Length-independent telomere damage drives post-mitotic cardiomyocyte senescence.

Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age-related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a, and results in a non-canonical senescence-associated secretory phenotype, which is pro-fibrotic and pro-hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age-related myocardial dysfunction and in the wider setting to ageing in post-mitotic tissues.

DAP Kinase-Related Apoptosis-Inducing Protein Kinase 2 (DRAK2) Is a Key Regulator and Molecular Marker in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World and it is characterized by a marked degree of clinical heterogeneity. An impaired balance between pro- and anti-apoptotic stimuli determines chemorefractoriness and outcome. The low proliferation rate of CLL cells indicates that one of the primary mechanisms involved in disease development may be an apoptotic failure. Here, we study the clinical and functional significance of DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in CLL. We have analyzed CLL patient samples and showed that low expression levels of DRAK2 were significantly associated with unfavorable outcome in our CLL cohort. DRAK2 expression levels showed a positive correlation with the expression of DAPK1, and TGFBR1. Consistent with clinical data, the downregulation of DRAK2 in MEC-1 CLL cells strongly increased cell viability and proliferation. Further, our transcriptome data from MEC-1 cells highlighted MAPK, NF-κB, and Akt and as critical signaling hubs upon DRAK2 knockdown. Taken together, our results indicate DRAK2 as a novel marker of CLL survival that plays key regulatory roles in CLL prognosis.

RRAD, IL4I1, CDKN1A, and SERPINE1 genes are potentially co-regulated by NF-κB and p53 transcription factors in cells exposed to high doses of ionizing radiation.

BACKGROUND: The cellular response to ionizing radiation involves activation of p53-dependent pathways and activation of the atypical NF-κB pathway. The crosstalk between these two transcriptional networks include (co)regulation of common gene targets. Here we looked for novel genes potentially (co)regulated by p53 and NF-κB using integrative genomics screening in human osteosarcoma U2-OS cells irradiated with a high dose (4 and 10 Gy). Radiation-induced expression in cells with silenced TP53 or RELA (coding the p65 NF-κB subunit) genes was analyzed by RNA-Seq while radiation-enhanced binding of p53 and RelA in putative regulatory regions was analyzed by ChIP-Seq, then selected candidates were validated by qPCR. RESULTS: We identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes, namely IL4I1, SERPINE1, and CDKN1A, an antagonistic effect of the TP53 and RELA silencing was consistent with radiation-enhanced binding of both p53 and RelA. This suggested the possibility of a direct antagonistic (co)regulation by both factors: activation by NF-κB and inhibition by p53 of IL4I1, and activation by p53 and inhibition by NF-κB of CDKN1A and SERPINE1. On the other hand, radiation-enhanced binding of both p53 and RelA was observed in a putative regulatory region of the RRAD gene whose expression was downregulated both by TP53 and RELA silencing, which suggested a possibility of direct (co)activation by both factors. CONCLUSIONS: Four new candidates for genes directly co-regulated by NF-κB and p53 were revealed.

Irradiation with UV-C inhibits TNF-α-dependent activation of the NF-κB pathway in a mechanism potentially mediated by reactive oxygen species.

Pathways depending on the NF-κB transcription factor are essential components of cellular response to stress. Plethora of stimuli modulating NF-κB includes inflammatory signals, ultraviolet radiation (UV) and reactive oxygen species (ROS), yet interference between different factors affecting NF-κB remains relatively understudied. Here, we aim to characterize the influence of UV radiation on TNF-α-induced activity of the NF-κB pathway. We document inhibition of TNF-α-induced activation of NF-κB and subsequent suppression of NF-κB-regulated genes in cells exposed to UV-C several hours before TNF-α stimulation. Accumulation of ROS and subsequent activation of NRF2, p53, AP-1 and NF-κB-dependent pathways, with downstream activation of antioxidant mechanisms (e.g., SOD2 and HMOX1 expression), is observed in the UV-treated cells. Moreover, NF-κB inhibition is not observed if generation of UV-induced ROS is suppressed by chemical antioxidants. It is noteworthy that stimulation with TNF-α also generates a wave of ROS, which is suppressed in cells pre-treated by UV. We postulate that irradiation with UV-C activates antioxidant mechanisms, which in turn affect ROS-mediated activation of NF-κB by TNF-α. Considering a potential cross talk between p53 and NF-κB, we additionally compare observed effects in p53-proficient and p53-deficient cells and find the UV-mediated suppression of TNF-α-activated NF-κB in both types of cells.

Heart irradiation reduces microvascular density and accumulation of HSPA1 in mice.

PURPOSE: Improvement of radiotherapy techniques reduces the exposure of normal tissues to ionizing radiation. However, the risk of radiation-related late effects remains elevated. In the present study, we investigated long-term effects of radiation on heart muscle morphology. MATERIALS AND METHODS: We established a mouse model to study microvascular density (MVD), deposition of collagen fibers, and changes in accumulation of heat shock 70 kDa protein 1 (HSPA1) in irradiated heart tissue. Hearts of C57BL/6 mice received a single dose of X­ray radiation in the range 0.2-16 Gy. Analyses were performed 20, 40, and 60 weeks after irradiation. RESULTS: Reduction in MD was revealed as a long-term effect observed 20-60 weeks after irradiation. Moreover, a significant and dose-dependent increase in accumulation of HSPA1, both cytoplasmic and nuclear, was observed in heart tissues collected 20 weeks after irradiation. We also noticed an increase in collagen deposition in hearts treated with higher doses. CONCLUSIONS: This study shows that some changes induced by radiation in the heart tissue, such as reduction in microvessel density, increase in collagen deposition, and accumulation of HSPA1, are observed as long-term effects which might be associated with late radiation cardiotoxicity.

Long-term effects of low-dose mouse liver irradiation involve ultrastructural and biochemical changes in hepatocytes that depend on lipid metabolism.

The aim of the study was to investigate long-term effects of radiation on the (ultra)structure and function of the liver in mice. The experiments were conducted on wild-type C57BL/6J and apolipoprotein E knock-out (ApoE-/-) male mice which received a single dose (2 or 8 Gy) of X-rays to the heart with simultaneous exposure of liver to low doses (no more than 30 and 120 mGy, respectively). Livers were collected for analysis 60 weeks after irradiation and used for morphological, ultrastructural, and biochemical studies. The results show increased damage to mitochondrial ultrastructure and lipid deposition in hepatocytes of irradiated animals as compared to non-irradiated controls. Stronger radiation-related effects were noted in ApoE-/- mice than wild-type animals. In contrast, radiation-related changes in the activity of lysosomal hydrolases, including acid phosphatase, ß-glucuronidase, N-acetyl-ß-D-hexosaminidase, ß-galactosidase, and α-glucosidase, were observed in wild type but not in ApoE-deficient mice, which together with ultrastructural picture suggests a higher activity of autophagy in ApoE-proficient animals. Irradiation caused a reduction of plasma markers of liver damage in wild-type mice, while an increased level of hepatic lipase was observed in plasma of ApoE-deficient mice, which collectively indicates a higher resistance of hepatocytes from ApoE-proficient animals to radiation-mediated damage. In conclusion, liver dysfunctions were observed as late effects of irradiation with an apparent association with malfunction of lipid metabolism.

Late Effects of Ionizing Radiation on the Ultrastructure of Hepatocytes and Activity of Lysosomal Enzymes in Mouse Liver Irradiated In Vivo.

The study aimed to investigate late radiation-induced changes in the histology, ultrastructure, and activity of lysosomal enzymes in mouse liver exposed to ionizing radiation. The experiment was conducted on C57BL/6J male mice whose distal part of the liver was exposed occasionally to single doses of radiation (6 MV photons) during targeted heart irradiation; estimated doses delivered to analyzed tissue were 0.025 Gy, 0.25 Gy, 1 Gy, and 2 Gy. Tissues were collected 40 weeks after irradiation. We have observed that late effects of radiation have an adaptive nature and their intensity was dose-dependent. Morphological changes in hepatocytes included an increased number of primary lysosomes and autophagic vacuoles, which were visible in tissues irradiated with 0.25 Gy and higher doses. On the other hand, a significant increase in the activity of lysosomal hydrolases was observed only in tissues exposed to 2 Gy. The etiology of these changes may be multifactorial and result, among others, from unintentional irradiation of the distal part of the liver and/or functional interaction of the liver with an irradiated heart. In conclusion, we confirmed the presence of late dose-dependent ultrastructural and biochemical changes in mouse hepatocytes after liver irradiation in vivo.

Food Traceability as an Element of Sustainable Consumption-Pandemic-Driven Changes in Consumer Attitudes.

A conscious approach to the issue of food traceability on the part of consumers is essential for making rational food purchases, which in turn contributes to sustainable consumption and globally, is an element of sustainable development. The study aims to assess the changes in consumers' buying behaviors in the context of food traceability before and during the COVID-19 pandemic, as well as the impact of sociodemographic factors on those changes. Therefore, an online survey was conducted on a sample of 1000 respondents who were Polish food consumers. The study covered aspects related to the traceability of food by consumers before and during the pandemic. The results allowed for positive verification of the H1: Polish consumers attitudes related to food buying process changed during the COVID-19 pandemic. The results didn't allow for fully positive verification of the H2: Sociodemographic factors significantly influence Polish consumers attitudes to the food shopping during COVID-19 period compared to pre-pandemic period. The significant influence was supported in almost all (in 6 out of 8) analyzed aspects in case of age, education, and place of residence. However, in case of gender it was confirmed only in terms of two out of eight aspects: choosing product of national origin and using the online form of ordering purchases.

Senescence and senolytics in cardiovascular disease: Promise and potential pitfalls.

Ageing is the biggest risk factor for impaired cardiovascular health, with cardiovascular disease being the cause of death in 40 % of individuals over 65 years old. Ageing is associated with an increased prevalence of atherosclerosis, coronary artery stenosis and subsequent myocardial infarction, thoracic aortic aneurysm, valvular heart disease and heart failure. An accumulation of senescence and increased inflammation, caused by the senescence-associated secretory phenotype, have been implicated in the aetiology and progression of these age-associated diseases. Recently it has been demonstrated that compounds targeting components of anti-apoptotic pathways expressed by senescent cells can preferentially induce senescence cells to apoptosis and have been termed senolytics. In this review, we discuss the evidence demonstrating that senescence contributes to cardiovascular disease, with a particular focus on studies that indicate the promise of senotherapy. Based on these data we suggest novel indications for senolytics as a treatment of cardiovascular diseases which have yet to be studied in the context of senotherapy. Finally, while the potential benefits are encouraging, several complications may result from senolytic treatment. We, therefore, consider these challenges in the context of the cardiovascular system.

Low BACH2 Expression Predicts Adverse Outcome in Chronic Lymphocytic Leukaemia.

Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a highly variable clinical outcome. There are well-established CLL prognostic biomarkers that have transformed treatment and improved the understanding of CLL biology. Here, we have studied the clinical significance of two crucial B cell regulators, BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) and BCL6 (B-cell CLL/lymphoma 6), in a cohort of 102 CLL patients and determined the protein interaction networks that they participate in using MEC-1 CLL cells. We observed that CLL patients expressing low levels of BCL6 and BACH2 RNA had significantly shorter overall survival (OS) than high BCL6- and BACH2-expressing cases. Notably, their low expression specifically decreased the OS of immunoglobulin heavy chain variable region-mutated (IGHV-M) CLL patients, as well as those with 11q and 13q deletions. Similar to the RNA data, a low BACH2 protein expression was associated with a significantly shorter OS than a high expression. There was no direct interaction observed between BACH2 and BCL6 in MEC-1 CLL cells, but they shared protein networks that included fifty different proteins. Interestingly, a prognostic index (PI) model that we generated, using integrative risk score values of BACH2 RNA expression, age, and 17p deletion status, predicted patient outcomes in our cohort. Taken together, these data have shown for the first time a possible prognostic role for BACH2 in CLL and have revealed protein interaction networks shared by BCL6 and BACH2, indicating a significant role for BACH2 and BCL6 in key cellular processes, including ubiquitination mediated B-cell receptor functions, nucleic acid metabolism, protein degradation, and homeostasis in CLL biology.

