Premature Chromosome Condensation Research Articles

Optimizing chemical-induced premature chromosome condensation assay for rapid estimation of high-radiation doses.

In the event of exposure to high doses of radiation, prompt dose estimation is crucial for selecting appropriate treatment modalities, such as cytokine therapy or stem cell transplantation. The chemical-induced premature chromosome condensation (PCC) method offers a simple approach for such dose estimation with significant radiation exposure, but its 48-h incubation time poses challenges for early dose assessment. In this study, we optimized the chemical-induced PCC assay for more rapid dose assessment. A sufficient number of PCC and G2/M-PCC cells were obtained after 40 h of culture for irradiated human peripheral blood up to 20Gy. By adding caffeine (final concentration of 1 mM) at 34 h from the start of culture, G2/M-PCC index increased by 1.4-fold in 10Gy cultures. There was also no significant difference in the G2/M-PCC ring frequency induced for doses 0 to 15Gy between our 40-h caffeine-supplemented chemical-induced PCC method and the conventional 48-h PCC assay.

Applicability of Scoring Calyculin A-Induced Premature Chromosome Condensation Objects for Dose Assessment Including for Radiotherapy Patients

As an extension to a previous study, a linear calibration curve covering doses from 0 to 10 Gy was constructed and evaluated in the present study using calyculin A-induced premature chromosome condensation (PCC) by scoring excess PCC objects. The main aim of this study was to assess the applicability of this PCC assay for doses below 2 Gy that are critical for triage categorization. Two separate blind tests involving a total of 6 doses were carried out; 4 out of 6 dose estimates were within the 95% confidence limits (95% CL) with the other 2 just outside. In addition, blood samples from five cancer patients undergoing external beam radiotherapy (RT) were also analyzed, and the results showed whole-body dose estimates statistically comparable to the dicentric chromosome assay (DCA) results. This is the first time that calyculin A-induced PCC was used to analyze clinical samples by scoring excess objects. Although dose estimates for the pre-RT patient samples were found to be significantly higher than the mean value for the healthy donors and were also significantly higher than those obtained using DCA, all these pre-treatment patients fell into the same category as those who may have received a low dose (<1 Gy) and do not require immediate medical care during emergency triage. Additionally, for radiological accidents with unknown exposure scenario, PCC objects and rings can be scored in parallel for the assessment of both low- and high-dose exposures. In conclusion, scoring excess objects using calyculin A-induced PCC is confirmed to be another potential biodosimetry tool in radiological emergency particularly in mass casualty scenarios, even though the data need to be interpreted with caution when cancer patients are among the casualties.

Fluorescence in situ hybridisation for interphase chromosomal aberration-based biological dosimetry.

Metaphase spreads stained with Giemsa or painted with chromosome-specific probes by fluorescence in situ hybridisation (FISH) have been in use since long for retrospective dose assessment (biological dosimetry). However, in cases of accidental exposure to ionising radiation, the culturing of lymphocytes to obtain metaphase chromosomes and analysis of chromosomal aberrations is time-consuming and problematic after high radiation doses. Similarly, analysing chromosomal damage in G0/G1 cells or nondividing cells by premature chromosome condensation is laborious. Following large-scale radiological emergencies, the time required for analysis is more important than precision of dose estimate. Painting of whole chromosomes using chromosome-specific probes in interphase nuclei by the FISH technique will eliminate the time required for cell culture and allow a fast dose estimate, provided that a meaningful dose-response can be obtained by scoring the number of chromosomal domains visible in interphase nuclei. In order to test the applicability of interphase FISH for quick biological dosimetry, whole blood from a healthy donor was irradiated with 8Gy of gamma radiation. Irradiated whole blood was kept for 2h at 37°C to allow DNA repair and thereafter processed for FISH with probes specific for Chromosomes-1 and 2. Damaged chromosomal fragments, distinguished by extra color domains, were observed in interphase nuclei of lymphocytes irradiated with 8Gy. These fragments were efficiently detected and quantified by the FISH technique utilising both confocal and single plane fluorescence microscopy. Furthermore, a clear dose-response curve for interphase fragments was achieved following exposure to 0, 1, 2, 4 and 8Gy of gamma radiation. These results demonstrate interphase FISH as a promising test for biodosimetry and for studying cytogenetic effects of radiation in nondividing cells.

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

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

High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview.

In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients.

Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (60Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters β/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

Transformed Cell Ratio (TCR): A Novel Parameter for Radiation Dose Estimation in Rapid Premature Chromosome Condensation (PCC) Assay Induced by 0–40 Gy Co-60 Gamma Rays

Abstract Calyculin A-induced premature chromosome condensation (PCC) assay is considered a method for estimating radiation dose. In this study, a novel parameter, the transformed cell ratio (TCR), which is the ratio of irradiated and unirradiated lymphocyte transformation rates in the PCC assay, was suggested for dose estimation since it may be correlated with ionizing radiation dose. Human peripheral lymphocytes were irradiated in vitro with 60Co gamma ray doses of 0, 10, 20, 30, and 40 Gy, stimulated with phytohemagglutinin, and incubated for 3, 6, 12, or 48 h, division-blocked with colcemid, and PCC-induced by calyculin A or addition of extra cyclin-dependent kinase 1/cyclin B (CDK1/cyclinB). Results showed that G1-, S-, G2-, and M-PCC cells appeared in human lymphocyte culture, dose-response curves between the absorbed dose and lymphocyte transformation rate or TCR was fitted. TCR presented the best goodness-of-fit to a linear model (y = 0.0097 X +0.9952, R2 = 0.971, P &lt; 0.05) within 0 to 40 Gy. Observations also suggested that TCR is a potential biological dosimeter for assessing received radiation doses ranging from 0 to 40 Gy during radiation exposure.

Chemical-Induced Premature Chromosome Condensation Protocol.

Chromosome analysis is one of most fundamental techniques for cytogenetic studies. Chromosomes are conventionally prepared from mitotic cells arrested by colcemid block protocol. Premature chromosome condensation (PCC) technique is an alternative to obtain chromosomes. It was more than half century ago that the first observation of PCC phenomena reported. Since then, cell-fusion-mediated PCC method has been developed and introduced in many fields of chromosome analysis. More than quarter century ago, novel PCC technique using chemical drug has been developed. Afterwards, this simple and efficient drug-induced PCC technique becomes a standard protocol for preparing chromosomes. Thus, it seems to be the good time to introduce PCC technique protocol for the artisans in the field of cytogenetic studies.

DNA Damage Foci on Metaphase Chromosomes.

After DNAs are damaged, DNA repair proteins accumulate and areactivated at the DNA damaged site. These accumulated proteins are visualized as fociby fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci onchromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.

The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

MCPH1 is the first gene identified to be responsible for the human autosomal recessive disorder primary microcephaly (MCPH). Mutations in the N-terminal and central domains of MCPH1 are strongly associated with microcephaly in human patients. A recent study showed that the central domain of MCPH1, which is mainly encoded by exon 8, interacts with E3 ligase βTrCP2 and regulates the G2/M transition of the cell cycle. In order to investigate the biological functions of MCPH1’s central domain, we constructed a mouse model that lacked the central domain of MCPH1 by deleting its exon 8 (designated as Mcph1-Δe8). Mcph1-Δe8 mice exhibited a reduced brain size and thinner cortex, likely caused by a compromised self-renewal capacity and premature differentiation of Mcph1-Δe8 neuroprogenitors during corticogenesis. Furthermore, Mcph1-Δe8 mice were sterile because of a loss of germ cells in the testis and ovary. The embryonic fibroblasts of Mcph1-Δe8 mice exhibited premature chromosome condensation (PCC). All of these findings indicate that Mcph1-Δe8 mice are reminiscent of MCPH1 complete knockout mice and Mcph1-ΔBR1 mice. Our study demonstrates that the central domain of MCPH1 represses microcephaly, and is essential for gonad development in mammals.

