Cell survival following radiation exposure requires miR-525-3p mediated suppression of ARRB1 and TXN1.

BACKGROUND: microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer. PRINCIPAL FINDINGS: Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity. CONCLUSION: The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.

Quantitative phosphoproteomic analysis of early alterations in protein phosphorylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥ 1.5 in one of the experiments per time point. The vast majority of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers and heterodimers to different enhancers suggesting that this phosphorylation represents a novel mechanism contributing to the alteration of gene expression by TCDD. Other proteins with altered phosphorylation included, among others, various transcriptional coregulators previously unknown to participate in TCDD-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity.

Evaluation of different biomarkers to predict individual radiosensitivity in an inter-laboratory comparison--lessons for future studies.

Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity.

A radiation-induced gene expression signature as a tool to predict acute radiotherapy-induced adverse side effects.

The majority of patients tolerate radiotherapy well, but some of them suffer from severe side effects. To find genes possibly predictive for radiosensitivity, mRNA profiles were generated before and 6h after in vitro irradiation with 5Gy. We analyzed lymphocytes from four head and neck and eight breast cancer patients with strong acute radiation toxicity and from 12 matching normal reacting patients in a blind study. Expression was also measured in lymphocyte subpopulations and Epstein-Barr transformed lymphocytes. Radiation response in whole lymphocyte populations was most similar to that of B cells. In peripheral blood lymphocytes of all patients; 153 genes were identified which were statistically significantly altered by a fold change of more than 50% by irradiation. The signatures of radio-responsive genes differed tremendously between primary and transformed cells. Pathway analysis revealed genes involved in p53 signalling, cell cycle control and apoptosis in response to radiation in primary lymphocytes. In these cells, a set of 67 radiation-induced genes was identified capable of differentiating between severe radiosensitive and normal reacting patients. More than one third of such classifying genes belong to the group of apoptosis or cell cycle regulating genes. The classifying potential of the expression signature has now to be validated in further patient cohorts.

A novel function of Ubc13 in TNFR1 receptor activation.

The tumour necrosis factor receptor 1 (TNFR1) activates prosurvival pathways by induction of the NFkappaB pathway and induces cell death via apoptosis. The ubiquitin-conjugating enzyme, Ubc13, mediates the ubiquitylation-dependent formation of protein complexes crucial for the activation and regulation of both pathways. We describe a new role for Ubc13 in the regulation of TNFR1 activity after UV stimulation. Depletion of Ubc13 by RNAi produced a decreased NFkappaB activity and increased apoptosis after stimulation by TNFalpha and UV-C light. These results are consistent with the function of Ubc13 in the ubiquitylation of RIP1, which controls the proapoptotic or prosurvival response after TNFR1 activation. Moreover, we demonstrated that UV-C light induces a close interaction between the Ubc13 protein and the TNFR1 receptor. In the absence of Ubc13 TNFR1 clustering was increased. We conclude that Ubc13 has a regulatory role for the activation of TNFR1 and hence, apoptotic cell death. Thus, our results elucidated a new role for Ubc13 in the regulation of prosurvival or proapoptotic processes, which is upstream of so far investigated functions.

Chromosome analysis of the differential radiosensitivity of an Epstein-Barr virus (EBV)-transformed B cell line and B and T lymphocytes from the same blood donor.

PURPOSE: To date, simultaneously performed investigations on the differential radiosensitivity of an Epstein-Barr virus (EBV)-transformed B cell line as well as B and T lymphocytes of human peripheral blood are not available. Thus the aim of the present study was to fill this gap by directly comparing the corresponding dose-response relationships of dicentrics obtained in blood samples from the same donor. MATERIAL AND METHODS: Cell samples of whole blood or low passage cells of an EBV-transformed B cell line were irradiated by 120 kV X-rays in chambers tightly embedded in a polymethylmethacrylate phantom. Chromosome analysis was performed in phytohemagglutinin-stimulated T lymphocytes, in pokeweed mitogen-stimulated B lymphocytes and in the EBV-transformed B cell line. RESULTS: Based on dose-response relationships of dicentrics, different radiosensitivity values relative to T lymphocytes were found from 1.53-1.46 for the EBV-transformed cell line, from 0.76-0.80 for resting B lymphocytes and from 2.36-2.20 for cycling B lymphocytes within the dose range from 0.25-4 Gy. CONCLUSIONS: Owing to these different radiosensitivity values, care has to be taken when dose-response relationships of dicentrics determined in B cell lines are used in biological dosimetry to estimate any dose levels for radiation protection purposes.

The DNA repair protein NBS1 influences the base excision repair pathway.

NBS1 fulfills important functions for the maintenance of genomic stability and cellular survival. Mutations in the NBS1 (Nijmegen Breakage Syndrome 1) gene are responsible for the Nijmegen breakage syndrome (NBS) in humans. The symptoms of this disease and the phenotypes of NBS1-defective cells, especially their enhanced radiosensitivity, can be explained by an impaired DNA double-strand break-induced signaling and a disturbed repair of these DNA lesions. We now provide evidence that NBS1 is also important for cellular survival after oxidative or alkylating stress where it is required for the proper initiation of base excision repair (BER). NBS1 downregulated cells show reduced activation of poly-(adenosine diphosphate-ribose)-polymerase-1 (PARP1) following genotoxic treatment with H(2)O(2) or methyl methanesulfonate, indicating impaired processing of damaged bases by BER as PARP1 activity is stimulated by the single-strand breaks intermediately generated during this repair pathway. Furthermore, extracts of these cells have a decreased capacity for the in vitro repair of a double-stranded oligonucleotide containing either uracil or 8-oxo-7,8-dihydroguanine to trigger BER. Our data presented here highlight for the first time a functional role for NBS1 in DNA maintenance by the BER pathway.

Multicentric investigation of ionising radiation-induced cell death as a predictive parameter of individual radiosensitivity.

In the present study, the predictive value of ionising radiation (IR)-induced cell death was tested in peripheral blood lymphocytes (PBLs) and their corresponding Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) in an interlaboratory comparison. PBLs and their corresponding LCLs were derived from 15 tumour patients, that were considered clinically radiosensitive based on acute side-effects, and matched controls. Upon coding of the samples, radiosensitivity of the matched pairs was analysed in parallel in three different laboratories by assessing radiation-induced apoptotic and necrotic cell death using annexin V. All participating laboratories detected a dose-dependent increase of apoptosis and necrosis in the individual samples, to a very similar extent. However, comparing the mean values of apoptotic and necrotic levels derived from PBLs of the radiosensitive cohort with the mean values of the control cohort did not reveal a significant difference. Furthermore, within 15 matched pairs, no sample was unambiguously and independently identified by all three participating laboratories to demonstrate in vitro hypersensitivity that matched the clinical hypersensitivity. As has been reported previously, apoptotic and necrotic cell death is barely detectable in immortalised LCL derivatives using low doses of IR. Concomitantly, the differences in apoptosis or necrosis levels found in primary cells of different individuals were not observed in the corresponding LCL derivatives. All participating laboratories concordantly reasoned that, with the methods applied here, IR-induced cell death in PBLs is unsuitable to unequivocally predict the individual clinical radiosensitivity of cancer patients. Furthermore, LCLs do not reflect the physiological properties of the corresponding primary blood lymphocytes with regard to IR-induced cell death. Their value to predict clinical radiosensitivity is thus highly questionable.

Reduced expression of SRC family kinases decreases PI3K activity in NBS1-/- lymphoblasts.

SRC family kinases (SFKs) are involved in the activation of phosphatidylinositol-3-kinase (PI3K). In addition, the activity of this lipid kinase can be regulated by the DNA repair protein NBS1. Here, we describe a disturbed expression of some members of the non-receptor tyrosine kinase family in lymphoblastoid cell lines generated from cells of Nijmegen breakage syndrome (NBS) patients. Especially, only minor amounts of the kinases LCK and HCK are expressed in the NBS1(-/-) cell lines as compared to the consanguineous NBS1(+/-) cells. We demonstrate that SFK activity is important for a proper activation of PI3K in these cells and that it is reduced in NBS1(-/-) cells. We provide evidence that the observed reduced PI3K activity in NBS lymphoblasts is caused by an impaired expression of the SFKs LCK and/or HCK. Thus, our data establish a new function for the NBS1 protein as a regulator of PI3K activity via SFK members.

Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription.

During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

NBS1 is required for IGF-1 induced cellular proliferation through the Ras/Raf/MEK/ERK cascade.

NBS1 is a member of the Mre11-Rad50-NBS1 complex, which plays a role in cellular responses to DNA damage and the maintenance of genomic stability. Transgenic mice models and clinical symptoms of NBS patients have shown that NBS1 exerts pleiotropic actions on the growth and development of mammals. The present study showed that after repression of endogenous NBS1 levels using short interfering RNA, hTERT-RPE cells demonstrated impaired proliferation and a poor response to IGF-1. NBS1 down-regulated cells displayed disturbances in periodical oscillations of cyclin E and A and delayed cell cycle progression. Remarkably, lower phosphorylation levels of c-Raf and diminished activity of Erk1/2 in response to IGF-1 suggest a link among NBS1, IGF-1 signaling and the Ras/Raf/MEK/ERK cascade. The functional relevance of NBS1 in mitogenic signaling and initiation of cell cycle progression were demonstrated in NBS1 down-regulated cells where IGF-1 had a limited ability to induce the FOS and CCND1 expressions. In conclusion, our findings provide strong evidence that NBS1 has a functional role in IGF-1 signaling for the promotion of cell proliferation via the Ras/Raf/MEK/ERK cascade.

