Effectiveness of first-line anticancer treatment may predict treatment response in further lines in stage III/IV patients with non-small cell lung cancer.

PURPOSE: The aim of our study was to evaluate if therapeutic success in the first-line of anticancer treatments in patients with NSCLC may predict treatment success in the following lines. METHODS: We analyzed the data of patients with NSCLC stage III/IV from the TULUNG registry separately for chemotherapy, TKIs, ALK inhibitors, and immunotherapy in the first line during the years 2011-2019. "Succesful treatment " was defined as PFS ≥ 6 months, a "good responder " was a patient with ˃50% of "successful treatment " lines. Treatment responses were analyzed separately for each drug group. Descriptive statistics, Fisher exact test, Pearson Chi-Squared test, log-rank test, and univariate/multivariate logistic regression models were used. RESULTS: The first-line TKI therapy was successful in 66.2%, while good responders accounted for 50.7% of the cohort and their rates were similar for all types of TKIs. First-line platinum-based chemotherapy was successful in 43.1% and 48.6% for combinations with pemetrexed and bevacizumab, respectively. Good responders accounted for 29.5% and 25.9%, respectively. In the group of ALK inhibitors, we observed treatment success in 52.3% of cases, while alectinib showed the highest effectiveness (up to 70%). Good responders constituted 50% of the group. In the first-line immunotherapy group, survival benefit was observed in 52.3%, and good responders constituted 52.3% of the cohort. CONCLUSION: We concluded that the treatment success in first-line therapies in patients with NSCLC may predict survival benefits in the subsequent lines, particularly in EGFR- or ALK-positive disease and immunotherapy-treated patients.

Serious Immune-related Adverse Events Are Associated With Greater Efficacy of Nivolumab Therapy Against Non-small Cell Lung Cancer.

BACKGROUND/AIM: The aim of this study was to investigate possible association between adverse events of nivolumab therapy and the effectiveness of treatment in patients with non-small cell lung cancer (NSCLC). Focusing on serious adverse events (i.e., those of grade ≥3), we evaluated overall survival (OS), progression-free survival (PFS), as well as objective response rate (ORR) to treatment. PATIENTS AND METHODS: We retrospectively analyzed a set of patients from the TULUNG database of NSCLC treated with nivolumab in eight oncology centers. We evaluated OS data based upon this set. To reduce possible bias, we further evaluated a subgroup of patients treated at the University Hospital in Pilsen, where the occurrence of adverse events, PFS, and ORR were independently examined by two experienced physicians. Survival statistics were evaluated using the Kaplan-Meier method and Cox analysis. RESULTS: We observed significantly greater OS, PFS, and ORR in the group of patients experiencing adverse events upon nivolumab treatment versus in those patients without such events. Although the univariable model analyzing the data set of all patients demonstrated higher OS in patients with serious adverse events, only a nonsignificant trend was observed in the Cox multivariable model. In a subgroup of patients with PFS and ORR evaluation, we did observe significant, favorable effects for patients having had serious adverse effects. CONCLUSION: Patients experiencing severe adverse events show a tendency toward better OS, PFS, and ORR compared to patients without or having only mild adverse events with nivolumab treatment.

110 µm thin endo-microscope for deep-brain in vivo observations of neuronal connectivity, activity and blood flow dynamics.

Light-based in-vivo brain imaging relies on light transport over large distances of highly scattering tissues. Scattering gradually reduces imaging contrast and resolution, making it difficult to reach structures at greater depths even with the use of multiphoton techniques. To reach deeper, minimally invasive endo-microscopy techniques have been established. These most commonly exploit graded-index rod lenses and enable a variety of modalities in head-fixed and freely moving animals. A recently proposed alternative is the use of holographic control of light transport through multimode optical fibres promising much less traumatic application and superior imaging performance. We present a 110 µm thin laser-scanning endo-microscope based on this prospect, enabling in-vivo volumetric imaging throughout the whole depth of the mouse brain. The instrument is equipped with multi-wavelength detection and three-dimensional random access options, and it performs at lateral resolution below 1 µm. We showcase various modes of its application through the observations of fluorescently labelled neurones, their processes and blood vessels. Finally, we demonstrate how to exploit the instrument to monitor calcium signalling of neurones and to measure blood flow velocity in individual vessels at high speeds.

Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes.