Dose Response Curve Using Chemically Induced PCC Assay in Peripheral Cells Irradiated In-vitro to 5 MeV Alpha Particles Emitted From Radon Source

Bio-dosimetry is defined as estimation of dose received by individuals based on biological end points induced by ionizing radiation. By using cytogenetic assay, it is possible to quantify the absorbed dose. High linear energy transfer (LET) radiation produces dense ionizations along their path and causes more clustered DNA damage compared to low LET. Radon is a natural radioactive element present in the atmosphere mainly produced during decay of uranium. The main objective of the present study is to construct dose response curve by using Premature Chromosome Condensation (PCC) techniques in blood cells exposed in-vitro to radon exposure. Blood samples were drawn from healthy non-smokers (n=4), aged between 28 to 42 years and exposed to twenty doses of radon ranged between 0 to 5.5 mGy. A total of about 24000 metaphase spreads were counted to find the DNA damages include ring chromosomes (RC), dicentric chromosomes (DC) and acentric fragments (AF). As the dose increases gradual increase was observed in RC, DC and AF with the slope of 0.001, 0.005 and 0.064 respectively. So far, no calibration reference curve was generated for low dose radon using chemically induced PCC assay and hence this study is considered as first of its kind.

Chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose ionizing radiation

The premature chromosome condensation (PCC) assay is considered as complementary bio-dosimetry tool for chromosome aberration assay and the PCC assay can be used to estimate high dose exposure. Though the PCC ring is considered as prospective biomarker, chromosome length ratio (ratio of longest and shortest chromosome length in PCC spreads) of chemically induced PCC is shown to be very good indicator of ionizing radiation. In view of this, an in-vitro study has been performed using PCC assay to suggest chromosome length ratio (LR) as potential bio-dosimeter induced by high dose ionizing radiation. Blood samples were collected from healthy subjects (n=3) after prior consent and irradiated to ten different doses ranging between 0 and 20Gy using 6MV LINAC X-rays with dose rate of 5.6Gy/min. Irradiated lymphocytes were cultured and calyculin induced PCC spreads were prepared. PCC spreads were captured using image analysis system and chromosome lengths were measured using open-source ImageJ software. For each dose, LR for 50 chromosome spreads were computed and mean LR value was calculated. LR varies between 6.0±0.08 and23.6±0.55 for the dose range between 2 and 20Gy. The dose response curve for LR was observed to be linear with y=1.02x+3.36, R2 =0.97. Linear dose response relationship obtained in the present study confirms the prospective use of LR measurement. This study is first of its kind to examine chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose X-ray exposure.

Genotoxicity Associated with 131I and 99mTc Exposure in Nuclear Medicine Staff: A Physical and Biological Monitoring Study.

Nuclear medicine staff are constantly exposed to low doses of ionizing radiation. This study investigated the level of genotoxic effects in hospital employees exposed to routinely used 131I and 99mTc in comparison with a control group. The study compared the results of physical and biological monitoring in peripheral blood lymphocytes. The effects of confounding factors, such as smoking status and physical activity, were also considered. Physical dosimetry monitoring revealed differences in the individual annual effective dose as measured by finger ring dosimeter and whole-body dosimeter between the 131I- and 99mTc-exposed groups. The DNA damage studies revealed differences between the groups in terms of excess premature chromosome condensation (PCC) fragments and tail DNA. Physical activity and smoking status differentiated the investigated groups. When assessed by the level of physical activity, the highest mean values of tail DNA were observed for the 99mTc group. When assessed by work-related physical effort, excess PCC fragments were significantly higher in the 131I group than in the control group. In the investigated groups, the tail DNA values were significantly different between non-smokers and past or current smokers, but excess PCC fragments did not significantly differ by smoking status. It is important to measure exposure to low doses of ionizing radiation and assess the potential risk from this exposure. Such investigations support the need to continue epidemiological and experimental studies to improve our understanding of the mechanisms of the health effects of radionuclides and to develop predictive models of the behavior of these complex systems in response to low-dose radiation.

MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface.

Dramatic change in chromosomal DNA morphology between interphase and mitosis is a defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin's loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, accompanied by enhanced mixing of A and B chromatin compartments, and this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II's NCAPG2 subunit. MCPH1's ability to block condensin II's association with chromatin is abrogated by the fusion of SMC2 with NCAPH2, hence may work by a mechanism similar to cohesin. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis.

Refined premature chromosome condensation (G0-PCC) with cryo-preserved mitotic cells for rapid radiation biodosimetry

Mitotic cell fusion induced Premature Chromosome Condensation (G0-PCC) assay in human lymphocytes allows rapid detection of cytogenetic damage in interphase stage, within few hours after blood collection. Hence, it is the most suitable method for rapid and high dose biodosimetry. Mitotic cells, used for G0-PCC could be either freshly isolated or previously cryo-preserved. However, under emergency scenarios, only cryo-preserved cells can be relied upon, fresh isolation will only delay the process by 18–24 h. Impact of cryopreservation on mitotic cells and their efficacy to induce PCC are not reported. In the present study, we investigated effect of cryopreservation on mitotic cells and refined the parameters for G0-PCC. More than 95% of the cells were recoverable after 4 months of cryopreservation, within 20 min recovery at 37 °C, without significant change in the mitotic index or viability. Recovered mitotic cells have shown mitotic index of 89 ± 4% and viability of 90 ± 4%, similar to that of freshly isolated cells. Decrease in metaphases was observed within 40 min after recovery as the mitotic cells progressed through cell cycle and reduced to 21% at 1 h. Nevertheless, in presence of Colcemid, the cells progressed slowly and considerably high metaphase index (60%) persisted up to ~ 2 h. The recovered cells efficiently fused with lymphocytes and induced PCC. Average PCC index varied from 10 to 20%, which did not change with cryopreservation duration. Post fusion incubation duration of 2 h was found to be optimum for proper chromosome condensation. In conclusion, use of cryo-preserved mitotic cells is the most practical approach for rapid biodosimetry. The cells can be recovered quickly and efficiently without alteration in viability or mitotic index. Recovered cells are fully competent to induce G0-PCC.

Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba’s ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

Construction of dose response curve for 6 MV LINAC X-rays using Premature Chromosome Condensation assay for radiation dosimetry

Quantification of chromosomal aberrations in the exposed personnel blood samples is considered as a ‘gold standard’ and sensitive biomarker in biological dosimetry. Despite technological developments, culture of cells for 48–52 h remains an unmet need in case of triage biodosimetry. Moreover, it is difficult to get sufficient number of metaphase spreads for scoring after high doses of exposures. The technique which causes condensation of chromatin before mitosis using biological or chemical agent is named as Premature Chromosome Condensation (PCC) assay. This assay is considered as an alternative to chromosome aberration assay, particularly at high acute doses of low and high LET radiation. To establish the PCC assay, blood samples were collected from healthy non-smoking individuals (n = 3) and exposed to various doses (0–20 Gy) of 6 MV X-rays at a dose rate of 5.6 Gy/min, using a high energy Linear accelerator (LINAC). Irradiated blood samples were subjected to Calyculin-A induced PCC. About 500 cells or more than 100 Ring Chromosomes (RC) were scored at each dose. Dicentric chromosomes (DC) and acentric fragments were also scored at each dose; the number of chromosomal aberrations in G1, M, G2/M and M/A phase of cell cycle were recorded and the frequency was used to construct the dose response curve. A dose dependent increase in RC and DC frequency were observed with a slope of 0.049 ± 0.002 and 0.30 ± 0.02 respectively. This study is first of its kind to construct a dose response curve for LINAC X-rays using a PCC assay.

Investigation of DNA Damage and Cell-Cycle Distribution in Human Peripheral Blood Lymphocytes under Exposure to High Doses of Proton Radiotherapy.

Simple SummaryRadiotherapy is a cornerstone care therapy for many tumors. Despite permanent advances in ra-diation dose delivery, there are unmet needs for further improvement. In principle, proton therapy offers a substantial clinical advantage over conventional modalities using photons in uniform-dose delivery of radiation to tumors, along with significant reductions in the harmful effects on normal tissue. However, the effect and mechanisms of a single high-dose delivery remain unclear. The study aimed to systematically observe and compare the biological effects of DNA damage and cell-cycle phase distribution in the human peripheral blood lymphocytes ex vivo irradiation model of normal tissue after proton versus conventional radiotherapy (X-rays). The effects induced at a single high-dose radiation exposure at a dose range of 8.00–20.00 Gy were studied. The results in-dicate a different distribution of DNA damage following high doses of irradiation with protons versus photons between donors, types of radiation, and doses. The results illuminate the cellular and molecular mechanisms that underlie differences in the distribution of DNA damage and cell-cycle phases. An understanding of the mechanisms in the distinct pathways induced by radiation can facilitate the development of more efficient radiotherapies with beneficial immunological conse-quences.This study systematically investigates how a single high-dose therapeutic proton beam versus X-rays influences cell-cycle phase distribution and DNA damage in human peripheral blood lymphocytes (HPBLs). Blood samples from ten volunteers (both male and female) were irradiated with doses of 8.00, 13.64, 15.00, and 20.00 Gy of 250 kV X-rays or 60 MeV protons. The dose–effect relations were calculated and distributed by plotting the frequencies of DNA damage of excess Premature Chromosome Condensation (PCC) fragments and rings in the G2/M phase, obtained via chemical induction with calyculin A. The Papworth’s u test was used to evaluate the distribution of DNA damage. The study shows that high doses of protons induce HPBL DNA damage in the G2/M phase differently than X-rays do. The results indicate a different distribution of DNA damage following high doses of irradiation with protons versus photons between donors, types of radiation, and doses. The proliferation index confirms the impact of high doses of mitosis and the influence of radiotherapy type on the different HPBL response. The results illuminate the cellular and molecular mechanisms that underlie differences in the distribution of DNA damage and cell-cycle phases; these findings may yield an improvement in the efficacy of the radiotherapies used.

The N-terminal BRCT domain determines MCPH1 function in brain development and fertility

MCPH1 is a causal gene for the neurodevelopmental disorder, human primary microcephaly (MCPH1, OMIM251200). Most pathogenic mutations are located in the N-terminal region of the gene, which encodes a BRCT domain, suggesting an important function of this domain in brain size determination. To investigate the specific function of the N-terminal BRCT domain in vivo, we generated a mouse model lacking the N’-BRCT domain of MCPH1 (referred as Mcph1-ΔBR1). These mutant mice are viable, but exhibit reduced brain size, with a thinner cortex due to a reduction of neuroprogenitor populations and premature neurogenic differentiation. Mcph1-ΔBR1 mice (both male and female) are infertile; however, almost all female mutants develop ovary tumours. Mcph1-ΔBR1 MEF cells exhibit a defect in DNA damage response and DNA repair, and show the premature chromosome condensation (PCC) phenotype, a hallmark of MCPH1 patient cells and also Mcph1 knockout cells. In comparison with Mcph1 complete knockout mice, Mcph1-ΔBR1 mice faithfully reproduce all phenotypes, indicating an essential role of the N-terminal BRCT domain for the physiological function of MCPH1 in the control of brain size and gonad development as well as in multiple cellular processes.

Kinetics of DNA Repair in Vicia faba Meristem Regeneration Following Replication Stress.

The astonishing survival abilities of Vicia faba, one the earliest domesticated plants, are associated, among other things, to the highly effective replication stress response system which ensures smooth cell division and proper preservation of genomic information. The most crucial pathway here seems to be the ataxia telangiectasia-mutated kinase (ATM)/ataxia telangiectasia and Rad3-related kinase (ATR)-dependent replication stress response mechanism, also present in humans. In this article, we attempted to take an in-depth look at the dynamics of regeneration from the effects of replication inhibition and cell cycle checkpoint overriding causing premature chromosome condensation (PCC) in terms of DNA damage repair and changes in replication dynamics. We were able to distinguish a unique behavior of replication factors at the very start of the regeneration process in the PCC-induced cells. We extended the experiment and decided to profile the changes in replication on the level of a single replication cluster of heterochromatin (both alone and with regard to its position in the nucleus), including the mathematical profiling of the size, activity and shape. The results obtained during these experiments led us to the conclusion that even “chaotic” events are dealt with in a proper degree of order.