TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation.

Background/aim: Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Materials and methods: Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. Results: In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. Conclusion: TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a "noncanonical" pathway, that activates ERK and Akt.

Molecular prevalence of Pneumocystis jirovecii and Cryptosporidium in patients with asthma.

Asthma is characterized by chronic airway inflammation. In addition to allergens, microorganisms can affect the clinical course of asthma. It has been shown that some fungi play an important role in the progression of asthma. However, the effects of Pneumocystis jirovecii and Cryptosporidium spp., on the disease are little known. We investigated P. jirovecii and Cryptosporidium spp. in the sputum and stool sample of patients with asthma (n = 40) by microscopy and PCR compared to the healthy group (n = 40). P. jirovecii (12.5 %), and Cryptosporidium spp. (12.5 %) were detected in the sputum samples of only asthmatic patients (p = 0.029 and 0.029 respectively). However, Crpytosporidium spp. was detected equally in stool samples of both groups (p = 0.682). Our results indicate that P. jirovecii and Cryptosporidium spp. should be considered in patients with asthma and molecular screening of these neglected eukaryotes in respiratory tract samples may be beneficial in the clinical management of the disease.

Salivary Lipids of Patients with Non-Small Cell Lung Cancer Show Perturbation with Respect to Plasma.

A comprehensive lipid profile was analyzed in patients with non-small cell lung cancer (NSCLC) using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. This study investigated 297 and 202 lipids in saliva and plasma samples, respectively, comparing NSCLC patients to healthy controls. Lipids with significant changes (>2-fold, p < 0.05) were further analyzed in each sample type. Both saliva and plasma exhibited similar lipid alteration patterns in NSCLC, but saliva showed more pronounced changes. Total triglycerides (TGs) increased (>2-3-fold) in plasma and saliva samples. Three specific TGs (50:2, 52:5, and 54:6) were significantly increased in NSCLC for both sample types. A common ceramide species (d18:1/24:0) and phosphatidylinositol 38:4 decreased in both plasma and saliva by approximately two-fold. Phosphatidylserine 36:1 was selectively detected in saliva and showed a subsequent decrease, making it a potential biomarker for predicting lung cancer. We identified 27 salivary and 10 plasma lipids as candidate markers for NSCLC through statistical evaluations. Moreover, this study highlights the potential of saliva in understanding changes in lipid metabolism associated with NSCLC.

Profiling of circRNA expressions in radiation-treated head and neck cancer cells and the potential role of circPVT1.

OBJECTIVE: Radiotherapy is an indispensable treatment modality for head and neck cancers (HNCs). Due to their stable structure, circular RNAs (circRNA) have been implicated as potential biomarkers for clinical use in cancers. The purpose of this study was profiling the circRNA in radiation-treated head and neck cancer cells to identify potential differentially expressed circRNAs. DESIGN: The effects of radiation on the expression level of circRNAs were investigated in HNCs cells, compared to healthy cell lines. To predict the potential role of circRNAs in HNC patients, tissue expression levels, survival analyses of circRNAs, and circRNA-miRNA network were evaluated using TCGA/CPTAC datasets. Based on expression level in irradiated cells, circPVT1 (plasmacytoma variant translocation 1) was further investigated by sequence analysis. RESULTS: The study revealed the characterization of differentially expressed circRNAs in cancer cells and that irradiation made significant changes in the expression of circRNAs. These findings suggest that certain circRNAs, especially circPVT1, may be potential biomarkers to monitor radiotherapy effects in patients with HNCs. CONCLUSIONS: CircRNAs may be promising molecules for improving and understanding radiotherapy efficacy in HNCs.

Detection of Pneumocystis jirovecii by PCR in patients with lung cancer: A preliminary study.

INTRODUCTION: Infection complications in lung cancer (LC), one of the most common cancers in the world, are still among the most important causes of death. Of them, P. jirovecii, which is as an opportunistic infection, causes a life-threatening type of pneumonia in cancer patients. This preliminary study aimed to determine the incidence and clinical status of P. jirovecii by PCR in lung cancer patients compared to the conventional method. MATERIAL AND METHODS: Sixty-nine lung cancer patients and fSorty healthy individuals were included in the study. After sociodemographical and clinical features were recorded, sputum samples were collected from attenders. Firstly, microscopic examination was made with Gomori's methenamine silver stain and then PCR was performed. RESULTS: P. jirovecii was detected in three of 69 lung cancer patients by PCR (4.3%), but not by microscopy. However, healthy individuals were negative for P. jirovecii by both methods. Based on clinical and radiological findings, P. jirovecii was evaluated as probable infection in one patient and colonization in the other two patients. Although PCR is more sensitive than conventional staining methods, it cannot distinguish probable and proven infections from pulmonary colonization. DISCUSSION: It is important to evaluate the decision of infection together with laboratory, clinical and radiological findings. Moreover, PCR may enable to know the colonization and to take precautions such as prophylaxis, due to the risk of colonization turning into an infection in immunocompromised patient groups. Further studies involving larger populations and evaluating the colonization-infection relationship in patients with solid tumors are needed.

Molecular Prevalence of Microsporidia Infection in Patients with Lung Cancer.

Infections are still among the most important causes of morbidity and mortality in patients with lung cancer, which has the highest rate of cancer-related deaths in the world. Microsporidia, which are opportunistic parasitic fungi, primarily localize to the intestine by ingestion but can disseminate to the respiratory tract or can be acquired by spore inhalation. Cancer patients are at higher risk for microsporidia, a life-threatening infection, than the normal population is. We aimed to characterize the prevalence of microsporidia infection for the first time by evaluating the intestinal and respiratory tracts of patients with lung cancer. In this study, we investigated 98 patients with lung cancer and 103 healthy individuals for microsporidia infection and evaluated the clinical findings of patients who were found to be positive. Sputum and stool samples were tested by microscopic examination, in addition to pan-microsporidia and genus-specific polymerase chain reactions. Nine patients with lung cancer had positive results for microsporidia (9.2%), which was significantly higher than the rate in healthy individuals (P = 0.008), and most of them had clinical findings. Among these positive patients, polymerase chain reaction revealed microsporidia in the sputum samples of seven patients, the stool sample of one patient, and both the sputum and stool samples of one patient. Encephalitozoon cuniculi was identified as the predominant pathogen in 87.5% (7/8) of positive sputum samples. Microsporidia infection was significantly associated with advanced stages of cancer. However, in the control group, Encephalitozoon intestinalis was detected in the stool sample of an individual without clinical symptoms. Microsporidia, especially E. cuniculi, should be considered as a cause of respiratory tract infection as well as intestinal infection in cancer patients and should be screened in respiratory samples of these patients when they have pulmonary symptoms.

The Effect of BTK Inhibitor Ibrutinib on Leishmania infantum Infection In Vitro.

PURPOSE: Leishmaniasis is a neglected infectious disease affecting millions of people worldwide. Visceral leishmaniasis (VL), caused by Leishmania infantum and Leishmania donovani, is one of the main clinical forms of the disease and fatal if not treated promptly and properly. Despite being available for the last 70 years, current drugs used in the treatment of leishmaniasis have serious problems as they have high toxicity, require long-term administration and cause serious side-effects, leading to the emergence of resistant and relapse cases. Therefore, there is an urgent need for the discovery of novel antileishmanial molecules and the development of new treatment regimens. The drug used for chemotherapy of B-cell malignancies, Ibrutinib, an inhibitor of Bruton's Tyrosine Kinase (BTK), can offer a new therapeutic perspective due to the functions of BTK on intracellular signaling mechanism of macrophages, which are the primary resident cell for Leishmania. Hence, the study aimed to evaluate ibrutinib as a potential anti-Leishmanial drug. METHOD: In this study, we evaluated the antileishmanial effect of Ibrutinib by in vitro L. infantum infection model using macrophages, with cell viability assay, parasite rescue assay, real-time qPCR. RESULTS: We showed that Ibrutinib was significantly more effective than the Glucantime against L. infantum. In addition, our data revealed that Ibrutinib inhibited parasite growth and load without impairing macrophage viability. CONCLUSIONS: Consequently, due to its efficacy and safety, Ibrutinib may be a promising candidate for the treatment of VL caused by L. infantum as a host-targeted drug.

A Pilot Study on the Evaluation of Cryptosporidium Infection in Patients with Lung Cancer: Respiratory Cryptosporidiosis.

Lung carcinoma is one of the most common cancers and the leading cause of cancer-related death worldwide. Increasing evidence has shown that Cryptosporidium spp., an opportunistic parasite, is associated with cancers, causing life-threatening infections. The most common clinical form of Cryptosporidium is intestinal infections. However, respiratory cryptosporidiosis has rarely been documented, although the parasite infects respiratory epithelial cells and gastrointestinal (GIS) epithelial cells. To evaluate respiratory cryptosporidiosis in patients with lung cancer, we investigated Cryptosporidium spp. in patients with lung cancer (n = 69) in comparison with healthy groups (n = 40). Sputum and stool samples were examined microscopically and by polymerase chain reaction (PCR). Two cancer patients were diagnosed with respiratory cryptosporidiosis (2.9%), on PCR examination of the sputum samples. Cryptosporidium spp. was detected in the stool samples of one patient (1.5%) and 2 healthy individuals (5.4%) by PCR and microscopy. First, respiratory cryptosporidiosis was documented in 2 patients with lung cancer. Cryptosporidium is an important agent of the respiratory tract and GIS infections in cancer patients. These new findings highlight the molecular prevalence of Cryptosporidium spp., an opportunistic infection, in patients with lung cancer. Respiratory cryptosporidiosis should also be considered when patients have respiratory symptoms.

Blood Meal Analysis and Molecular Detection of Leishmania DNA in Wild-Caught Sand Flies in Leishmaniasis Endemic Areas of Turkey and Northern Cyprus.

INTRODUCTION: Phlebotomine sand flies (Diptera: Psychodidae) are known as the vector of diseases such as leishmaniasis, bartonellosis and viral diseases. The aim of this study is to detect the host feeding pattern of sand flies in the endemic areas for leishmaniasis in Turkey (Antalya, Kayseri) and Northern Cyprus (TRNC) as well as the presence of Leishmania DNA in the specimens. METHODS: One-hundred seventy-six blood-fed sand fly specimens were examined for blood meal analysis. A SYBR Green-PCR assay was performed with specific forward primers for each host and a universal reverse primer. Primers of human and goat were used together in multiplex PCR while goat and cow were studied separately. ITS-1 qPCR assay was also performed on both blood-fed and non-blood-fed females to detect Leishmania parasites. RESULTS: Blood sources could be detected in 69 out of 176 blood-fed sand fly specimens. The results of blood meal analysis showed that specimens were fed mostly on cows (22.2%) followed by humans (5.7%), goats (2.8%) and dogs (0.6%). Multiple feeding patterns were also detected as human + cow (3.4%), cow + goat (2.8%) and human + goat (1.7%). Five of the blood-fed specimens were Leishmania spp. positive: P. major s.l. (n = 1), P. tobbi (n = 2) were L. tropica positive from Antalya, P. simici was positive for L. infantum from Kayseri and P. papatasi (n = 1) was positive for L. major from Cyprus. Leishmania infection rates were determined as 3.79%, 1.69% and 2.63% among the blood-fed sand fly specimens in Antalya, Kayseri and TRNC, respectively. CONCLUSION: The SYBR-Green-based multiplex PCR assay is a cost-effective and promising tool for blood meal identification of wild-caught sand flies as well as other blood-sucking arthropods. Feeding patterns of important vector species detected in the present study show the high risk in these endemic areas. As a next step, to identify the blood source in a shorter time and to make the test more sensitive, development of this assay to probe-based and multiplex PCR will be also planned.

Identification of gene expression profiles in Leishmania major infection by integrated bioinformatics analyses.

Gene expression profiling in mouse models of leishmaniasis has given useful information to understand the molecular pathways active in lesions and to discover new diagnostic/therapeutic targets. Although the host response plays a critical role in protection from leishmaniasis and promoting disease severity, there are still unexplained aspects in the mechanism of non-healing cutaneous lesions, which need biomarkers for both targeted- therapy and diagnosis. To address this, transcriptional profiling of the skin lesions obtained from BALB/c mice infected with Leishmania major and healthy skin from naïve mice were evaluated by bioinformatics analysis, and then the results were validated by Revers Transcriptase-PCR. Five genes among the up-regulated differentially expressed genes named FCGR4, CCL4, CXCL9, Arg1 and IL-1ß were found to have relatively high diagnostic value for CL due to L. major. Pathway analysis revealed that Triggering Receptor Expressed On Myeloid Cells 1 (TREM1) signaling pathways are active in cutaneous lesions, providing new insights for the understanding and treatment of leishmaniasis.

Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1.

Tumor necrosis factor alpha (TNF-α) plays a paramount role in homeostasis by inducing tumor cytotoxicity and activating immune system. The signaling complexes formed by TNFR1 to activate JNK, p38, and nuclear factor-kappa B pathways and to subsequently induce apoptosis and necroptosis are well known. However, this "canonical TNF-α signaling" does not explain how ERK, AKT, and STAT3 can be activated by TNF-α. In addition, little to nothing is known about negative regulation of TNFR1 signaling. Because cyclic AMP-activated kinase (PKA) shows anti-TNF and anti-inflammatory activities, we postulated that PKA might affect TNF-α signaling by directly phosphorylating TNFR1. In line with this, we identified 2 putative PKA-phosphorylation motifs RRRT411 and REAT417 within the death domain of TNFR1, and investigated whether "canonical" and "noncanonical" TNFR1 signaling is regulated by modifications of T411 and T417. In this study, we demonstrate for the first time that PKA directly binds to and phosphorylates TNFR1 after TNF-α stimulation. To further support our hypothesis, we generated alanine and phosphomimetic (aspartic acid) mutants of TNFR1 at positions T411 and T417, ectopically expressed these mutants, and determined their influence on TNF-α-induced activations of ERKs, AKT, STAT3, p38α, and JNK1/2. Our results clearly showed that phosphomimetic mutants significantly suppressed and alanine mutants augmented TNF-α-induced phosphorylations of ERKs, AKT, Stat3, p38α, and JNKs. These findings strongly suggest that PKA-mediated phosphorylation of T411 and T417 of TNFR1 interferes with both "canonical" and "noncanonical" TNF-α signaling. [Figure: see text].

TRAIL Induces Nuclear Translocation and Chromatin Localization of TRAIL Death Receptors.

Binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to the plasma membrane TRAIL-R1/-R2 selectively kills tumor cells. This discovery led to evaluation of TRAIL-R1/-R2 as targets for anti-cancer therapy, yet the corresponding clinical trials were disappointing. Meanwhile, it emerged that many cancer cells are TRAIL-resistant and that TRAIL-R1/-R2-triggering may lead to tumor-promoting effects. Intriguingly, recent studies uncovered specific functions of long ignored intracellular TRAIL-R1/-R2, with tumor-promoting functions of nuclear (n)TRAIL-R2 as the regulator of let-7-maturation. As nuclear trafficking of TRAIL-Rs is not well understood, we addressed this issue in our present study. Cell surface biotinylation and tracking of biotinylated proteins in intracellular compartments revealed that nTRAIL-Rs originate from the plasma membrane. Nuclear TRAIL-Rs-trafficking is a fast process, requiring clathrin-dependent endocytosis and it is TRAIL-dependent. Immunoprecipitation and immunofluorescence approaches revealed an interaction of nTRAIL-R2 with the nucleo-cytoplasmic shuttle protein Exportin-1/CRM-1. Mutation of a putative nuclear export sequence (NES) in TRAIL-R2 or the inhibition of CRM-1 by Leptomycin-B resulted in the nuclear accumulation of TRAIL-R2. In addition, TRAIL-R1 and TRAIL-R2 constitutively localize to chromatin, which is strongly enhanced by TRAIL-treatment. Our data highlight the novel role for surface-activated TRAIL-Rs by direct trafficking and signaling into the nucleus, a previously unknown signaling principle for cell surface receptors that belong to the TNF-superfamily.

Impact of p53 status on TRAIL-mediated apoptotic and non-apoptotic signaling in cancer cells.

Due to their ability to preferentially induce cell death in tumor cells, while sparing healthy cells, TNF-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL-R1 or anti-TRAIL-R2-specific antibodies are under clinical investigations for cancer-treatment. However, TRAIL-Rs may also induce signaling pathways, which result in malignant progression. TRAIL receptors are transcriptionally upregulated via wild-type p53 following radio- or chemotherapy. Nevertheless, the impact of p53 status on the expression and signaling of TRAIL-Rs is not fully understood. Therefore, we analyzed side by side apoptotic and non-apoptotic signaling induced by TRAIL or the agonistic TRAIL-R-specific antibodies Mapatumumab (anti-TRAIL-R1) and Lexatumumab (anti-TRAIL-R2) in the two isogenic colon carcinoma cell lines HCT116 p53+/+ and p53-/-. We found that HCT116 p53+/+ cells were significantly more sensitive to TRAIL-R-triggering than p53-/- cells. Similarly, A549 lung cancer cells expressing wild-type p53 were more sensitive to TRAIL-R-mediated cell death than their derivatives with knockdown of p53. Our data demonstrate that the contribution of p53 in regulating TRAIL-R-induced apoptosis does not correlate to the levels of TRAIL-Rs at the plasma membrane, but rather to p53-mediated upregulation of Bax, favouring the mitochondrial amplification loop. Consistently, stronger caspase-9 and caspase-3 activation as well as PARP-cleavage was observed following TRAIL-R-triggering in HCT116 p53+/+ compared to HCT116 p53-/- cells. Interestingly, HCT116 p53+/+ cells showed also a more potent activation of non-canonical TRAIL-R-induced signal transduction pathways like JNK, p38 and ERK1/ERK2 than p53-/- cells. Likewise, these cells induced IL-8 expression in response to TRAIL, Mapatumumab or Lexatumumab significantly stronger than p53-/- cells. We obtained similar results in A549 cells with or without p53-knockdown and in the two isogenic colon cancer cell lines RKO p53+/+ and p53-/-. In both cellular systems, we could clearly demonstrate the potentiating effects of p53 on TRAIL-R-mediated IL-8 induction. In conclusion, we found that wild-type p53 increases TRAIL-R-mediated apoptosis but simultaneously augments non-apoptotic signaling.

Intracellular localization of DR5 and related regulatory pathways as a mechanism of resistance to TRAIL in cancer.

TNF-related apoptosis-inducing ligand (TRAIL) is a prominent cytokine capable of inducing apoptosis. It can bind to five different cognate receptors, through which diverse intracellular pathways can be activated. TRAIL's ability to preferentially kill transformed cells makes it a promising potential weapon for targeted tumor therapy. However, recognition of several resistance mechanisms to TRAIL-induced apoptosis has indicated that a thorough understanding of the details of TRAIL biology is still essential before this weapon can be confidently unleashed. Critical to this aim is revealing the functions and regulation mechanisms of TRAIL's potent death receptor DR5. Although expression and signaling mechanisms of DR5 have been extensively studied, other aspects, such as its subcellular localization, non-signaling functions, and regulation of its membrane transport, have only recently attracted attention. Here, we discuss different aspects of TRAIL/DR5 biology, with a particular emphasis on the factors that seem to influence the cell surface expression pattern of DR5, along with factors that lead to its nuclear localization. Disturbance of this balance apparently affects the sensitivity of cancer cells to TRAIL-mediated apoptosis, thus constituting an eligible target for potential new therapeutic agents.

Differential expression of long non-coding RNAs during genotoxic stress-induced apoptosis in HeLa and MCF-7 cells.

Long non-coding RNAs (lncRNAs) are emerging as new players in cancer as they are implicated in diverse biological processes and aberrantly expressed in a variety of human cancers. No data are available on their function under genotoxic stress-induced apoptosis. In this work, we assessed the behavior of some candidate lncRNAs (HOTAIR, MALAT1, TUG1, lincRNA-p21, GAS5, MEG3, PANDA, UCA1, ANRIL, and CCND1) during DNA damage-induced cell death in HeLa and caspase-3-deficient MCF-7 cells using bleomycin (BLM) and γ-radiation to induce DNA damage. Cells were incubated in the presence of BLM for 24 h or irradiated. Apoptosis was analyzed by measurement of oligonucleosomal fragmentation of nuclear DNA. Our results reveal that basal RNA expression levels as well as the changes in the lncRNA expression rates during genotoxic stress-induced apoptosis were cell-type and/or DNA-damaging agent-specific. Generally, we found that some of the RNA molecules (HOTAIR and MALAT1) are down-regulated while many of them (lincRNA-p21, GAS5, MEG3, ANRIL, and ncRNA-CCND1) are up-regulated and some others (TUG1, UCA1, and PANDA) not affected. The decline in the expression of HOTAIR (approx. twofold, p < 0.01) and MALAT1 (approx 1.6-fold, p < 0.01) was clearly evident in BLM-treated HeLa and MCF cells (only HOTAIR, fivefold, p < 0.01). For lincRNA-p21, ncRNA-CCND1, and MEG3, a similar up-regulation pattern was obvious in both cell lines where the increase was generally more pronounced in BLM-treated cells. Interestingly, the induction of ANRIL and GAS5 was mainly restricted to irradiated cells. In conclusion, our findings reveal a differential regulation of individual lncRNAs during genotoxic stress-induced apoptosis.

Promoter and histone methylation and p16(INK4A) gene expression in colon cancer.

The inactivation of the cyclin-dependent kinase inhibitor p16(INK4A) gene by hypermethylation is observed in numerous types of cancer. New findings indicate that DNA and histone methylation act in concert in gene silencing. In this study, we investigated the methylation status of the p16(INK4A) gene promoter and the histone 3 lysine 9 residue in the tumors and matched normal tissue samples from patients with colorectal cancer and analyzed their association with gene expression. The methylation and expression of the p16(INK4A) gene were analyzed by real-time PCR, and histone methylation was analyzed by chromatin immunoprecipitation followed by real-time PCR. p16(INK4A) expression was significantly higher in the tumors compared to normal tissue. Mono-, di- and trimethylation levels of the H3K9 residue were similar in the tumor and normal tissue samples. We did not observe any significant correlation between p16(INK4A) methylation or expression and clinical parameters. Our results suggest that epigenetic modifications of the p16(INK4A) gene and histone lysine methylation do not play a major role in colon carcinogenesis.

Induction of p53-inducible microRNA miR-34 by gamma radiation and bleomycin are different.

microRNAs (miRNAs) are small molecules in their mature form and master regulators of gene expression. Recent work has shown that miRNAs are involved in the p53 network. Of the various miRNAs, miR-34 is regulated by the p53 protein. miR-34 can be induced by ionizing radiation (IR) in vitro and in vivo. However, there is no data in the literature for induction of miR-34 by a chemical agent inducing DNA damage. Here we studied the expression of miR-34 in HeLa and MCF-7 cells exposed to genotoxic stress-induced by bleomycin (BLM) or γ-radiation. We first analyzed p53 accumulation upon DNA damage induction. The basal level of p53 in MCF-7 cells was higher (approx. 6-fold) than in HeLa cells, and its accumulation was similar for both DNA-damaging agents in both cell lines. We have shown that miR-34 is significantly induced by γ-radiation in HeLa cells, but not in MCF-7 cells. BLM did not significantly affect miR-34 expression in both cell types. In conclusion, our findings reveal that miR-34 induction by genotoxic stress may be cell-type specific.

Correlation of histone methyl marks with circulating nucleosomes in blood plasma of cancer patients.

Circulating DNA is present in plasma/serum, mainly complexed with histones as nucleosomes. The detection of circulating nucleosomes (cNUCs) in the peripheral blood may be a diagnostic modality for cancer-associated changes of modified histone tails in blood circulation. In the present study, we investigated the correlation between the trimethylation of H3 lysine 9 (H3K9me3) and H4 lysine 20 (H4K20me3), which are hallmarks of pericentric heterochromatin, and cNUCs in healthy subjects and patients with colorectal cancer (CRC) and multiple myeloma (MM). The plasma concentration of cNUCs was measured using the Cell-Death Detection ELISA kit. Histone methylation marks were detected using chromatin immunoprecipitation (ChIP), followed by quantitative PCR with pericentric satellite 2 as the target sequence. The results showed a high variation in the concentrations of cNUCs, with healthy subjects exhibiting the lowest levels (median 0.194), the CRC patients intermediate (median 0.25) and the MM patients the highest levels (median 0.648). However, the differences between the groups did not reach statistical significance (p>0.05). Analysis using the Pearson's correlation test revealed a significant positive correlation between the concentration of cNUCs and H3K9me3 and H4K20me3 in the whole study group (N=57, p<0.001 for both histone marks). A study of the correlation between cNUCs and histone marks in the individual study groups demonstrated the correlation between cNUCs and H3K9me3 in CRC patients to be weak (p=0.046), indicating that circulating H3K9me3 may be modified in CRC patients. The histone marks were normalized using the values of cNUCs. In agreement with the weak correlation between cNUCs and H3K9me3 in CRC patients, H3K9me3 levels (median 0.047) were lowest in this group compared with the other two groups (0.06 in healthy subjects, 0.2 in MM patients, p = not significant). For H4K20me3, the median values were 0.022 in healthy subjects, 0.052 in CRC patients and 0.056 in MM patients. In conclusion, our findings indicate a marked correlation between cNUCs and histone methyl marks.