Involvement of adenosine A2B receptor in radiation-induced translocation of epidermal growth factor receptor and DNA damage response leading to radioresistance in human lung cancer cells.

BACKGROUND: Adenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR. METHODS: We investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation. RESULTS: A2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression. CONCLUSION: Activation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage. GENERAL SIGNIFICANCE: Our results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.

Detection of Simple, Complex, and Clonal Chromosome Translocations Induced by Internal Radioiodine Exposure: A Cytogenetic Follow-Up Case Study after 25 Years.

Here, we report the findings of a 25-year cytogenetic follow-up study on a male patient who received 2 rounds of radioiodine treatment within a span of 26 months (1.78 GBq in 1992 and 14.5 GBq in 1994). The patient was 34 years old with a body mass index of 25 at the time of the first radioiodine treatment. Multicolor FISH and multicolor banding (mBAND) techniques performed on the patient detected inter- and intrachromosomal exchanges. Although the frequency of chromosome translocations remained essentially the same as reported in our earlier study (0.09/cell), the percentage of reciprocal (balanced) translocations increased from 54.38 to 80.30% in the current study. In addition to simple chromosome translocations, complex exchanges (0.29%) involving more than 2 chromosomes were detected for the first time in this patient. Strikingly, a clonal translocation involving chromosomes 14 and 15, t(14p;15q), was found in 7 of the 677 cells examined (1.03%). The presence of complex and clonal translocations indicates the onset of chromosomal instability induced by internal radioiodine exposure. mBAND analysis using probes specific for chromosomes 1, 2, 4, 5, and 10 revealed 5 inversions in a total of 717 cells (0.69%), and this inversion frequency is several-fold higher than the baseline frequency reported in healthy individuals using the classical G-banding technique. Collectively, our study suggests that stable chromosome aberrations such as translocations and inversions can be useful not only for retrospective biodosimetry but also for long-term monitoring of chromosomal instability caused by past radioiodine exposure.

Construction of fluorescence in situ hybridization (FISH) translocation dose-response calibration curve with multiple donor data sets using R, based on ISO 20046:2019 recommendations.

Purpose: Dose-response curve (DRC) generation is an important aspect in cytogenetic biodosimetry for accurate dose estimation for individuals suspected of prior irradiation. DRC construction with dicentric chromosomes after acute radiation is well-established following the publication of the IAEA EPR-Biodosimetry 2011 and ISO 19238:2014. However, the short half-life of dicentrics might not be suitable for retrospective dose estimation in radiation medical workers, radiation accident clean-up workers and the general public living in areas with higher than average amount of radiation. There is an urgent need for a chromosome translocation-based DRC, which is constructed based on translocation identification with fluorescence in situ hybridization (FISH). Despite several attempts to generate such a DRC in the past 40 years, no internationally standardized protocol has been developed until 2019, resulting in possible statistical uncertainties between DRCs previously generated.Materials and methods: Using the recently published ISO 20049:2019, a DRC from five healthy donors (four males: 23, 35, 44, 55 years old, one female: 33 years old) was generated with age-adjusted translocations scored per cell equivalent (age-adjusted Tr/CE), using a modified R-script previously published in EPR-Biodosimetry, for 60Co gamma-ray doses of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 Gy. The translocation data set used, based on probes used for chromosomes number 1, 2, and 4, was previously published by Abe et al. in 2018.Results: The results output from R include the DRC coefficients (C, α, ß), their p-values, the goodness-of-fit Pearson's chi square value and its corresponding p-value, and the DRC with its 95% confidence interval (CI). The equation of the DRC obtained was 0.0005 (±0.0001) +0.0178 (±0.0037) D + 0.0901 (±0.0054) D2. DRC generated with averaged Tr/CE had a wider 95% CI than DRC generated with pooled Tr/CE, resulting in a 1.3-1.5 times increase in estimated dose range. No outliers between α coefficients from previously published modified DRCs and our DRC were detected with robust Z-score.Conclusions: ISO 20046:2019 should be referenced for future FISH translocation-based DRC generation to ensure statistical reliability of dose estimation. Important considerations for FISH translocation-based DRC up to 1 Gy include scoring more than 2000 CE per dose, the use of multiple donors, age-adjustment of observed translocations, the use of a minimum of 5 dose points including 0 Gy, scoring of total simple translocations in only stable cells and the decision of using pooled or averaged age-adjusted Tr/CE.

1,3-Butadiene metabolite 1,2,3,4 diepoxybutane induces DNA adducts and micronuclei but not t(9;22) translocations in human cells.

Epidemiological studies of 1,3-butadiene (BD) exposures have reported a possible association with chronic myelogenous leukemia (CML), which is defined by the presence of the t(9;22) translocation (Philadelphia chromosome) creating an oncogenic BCR-ABL fusion gene. Butadiene diepoxide (DEB), the most mutagenic of three epoxides resulting from BD, forms DNA-DNA crosslink adducts that can lead to DNA double-strand breaks (DSBs). Thus, a study was designed to determine if (±)-DEB exposure of HL60 cells, a promyelocytic leukemia cell line lacking the Philadelphia chromosome, can produce t(9;22) translocations. In HL60 cells exposed for 3 h to 0-10 µM DEB, overlapping dose-response curves suggested a direct relationship between 1,4-bis-(guan-7-yl)-2,3-butanediol crosslink adduct formation (R = 0.977, P = 0.03) and cytotoxicity (R = 0.961, P = 0.002). Experiments to define the relationships between cytotoxicity and the induction of micronuclei (MN), a dosimeter of DNA DSBs, showed that 24 h exposures of HL60 cells to 0-5.0 µM DEB caused significant positive correlations between the concentration and (i) the degree of cytotoxicity (R = 0.998, p = 0.002) and (ii) the frequency of MN (R = 0.984, p = 0.016) at 48 h post exposure. To determine the relative induction of MN and t(9;22) translocations following exposures to DEB, or x-rays as a positive control for formation of t(9;22) translocations, HL60 cells were exposed for 24 h to 0, 1, 2.5, or 5 µM DEB or to 0, 2.0, 3.5, or 5.0 Gy x-rays, or treatments demonstrated to yield 0, 20%, 50%, or 80% cytotoxicity. Treatments between 0 and 3.5 Gy x-rays caused significant dose-related increases in both MN (p < 0.001) and t(9;22) translocations (p = 0.01), whereas DEB exposures causing similar cytotoxicity levels did not increase translocations over background. These data indicate that, while DEB induces DNA DSBs required for formation of MN and translocations, acute DEB exposures of HL60 cells did not produce the Philadelphia chromosome obligatory for CML.

Identification of novel breakpoints for locus- and region-specific translocations in 293 cells by molecular cytogenetics before and after irradiation.

The human kidney embryonic 293 cell line (293 cells) is extensively used in biomedical and pharmaceutical research. These cells exhibit a number of numerical and structural chromosomal anomalies. However, the breakpoints responsible for these structural chromosomal rearrangements have not been comprehensively characterized. In addition, it is not known whether chromosomes with structural rearrangement are more sensitive to external toxic agents, such as ionizing radiation. We used G-banding, spectral karyotyping (SKY), and locus- and region-specific fluorescence in situ hybridization (FISH) probes designed in our lab or obtained from commercial vendor to address this gap. Our G-banding analysis revealed that the chromosome number varies from 66 to 71, with multiple rearrangements and partial additions and deletions. SKY analysis confirmed 3 consistent rearrangements, two simple and one complex in nature. Multicolor FISH analysis identified an array of breakpoints responsible for locus- and region-specific translocations. Finally, SKY analysis revealed that radio-sensitivity of structurally rearranged chromosomes is dependent on radiation dose. These findings will advance our knowledge in 293 cell biology and will enrich the understanding of radiation biology studies.

Translocation Frequency in Patients with Repeated CT Exposure: Comparison with CT-Naive Patients.

Epidemiologic studies using clinical indicators are limited in the assessment of the biological effects of low-dose ionizing radiation for medical purposes. We evaluated the biological effect of low-dose radiation by comparing translocation frequencies in patients with repeated computed tomography (CT) exposure and CT-naïve patients. The goal of this prospective case-control study was to determine whether repeated CT exposure is associated with increased frequency in chromosomal translocations. Two cohorts, comprised of case patients with a history of repeated CT exposure and age- and sex-matched CT-naïve control patients (n = 48 per cohort), were consecutively enrolled in this single-institution study. CT-radiation exposure was estimated using dose-length products, and translocation frequencies of peripheral blood lymphocytes were assessed using whole chromosome paints by fluorescence in situ hybridization (FISH). Comparison of translocation frequencies between cases and controls was performed using the Wilcoxon rank sum test (paired samples), and the relationship between cumulative radiation exposure and translocation frequency was assessed using a partial correlation analysis. Translocation frequencies were significantly different between cases and controls (P = 0.0003). The median translocation frequency was 7 [95% confidence interval (CI): 6, 8] for cases and 4 (95% CI: 3, 6) for controls. By using cumulative radiation exposure as the effect variable and translocation frequency as the response variable, we found a significant correlation between cumulative radiation exposure and translocation frequency (r = 0.6579, P < 0.0001). Chromosomal translocations were more frequent with repeated CT-exposed patients than in CT-naïve patients, and a positive dose-response relationship was present between cumulative radiation exposure and translocation frequency.

Chromosome Translocations, Inversions and Telomere Length for Retrospective Biodosimetry on Exposed U.S. Atomic Veterans.

It has now been over 60 years since U.S. nuclear testing was conducted in the Pacific islands and Nevada, exposing military personnel to varying levels of ionizing radiation. Actual doses are not well-established, as film badges in the 1950s had many limitations. We sought a means of independently assessing dose for comparison with historical film badge records and dose reconstruction conducted in parallel. For the purpose of quantitative retrospective biodosimetry, peripheral blood samples from 12 exposed veterans and 12 age-matched (>80 years) veteran controls were collected and evaluated for radiation-induced chromosome damage utilizing directional genomic hybridization (dGH), a cytogenomics-based methodology that facilitates simultaneous detection of translocations and inversions. Standard calibration curves were constructed from six male volunteers in their mid-20s to reflect the age range of the veterans at time of exposure. Doses were estimated for each veteran using translocation and inversion rates independently; however, combining them by a weighted-average generally improved the accuracy of dose estimations. Various confounding factors were also evaluated for potential effects on chromosome aberration frequencies. Perhaps not surprisingly, smoking and age-associated increases in background frequencies of inversions were observed. Telomere length was also measured, and inverse relationships with both age and combined weighted dose estimates were observed. Interestingly, smokers in the non-exposed control veteran cohort displayed similar telomere lengths as those in the never-smoker exposed veteran group, suggesting that chronic smoking had as much effect on telomere length as a single exposure to radioactive fallout. Taken together, we find that our approach of combined chromosome aberration-based retrospective biodosimetry provided reliable dose estimation capability, particularly on a group average basis, for exposures above statistical detection limits.

Dose Estimation Curves Following In Vitro X-ray Irradiation Using Blood From Four Healthy Korean Individuals.

Cytogenetic dosimetry is useful for evaluating the absorbed dose of ionizing radiation based on analysis of radiation-induced chromosomal aberrations. We created two types of in vitro dose-response calibration curves for dicentric chromosomes (DC) and translocations (TR) induced by X-ray irradiation, using an electron linear accelerator, which is the most frequently used medical device in radiotherapy. We irradiated samples from four healthy Korean individuals and compared the resultant curves between individuals. Aberration yields were studied in a total of 31,800 and 31,725 metaphases for DC and TR, respectively, obtained from 11 X-ray irradiation dose-points (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 Gy). The dose-response relationship followed a linear-quadratic equation, Y=C+αD+ßD², with the coefficients C=0.0011 for DC and 0.0015 for TR, α=0.0119 for DC and 0.0048 for TR, and ß=0.0617 for DC and 0.0237 for TR. Correlation coefficients between irradiation doses and chromosomal aberrations were 0.971 for DC and 0.6 for TR, indicating a very strong and a moderate correlation, respectively. This is the first study implementing cytogenetic dosimetry following exposure to ionizing X-radiation.

DEFINED BIOLOGICAL MODELS OF HIGH-LET RADIATION LESIONS.

DNA double-strand break (DSB) complexity is invoked to explain the increased efficacy of high-linear energy transfer (LET) radiation. Complexity is usually defined as presence of additional lesions in the immediate proximity of the DSB. DSB-clusters represent a different level of complexity that can jeopardize processing by destabilizing chromatin in the vicinity of the cluster. DSB-clusters are generated after exposure of cells to ionizing radiation (IR), particularly high-LET radiation, and have been considered as particularly consequential in several mathematical models of IR action. Yet, experimental demonstration of their relevance to the adverse IR effects, as well as information on the mechanisms underpinning their severity as DNA lesions is lacking. We addressed this void by developing cell lines with especially designed, multiply integrated constructs modeling defined combinations of DSB-clusters through appropriately engineered I-SceI meganuclease recognition sites. Using this model system, we demonstrate efficient activation of the DNA damage response, as well as a markedly increased potential of DSB-clusters, as compared to single-DSBs, to kill cells, and cause Parp1- dependent chromosomal translocations. We propose that DSB repair relying on first line DSB-processing pathways (canonical non-homologous end joining and to some degree homologous recombination repair) is compromised within DSB clusters, presumably through the associated chromatin destabilization, leaving alternative end joining as last option and translocation formation as a natural consequence. Our observations offer a mechanistic explanation for the increased efficacy of high-LET radiation.

Lessons from the accident with 137Cesium in Goiania, Brazil: Contributions to biological dosimetry in case of human exposure to ionizing radiation.

Human exposure to ionizing radiation has increased over time, mainly due to medical applications, occupational and environmental exposure, as well as accidents involving radioactive materials. In September 1987, an accident with 137Cesium occurred in Goiânia city, Brazil; the accident started with the removal of a 50.9-TBq 137Cesium source from an abandoned radiotherapy unit. Among the radiation-exposed victims, at least 50 individuals showed symptoms of whole-body and local acute irradiation, and also external or internal contamination. In this report, the purpose was to review and summarize the main results of cytogenetic studies carried out with victims of 137Cesium, for blood collection performed shortly after the accident, and following several years post-exposure. The importance of dose estimates by biological dosimetry is highlighted, and also several lessons that were learned from the initial to follow-up (7-10 years after the accident) studies, mainly by applying the fluorescence in situ hybridization (FISH) method. A relevant aspect discussed on the basis of the results obtained in those studies refers to the incidence of chromosomal translocations, which were directly compared to the initial frequencies of dicentrics that were previously used to estimate the absorbed doses. In general, translocation frequencies were two to three times lower than the dicentric frequencies, and the differences were dose-dependent. Furthermore, regarding attempts to perform retrospective dosimetry (10 years post-accident), the dose estimates using translocation frequencies for victims of 137Cesium indicate the feasibility of this approach only for low level exposure (below 0.5 Gy), while for higher doses there are some limitations, and the requirement to apply appropriate correction factors, which were discussed on the basis of literature data. Apart of this, in general terms, important aspects to be mentioned refer to the need for better care and control of radioactive devices, as well as adequate education programs for professionals and also the population.

Inhibition of Parp1 by BMN673 Effectively Sensitizes Cells to Radiotherapy by Upsetting the Balance of Repair Pathways Processing DNA Double-Strand Breaks.

Parp inhibitors (Parpi) are commonly used as single agents for the management of tumors with homologous recombination repair (HRR) deficiencies, but combination with radiotherapy (RT) is not widely considered due to the modest radiosensitization typically observed. BMN673 is one of the most recently developed Parpi and has been shown to mediate strong cell sensitization to methylating agents. Here, we explore the mechanisms of BMN673 radiosensitization to killing, aiming to combine it with RT. We demonstrate markedly stronger radiosensitization by BMN673 at concentrations substantially lower (50 nmol/L) than olaparib (3 µmol/L) or AG14361 (0.4 µmol/L) and dramatically lower as compared with second-generation inhibitors such as PJ34 (5 µmol/L). Notably, BMN673 radiosensitization peaks after surprisingly short contact times (∼1 hour) and at pharmacologically achievable concentrations in vivo BMN673 exerts a complex set of effects on DNA double-strand break (DSB) processing, including inhibition of classic nonhomologous end-joining (cNHEJ) and alternative end-joining (altEJ) pathway at high doses of ionizing radiation (IR). BMN673 enhances resection at DSB and favors HRR and altEJ at low clinically relevant IR doses. The combined outcome of these effects is an abrogation in the inherent balance of DSB processing culminating in the formation of chromosomal translocations that underpin radiosensitization. Our observations pave the way to clinical trials exploring inherent benefits in combining BMN673 with RT for the treatment of various forms of cancer. Mol Cancer Ther; 17(10); 2206-16. ©2018 AACR.

Molecular Cytogenetics Guides Massively Parallel Sequencing of a Radiation-Induced Chromosome Translocation in Human Cells.

Chromosome rearrangements are large-scale structural variants that are recognized drivers of oncogenic events in cancers of all types. Cytogenetics allows for their rapid, genome-wide detection, but does not provide gene-level resolution. Massively parallel sequencing (MPS) promises DNA sequence-level characterization of the specific breakpoints involved, but is strongly influenced by bioinformatics filters that affect detection efficiency. We sought to characterize the breakpoint junctions of chromosomal translocations and inversions in the clonal derivatives of human cells exposed to ionizing radiation. Here, we describe the first successful use of DNA paired-end analysis to locate and sequence across the breakpoint junctions of a radiation-induced reciprocal translocation. The analyses employed, with varying degrees of success, several well-known bioinformatics algorithms, a task made difficult by the involvement of repetitive DNA sequences. As for underlying mechanisms, the results of Sanger sequencing suggested that the translocation in question was likely formed via microhomology-mediated non-homologous end joining (mmNHEJ). To our knowledge, this represents the first use of MPS to characterize the breakpoint junctions of a radiation-induced chromosomal translocation in human cells. Curiously, these same approaches were unsuccessful when applied to the analysis of inversions previously identified by directional genomic hybridization (dGH). We conclude that molecular cytogenetics continues to provide critical guidance for structural variant discovery, validation and in "tuning" analysis filters to enable robust breakpoint identification at the base pair level.

Twenty years of FISH-based translocation analysis for retrospective ionizing radiation biodosimetry.

PURPOSE: The fluorescent in situ hybridization (FISH) technique, which easily detects reciprocal translocations, is currently used to estimate doses in retrospective biological dosimetry, after suspected accidental overexposure to ionizing radiation (IR). This study of 42 cases aimed to verify the appropriateness of this assay for radiation dose reconstruction, compared to the dicentric assay, and to evaluate other limitations. MATERIAL AND METHODS: We labeled chromosomes 2, 4, and 12 by 3-color FISH painting to detect translocations on lymphocytes of patients with suspected past IR overexposure. RESULT: Translocation dose estimation showed doses significantly different from 0 Gy in 25 of the 42 cases. The lowest positive dose measured was 0.3 Gy. Several months after IR exposure, the doses measured by translocation and dicentric assays are quite similar. For a year, dose estimation by translocation assay becomes more relevant as dicentric frequency starts to decrease, coming close to 0 for more than a year after the exposure. The persistence of translocations enabled us to corroborate an overexposure 44 years earlier. Interpretation of the observed translocation yield requires the knowledge of the patient's other radiation exposures. A dose assessment by this biomarker is relevant only if the radiation exposure is confirmed. CONCLUSIONS: This technique is appropriate for corroborating a former IR exposure of individuals. When the radiation dose is greater than 1 Gy, the translocations in complex exchanges must be considered. Another relevant point is the use of an appropriate background yield of translocations. The dose assessment, however, also depends on exposure to various genotoxic agents besides IR. If no evidence about the existence of radiation exposure is available, dose assessment is not useful. For this reason, report only the translocation frequency and its comparison with the background yield by age class is preferable.

Dose-response curves for analyzing of dicentric chromosomes and chromosome translocations following doses of 1000 mGy or less, based on irradiated peripheral blood samples from five healthy individuals.

In terms of biological dosimetry at the time of radiation exposure, the dicentric chromosome (Dic) assay (DCA) is the gold standard for assessing for the acute phase and chromosome translocation (Tr) analysis is the gold standard for assessing the chronic phase. It is desirable to have individual dose-response curves (DRCs) for each laboratory because the analysis criteria differ between laboratories. We constructed the DRCs for radiation dose estimation (with three methods) using peripheral blood (PB) samples from five healthy individuals. Aliquots were irradiated with one of eight gamma-ray doses (0, 10, 20, 50, 100, 200, 500 or 1000 mGy), then cultured for 48 h. The number of chromosome aberrations (CAs) was analyzed by DCA, using Giemsa staining and centromere-fluorescence in situ hybridization (centromere-FISH) and by chromosome painting (chromosome pairs 1, 2 and 4) for Tr analysis. In DCA, there was large variation between individuals in the frequency of Dics formed, and the slopes of the DRCs were different. In Tr analysis, although variation was observed in the frequency of Tr, the slopes of the DRCs were similar after adjusting the background for age. Good correlation between the irradiation dose and the frequency of CAs formed was observed with these three DRCs. However, performing three different biological dosimetry assays simultaneously on PB from five donors nonetheless results in variation in the frequency of CAs formed, especially at doses of 50 mGy or less, highlighting the difficulty of biological dosimetry using these methods. We conclude that it might be difficult to construct universal DRCs.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/µm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/µm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Ausencia de translocaciones cromosómicas y de expresión proteica de ALK en los adenocarcinomas nasosinusales / Absence of chromosomal translocations and protein expression of ALK in sinonasal adenocarcinomas

Introducción. Las translocaciones de la región cromosómica 2p23 causan la sobreexpresión del gen de la quinasa del linfoma anaplásico (ALK), un receptor tirosinquinasa involucrado en rutas de señalización celular que regulan la proliferación. Dicha alteración se identifica en el 5% de los adenocarcinomas de pulmón, representando una diana terapéutica en dicho subgrupo de tumores. Debido a que los adenocarcinomas nasosinusales (ACNS) tienen una histología similar a los adenocarcinomas de pulmón, el objetivo de este estudio fue evaluar si existen alteraciones en el gen ALK en los ACNS. Método. La presencia de translocaciones del gen ALK se analizó en 96 muestras de ACNS mediante fluorescence in situ hybridization usando unas sondas «break apart». Además se estudió la expresión proteica de ALK por inmunohistoquímica. Resultados. En ninguno de los casos se observó la presencia de translocaciones de ALK. Además, no se detectó expresión proteica en ninguno de los casos. Conclusiones. Los resultados obtenidos sugieren que ALK no desempeña un papel relevante en la oncogénesis de los ACNS (AU)

Introduction. Chromosomal translocations at 2p23 cause overexpression of anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase involved in signalling pathways that regulate cell proliferation. This translocation occurs in 5% of lung adenocarcinoma and has been demonstrated to be useful as a therapeutic target for crizotinib. sinonasal adenocarcinomas (SNAC) are histologically similar to lung adenocarcinomas; the aim of this study was to evaluate the presence of ALK alterations in SNAC. Method. Break-apart fluorescent in-situ hybridization was used to analyse the presence of ALK translocations in 96 tumour samples. In addition, ALK protein expression was studied by immunohistochemistry. Results. The samples of SNAC did not show ALK translocation. Moreover, ALK protein expression was absent in all cases. Conclusions. These results suggest that ALK is not involved in SNAC (AU)

RENEB biodosimetry intercomparison analyzing translocations by FISH.

PURPOSE: In the framework of RENEB, several biodosimetry exercises were conducted analyzing different endpoints. Among them, the analysis of translocations is considered the most useful method for retrospective biodosimetry due to the relative stability of their frequency with post irradiation time. The aim of this study was to harmonize the accuracy of translocation-based biodosimetry within the RENEB consortium. MATERIALS AND METHODS: An initial telescoring exercise analyzing FISH metaphase images was done to harmonize chromosome aberration descriptions. Then two blind intercomparison exercises (IE) were performed, by sending irradiated blood samples to each partner. Samples were cultured and stained by each partner using their standard protocol and translocation frequency was used to produce dose estimates. RESULTS: The coefficient of variation in the 1st IE (CV = 0.34) was higher than in the 2nd IE (CV = 0.16 and 0.23 in the two samples analyzed), for the genomic frequency of total translocations. Z-score analysis revealed that eight out of 10 and 17 out of 20 dose estimates were satisfactory in the 1st and 2nd IE, respectively. CONCLUSIONS: The results obtained indicate that, despite the problems identified in few partners, which can be corrected, the RENEB consortium is able to carry out retrospective biodosimetry analyzing the frequency of translocations by FISH.

Attenuated DNA damage repair delays therapy-related myeloid neoplasms in a mouse model.

Therapy-related cancers are potentially fatal late life complications for patients who received radio- or chemotherapy. So far, the mouse model showing reduction or delay of these diseases has not been described. We found that the disruption of Aplf in mice moderately attenuated DNA damage repair and, unexpectedly, impeded myeloid neoplasms after exposure to ionizing radiation (IR). Irradiated mutant mice showed higher rates of p53-dependent cell death, fewer chromosomal translocations, and a delay in malignancy-induced mortality. Simultaneous deficiency of p53 abrogated IR-induced apoptosis and the benefit of impaired DNA repair on mortality in irradiated Aplf­/­ mice. Depletion of APLF in non-tumorigenic human cells also markedly reduced the risk of radiation-induced chromosomal aberrations. We therefore conclude that proficient DNA damage repair may promote chromosomal aberrations in normal tissues after irradiation and induce malignant evolution, thus illustrating the potential benefit in sensitizing p53 function by manipulating DNA repair efficiency in cancer patients undergoing genotoxic therapies.

Irradiation at Different Fetal Stages Results in Different Translocation Frequencies in Adult Mouse Thyroid Cells.

While it is generally believed that fetuses are at high risk of developing cancers, including leukemia, after low doses of radiation, it has been reported that atomic bomb survivors exposed in utero did not show a dose response for translocations in blood T lymphocytes when they were examined at approximately 40 years of age. Subsequent mouse studies confirmed that animals irradiated during the fetal stage did not show evidence of radiation effects in lymphocytes and bone marrow cells when they were examined after reaching adulthood. However, in a study of rat mammary epithelial cells, radiation effects were clearly observed after fetal irradiation. These results indicate that the fate of chromosome aberrations induced in a fetus could vary among different tissues. Here we report on translocation frequencies in mouse thyroid cells, which were irradiated at different stages of fetal development. Cytogenetic examination was conducted using fluorescence n situ hybridization (FISH) painting of chromosomes 1 and 3. Adult mice, 2 Gy X-ray irradiated at 15.5-day-old fetuses (E15.5), showed a higher translocation frequency (30/1,155 or 25.3 × 10-3) than nonirradiated adult controls (0/1,007 or 0.1 × 10-3), and was near that experienced by irradiated mothers and non-pregnant adult females (43/1,244 or 33.7 × 10-3). These results are consistent with those seen in rat mammary cells. However, when fetuses were irradiated at an earlier stage of development (E6.5) before thyroid organogenesis, the resulting observed translocation frequency was much lower (3/502 or 5.8 × 10-3) than that in E15.5 mice. These results suggest that after fetal irradiation, tissue stem cells record radiation effects primarily when the exposure occurs in cells that have been integrated into tissue. Embryonic stem cells that have been damaged prior to integration into the niche may undergo negative selection due to apoptosis, mitotic death or stem cell-niche cell interactions. The implications of these results in interpreting cancer risks after fetal irradiation are also discussed.

Dynamics of chromosomal aberrations, induction of apoptosis, BRCA2 degradation and sensitization to radiation by hyperthermia.

Hyperthermia can transiently degrade BRCA2 and thereby inhibit the homologous recombination pathway. Induced DNA-double strand breaks (DSB) then have to be repaired via the error prone non-homologous end-joining pathway. In the present study, to investigate the role of hyperthermia in genotoxicity and radiosensitization, the induction of chromosomal aberrations was examined by premature chromosome condensation and fluorescence in situ hybridisation (PCC-FISH), and cell survival was determined by clonogenic assay shortly (0-1 h) and 24 h following exposure to hyperthermia in combination with ionizing radiation. Prior to exposure to 4 Gy γ-irradiation, confluent cultures of SW­1573 (human lung carcinoma) and RKO (human colorectal carcinoma) cells were exposed to mild hyperthermia (1 h, 41˚C). At 1 h, the frequency of chromosomal translocations was higher following combined exposure than following exposure to irradiation alone. At 24 h, the number of translocations following combined exposure was lower than following exposure to irradiation only, and was also lower than at 1 h following combined exposure. These dynamics in translocation frequency can be explained by the hyperthermia-induced transient reduction of BRCA2 observed in both cell lines. In both cell lines exposed to radiation only, potentially lethal damage repair (PLDR) correlated with a decreased number of chromosomal fragments at 24 h compared to 1 h. With combined exposure, PLDR did not correlate with a decrease in fragments, as in the RKO cells at 24 h following combined exposure, the frequency of fragments remained at the level found after 1 h of exposure and was also significantly higher than that found following exposure to radiation alone. This was not observed in the SW­1573 cells. Cell survival experiments demonstrated that exposure to hyperthermia radiosensitized the RKO cells, but not the SW­1573 cells. This radiosensitization was at least partly due to the induction of apoptosis, which was only observed in the RKO cells and which may have been induced by BRCA2 degradation or different types of chromosomal aberrations. An important observation of this study is that the genotoxic effect of hyperthermia shortly after combined epxosure (to hyperthermia and radiation) is not observed at 24 h after treatment.