Aging-Related Changes in the Ultrastructure of Hepatocytes and Cardiomyocytes of Elderly Mice Are Enhanced in ApoE-Deficient Animals.

Biological aging is associated with various morphological and functional changes, yet the mechanisms of these phenomena remain unclear in many tissues and organs. Hyperlipidemia is among the factors putatively involved in the aging of the liver and heart. Here, we analyzed morphological, ultrastructural, and biochemical features in adult (7-month-old) and elderly (17-month-old) mice, and then compared age-related features between wild type (C57Bl/6 strain) and ApoE-deficient (transgenic ApoE-/-) animals. Increased numbers of damaged mitochondria, lysosomes, and lipid depositions were observed in the hepatocytes of elderly animals. Importantly, these aging-related changes were significantly stronger in hepatocytes from ApoE-deficient animals. An increased number of damaged mitochondria was observed in the cardiomyocytes of elderly animals. However, the difference between wild type and ApoE-deficient mice was expressed in the larger size of mitochondria detected in the transgenic animals. Moreover, a few aging-related differences were noted between wild type and ApoE-deficient mice at the level of plasma biochemical markers. Levels of cholesterol and HDL increased in the plasma of elderly ApoE-/- mice and were markedly higher than in the plasma of elderly wild type animals. On the other hand, the activity of alanine transaminase (ALT) decreased in the plasma of elderly ApoE-/- mice and was markedly lower than in the plasma of elderly wild type animals.

Clearance of senescent cells during cardiac ischemia-reperfusion injury improves recovery.

A key component of cardiac ischemia-reperfusion injury (IRI) is the increased generation of reactive oxygen species, leading to enhanced inflammation and tissue dysfunction in patients following intervention for myocardial infarction. In this study, we hypothesized that oxidative stress, due to ischemia-reperfusion, induces senescence which contributes to the pathophysiology of cardiac IRI. We demonstrate that IRI induces cellular senescence in both cardiomyocytes and interstitial cell populations and treatment with the senolytic drug navitoclax after ischemia-reperfusion improves left ventricular function, increases myocardial vascularization, and decreases scar size. SWATH-MS-based proteomics revealed that biological processes associated with fibrosis and inflammation that were increased following ischemia-reperfusion were attenuated upon senescent cell clearance. Furthermore, navitoclax treatment reduced the expression of pro-inflammatory, profibrotic, and anti-angiogenic cytokines, including interferon gamma-induced protein-10, TGF-ß3, interleukin-11, interleukin-16, and fractalkine. Our study provides proof-of-concept evidence that cellular senescence contributes to impaired heart function and adverse remodeling following cardiac ischemia-reperfusion. We also establish that post-IRI the SASP plays a considerable role in the inflammatory response. Subsequently, senolytic treatment, at a clinically feasible time-point, attenuates multiple components of this response and improves clinically important parameters. Thus, cellular senescence represents a potential novel therapeutic avenue to improve patient outcomes following cardiac ischemia-reperfusion.

Mass spectrometry-based serum proteome pattern analysis in molecular diagnostics of early stage breast cancer.

BACKGROUND: Mass spectrometric analysis of the blood proteome is an emerging method of clinical proteomics. The approach exploiting multi-protein/peptide sets (fingerprints) detected by mass spectrometry that reflect overall features of a specimen's proteome, termed proteome pattern analysis, have been already shown in several studies to have applicability in cancer diagnostics. We aimed to identify serum proteome patterns specific for early stage breast cancer patients using MALDI-ToF mass spectrometry. METHODS: Blood samples were collected before the start of therapy in a group of 92 patients diagnosed at stages I and II of the disease, and in a group of age-matched healthy controls (104 women). Serum specimens were purified and the low-molecular-weight proteome fraction was examined using MALDI-ToF mass spectrometry after removal of albumin and other high-molecular-weight serum proteins. Protein ions registered in a mass range between 2,000 and 10,000 Da were analyzed using a new bioinformatic tool created in our group, which included modeling spectra as a sum of Gaussian bell-shaped curves. RESULTS: We have identified features of serum proteome patterns that were significantly different between blood samples of healthy individuals and early stage breast cancer patients. The classifier built of three spectral components that differentiated controls and cancer patients had 83% sensitivity and 85% specificity. Spectral components (i.e., protein ions) that were the most frequent in such classifiers had approximate m/z values of 2303, 2866 and 3579 Da (a biomarker built from these three components showed 88% sensitivity and 78% specificity). Of note, we did not find a significant correlation between features of serum proteome patterns and established prognostic or predictive factors like tumor size, nodal involvement, histopathological grade, estrogen and progesterone receptor expression. In addition, we observed a significantly (p = 0.0003) increased level of osteopontin in blood of the group of cancer patients studied (however, the plasma level of osteopontin classified cancer samples with 88% sensitivity but only 28% specificity). CONCLUSION: MALDI-ToF spectrometry of serum has an obvious potential to differentiate samples between early breast cancer patients and healthy controls. Importantly, a classifier built on MS-based serum proteome patterns outperforms available protein biomarkers analyzed in blood by immunoassays.

Pharmacological clearance of senescent cells improves survival and recovery in aged mice following acute myocardial infarction.

Cardiovascular disease is the leading cause of death in individuals over 60 years old. Aging is associated with an increased prevalence of coronary artery disease and a poorer prognosis following acute myocardial infarction (MI). With age, senescent cells accumulate in tissues, including the heart, and contribute to age-related pathologies. However, the role of senescence in recovery following MI has not been investigated. In this study, we demonstrate that treatment of aged mice with the senolytic drug, navitoclax, eliminates senescent cardiomyocytes and attenuates profibrotic protein expression in aged mice. Importantly, clearance of senescent cells improved myocardial remodelling and diastolic function as well as overall survival following MI. These data provide proof-of-concept evidence that senescent cells are major contributors to impaired function and increased mortality following MI and that senolytics are a potential new therapeutic avenue for MI.

Changes in activity and structure of lysosomes from liver of mouse irradiated in vivo.

PURPOSE: Lysosomes may have an important role in response to ionizing radiation. Moreover, radiation could affect autophagy, which process involves the activity of lysosomal enzymes. In the present study, the effect of ionizing radiation on the lysosomal compartment of mouse liver was investigated after in vivo exposure. MATERIALS AND METHODS: Morphology and ultrastructure of hepatocytes were assessed by light and electron microscopy, and activities of selected lysosomal enzymes were assessed in 12, 36 and 120 h after exposure to the mean dose of 1 Gy. The levels of autophagy-related proteins LC3-II and p62 were compared by Western blotting between untreated and irradiated animals (120 h after exposure). RESULTS: Increased number of autophagic vacuoles in hepatocytes from exposed animals was documented in the ultrastructural study; destroyed mitochondria were the dominant component of such vacuoles. Moreover, an increased activity of lysosomal hydrolases was observed after exposure. However, levels of autophagy substrates LC3-II and p62 were barely affected in exposed animals 120 h after irradiation when the accumulation of autophagic vacuoles was observed. CONCLUSION: Effects of irradiation included an increased number of autophagic vacuoles, especially of autophagosomes, and increased activity of lysosomal enzymes. However, putative markers of autophagic flux were not observed, which suggested suppression of the completion of the radiation-mediated autophagy pathway.

Pro-inflammatory cytokine and high doses of ionizing radiation have similar effects on the expression of NF-kappaB-dependent genes.

The NF-κB transcription factors are activated via diverse molecular mechanisms in response to various types of stimuli. A plethora of functions associated with specific sets of target genes could be regulated differentially by this factor, affecting cellular response to stress including an anticancer treatment. Here we aimed to compare subsets of NF-κB-dependent genes induced in cells stimulated with a pro-inflammatory cytokine and in cells damaged by a high dose of ionizing radiation (4 and 10 Gy). The RelA-containing NF-κB species were activated by the canonical TNFα-induced and the atypical radiation-induced pathways in human osteosarcoma cells. NF-κB-dependent genes were identified using the gene expression profiling (by RNA-Seq) in cells with downregulated RELA combined with the global profiling of RelA binding sites (by ChIP-Seq), with subsequent validation of selected candidates by quantitative PCR. There were 37 NF-κB-dependent protein-coding genes identified: in all cases RelA bound in their regulatory regions upon activation while downregulation of RELA suppressed their stimulus-induced upregulation, which apparently indicated the positive regulation mode. This set of genes included a few "novel" NF-κB-dependent species. Moreover, the evidence for possible negative regulation of ATF3 gene by NF-κB was collected. The kinetics of the NF-κB activation was slower in cells exposed to radiation than in cytokine-stimulated ones. However, subsets of NF-κB-dependent genes upregulated by both types of stimuli were essentially the same. Hence, one should expect that similar cellular processes resulting from activation of the NF-κB pathway could be induced in cells responding to pro-inflammatory cytokines and in cells where so-called "sterile inflammation" response was initiated by radiation-induced damage.

Therapy-Related Changes in the Serum Proteome Patterns of Early Stage Breast Cancer Patients with Different Outcomes.

Adjuvant chemo- and/or radiotherapy is applied in a majority of patients treated for early stage breast cancer, although only a small percentage of these individuals are at high risk of metastasis or recurrence. Hence, knowledge of the biomarkers associated with the risk of disease progression might facilitate the planning of an optimal therapy and protect many patients from the toxicity of unnecessary treatment. In this study, we characterized the serum proteome of patients diagnosed with early-stage breast cancer, exhibiting either no evidence of disease five years after the end of therapy or suffering from metastasis, relapse or a second cancer during the corresponding follow-up. Samples collected before treatment and one year after the end of therapy, when no clinical symptoms of a treatment failure was evidenced, were analyzed using two classical proteomics approaches: LC-MS/MS and 2D-PAGE. A total of 42 proteins with relative quantities that were significantly different between pre- and post-treatment samples were identified in either group of patients; however, the observed changes were more frequent in the treatment-failure group. Among the posttreatment samples, 30 proteins were upregulated, and 10 proteins were downregulated, while 11 proteins were upregulated, and eight proteins were downregulated in the control group. Moreover, several proteins exhibited different patterns of changes in both groups of patients. For example, haptoglobin expression increased in the treatment-failure group but decreased in the control group (this pattern of changes was confirmed using an immunoassay). Notably, proteins affected in posttreatment samples in either group of patients could be associated with different molecular and cellular functions, including angiogenesis, blood coagulation and wound healing in the treatment-failure group and cell adhesion and cell death in the control group.

Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology.

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH2A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation.

Association between plasma proteome profiles analysed by mass spectrometry, a lymphocyte-based DNA-break repair assay and radiotherapy-induced acute mucosal reaction in head and neck cancer patients.

PURPOSE: The plasma proteome was analysed as a potential source of markers of radiosensitivity in patients treated with definitive radiotherapy for head and neck cancer. MATERIALS AND METHODS: Acute mucosal reactions that developed during radiotherapy were assessed in 55 patients. Blood samples were collected from each patient before the treatment and also from 50 healthy donors. The low-molecular-weight fraction of the plasma proteome (2,000-10,000 Da range) was analysed by the Matrix-Assisted Laser Desorption Ionisation mass spectrometry. The capacity for DNA break repair was assessed by the comet assay using lymphocytes irradiated in vitro. RESULTS: Spectral components registered in plasma samples were used to build classifiers that discriminated patients from healthy individuals with about 90% specificity and sensitivity (components of 4469, 6929 and 8937 Da were the most essential for cancer classification). Four spectral components were identified (2219, 2454, 3431 and 5308 Da) whose abundances correlated with a maximal intensity of the acute reaction. Several spectral components whose abundances correlated with the rate of DNA repair in irradiated lymphocytes were also detected. Additionally, a more rapid escalation of an acute reaction was correlated with a higher level of unrepaired damage assessed by the comet assay. conclusions: The plasma proteome could be considered as a potential source of predictive markers of acute reaction in patients with head and neck cancer treated with radiotherapy.