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.

Homologous Recombination Subpathways: A Tangle to Resolve.

Homologous recombination (HR) is an essential pathway for DNA double-strand break (DSB) repair, which can proceed through various subpathways that have distinct elements and genetic outcomes. In this mini-review, we highlight the main features known about HR subpathways operating at DSBs in human cells and the factors regulating subpathway choice. We examine new developments that provide alternative models of subpathway usage in different cell types revise the nature of HR intermediates involved and reassess the frequency of repair outcomes. We discuss the impact of expanding our understanding of HR subpathways and how it can be clinically exploited.

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.

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.

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.

Differences in the Response to DNA Double-Strand Breaks between Rod Photoreceptors of Rodents, Pigs, and Humans.

Genome editing (GE) represents a powerful approach to fight inherited blinding diseases in which the underlying mutations cause the degeneration of the light sensing photoreceptor cells of the retina. Successful GE requires the efficient repair of DNA double-stranded breaks (DSBs) generated during the treatment. Rod photoreceptors of adult mice have a highly specialized chromatin organization, do not efficiently express a variety of DSB response genes and repair DSBs very inefficiently. The DSB repair efficiency in rods of other species including humans is unknown. Here, we used ionizing radiation to analyze the DSB response in rods of various nocturnal and diurnal species, including genetically modified mice, pigs, and humans. We show that the inefficient repair of DSBs in adult mouse rods does not result from their specialized chromatin organization. Instead, the DSB repair efficiency in rods correlates with the level of Kruppel-associated protein-1 (KAP1) expression and its ataxia-telangiectasia mutated (ATM)-dependent phosphorylation. Strikingly, we detected robust KAP1 expression and phosphorylation only in human rods but not in rods of other diurnal species including pigs. Hence, our study provides important information about the uniqueness of the DSB response in human rods which needs to be considered when choosing model systems for the development of GE strategies.

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.

The pendulum of the Ku-Ku clock.

Canonical DNA non-homologous end-joining (c-NHEJ) and homologous recombination (HR), the two major DNA double-strand break (DSB) repair pathways, have long been depicted as competitors, fighting a race to rejoin DSBs. In human cells, Ku, an upstream component of NHEJ, is highly abundant and has exquisite end-binding capacity. Emerging evidence has suggested that Ku is the first protein binding most, if not all, DSBs, and creates a block to resection. Although most c-NHEJ proceeds without resection, recent studies have provided strong evidence for a process of resection-dependent c-NHEJ, that repairs a subset of DSBs. HR also repairs a subset of two-ended DSBs in G2 phase and processes one-ended DSBs that arise following replication fork stalling or collapse to promote replication restart. HR also necessitates end-resection. This raises the question of how end-resection takes place despite Ku's avid end-binding capacity. Insight into this enigma has been gained from the analysis of DSBs generated by Spo11 or TOP2, which create protein-bridged DSBs. The progression of repair by HR or NHEJ requires removal of the end-blocking lesions. The MRE11-RAD50-NBS1 (MRN) complex, CtIP and EXO1 play critical roles in this process. Here, we review our current understanding of how resection arises at lesions blocked by covalently bound Spo11 or TOP2 or following Ku binding, which effectively creates a distinct resection-blocking lesion due to its avid end-binding activity and abundance. Our review reveals that Ku plays an active role in determining pathway choice and exposes similarities yet distinctions in the progression of resection that is suited to the optimal repair pathway choice.

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.

A Process of Resection-Dependent Nonhomologous End Joining Involving the Goddess Artemis.

DNA double-strand breaks (DSBs) are a hazardous form of damage that can potentially cause cell death or genomic rearrangements. In mammalian G1- and G2-phase cells, DSBs are repaired with two-component kinetics. In both phases, a fast process uses canonical nonhomologous end joining (c-NHEJ) to repair the majority of DSBs. In G2, slow repair occurs by homologous recombination. The slow repair process in G1 also involves c-NHEJ proteins but additionally requires the nuclease Artemis and DNA end resection. Here, we consider the nature of slow DSB repair in G1 and evaluate factors determining whether DSBs are repaired with fast or slow kinetics. We consider limitations in our current knowledge and present a speculative model for Artemis-dependent c-NHEJ and the environment underlying its usage.

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.

Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability.