Integrated Microarray-Based Data Analysis of miRNA Expression Profiles: Identification of Novel Biomarkers of Cisplatin-Resistance in Testicular Germ Cell Tumours.

Testicular germ cell tumours (TGCTs) are the most common solid malignancy among young men, and their incidence is still increasing. Despite good curability with cisplatin (CDDP)-based chemotherapy, about 10% of TGCTs are non-responsive and show a chemoresistant phenotype. To further increase TGCT curability, better prediction of risk of relapse and early detection of refractory cases is needed. Therefore, to diagnose this malignancy more precisely, stratify patients more accurately and improve decision-making on treatment modality, new biomarkers are still required. Numerous studies showed association of differential expressions of microRNAs (miRNAs) with cancer. Using microarray analysis followed by RT-qPCR validation, we identified specific miRNA expression patterns that discriminate chemoresistant phenotypes in TGCTs. Comparing CDDP-resistant vs. -sensitive TGCT cell lines, we identified miR-218-5p, miR-31-5p, miR-125b-5p, miR-27b-3p, miR-199a-5p, miR-214-3p, let-7a and miR-517a-3p as significantly up-regulated and miR-374b-5p, miR-378a-3p, miR-20b-5p and miR-30e-3p as significantly down-regulated. In patient tumour samples, we observed the highest median values of relative expression of miR-218-5p, miR-31-5p, miR-375-5p and miR-517a-3p, but also miR-20b-5p and miR-378a-3p, in metastatic tumour samples when compared with primary tumour or control samples. In TGCT patient plasma samples, we detected increased expression of miR-218-5p, miR-31-5p, miR-517a-3p and miR-375-5p when compared to healthy individuals. We propose that miR-218-5p, miR-31-5p, miR-375-5p, miR-517-3p, miR-20b-5p and miR-378a-3p represent a new panel of biomarkers for better prediction of chemoresistance and more aggressive phenotypes potentially underlying metastatic spread in non-seminomatous TGCTs. In addition, we provide predictions of the targets and functional and regulatory networks of selected miRNAs.

Mitochondrial Genetic and Epigenetic Regulations in Cancer: Therapeutic Potential.

Mitochondria are dynamic organelles managing crucial processes of cellular metabolism and bioenergetics. Enabling rapid cellular adaptation to altered endogenous and exogenous environments, mitochondria play an important role in many pathophysiological states, including cancer. Being under the control of mitochondrial and nuclear DNA (mtDNA and nDNA), mitochondria adjust their activity and biogenesis to cell demands. In cancer, numerous mutations in mtDNA have been detected, which do not inactivate mitochondrial functions but rather alter energy metabolism to support cancer cell growth. Increasing evidence suggests that mtDNA mutations, mtDNA epigenetics and miRNA regulations dynamically modify signalling pathways in an altered microenvironment, resulting in cancer initiation and progression and aberrant therapy response. In this review, we discuss mitochondria as organelles importantly involved in tumorigenesis and anti-cancer therapy response. Tumour treatment unresponsiveness still represents a serious drawback in current drug therapies. Therefore, studying aspects related to genetic and epigenetic control of mitochondria can open a new field for understanding cancer therapy response. The urgency of finding new therapeutic regimens with better treatment outcomes underlines the targeting of mitochondria as a suitable candidate with new therapeutic potential. Understanding the role of mitochondria and their regulation in cancer development, progression and treatment is essential for the development of new safe and effective mitochondria-based therapeutic regimens.

Response of the Urothelial Carcinoma Cell Lines to Cisplatin.

Cisplatin (CDDP)-based chemotherapy is the standard of care in patients with muscle-invasive bladder cancer. However, in a large number of cases, the disease becomes resistant or does not respond to CDDP, and thus progresses and disseminates. In such cases, prognosis of patients is very poor. CDDP manifests its cytotoxic effects mainly through DNA damage induction. Hence, response to CDDP is mainly dependent on DNA damage repair and tolerance mechanisms. Herein, we have examined CDDP response in a panel of the urothelial carcinoma cell (UCC) lines. We characterized these cell lines with regard to viability after CDDP treatment, as well as kinetics of induction and repair of CDDP-induced DNA damage. We demonstrate that repair of CDDP-induced DNA lesions correlates, at least to some extent, with CDDP sensitivity. Furthermore, we monitored expression of the key genes involved in selected DNA repair and tolerance mechanisms, nucleotide excision repair, homologous recombination and translesion DNA synthesis, and show that it differs in the UCC lines and positively correlates with CDDP resistance. Our data indicate that CDDP response in the UCC lines is dependent on DNA damage repair and tolerance factors, which may, therefore, represent valuable therapeutic targets in this malignancy.

Extract of Acanthopanax senticosus and Its Components Interacting with Sulfide, Cysteine and Glutathione Increase Their Antioxidant Potencies and Inhibit Polysulfide-Induced Cleavage of Plasmid DNA.

Aqueous root extract from Acanthopanax senticosus (ASRE) has a wide range of medicinal effects. The present work was aimed at studying the influence of sulfide, cysteine and glutathione on the antioxidant properties of ASRE and some of its selected phytochemical components. Reduction of the 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide (●cPTIO) stable radical and plasmid DNA (pDNA) cleavage in vitro assays were used to evaluate antioxidant and DNA-damaging properties of ASRE and its individual components. We found that the interaction of ASRE and its two components, caffeic acid and chlorogenic acid (but not protocatechuic acid and eleutheroside B or E), with H2S/HS-, cysteine or glutathione significantly increased the reduction of the ●cPTIO radical. In contrast, the potency of ASRE and its selected components was not affected by Na2S4, oxidized glutathione, cystine or methionine, indicating that the thiol group is a prerequisite for the promotion of the antioxidant effects. ASRE interacting with H2S/HS- or cysteine displayed a bell-shaped effect in the pDNA cleavage assay. However, ASRE and its components inhibited pDNA cleavage induced by polysulfides. In conclusion, we suggest that cysteine, glutathione and H2S/HS- increase antioxidant properties of ASRE and that changes of their concentrations and the thiol/disulfide ratio can influence the resulting biological effects of ASRE.

Are Changes in the Percentage of Specific Leukocyte Subpopulations Associated with Endogenous DNA Damage Levels in Testicular Cancer Patients?

Chemoresistance of germ cell tumors (GCTs) represents an intensively studied property of GCTs that is the result of a complicated multifactorial process. One of the driving factors in this process is the tumor microenvironment (TME). Intensive crosstalk between the DNA damage/DNA repair pathways and the TME has already been reported. This study aimed at evaluating the interplay between the immune TME and endogenous DNA damage levels in GCT patients. A cocultivation system consisting of peripheral blood mononuclear cells (PBMCs) from healthy donors and GCT cell lines was used in an in vitro study. The patient cohort included 74 chemotherapy-naïve GCT patients. Endogenous DNA damage levels were measured by comet assay. Immunophenotyping of leukocyte subpopulations was performed using flow cytometry. Statistical analysis included data assessing immunophenotypes, DNA damage levels and clinicopathological characteristics of enrolled patients. The DNA damage level in PBMCs cocultivated with cisplatin (CDDP)-resistant GCT cell lines was significantly higher than in PBMCs cocultivated with their sensitive counterparts. In GCT patients, endogenous DNA damage levels above the cutoff value were independently associated with increased percentages of natural killer cells, CD16-positive dendritic cells and regulatory T cells. The crosstalk between the endogenous DNA damage level and specific changes in the immune TME reflected in the blood of GCT patients was revealed. The obtained data contribute to a deeper understanding of ongoing interactions in the TME of GCTs.

Increased levels of XPA might be the basis of cisplatin resistance in germ cell tumours.

BACKGROUND: Germ cell tumours (GCTs) represent a highly curable malignity as they respond well to cisplatin (CDDP)-based chemotherapy. Nevertheless, a small proportion of GCT patients relapse or do not respond to therapy. As this might be caused by an increased capacity to repair CDDP-induced DNA damage, identification of DNA repair biomarkers predicting inadequate or aberrant response to CDDP, and thus poor prognosis for GCT patients, poses a challenge. The objective of this study is to examine the expression levels of the key nucleotide excision repair (NER) factors, XPA, ERCC1 and XPF, in GCT patients and cell lines. METHODS: Two hundred seven GCT patients' specimens with sufficient follow-up clinical-pathological data and pairwise combinations of CDDP-resistant and -sensitive GCT cell lines were included. Immunohistochemistry was used to detect the ERCC1, XPF and XPA protein expression levels in GCT patients' specimen and Western blot and qRT-PCR examined the protein and mRNA expression levels in GCT cell lines. RESULTS: GCT patients with low XPA expression had significantly better overall survival than patients with high expression (hazard ratio = 0.38, 95% confidence interval: 0.12-1.23, p = 0.0228). In addition, XPA expression was increased in the non-seminomatous histological subtype, IGCCCG poor prognosis group, increasing S stage, as well as the presence of lung, liver and non-pulmonary visceral metastases. Importantly, a correlation between inadequate or aberrant CDDP response and XPA expression found in GCT patients was also seen in GCT cell lines. CONCLUSIONS: XPA expression is an additional independent prognostic biomarker for stratifying GCT patients, allowing for improvements in decision-making on treatment for those at high risk of refractoriness or relapse. In addition, it could represent a novel therapeutic target in GCTs.

XPA: DNA Repair Protein of Significant Clinical Importance.

The nucleotide excision repair (NER) pathway is activated in response to a broad spectrum of DNA lesions, including bulky lesions induced by platinum-based chemotherapeutic agents. Expression levels of NER factors and resistance to chemotherapy has been examined with some suggestion that NER plays a role in tumour resistance; however, there is a great degree of variability in these studies. Nevertheless, recent clinical studies have suggested Xeroderma Pigmentosum group A (XPA) protein, a key regulator of the NER pathway that is essential for the repair of DNA damage induced by platinum-based chemotherapeutics, as a potential prognostic and predictive biomarker for response to treatment. XPA functions in damage verification step in NER, as well as a molecular scaffold to assemble other NER core factors around the DNA damage site, mediated by protein-protein interactions. In this review, we focus on the interacting partners and mechanisms of regulation of the XPA protein. We summarize clinical oncology data related to this DNA repair factor, particularly its relationship with treatment outcome, and examine the potential of XPA as a target for small molecule inhibitors.

Inorganic Polysulfides and Related Reactive Sulfur­Selenium Species from the Perspective of Chemistry.

Polysulfides (H2Sx) represent a class of reactive sulfur species (RSS) which includes molecules such as H2S2, H2S3, H2S4, and H2S5, and whose presence and impact in biological systems, when compared to other sulfur compounds, has only recently attracted the wider attention of researchers. Studies in this field have revealed a facet-rich chemistry and biological activity associated with such chemically simple, still unusual inorganic molecules. Despite their chemical simplicity, these inorganic species, as reductants and oxidants, metal binders, surfactant-like "cork screws" for membranes, components of perthiol signalling and reservoirs for inorganic hydrogen sulfide (H2S), are at the centre of complicated formation and transformation pathways which affect numerous cellular processes. Starting from their chemistry, the hidden presence and various roles of polysulfides in biology may become more apparent, despite their lack of clear analytical fingerprints and often murky biochemical footprints. Indeed, the biological chemistry of H2Sx follows many unexplored paths and today, the relationship between H2S and its oxidized H2Sx species needs to be clarified as a matter of "unmistaken identity". Simultaneously, emerging species, such as HSSeSH and SenS8-n, also need to be considered in earnest.

Sulfide (Na2S) and Polysulfide (Na2S2) Interacting with Doxycycline Produce/Scavenge Superoxide and Hydroxyl Radicals and Induce/Inhibit DNA Cleavage.

Doxycycline (DOXY) is an antibiotic routinely prescribed in human and veterinary medicine for antibacterial treatment, but it has also numerous side effects that include oxidative stress, inflammation, cancer or hypoxia-induced injury. Endogenously produced hydrogen sulfide (H2S) and polysulfides affect similar biological processes, in which reactive oxygen species (ROS) play a role. Herein, we have studied the interaction of DOXY with H2S (Na2S) or polysulfides (Na2S2, Na2S3 and Na2S4) to gain insights into the biological effects of intermediates/products that they generate. To achieve this, UV-VIS, EPR spectroscopy and plasmid DNA (pDNA) cleavage assay were employed. Na2S or Na2S2 in a mixture with DOXY, depending on ratio, concentration and time, displayed bell-shape kinetics in terms of producing/scavenging superoxide and hydroxyl radicals and decomposing hydrogen peroxide. In contrast, the effects of individual compounds (except for Na2S2) were hardly observable. In addition, DOXY, as well as oxytetracycline and tetracycline, interacting with Na2S or other studied polysulfides reduced the •cPTIO radical. Tetracyclines induced pDNA cleavage in the presence of Na2S. Interestingly, they inhibited pDNA cleavage induced by other polysulfides. In conclusion, sulfide and polysulfides interacting with tetracyclines produce/scavenge free radicals, indicating a consequence for free radical biology under conditions of ROS production and tetracyclines administration.

Polysulfides and products of H2S/S-nitrosoglutathione in comparison to H2S, glutathione and antioxidant Trolox are potent scavengers of superoxide anion radical and produce hydroxyl radical by decomposition of H2O2.

Exogenous and endogenously produced sulfide derivatives, such as H2S/HS-/S2-, polysulfides and products of the H2S/S-nitrosoglutathione interaction (S/GSNO), affect numerous biological processes in which superoxide anion (O2-) and hydroxyl (OH) radicals play an important role. Their cytoprotective-antioxidant and contrasting pro-oxidant-toxic effects have been reported. Therefore, the aim of our work was to contribute to resolving this apparent inconsistency by studying sulfide derivatives/free radical interactions and their consequent biological effects compared to the antioxidants glutathione (GSH) and Trolox. Using the electron paramagnetic resonance (EPR) spin trapping technique and O2-, we found that a polysulfide (Na2S4) and S/GSNO were potent scavengers of O2- and cPTIO radicals compared to H2S (Na2S), GSH and Trolox, and S/GSNO scavenged the DEPMPO-OH radical. As detected by the EPR spectra of DEPMPO-OH, the formation of OH in physiological solution by S/GSNO was suggested. All the studied sulfide derivatives, but not Trolox or GSH, had a bell-shaped potency to decompose H2O2 and produced OH in the following order: S/GSNO > Na2S4 ≥ Na2S > GSH = Trolox = 0, but they scavenged OH at higher concentrations. In studies of the biological consequences of these sulfide derivatives/H2O2 properties, we found the following: (i) S/GSNO alone and all sulfide derivatives in the presence of H2O2 cleaved plasmid DNA; (ii) S/GSNO interfered with viral replication and consequently decreased the infectivity of viruses; (iii) the sulfide derivatives induced apoptosis in A2780 cells but inhibited apoptosis induced by H2O2; and (iv) Na2S4 modulated intracellular calcium in A87MG cells, which depended on the order of Na2S4/H2O2 application. We suggest that the apparent inconsistency of the cytoprotective-antioxidant and contrasting pro-oxidant-toxic biological effects of sulfide derivatives results from their time- and concentration-dependent radical production/scavenging properties and their interactions with O2-, OH and H2O2. The results imply a direct involvement of sulfide derivatives in O2- and H2O2/OH free radical pathways modulating antioxidant/toxic biological processes.

Resveratrol-Inspired Benzo[b]selenophenes Act as Anti-Oxidants in Yeast.

Resveratrol is a natural (poly)phenol primarily found in plants protecting them against pathogens, as well as harmful effects of physical and chemical agents. In higher eukaryotic cells and organisms, this compound displays a remarkable range of biological activities, such as anti-oxidant, anti-inflammatory, anti-cancer, anti-aging, cardio- and neuro-protective properties. Here, biological activities of synthetic selenium-containing derivatives of resveratrol-benzo[b]selenophenes-have been studied in lower eukaryotes Saccharomyces cerevisiae. Their toxicity, as well as DNA damaging and reactive oxygen species (ROS) inducing potencies, manifested through their ability to act as redox active anti-microbial agents, have been examined. We show that some benzo[b]selenophenes can kill yeast cells and that the killing effects are not mediated by DNA damage types that can be detected as DNA double-strand breaks. These benzo[b]selenophenes could potentially be used as anti-fungal agents, although their concentrations relevant to application in humans need to be further evaluated. In addition, most of the studied benzo[b]selenophenes display redox-modulating/anti-oxidant activity (comparable or even higher than that of resveratrol or Trolox) causing a decrease in the intracellular ROS levels in yeast cells. Therefore, after careful re-evaluation in other biological systems these observations might be transferred to humans, where resveratrol-inspired benzo[b]selenophenes could be used as supra-anti-oxidant supplements.

Regulation of non-homologous end joining via post-translational modifications of components of the ligation step.

DNA double-strand breaks are the most serious type of DNA damage and non-homologous end joining (NHEJ) is an important pathway for their repair. In Saccharomyces cerevisiae, three complexes mediate the canonical NHEJ pathway, Ku (Ku70/Ku80), MRX (Mre11/Rad50/Xrs2) and DNA ligase IV (Dnl4/Lif1). Mammalian NHEJ is more complex, primarily as a consequence of the fact that more factors are involved in the process, and also because higher chromatin organization and more complex regulatory networks exist in mammals. In addition, a stronger interconnection between the NHEJ and DNA damage response (DDR) pathways seems to occur in mammals compared to yeast. DDR employs multiple post-translational modifications (PTMs) of the target proteins and mutual crosstalk among them to ensure highly efficient down-stream effects. Checkpoint-mediated phosphorylation is the best understood PTM that regulates DDR, although recently SUMOylation has also been shown to be involved. Both phosphorylation and SUMOylation affect components of NHEJ. In this review, we discuss a role of these two PTMs in regulation of NHEJ via targeting the components of the ligation step.

Lif1 SUMOylation and its role in non-homologous end-joining.

Non-homologous end-joining (NHEJ) repairs DNA double-strand breaks by tethering and ligating the two DNA ends. The mechanisms regulating NHEJ efficiency and interplay between its components are not fully understood. Here, we identify and characterize the SUMOylation of budding yeast Lif1 protein, which is required for the ligation step in NHEJ. We show that Lif1 SUMOylation occurs throughout the cell cycle and requires the Siz SUMO ligases. Single-strand DNA, but not double-strand DNA or the Lif1 binding partner Nej1, is inhibitory to Lif1 SUMOylation. We identify lysine 301 as the major conjugation site and demonstrate that its replacement with arginine completely abolishes Lif1 SUMOylation in vivo and in vitro. The lif1-K301R mutant cells exhibit increased levels of NHEJ repair compared with wild-type cells throughout the cell cycle. This is likely due to the inhibitory effect of Lif1 SUMOylation on both its self-association and newly observed single-strand DNA binding activity. Taken together, these findings suggest that SUMOylation of Lif1 represents a new regulatory mechanism that downregulates NHEJ in a cell cycle phase-independent manner.

Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast.

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

Intracellular diagnostics: hunting for the mode of action of redox-modulating selenium compounds in selected model systems.

Redox-modulating compounds derived from natural sources, such as redox active secondary metabolites, are currently of considerable interest in the field of chemoprevention, drug and phytoprotectant development. Unfortunately, the exact and occasionally even selective activity of such products, and the underlying (bio-)chemical causes thereof, are often only poorly understood. A combination of the nematode- and yeast-based assays provides a powerful platform to investigate a possible biological activity of a new compound and also to explore the "redox link" which may exist between its activity on the one side and its chemistry on the other. Here, we will demonstrate the usefulness of this platform for screening several selenium and tellurium compounds for their activity and action. We will also show how the nematode-based assay can be used to obtain information on compound uptake and distribution inside a multicellular organism, whilst the yeast-based system can be employed to explore possible intracellular mechanisms via chemogenetic screening and intracellular diagnostics. Whilst none of these simple and easy-to-use assays can ultimately substitute for in-depth studies in human cells and animals, these methods nonetheless provide a first glimpse on the possible biological activities of new compounds and offer direction for more complicated future investigations. They may also uncover some rather unpleasant biochemical actions of certain compounds, such as the ability of the trace element supplement selenite to induce DNA strand breaks.

Products of Selenite/Thiols Interaction Have Reducing Properties, Cleave Plasmid DNA and Decrease Rat Blood Pressure and Tension of Rat Mesenteric Artery.

Selenium compounds exert their antioxidant activity mostly when the selenium atom is incorporated into selenoproteins. In our work, we tested the possibility that selenite itself interacts with thiols to form active species that have reducing properties. Therefore, we studied the reduction of 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide radical (•cPTIO), damage of plasmid DNA (pDNA), modulation of rat hemodynamic parameters and tension of isolated arteries induced by products of interaction of selenite with thiols. We found that the products of selenite interaction with thiols had significant reducing properties that could be attributed mainly to the selenide and that selenite had catalytic properties in the access of thiols. The potency of thiols to reduce •cPTIO in the interaction with selenite was cysteine > homocysteine > glutathione reduced > N-acetylcysteine. Thiol/selenite products cleaved pDNA, with superoxide dismutase enhancing these effects suggesting a positive involvement of superoxide anion in the process. The observed •cPTIO reduction and pDNA cleavage were significantly lower when selenomethionine was used instead of selenite. The products of glutathione/selenite interaction affected several hemodynamic parameters including rat blood pressure decrease. Notably, the products relaxed isolated mesenteric artery, which may explain the observed decrease in rat blood pressure. In conclusion, we found that the thiol/selenite interaction products exhibited significant reducing properties which can be used in further studies of the treatment of pathological conditions caused by oxidative stress. The results of decreased rat blood pressure and the tension of mesenteric artery may be perspective in studies focused on cardiovascular disease and their prevention.

DNA damage levels in peripheral blood mononuclear cells before and after first cycle of chemotherapy have comparable prognostic values in germ cell tumor patients.

Background: Germ cell tumors (GCTs) represent the most frequent solid malignancy in young men. This malignancy is highly curable by cisplatin (CDDP)-based chemotherapy. However, there is a proportion of patients having a poor prognosis due to refractory disease or its relapse. No reliable biomarkers being able to timely and accurately stratify poor prognosis GCT patients are currently available. Previously, we have shown that chemotherapy-naïve GCT patients with higher DNA damage levels in peripheral blood mononuclear cells (PBMCs) have significantly worse prognosis compared to patients with lower DNA damage levels. Methods: DNA damage levels in PBMCs of both chemotherapy-naïve and first cycle chemotherapy-treated GCT patients have been assessed by standard alkaline comet assay and its styrene oxide (SO)-modified version. These levels were correlated with clinico-pathological characteristics. Results: We re-confirm prognostic value of DNA damage level in chemotherapy-naïve GCT patients and reveal that this prognosticator is equally effective in GCT patients after first cycle of CDDP-based chemotherapy. Furthermore, we demonstrate that SO-modified comet assay is comparably sensitive as standard alkaline comet assay in case of patients who underwent first cycle of CDDP-based chemotherapy, although it appears more suitable to detect DNA cross-links. Conclusion: We propose that DNA damage levels in PBMCs before and after first cycle of CCDP-based chemotherapy are comparable independent prognosticators for progression-free and overall survivals in GCT patients. Therefore, their clinical use is highly advised to stratify GCT patients to identify those who are most at risk of developing disease recurrence or relapse, allowing tailoring therapeutic interventions to poor prognosis individuals, and optimizing their care management and treatment regimen.

Selenium toxicity toward yeast as assessed by microarray analysis and deletion mutant library screen: a role for DNA repair.

Selenium (Se) is a trace element that is essential for human health as it takes part in many cellular processes. The cellular response to this compound elicits very diverse processes including DNA damage response and repair. Because an inorganic form of Se, sodium selenite (SeL), has often been a part of numerous studies and because this form of Se is used as a dietary supplement by the public, here, we elucidated mechanisms of SeL-induced toxicity in yeast Saccharomyces cerevisiae using a combination of systematic genetic and transcriptome analysis. First, we screened the yeast haploid deletion mutant library for growth in the presence of this Se compound. We identified 39 highly SeL sensitive mutants. The corresponding deleted genes encoded mostly proteins involved in DNA damage response and repair, vacuole function, glutathione (GSH) metabolism, transcription, and chromatin metabolism. DNA damage response and repair mutants were examined in more detail: a synergistic interaction between postreplication (PRR) and homologous recombination (HRR) repair pathways was revealed. In addition, the effect of combined defects in HRR and GSH metabolism was analyzed, and again, the synergistic interaction was found. Second, microarray analysis was used to reveal expression profile changes after SeL exposure. The gene process categories "amino acid metabolism" and "generation of precursor metabolites and energy" comprised the greatest number of induced and repressed genes, respectively. We propose that SeL-induced toxicity markedly results from DNA injury, thereby highlighting the importance of DNA damage response and repair pathways in protecting cells against toxic effects of this Se compound. In addition, we suggest that SeL toxicity also originates from damage to cellular proteins, including those acting in DNA damage response and repair.

Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism.

Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair.

Detection of Specific Immune Cell Subpopulation Changes Associated with Systemic Immune Inflammation-Index Level in Germ Cell Tumors.

The tumor microenvironment (TME) and the host inflammatory response are closely interconnected. The interplay between systemic inflammation and the local immune response may influence tumor development and progression in various types of cancer. The systemic immune-inflammation index (SII) represents a prognostic marker for germ cell tumors (GCTs). The aim of the present study was to detect specific immune cell subpopulation changes which were associated with the SII level in chemotherapy-naïve GCT patients. In total, 51 GCT patients, prior to cisplatin-based chemotherapy, were included in the present study. Immunophenotyping of peripheral blood leukocyte subpopulations was performed using flow cytometry. The SII level was correlated with the percentage of various leukocyte subpopulations. The obtained results demonstrated that SII levels above the cut-off value of SII ≥ 1003 were associated with higher neutrophil percentages. An inverse correlation was found between the SII and the peripheral lymphocyte percentage that logically reflects the calculations of the SII index. Furthermore, the presented data also showed that in the lymphocyte subpopulation, the association with the SII was driven by T-cell subpopulations. In innate immunity-cell subpopulations, we observed a correlation between SII level and neutrophils as well as associations with eosinophil, basophil, natural killer cell and dendritic cell percentages. We suppose that the described interactions represent a manifestation of cancer-induced immune suppression. The results of the present study contribute to the elucidation of the interrelationship between tumor cells and the innate/adaptive immune system of the host.