Genome duplication is safeguarded by constantly adjusting the activity of the replicative CMG (CDC45-MCM2-7-GINS) helicase. However, minichromosome maintenance proteins (MCMs)-the structural core of the CMG helicase-have never been visualized at sites of DNA synthesis inside a cell (the so-called MCM paradox). Here, we solve this conundrum by showing that anti-MCM antibodies primarily detect inactive MCMs. Upon conversion of inactive MCMs to CMGs, factors that are required for replisome activity bind to the MCM scaffold and block MCM antibody binding sites. Tagging of endogenous MCMs by CRISPR-Cas9 bypasses this steric hindrance and enables MCM visualization at active replisomes. Thus, by defining conditions for detecting the structural core of the replicative CMG helicase, our results explain the MCM paradox, provide visual proof that MCMs are an integral part of active replisomes in vivo, and enable the investigation of replication dynamics in living cells exposed to a constantly changing environment.

Worse Prognosis in the Symptomatic Patients With Lung Cancer - Czech Multicentric Study.

Background/Aim: This study aimed at contributing to a better diagnosis of lung cancer by analyzing the patient's symptoms and their linkage to other characteristics. Patients and Methods: We analyzed the data of 3,322 patients from LUCAS (LUngCAncerfocuS) National Registry of the Czech Republic. Overall survival was assessed using the Kaplan-Meier method. Results: The most common symptoms were cough (47.5%), dyspnea (45.6%), pain (27.3%), and weight loss (25.7%). Among all patients, 16% were asymptomatic. We demonstrated the negative prognostic significance of increasing number of lung cancer symptoms, that was significant after adjustment for age, TNM stages, and performance status, and morphological types of the cancer. Conclusion: Monitoring the severity and type of symptoms in patients with lung cancer can help in the diagnostics of the disease and the estimation of prognosis.

Can Previous Chemotherapy Affect the Outcome of Nivolumab Treatment in Non-small Cell Lung Cancer?

AIM: This study compared the results of nivolumab treatment in patients with pulmonary adenocarcinomas based upon previous chemotherapeutic regimens. PATIENTS AND METHODS: The data source for this retrospective study was the Czech VILP registry of patients with nivolumab-treated adenocarcinomas in second and higher lines of treatment. In relation to objective response rate, progression-free interval, and overall survival, three comparisons of patient were made: A: Those treated in first line with cisplatin and pemetrexed versus carboplatin with paclitaxel or vinorelbine; B: treatment with cisplatin and pemetrexed versus carboplatin with paclitaxel/vinorelbine and bevacizumab; and C: treatment in previous lines with pemetrexed (first-line cisplatin and pemetrexed plus those treated in second line with pemetrexed) versus treatment with taxane (first-line carboplatin and paclitaxel only plus those treated with second-line docetaxel). RESULTS: We observed no differences in objective response rate or progression-free survival between patients treated with the stated chemotherapeutic regimens. We observed a trend towards better overall survival for patients treated with carboplatin plus taxanes or vinorelbine with/without bevacizumab. CONCLUSION: From our overall survival data, a chemotherapeutic regimen of carboplatin plus taxanes or vinorelbine with/without bevacizumab might be a better partner for immunotherapy than a cisplatin and pemetrexed-based one.

Lung Cancer Versus "Young Cancer": Is Non-Small Cell Lung Cancer in Young Patients a Different Entity?

Purpose: Aim was to analyze demographic and tumor characteristics, treatment, and survival of patients with lung cancer younger than 40 years of age (U40) compared to older subgroups (41-70 and >70 years). Methods: We analyzed data of young patients diagnosed and treated in 2011-2019 in five pneumo-oncology centers in Czechia. Standard descriptive statistics, chi-squared test, Fisher exact test, and Kaplan-Meier survival analysis were used. p-Values <0.05 were considered significant. These data were compared with two control subgroups (cohort 1: 41-70 years, cohort 2: >70 years). Results: We identified 66 patients U40, 61 with non-small cell lung cancer (NSCLC)-50.8% men, mean age 34.6 years, 54.1% nonsmokers, daily good performance status, and 82% in stage IV. Adenocarcinomas dominated, endothelial growth factor receptor (EGFR) positivity was less common than in older groups contrary to anaplastic lymphoma kinase (ALK) mutations. Median progression-free survival was 3.7 months (vs. 4.9 and 6.2 months; p = 0.006) and overall survival reached 11.7 months (vs. 22.3 and 27.3 months; p < 0.001). Young patients in stage IV and never-smokers had shorter survival than older patients. Conclusion: Patients with NSCLC U40 had significantly worse prognosis than older patients.

Lung Cancer in Non-smokers in Czech Republic: Data from LUCAS Lung Cancer Clinical Registry.

BACKGROUND/AIM: LUCAS is a clinical lung cancer registry (ClinicalTrials.gov identifier is NCT04228237), prospectively collecting data from newly diagnosed lung cancer patients in seven pneumooncology centers in the Czech Republic, since June 1, 2018. The aim of the study was to assess the stage of the disease at the time of diagnosis, percentage of morphological types, survival, percentage of driving mutations, eligibility for radical surgery, and percentage of patients who undergo radical surgery, in the non-smoking population in comparison with smokers and former smokers. PATIENTS AND METHODS: The total number of patients in the registry at the time of the analysis was 2,743. Only 2,439 patients with complete records (smoking status, stage, and type of tumor) were included in this study. RESULTS: The analysis indicated that non-smokers are diagnosed at a later stage of the disease but they have a better survival rate than smokers. Fewer smokers with stage III disease who are eligible for radical surgery will undergo surgery compared to non-smokers with the same clinical stage. Driving mutations are more common in non-smokers, even after adjustment for the more frequent occurrence of adenocarcinoma in the group of non-smokers. CONCLUSION: The data from LUCAS registry are consistent with already known facts, suggesting that the LUCAS registry is a useful clinical tool.

Prognostic Value of EGFR Exon-20 Insertions in Czech Patients With Advanced Non-small Cell Lung Cancer.

BACKGROUND/AIM: Per literature, patients with epidermal growth factor receptor (EGFR) exon-20 insertions respond poorly to tyrosine kinase inhibitors (TKIs). This study analyzed real-world data to examine the prognostic and predictive value of these mutations. PATIENTS AND METHODS: We conducted a retrospective cohort study using Czech TULUNG Registry data, with data on multiple mutation types, collected in 2011-2020. RESULTS: We analyzed 554 (95.85%) patients with EGFR exon-19 deletions or exon-21 L858R substitutions and 24 (4.15%) patients with exon-20 insertions who received first-line high-value therapies. We summarized clinical characteristics and outcomes in all patients and by cohort. The risk of progression was statistically significantly higher (86%) in the exon-20 insertion cohort compared to the cohort with other mutations. Although not statistically significant, the risk of death was 44% higher in patients with exon-20 insertions. CONCLUSION: Advanced NSCLC patients with rare EGFR exon-20 insertions have a high risk of progression.

Comparison of Chemotherapeutic Regimens Frequently Used in Metastatic Non-squamous NSCLC Treatment.

BACKGROUND/AIM: Platinum-based chemotherapy with pemetrexed or paclitaxel/bevacizumab are regimens used in combination with checkpoint inhibitors in non-squamous non-small cell lung cancer (NSCLC) treatment. We conducted a real-world study to compare the outcomes of these chemotherapeutic regimens. PATIENTS AND METHODS: We investigated 1,534 patients with advanced non-squamous NSCLC treated with platin/pemetrexed (n=1212) or platin/paclitaxel/bevacizumab (n=322) in 9 cancer centres in the Czech Republic. RESULTS: The regimen containing platin/paclitaxel/bevacizumab showed significantly better overall response rate (ORR) compared to the platin/pemetrexed [40.8% vs. 32.7% (p=0.008)] in the overall population and [55.0% vs. 38.8% (p=0.002)] in the Eastern Cooperative Oncology Group performance status 0 group. There was no significant improvement in progression-free survival (PFS) and overall survival (OS) in either of these two groups of patients. CONCLUSION: In our real-world data analysis, patients treated with platin/paclitaxel/bevacizumab had better overall response rate (ORR), but not PFS or OS. Thus, both treatment regimens are similarly effective. Their selection should therefore be based on the potential side effects.

Real-life Effectiveness of Afatinib Versus Gefitinib in Patients With Non-small-cell Lung Cancer: A Czech Multicentre Study.

BACKGROUND/AIM: We investigated efficacy differences for afatinib versus gefitinib in non-small-cell lung cancer (NSCLC) according to epidermal growth factor receptor (EGFR) mutations. PATIENTS AND METHODS: We retrospectively analysed data for 343 patients with NSCLC with performance status 1 having EGFR mutations treated with gefitinib or afatinib. Overall response rate (ORR) was tested by Fisher's exact test. Overall (OS) and progression-free (PFS) survival were estimated by Kaplan-Meier method. RESULTS: ORR did not differ in any group or subgroup. Among all patients, we observed significantly longer PFS for those treated with afatinib vs. gefitinib (median 13.4 vs. 9.5 months, p=0.026), but only a nonsignificant trend was observed for OS. We showed nonsignificant trends of better PFS and OS using afatinib for exon 19 deletion and L858R subgroups. We observed no significant PFS differences for other EGFR mutations but a nonsignificant trend towards better OS for those treated with afatinib. CONCLUSION: Afatinib led to longer PFS for patients with common EGFR mutations but not for those with rare mutations.

Cell differentiation and aging accompanied by depletion of the ACE2 protein.

ACE2 was observed as the cell surface receptor of the SARS-CoV-2 virus. Interestingly, we also found ACE2 positivity inside the cell nucleus. The ACE2 levels changed during cell differentiation and aging and varied in distinct cell types. We observed ACE2 depletion in the aortas of aging female mice, similarly, the aging caused ACE2 decrease in the kidneys. Compared with that in the heart, brain and kidneys, the ACE2 level was the lowest in the mouse lungs. In mice exposed to nicotine, ACE2 was not changed in olfactory bulbs but in the lungs, ACE2 was upregulated in females and downregulated in males. These observations indicate the distinct gender-dependent properties of ACE2. Differentiation into enterocytes, and cardiomyocytes, caused ACE2 depletion. The cardiomyogenesis was accompanied by renin upregulation, delayed in HDAC1-depleted cells. In contrast, vitamin D2 decreased the renin level while ACE2 was upregulated. Together, the ACE2 level is high in non-differentiated cells. This protein is more abundant in the tissues of mouse embryos and young mice in comparison with older animals. Mostly, downregulation of ACE2 is accompanied by renin upregulation. Thus, the pathophysiology of COVID-19 disease should be further studied not only by considering the ACE2 level but also the whole renin-angiotensin system.

The Distinct Function and Localization of METTL3/METTL14 and METTL16 Enzymes in Cardiomyocytes.

It has become evident that epitranscriptome events, mediated by specific enzymes, regulate gene expression and, subsequently, cell differentiation processes. We show that methyltransferase-like proteins METTL3/METTL14 and N6-adenosine methylation (m6A) in RNAs are homogeneously distributed in embryonic hearts, and histone deacetylase (HDAC) inhibitors valproic acid and Trichostatin A (TSA) up-regulate METTL3/METTL14 proteins. The levels of METTL3 in mouse adult hearts, isolated from male and female animals, were lower in the aorta and pulmonary trunks when compared with atria, but METT14 was up-regulated in the aorta and pulmonary trunk, in comparison with ventriculi. Aging caused METTL3 down-regulation in aorta and atria in male animals. Western blot analysis in differentiated mouse embryonic stem cells (mESCs), containing 10-30 percent of cardiomyocytes, showed METTL3/METTL14 down-regulation, while the differentiation-induced increased level of METTL16 was observed in both wild type (wt) and HDAC1 depleted (dn) cells. In parallel, experimental differentiation in especially HDAC1 wild type cells was accompanied by depletion of m6A in RNA. Immunofluorescence analysis of individual cells revealed the highest density of METTL3/METTL14 in α-actinin positive cardiomyocytes when compared with the other cells in the culture undergoing differentiation. In both wt and HDAC1 dn cells, the amount of METTL16 was also up-regulated in cardiomyocytes when compared to co-cultivated cells. Together, we showed that distinct anatomical regions of the mouse adult hearts are characterized by different levels of METTL3 and METTL14 proteins, which are changed during aging. Experimental cell differentiation was also accompanied by changes in METTL-like proteins and m6A in RNA; in particular, levels and distribution patterns of METTL3/METTL14 proteins were different from the same parameters studied in the case of the METTL16 protein.

Real-life effectiveness of first-line anticancer treatments in stage IIIB/IV NSCLC patients: Data from the Czech TULUNG Registry.

BACKGROUND: Data regarding real-life effectiveness of any treatment may improve clinical decision-making. The aim of this study was to evaluate real-life effectiveness of tyrosin-kinase inhibitors, bevacizumab and pemetrexed as first-line treatments in patients with advanced/metastatic non-small cell lung cancer (NSCLC). METHODS: We analyzed data of 2157 patients of the Czech TULUNG Registry of patients with advanced/metastatic NSCLC who received modern-era treatments between 2011 and 2018. Patients treated with gefitinib, erlotinib, afatinib, bevacizumab (+ maintenance), pemetrexed (+ maintenance) as first-line therapy were included in the study. A systematic literature search separately identified clinical trials suitable for calculation of comparator pooled OS and PFS for each regimen. For each subgroup, basic characteristics and survival data (Kaplan-Meier estimates) are shown. We propose the "index of real-life effectiveness" (IRE), a ratio of real-life OS/PFS and comparator pooled OS/PFS. Univariate and multivariate logistic regression identified factors were associated with longer OS (ie, IRE>1.1). RESULTS: Survival analysis showed median OS of 23 months for erlotinib, 29.3 months for afatinib, 19.6 months for gefitinib, 12.2 months for pemetrexed, 17.5 months for pemetrexed maintenance, 15.8 months for bevacizumab and 15.8 months for bevacizumab maintenance. Calculated IREs for OS for the regimens were: erlotinib 1.013, afatinib 1.184, gefitinib 0.736, pemetrexed 1.188, pemetrexed maintenance 1.294, bevacizumab 1.178, and bevacizumab maintenance 1.189. Multivariate regression analysis showed that these factors were associated with longer OS: lower PS for afatinib; lower PS, absence of adverse events and female sex for bevacizumab; and lower PS and female sex for pemetrexed. CONCLUSIONS: This study clearly demonstrated that real-life effectiveness of certain treatment regimens may strongly differ in various populations/health care systems, and comparison between TULUNG data and pooled survival data from trials showed higher real-life effectiveness for most of the studied first-line regimens. Lower ECOG PS, younger age, female sex and adverse events were associated with longer survival in most regimens. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: Comparison between TULUNG data and pooled survival data from trials showed higher real-life effectiveness for most of the studied first-line regimens; for most regimens, lower ECOG PS, younger age, female sex and adverse events were associated with longer survival. WHAT THIS STUDY ADDS: Real-life effectiveness of certain treatment regimens may strongly differ in various populations/health care systems.

Impact of Concomitant Medication Administered at the Time of Initiation of Nivolumab Therapy on Outcome in Non-small Cell Lung Cancer.

AIM: To investigate potential association between administration of corticosteroids, antibiotics, probiotics, proton pump inhibitors, non-steroidal anti-inflammatory drugs (NSAID), statins and metformin and outcome in patients with non-small cell lung cancer (NSCLC) treated with nivolumab. PATIENTS AND METHODS: A total of 224 patients with advanced NSCLC treated at nine comprehensive cancer centers were analyzed in this national retrospective study. Survival statistics were evaluated using Kaplan-Meier method and Cox analysis. RESULTS: Only corticosteroid use had a significant negative effect on the objective response rate. In the univariate analysis, there was no significant effect of the studied concomitant medications on the efficacy of nivolumab. In a subsequent multifactorial analysis, a possible positive effect of the concomitant use of NSAID at the initiation of nivolumab treatment was revealed. CONCLUSION: The results of the present retrospective exploratory analysis underscore the importance of knowing the exact type of concomitant medication, the route of administration, the dose of medication, and the region of the ongoing study. The present data indicated a significantly higher rate of progression in patients treated with corticosteroids and the possible positive effect of NSAID use at the initiation of nivolumab treatment.

Non-small Cell Lung Cancer as a Chronic Disease - A Prospective Study from the Czech TULUNG Registry.

AIM: To compare survival outcomes in patients with non-small cell lung cancer (NSCLC) treated with modern-era drugs (antifolates, antiangiogenics, tyrosine kinase and anaplastic lymphoma kinase inhibitors, immunotherapy) with treatment initiation in 2011-12 and 2015-16, respectively. PATIENTS AND METHODS: Prospective data from Czech TULUNG Registry (960 patients from 2011-12 and 512 patients from 2015-16) were analyzed. Kaplan-Meier analysis was used to estimate overall survival (OS) and progression-free survival (PFS); Cox proportional hazards model to assess factors associated with 2-year survival. RESULTS: Survival at 2 years was more frequent in cohort 2015-16 compared to cohort 2011-12 (43.2% vs. 24% for adenocarcinoma; p<0.001 and 28.7% vs. 11.8% for squamous-cell lung carcinoma; p=0.002). Assignment to cohort 2015-16 and treatment multilinearity (two or more lines in sequence) were associated with higher probability of 2-year survival (hazard ratio=0.666 and hazard ratio=0.597; p<0.001). Comparison of 2-year survivors from both cohorts showed no differences. CONCLUSION: Survival at 2 years probability in stage IIIB-IV NSCLC doubled between 2011-12 and 2015-16; advanced-stage NSCLC may be considered a chronic disease in a large proportion of patients.

H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions.

Methylation of histones H4 at lysine 20 position (H4K20me), which is functional in DNA repair, represents a binding site for the 53BP1 protein. Here, we show a radiation-induced increase in the level of H4K20me3 while the levels of H4K20me1 and H4K20me2 remained intact. H4K20me3 was significantly pronounced at DNA lesions in only the G1 phase of the cycle, while this histone mark was reduced in very late S and G2 phases when PCNA was recruited to locally micro-irradiated chromatin. H4K20me3 was diminished in locally irradiated Suv39h1/h2 double knockout (dn) fibroblasts, and the same phenomenon was observed for H3K9me3 and its binding partner, the HP1ß protein. Immunoprecipitation showed the existence of an interaction between H3K9me3-53BP1 and H4K20me3-53BP1; however, HP1ß did not interact with 53BP1. Together, H3K9me3 and H4K20me3 represent epigenetic markers that are important for the function of the 53BP1 protein in non-homologous end joining (NHEJ) repair. The very late S phase represents the cell cycle breakpoint when a DDR function of the H4K20me3-53BP1 complex is abrogated due to recruitment of the PCNA protein and other DNA repair factors of homologous recombination to DNA lesions.

Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells.

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.

Irradiation by γ-rays reduces the level of H3S10 phosphorylation and weakens the G2 phase-dependent interaction between H3S10 phosphorylation and γH2AX.

Histone posttranslational modifications regulate diverse nuclear functions, including DNA repair. Here, we use mass spectrometry, western blotting, immunohistochemistry and advanced confocal microscopy in order to show radiation-specific changes in the histone signature. We studied wild-type mouse embryonic stem cells (mESCs) and mESCs with a depletion of histone deacetylase 1 (HDAC1), which plays a role in DNA repair. Irradiation by γ-rays increased the S139 phosphorylation of histone H2AX but reduced the level of the H3K9-R17 peptide, which contains S10 phosphorylation (H3S10ph). On an individual cellular level, H3S10ph was low in highly γH2AX-positive UV laser-induced DNA lesions, and this nuclear distribution pattern was not changed by HDAC1 depletion. Despite this fact, spontaneous γH2AX-positive DNA lesions colocalized with large H3S10ph-positive nuclear bodies that appear in the G2 phase of the cell cycle. Similarly, by FLIM-FRET analysis, we observed an interaction between H3S10ph and γH2AX in the G2 phase. However, this interaction was reduced when cells were exposed to γ-rays. A mutual link between H3S10ph and γH2AX was not observed in the G1 phase of the cell cycle. Together, our data show that despite the fact that H3S10ph is not directly involved in DNA repair, a decrease in H3S10 phosphorylation and weakened interaction between H3S10ph and γH2AX is a result of radiation-induced damage of the genome. In this case, γ-irradiation also decreased the number of cells in the G1 phase, characterized by no interaction between H3S10ph and γH2AX.

Depletion of A-type lamins and Lap2α reduces 53BP1 accumulation at UV-induced DNA lesions and Lap2α protein is responsible for compactness of irradiated chromatin.

We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light. We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2α deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin. However, a well-known marker of double strand breaks, γH2AX, was highly abundant in the lesion-surrounding genome of Lap2α deficient cells. Described changes, induced by irradiation in Lap2α dn cells, were not accompanied by cell cycle changes. In Lap2α dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein. Two of them showed identical exponential decay times (τ1 and τ3), but the decay rate of τ2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2α dn fibroblasts. Moreover, γ-irradiation weakened an interaction between A-type lamins and Lap2α. Together, our results demonstrate how depletion of Lap2α affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2α deficient cells exposed to radiation.