Enhanced CD95-mediated apoptosis contributes to radiation hypersensitivity of NBS lymphoblasts.

The molecular causes for enhanced radiosensitivity of Nijmegen Breakage Syndrome cells are unclear, especially as repair of DNA damage is hardly impeded in these cells. We clearly demonstrate that radiation hypersensitivity is accompanied by enhanced gamma-radiation-induced apoptosis in NBS1 deficient lymphoblastoid cell lines. Differences in the apoptotic behavior of NBS1 (-/-) and NBS1 (+/-) cells are not due to an altered p53 stabilization or phosphorylation in NBS1 (-/-) cells. gamma-radiation-induced caspase-8 activity is increased and visualization of CD95 clustering by laser scanning microscopy shows a significant higher activation of the death receptor in NBS1 (-/-) cells. Further investigation of the molecular mechanisms reveals a role for reactive oxygen species-triggered activation of CD95. These results demonstrate that NBS1 suppresses the CD95 death receptor-dependent apoptotic pathway after gamma-irradiation and evidence is given that this is achieved by regulation of the PI3-K/AKT survival pathway.

The rate of extrachromosomal homologous recombination within a novel reporter plasmid is elevated in cells lacking functional ATM protein.

Homologous recombination between identical stretches of DNA depends on the coordinated action of many tightly regulated proteins. Cellular defects in homologous recombination are strongly associated with increased genomic instability and tumorigenesis. In cells of the cancer-prone syndrome ataxia telangiectasia (A-T), increased intrachromosomal recombination has been demonstrated, while extrachromosomal recombination has been discussed controversially. We constructed a novel, episomally replicating pGrec recombination vector containing two mutated alleles of the enhanced green fluorescent protein (eGFP) gene. Homologous recombination can reconstitute functional wildtype eGFP, thus allowing detection of recombination events based on cellular eGFP fluorescence. Using an isogenic cell pair of A-T fibroblasts and derivatives complemented by an ATM expression vector, we were able to demonstrate in A-T cells high extrachromosomal recombination rates, which are suppressed upon ectopic ATM expression. We thus found that ATM deficiency increases spontaneous recombination not only in intrachromosomal but also in extrachromosomal substrates, suggesting that lack of ATM increases homologous recombination independent of the chromatin structure.

Tumor-associated E-cadherin mutations do not induce Wnt target gene expression, but affect E-cadherin repressors.

E-cadherin is a cell-cell adhesion molecule and tumor invasion suppressor gene that is frequently altered in human cancers. It interacts through its cytoplasmic domain with beta-catenin which in turn interacts with the Wnt (wingless) signaling pathway. We have compared the effects of different tumor-derived E-cadherin variants with those of normal E-cadherin on Wnt signaling and on genes involved in epithelial mesenchymal transition. We established an in-house cDNA microarray composed of 1105 different, sequence verified cDNA probes corresponding to 899 unique genes that represent the majority of genes known to be involved in cadherin-dependent cell adhesion and signaling ('Adhesion/Signaling Array'). The expression signatures of E-cadherin-negative MDA-MB-435S cancer cells transfected with E-cadherin variants (in frame deletions of exon 8 or 9, D8 or D9, respectively, or a point mutation in exon 8 (D370A)) were compared to that of wild-type E-cadherin (WT) transfected cells. From the differentially expressed genes, we selected 38 that we subsequently analyzed by quantitative real-time RT-PCR and/or Northern Blot. A total of 92% of these were confirmed as differentially expressed. Most of these genes encode proteins of the cytoskeleton, cadherins/integrins, oncogenes and matrix metalloproteases. No significant expression differences of genes downstream of the Wnt-pathway were found, except in E-cadherin D8 transfected cells where upregulation of three Tcf/Lef-transcribed genes was seen. One possible reason for the lack of expression differences of the Tcf/Lef-regulated genes is upregulation of SFRP1 and SFRP3; both of which are competitive inhibitors of the Wnt proteins. Interestingly, known E-cadherin transcriptional repressors, such as SLUG (SNAI2), SIP1 (ZEB2), TWIST1, SNAIL (SNAI1) and ZEB1 (TCF8), but not E12/E47 (TCF3), had a lack of upregulation in cells expressing mutated E-cadherin compared to WT. In conclusion, E-cadherin mutations have no influence on expression of genes involved in Wnt-signaling, but they may promote their own expression by blocking upregulation of E-cadherin repressors.

Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion.

Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low. Here we report that its efficiency can be increased by gamma-irradiation of recipient cells at the time of transformation. Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52. In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30 degrees C, rich medium). The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation. A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens. This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements.