of some but not all two-ended DSBs.

AutoFoci, an automated high-throughput foci detection approach for analyzing low-dose DNA double-strand break repair.

Double-strand breaks (DSBs) are the most lethal DNA damages induced by ionising radiation (IR) and their efficient repair is crucial to limit genomic instability. The cellular DSB response after low IR doses is of particular interest but its examination requires the analysis of high cell numbers. Here, we present an automated DSB quantification method based on the analysis of γH2AX and 53BP1 foci as markers for DSBs. We establish a combination of object properties, combined in the object evaluation parameter (OEP), which correlates with manual object classification. Strikingly, OEP histograms show a bi-modal distribution with two maxima and a minimum in between, which correlates with the manually determined transition between background signals and foci. We used algorithms to detect the minimum, thus separating foci from background signals and automatically assessing DSB levels. To demonstrate the validity of this method, we analyzed over 600.000 cells to verify results of previous studies showing that DSBs induced by low doses are less efficiently repaired compared with DSBs induced by higher doses. Thus, the automated foci counting method, called AutoFoci, provides a valuable tool for high-throughput image analysis of thousands of cells which will prove useful for many biological screening approaches.

DNA repair synthesis and histone deposition partner during homologous recombination.

Chromatin remodeling is critical for the regulation of the DNA damage response. We highlight findings from our recent study showing that the deposition of the histone variant H3.3 by the alpha-thalassemia mental retardation X-linked protein (ATRX) and the death domain associated protein (DAXX) chromatin remodeling complex regulates DNA repair synthesis during homologous recombination.

ATRX Promotes DNA Repair Synthesis and Sister Chromatid Exchange during Homologous Recombination.

ATRX is a chromatin remodeler that, together with its chaperone DAXX, deposits the histone variant H3.3 in pericentromeric and telomeric regions. Notably, ATRX is frequently mutated in tumors that maintain telomere length by a specific form of homologous recombination (HR). Surprisingly, in this context, we demonstrate that ATRX-deficient cells exhibit a defect in repairing exogenously induced DNA double-strand breaks (DSBs) by HR. ATRX operates downstream of the Rad51 removal step and interacts with PCNA and RFC-1, which are collectively required for DNA repair synthesis during HR. ATRX depletion abolishes DNA repair synthesis and prevents the formation of sister chromatid exchanges at exogenously induced DSBs. DAXX- and H3.3-depleted cells exhibit identical HR defects as ATRX-depleted cells, and both ATRX and DAXX function to deposit H3.3 during DNA repair synthesis. This suggests that ATRX facilitates the chromatin reconstitution required for extended DNA repair synthesis and sister chromatid exchange during HR.

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination.

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Direct measurement of the 3-dimensional DNA lesion distribution induced by energetic charged particles in a mouse model tissue.

Charged particles are increasingly used in cancer radiotherapy and contribute significantly to the natural radiation risk. The difference in the biological effects of high-energy charged particles compared with X-rays or γ-rays is determined largely by the spatial distribution of their energy deposition events. Part of the energy is deposited in a densely ionizing manner in the inner part of the track, with the remainder spread out more sparsely over the outer track region. Our knowledge about the dose distribution is derived solely from modeling approaches and physical measurements in inorganic material. Here we exploited the exceptional sensitivity of γH2AX foci technology and quantified the spatial distribution of DNA lesions induced by charged particles in a mouse model tissue. We observed that charged particles damage tissue nonhomogenously, with single cells receiving high doses and many other cells exposed to isolated damage resulting from high-energy secondary electrons. Using calibration experiments, we transformed the 3D lesion distribution into a dose distribution and compared it with predictions from modeling approaches. We obtained a radial dose distribution with sub-micrometer resolution that decreased with increasing distance to the particle path following a 1/r2 dependency. The analysis further revealed the existence of a background dose at larger distances from the particle path arising from overlapping dose deposition events from independent particles. Our study provides, to our knowledge, the first quantification of the spatial dose distribution of charged particles in biologically relevant material, and will serve as a benchmark for biophysical models that predict the biological effects of these particles.

How cancer cells hijack DNA double-strand break repair pathways to gain genomic instability.

DNA DSBs (double-strand breaks) are a significant threat to the viability of a normal cell, since they can result in loss of genetic material if mitosis or replication is attempted in their presence. Consequently, evolutionary pressure has resulted in multiple pathways and responses to enable DSBs to be repaired efficiently and faithfully. Cancer cells, which are under pressure to gain genomic instability, have a striking ability to avoid the elegant mechanisms by which normal cells maintain genomic stability. Current models suggest that, in normal cells, DSB repair occurs in a hierarchical manner that promotes rapid and efficient rejoining first, with the utilization of additional steps or pathways of diminished accuracy if rejoining is unsuccessful or delayed. In the present review, we evaluate the fidelity of DSB repair pathways and discuss how cancer cells promote the utilization of less accurate processes. Homologous recombination serves to promote accuracy and stability during replication, providing a battlefield for cancer to gain instability. Non-homologous end-joining, a major DSB repair pathway in mammalian cells, usually operates with high fidelity and only switches to less faithful modes if timely repair fails. The transition step is finely tuned and provides another point of attack during tumour progression. In addition to DSB repair, a DSB signalling response activates processes such as cell cycle checkpoint arrest, which enhance the possibility of accurate DSB repair. We consider the ways by which cancers modify and hijack these processes to gain genomic instability.

PTIP associates with Artemis to dictate DNA repair pathway choice.

PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1(-/-)53BP1(-/-) cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway.

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1.

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin.

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity.

ATM release at resected double-strand breaks provides heterochromatin reconstitution to facilitate homologous recombination.

Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.

Opposing roles for 53BP1 during homologous recombination.

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication.