Expression Of DNA Damage Response Genes Research Articles

SOG1-dependent NAC103 modulates the DNA damage response as a transcriptional regulator in Arabidopsis.

The plant-specific transcription factor (TF) NAC103 was previously reported to modulate the unfolded protein response in Arabidopsis under endoplasmic reticulum (ER) stress. Alternatively, we report here that NAC103 is involved in downstream signaling of SOG1, a master regulator for expression of DNA damage response (DDR) genes induced by genotoxic stress. Arabidopsis NAC103 expression was strongly induced by genotoxic stress and nac103 mutants displayed substantial inhibition of DDR gene expression after gamma radiation or radiomimetic zeocin treatment. DDR phenotypes, such as true leaf inhibition, root cell death and root growth inhibition, were also suppressed significantly in the nac103 mutants, but to a lesser extent than in the sog1-1 mutant. By contrast, overexpression of NAC103 increased DDR gene expression without genotoxic stress and substantially rescued the phenotypic changes in the sog1-1 mutant after zeocin treatment. The putative promoters of some representative DDR genes, RAD51, PARP1, RPA1E, BRCA1 and At4g22960, were found to partly interact with NAC103. Together with the expected interaction of SOG1 with the promoter of NAC103, our study suggests that NAC103 is a putative SOG1-dependent transcriptional regulator of plant DDR genes, which are responsible for DDR phenotypes under genotoxic stress.

DNA damage response genes mark the early transition from colitis to neoplasia in colitis-associated colon cancer

Chronic intestinal inflammation predisposes patients with Inflammatory Bowel Disease (IBD) to Colitis-Associated Cancer (CAC). In the setting of chronic inflammation, microsatellite instability (MSI) results from early loss of DNA damage response (DDR) genes, ultimately leading to tumor formation. Despite continued efforts to improve early detection of high risk, pre-dysplastic regions in IBD patients, current macroscopic and genetic surveillance modalities remain limited. Therefore, understanding the regulation of key DDR genes in the progression from colitis to cancer may improve molecular surveillance of CAC. To evaluate DDR gene regulation in the transition from colitis to tumorigenesis, we utilized the well-established Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) pre-clinical murine model of CAC in C57BL/6 mice. In order to assess colonic tumor burden in the setting of mutagen and intestinal irritation, tumors were visualized and graded in real time through high-resolution murine colonoscopy. Upon sacrifice, colons were opened and assessed for macroscopic tumor via high magnification surgical lenses (HMSL). Tissues were then sectioned and separated into groups based on the presence or absence of macroscopically visible tumor. Critical DDR genes were evaluated by semi-quantitative RT-PCR. Interestingly, colon tissue with macroscopically visible tumor (MVT) and colon tissue prior to observable tumor (the non-macroscopically visible tumor-developing group, NMVT) were identical in reduced mRNA expression of mlh1, anapc1, and ercc4 relative to colitic mice without mutagen, or those receiving mutagen alone. Colitis alone was sufficient to reduce colonic ercc4 expression when compared to NMVT mice. Therefore, reduced ercc4 expression may mark the early transition to CAC in a pre-clinical model, with expression reduced prior to the onset of observable tumor. Moreover, the expression of select DDR genes inversely correlated with chronicity of inflammatory disease. These data suggest ercc4 expression may define early stages in the progression to CAC.

Severe PATCHED1 Deficiency in Cancer-Prone Gorlin Patient Cells Results in Intrinsic Radiosensitivity

Gorlin syndrome (or basal-cell nevus syndrome) is a cancer-prone genetic disease in which hypersusceptibility to secondary cancer and tissue reaction after radiation therapy is debated, as is increased radiosensitivity at cellular level. Gorlin syndrome results from heterozygous mutations in the PTCH1 gene for 60% of patients, and we therefore aimed to highlight correlations between intrinsic radiosensitivity and PTCH1 gene expression in fibroblasts from adult patients with Gorlin syndrome. The radiosensitivity of fibroblasts from 6 patients with Gorlin syndrome was determined by cell-survival assay after high (0.5-3.5Gy) and low (50-250mGy) γ-ray doses. PTCH1 and DNA damage response gene expression was characterized by real-time polymerase chain reaction and Western blotting. DNA damage and repair were investigated by γH2AX and 53BP1 foci assay. PTCH1 knockdown was performed in cells from healthy donors by using stable RNA interference. Gorlin cells were genotyped by 2 complementary sequencing methods. Only cells from patients with Gorlin syndrome who presented severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For 2 of the radiosensitive cell strains, heterozygous mutations in the 5' end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DNA damage response pathways (ATM, CHK2, and P53 levels and activation by phosphorylation) were deregulated after irradiation, whereas DSB repair recognition was unimpaired. Furthermore, normal cells with RNA interference-mediated PTCH1 deficiency showed reduced survival after irradiation, directly linking this gene to high- and low-dose radiosensitivity. In the present study, we show an inverse correlation between PTCH1 expression level and cellular radiosensitivity, suggesting an explanation for the conflicting results previously reported for Gorlin syndrome and possibly providing a basis for prognostic screens for radiosensitive patients with Gorlin syndrome and PTCH1 mutations.

A transcriptional factor B paralog functions as an activator to DNA damage-responsive expression in archaea.

Previously it was shown that UV irradiation induces a strong upregulation of tfb3 coding for a paralog of the archaeal transcriptional factor B (TFB) in Sulfolobus solfataricus, a crenarchaea. To investigate the function of this gene in DNA damage response (DDR), tfb3 was inactivated by gene deletion in Sulfolobus islandicus and the resulting Δtfb3 was more sensitive to DNA damage agents than the original strain. Transcriptome analysis revealed that a large set of genes show TFB3-dependent activation, including genes of the ups operon and ced system. Furthermore, the TFB3 protein was found to be associated with DDR gene promoters and functional dissection of TFB3 showed that the conserved Zn-ribbon and coiled-coil motif are essential for the activation. Together, the results indicated that TFB3 activates the expression of DDR genes by interaction with other transcriptional factors at the promoter regions of DDR genes to facilitate the formation of transcription initiation complex. Strikingly, TFB3 and Ced systems are present in a wide range of crenarchaea, suggesting that the Ced system function as a primary DNA damage repair mechanism in Crenarchaeota. Our findings further suggest that TFB3 and the concurrent TFB1 form a TFB3-dependent DNA damage-responsive circuit with their target genes, which is evolutionarily conserved in the major lineage of Archaea.

Investigating Intracellular RecA Concentrations in the Emerging Pathogen Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen responsible for many hospital‐acquired infections. A. baumannii is known for its ability to rapidly gain antibiotic resistances and survive desiccation. This ability to survive adverse conditions has been linked to A. baumannii's unique DNA‐damage response (DDR), which is modulated by the evolutionarily conserved recombinase, RecA. We have previously shown that in A. baumannii, RecA is needed to upregulate the expression of error‐prone DNA polymerase genes, which in turn results in higher frequency of mutagenesis.A. baumannii lacks a functional homologue for the highly conserved global Escherichia coli DDR transcriptional repressor, indicating that their DDRs are regulated differently. In contrast to E. coli, the DDR in A. baumannii is bimodal. That is, when DNA damage is induced and expression of DDR genes is tracked, two distinct subpopulations are evident, one with low expression of DDR genes and one with high. The two populations survive differently, suggesting that the bacterium can fall back on the population that does survive. The details of the mechanism underlying this regulation remain largely unknown. Our lab has recently shown that the recA gene is regulated by a 5′untranslated region that is important for both its stability and expression. Moreover, preliminary evidence suggests that differential recA expression is needed to maintain the bimodal nature of the DDR. Because of RecA's role in the DDR, we sought to compare the levels of RecA in A. baumannii and E. coli. We hypothesized that RecA intracellular concentration in A. baumannii would be lower than E. coli's, consistent with the differences in regulatory roles. To test this hypothesis, we performed a semiquantitative Western blot using anti‐E. coli RecA polyclonal antibody. We first purified His‐tagged RecA from each species. The purified proteins were then used to prepare a standard curve for the Western blot. As expected, we observed a difference in affinity of the anti‐recA antibody based on differences in the primary protein sequence of RecA from the two species. (A. baumannii and E. coli RecA are approximately 74% identical). Remarkably, and in agreement with our hypothesis, we find that the levels of basal intracellular RecA were at least three times lower in A. baumannii than in E. coli. This difference in RecA protein concentration may have profound effects on A. baumannii physiology and is consistent with the different regulatory role for RecA in A. baumannii. These results show that the intracellular RecA concentration in bacteria may provide insights into the regulation of the DDR and the mechanism by which A. baumannii is able to survive adverse conditions and readily acquire antibiotic resistances.Support or Funding InformationFunding was provided by the National Institute of General Medical Sciences (RO1GM088230 to Veronica Godoy).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A transcriptional factor B paralog functions as an activator to DNA damage-responsive expression in archaea.

Previously it was shown that UV irradiation induces a strong upregulation of tfb3 coding for a paralog of the archaeal transcriptional factor B (TFB) in Sulfolobus solfataricus, a crenarchaea. To investigate the function of this gene in DNA damage response (DDR), tfb3 was inactivated by gene deletion in Sulfolobus islandicus and the resulting Δtfb3 was more sensitive to DNA damage agents than the original strain. Transcriptome analysis revealed that a large set of genes show TFB3-dependent activation, including genes of the ups operon and ced system. Furthermore, the TFB3 protein was found to be associated with DDR gene promoters and functional dissection of TFB3 showed that the conserved Zn-ribbon and coiled-coil motif are essential for the activation. Together, the results indicated that TFB3 activates the expression of DDR genes by interaction with other transcriptional factors at the promoter regions of DDR genes to facilitate the formation of transcription initiation complex. Strikingly, TFB3 and Ced systems are present in a wide range of crenarchaea, suggesting that the Ced system function as a primary DNA damage repair mechanism in Crenarchaeota. Our findings further suggest that TFB3 and the concurrent TFB1 form a TFB3-dependent DNA damage-responsive circuit with their target genes, which is evolutionarily conserved in the major lineage of Archaea.

Transcriptional Upregulation of DNA Damage Response Genes in Bank Voles (Myodes glareolus) Inhabiting the Chernobyl Exclusion Zone

Exposure to ionizing radiation (IR) from radionuclides released into the environment can damage DNA. An expected response to exposure to environmental radionuclides, therefore, is initiation of DNA damage response (DDR) pathways. Increased DNA damage is a characteristic of many organisms exposed to radionuclides but expression of DDR genes of wildlife inhabiting an area contaminated by radionuclides is poorly understood. We quantified expression of five central DDR genes Atm, Mre11, p53, Brca1 and p21 in the livers of the bank vole Myodes glareolus that inhabited areas within the Chernobyl Exclusion Zone (CEZ) that differed in levels of background radioactivity, and also from control areas outside the CEZ (i.e. sites with no detectable environmental radionuclides) in Ukraine. Expression of these DDR genes did not significantly differ between male and female bank voles, nor among sites within the CEZ. We found a near two-fold upregulation in the DDR initiators Mre11 and Atm in animals collected from the CEZ compared with samples from control sites. As Atm is an important regulator of oxidative stress, our data suggest that antioxidant activity may be a key component of the defense against exposure to environmental radioactivity.

Polo-like kinase 1 (Plk1) overexpression enhances ionizing radiation-induced cancer formation in mice

Polo-like kinase 1 (Plk1), a serine/threonine protein kinase normally expressed in mitosis, is frequently up-regulated in multiple types of human tumors regardless of the cell cycle stage. However, the causal relationship between Plk1 up-regulation and tumorigenesis is incompletely investigated. To this end, using a conditional expression system, here we generated Plk1 transgenic mouse lines to examine the role of Plk1 in tumorigenesis. Plk1 overexpression in mouse embryonic fibroblasts prepared from the transgenic mice led to aberrant mitosis followed by aneuploidy and apoptosis. Surprisingly, Plk1 overexpression had no apparent phenotypes in the mice. Given that no malignant tumor formation was observed even after a long period of Plk1 overexpression, we reasoned that additional factors are required for tumorigenesis in Plk1-overexpressing mice. Because Plk1 can directly participate in the regulation of the DNA damage response (DDR) pathway, we challenged Plk1-overexpressing mice with ionizing radiation (IR) and found that Plk1-overexpressing mice are much more sensitive to IR than their wild-type littermates. Analysis of tumor development in the Plk1-overexpressing mice indicated a marked decrease in the time required for tumor emergence after IR. At the molecular level, Plk1 overexpression led to reduced phosphorylation of the serine/threonine kinases ATM and Chk2 and of histone H2AX after IR treatment both in vivo and in vitro Furthermore, RNA-Seq analysis suggested that Plk1 elevation decreases the expression of several DDR genes. We conclude that Plk1 overexpression may contribute to tumor formation by both inducing chromosomal instability and suppressing the DDR pathway.

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation.

In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii Here, we identify a structured 5' untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5' UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5' UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5' UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5' UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis-acting 5' UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5' UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability.

Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

ObjectiveOvarian steroid hormones (mainly E2 and P4) regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models.MethodsA cell line derived from the porcine oviductal epithelium (CCLV-RIE270) was characterized (lineage markers, proliferation characteristics and transformation status). Primary porcine oviduct epithelial cells (POEC) were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml). Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR) and inflammation was quantified.ResultsCCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml) upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8). Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2.ConclusionsCCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

The loss of ATP2C1 impairs the DNA damage response and induces altered skin homeostasis: Consequences for epidermal biology in Hailey-Hailey disease.

Mutation of the Golgi Ca2+-ATPase ATP2C1 is associated with deregulated calcium homeostasis and altered skin function. ATP2C1 mutations have been identified as having a causative role in Hailey-Hailey disease, an autosomal-dominant skin disorder. Here, we identified ATP2C1 as a crucial regulator of epidermal homeostasis through the regulation of oxidative stress. Upon ATP2C1 inactivation, oxidative stress and Notch1 activation were increased in cultured human keratinocytes. Using RNA-seq experiments, we found that the DNA damage response (DDR) was consistently down-regulated in keratinocytes derived from the lesions of patients with Hailey-Hailey disease. Although oxidative stress activates the DDR, ATP2C1 inactivation down-regulates DDR gene expression. We showed that the DDR response was a major target of oxidative stress-induced Notch1 activation. Here, we show that this activation is functionally important because early Notch1 activation in keratinocytes induces keratinocyte differentiation and represses the DDR. These results indicate that an ATP2C1/NOTCH1 axis might be critical for keratinocyte function and cutaneous homeostasis, suggesting a plausible model for the pathological features of Hailey-Hailey disease.

Cdk12 Regulates Neurogenesis and Late-Arising Neuronal Migration in the Developing Cerebral Cortex.

DNA damage response (DDR) pathways are critical for ensuring that replication stress and various types of DNA lesion do not perturb production of neural cells during development. Cdk12 maintains genomic stability by regulating expression of DDR genes. Mutant mice in which Cdk12 is conditionally deleted in neural progenitor cells (NPCs) die after birth and exhibit microcephaly with a thinner cortical plate and an aberrant corpus callosum. We show that NPCs of mutant mice accumulate at G2 and M phase, and have lower expression of DDR genes, more DNA double-strand breaks and increased apoptosis. In addition to there being fewer neurons, there is misalignment of layers IV-II neurons and the presence of abnormal axonal tracts of these neurons, suggesting that Cdk12 is also required for the migration of late-arising cortical neurons. Using in utero electroporation, we demonstrate that the migrating mutant cells remain within the intermediate zone and fail to adopt a bipolar morphology. Overexpression of Cdk5 brings about a partially restoration of the neurons reaching layers IV-II in the mutant mice. Thus, Cdk12 is crucial to the repair of DNA damage during the proliferation of NPCs and is also central to the proper migration of late-arising neurons.

MP90-04 A ROLE FOR HISTONE DEMETHYALASE JMJD1A IN THE DNA DAMAGE RESPONSE OF PROSTATE CANCER CELLS

INTRODUCTION AND OBJECTIVES: The major therapies for prostate cancer (PCa) such as radio-, chemoor androgen deprivation therapy induce DNA damage. Thus the DNA damage response (DDR) and expression of DDR genes are key factors in determining outcome of PCa therapies. Histone demethylase JMJD1A regulates the gene expression by demethylating H3K9. Our previous work shows that JMJD1A is essential for the long-term proliferation and survival of PCa cells in vitro or in the xenograft prostate tumor model, partly via its effect on the activities of androgen receptor (AR) and c-Myc transcription factors. Here, we find a new role for JMJD1A in the expression of DDR genes and sensitivity of PCa cells to irradiation and androgen deprivation. METHODS: Real-time PCR was used to measure the mRNA levels of some DDR genes in the PCa cells (control or siRNA knockdown (KD) of JMJD1A, AR or c-Myc). Chromatin immunoprecipitation (ChIP) was used to evaluate the binding of JMJD1A to the AR or MYC binding sites on these DDR genes. JMJD1A-KD PCa cells were treated with or without ionizing radiation (IR), and analyzed by the r-H2AX staining (a widely used marker for the DNA double strand breaks (DSBs)), western blot analysis of DNA damage signaling proteins, flow cytometry analysis of cell cycle, and GFP reporter assays for the DSB repair via the homologous recombination (HR) or non-homologous end joining (NHEJ) pathways. The growth of these PCa cells was also measured under the IR and androgen deprivation conditions. RESULTS: JMJD1A was found to promote the expression of some DDR genes by co-activating AR and/or c-Myc. The DDR genes regulated by JMJD1A encode factors mediating the DSB repair by both HR and NHEJ pathways. JMJD1A KD led to increased r-H2AX foci in the non-irradiated cells, and the delayed clearance of r-H2AX foci in the irradiated cells. JMJD1A KD also increased the levels of phospho-DNAPK or phospho-CHEK2 in the western blot analysis and caused G2 cell cycle arrest. Consistently, JMJD1A KD reduced the percentage of GFPpositive cells that reflects the DSB repair by both HR and NHEJ pathways. JMJD1A-KD cells showed a reduced growth under IR and androgen deprivation conditions. CONCLUSIONS: JMJD1A promotes the DSB repair via regulation of DDR gene expression through AR and/or c-Myc. The role of JMJD1A in the DSB repair may contribute to the resistance of PCa cells to the radioand androgen deprivation therapies.

Partial sleep deprivation activates the DNA damage response (DDR) and the senescence-associated secretory phenotype (SASP) in aged adult humans

Age-related disease risk has been linked to short sleep duration and sleep disturbances; however, the specific molecular pathways linking sleep loss with diseases of aging are poorly defined. Key cellular events seen with aging, which are thought to contribute to disease, may be particularly sensitive to sleep loss. We tested whether one night of partial sleep deprivation (PSD) would increase leukocyte gene expression indicative of DNA damage responses (DDR), the senescence-associated secretory phenotype (SASP), and senescence indicator p16INK4a in older adult humans, who are at increased risk for cellular senescence. Community-dwelling older adults aged 61–86years (n=29; 48% male) underwent an experimental partial sleep deprivation (PSD) protocol over 4 nights, including adaptation, an uninterrupted night of sleep, partial sleep deprivation (sleep restricted 3–7AM), and a subsequent full night of sleep. Blood samples were obtained each morning to assess peripheral blood mononuclear cell (PBMC) gene expression using Illumina HT-12 arrays. Analyses of microarray results revealed that SASP (p<.05) and DDR (p=.08) gene expression were elevated from baseline to PSD nights. Gene expression changes were also observed from baseline to PSD in NFKB2, NBS1 and CHK2 (all p’s<.05). The senescence marker p16INK4a (CDKN2A) was increased 1day after PSD compared to baseline (p<.01), however confirmatory RT-PCR did not replicate this finding. One night of partial sleep deprivation activates PBMC gene expression patterns consistent with biological aging in this older adult sample. PSD enhanced the SASP and increased the accumulation of damage that initiates cell cycle arrest and promotes cellular senescence. These findings causally link sleep deprivation to the molecular processes associated with biological aging.

Hsa-let-7g miRNA regulates the anti-tumor effects of gastric cancer cells under oxidative stress through the expression of DDR genes.

Oxidative stress is linked to increased risk of gastric cancer (GC). Recent reports have found that hsa-let-7 g microRNA (miRNA) has properties of anti-tumor and resistance to damages induced by oxidized low-density lipoprotein (ox-LDL). Dysregulation of hsa-let-7 g was present in GC in vivo and in vitro under exogenous stress. However, we didn't know whether there are regulatory mechanisms of hsa-let-7 g in GC under oxidative stress. This study was aimed at investigating the effects of hsa-let-7 g microRNA (miRNA) on GC under oxidative stress. The results showed that H2O2 induced the increase of DNA damage response (DDR) genes (ATM, H2AX and Chk1) and downregulation of hsa-let-7 g in GC cells. Further study confirmed Hsa-let-7 g caused the apoptosis and loss of proliferation in GC cells exposed to H2O2 associated with repression of DDR system. Yet, we found let-7 g didn't target DDR genes (ATM, H2AX and Chk1) directly. In addition, data revealed hsa-let-7 g miRNA increased the sensitivity of GC to X-rays involving in ATM regulation as well according to application of X-rays (another DDR inducer). In conclusion, Hsa-let-7 g miRNA increased the sensitivity of GC to oxidative stress by repression activation of DDR indirectly. Let-7 g improved the effects of X-rays on GC cells involving in DDR regulation as well.

BCL6 Dose Not Negatively Regulate DNA Damage Response Genes in Multiple Myeloma Cells

Background: A proto-oncogene BCL6 is a known transcriptional repressor and a master regulator of germinal center B cell program. It represses expression of DNA damage response genessuch as p53, ATR, CHEK1 and p21, which helps B cells to survive and expand during antigen receptor-diversification reactions. It also plays a pivotal role in the formation of germinal centers and lymphomagenesis. BCL6 is down-regulated in normal plasma cells while it is aberrantly expressed in bone marrow residing myeloma cells. Although the role of BCL6 in B cell lymphomas has been intensively studied, its role in plasma cell neoplasms remains to be elucidated. In this study we asked whether BCL6 plays a role in DNA damage response of myeloma cells.Methods: Bone marrow samples obtained from 36 of primary plasma cell dyscrasia patients (5 cases of MGUS, 30 of multiple myeloma (MM) and 1 plasma cell leukemia (PCL)) were subjected to the study after informed consent. This study was approved by IRBs of Gunma University Hospital and Nishigunma National Hospital. CD138-positive bone marrow plasma cells were isolated as a purity of >95% using magnetic beads and RNAs were extracted. Expression levels of BCL6, p53, ATR, CHEK1 and p21 were quantified by real time PCR using Taqman-probes. For retroviral infection, BCL6 was cloned into pMY-IRES-GFP vector and transfected to PlatA cells using lipofection reagents. 48 hours later, supernatants were collected and centrifuged for 16 hours and the pellets were used for infection. GFP positive cells were collected and used for following experiments. For γH2AX foci formation analysis, the cells were given ionized irradiation at doses of 0, 3, 5 and 10Gy, and used after an hour incubation. Cells were also exposed to different concentration of etoposide (0, 1, 5, 10, 50, 100μM) for 30minute, then washed with fresh media and incubated for an hour. Cells were stained with a FITC-labeled anti-γH2AX antibody as reported (Muslimovic et al, Nat. Protoc. 2008). For cell cycle analysis by flow cytometry, EdU uptake and 7AAD DNA staining were performed according to a manufacture’s protocol (Life Technologies). Correlation of expression levels between each of genes was assessed using Spearman’s non-parametric correlation analysis.Results: Median of BCL6 expression levels of MM and PCL cells (Qty median=1.47, range 0.09-17.2) was not significantly different from that of MGUS cells (Qty median=1.84, range 0.8-4.2, p=0.51). In marked contrast to germinal center B cells, expression levels of BCL6 and p53 were positively correlated in MM, PCL and MGUS (r=0.457, p=0.007). The correlation between expression of BCL6 and ATR did not reach statistical significance (r=0.323, p=0.062). ATR and p53 were also positively correlated (r=0.549, p=0.001). The expression level of p21, a well-known target of p53, was positively correlated to that of p53 (r=0.681, p=0.03), which supports our data qualification. We also examined mRNA expression levels of BCL6 in MM cell lines, RPMI8226, KMS11, KMS12PE, KMS12BM, KMS18, KMS26, ARH 77 and U266. U266, KMS12PE, KMS26 expressed little amount of BCL6 compared to the patient samples. The other five cell lines did not express BCL6. In order to study BCL6 functions in MM cells, we retrovirally expressed BCL6 in the KMS12BM cell line. The expression level of BCL6 was comparable to the patient samples after the retroviral expression (QTy 6.70). Since BCL6 is known to be a transcriptional repressor and supposed to directly repress p53, ATR, CHEK1 and p21 in B cells, we analyzed expression levels of these genes. Intriguingly, p53, ATR, CHEK1 and p21 are not repressed by overexpression of BCL6 in KMS12BM. These results suggest that alternative regulatory mechanisms of transcriptional regulation by BCL6 are present in MM cells. For further evaluation of the DNA damage response by BCL6 expression, we irradiated or treated these cells with etoposide and analyzed for γH2AX foci formation, a hallmark of DNA double strand break. No differences in the foci formation between mock-infected and BCL6-infected-KSM12BM were detected either with irradiation or etoposide exposure. We also studied cell cycle progressionin these cells. Flow cytometry analysis showed no significant difference between these cells.Conclusion: Unlike B cells in germinal centers, BCL6 expression in myeloma cells did not repress DNA damage response gene expression. DisclosuresNo relevant conflicts of interest to declare.

SC-16 * DIVERGENT CELL CYCLE REGULATION IN GBM CELLS PROVIDES A UNIQUE POINT OF INTERVENTION IN POLARIZED CD133+ CELLS

Recent studies in distinct glioma types have separately identified phenotypically distinct populations of tumor propagating cells (TPCs). Enrichment of TPCs by cell surface marker expression (CD133 [PMID:17051156], NG2 [PMID: 21907924]) has revealed the possibility of multiple TPC populations, within a single tumor, that have distinct properties and therapy sensitivities. Here we identify and compare the coexisting populations of glioblastoma multiforme (GBM) cells expressing the stem marker CD133 and the progenitor marker NG2. We compare the proliferative properties of CD133+ and NG2+ cells, and investigate how polarity and mode of division may determine other functional properties including cell cycle regulation and therapy sensitivity. Established and primary GBM cell cultures were analyzed by immunofluorescence and flow cytometry for expression of CD133 and NG2, alongside markers of polarity and proliferation. We showed that CD133+ and NG2+ GBM cells coexist as predominantly separate subpopulations. CD133+ cells are highly polarized and, compared to NG2+ or marker-negative subpopulations, are slower dividing. Cell cycle and gene expression analyses showed that the increase in cell cycle length in CD133+ cells is due to an extended G2-M period. As expected CD133+ cells have increased expression of DNA-damage response genes, however paradoxically we also detected increased activation of polo-like kinase 1 (PLK1). CD133+ cells were uniquely sensitive to the PLK1 inhibitor BI2536 in vivo, and treatment of intracranial GBM xenografts with the BRAF inhibitor PLX4720 and BI2536 showed greater anti-tumor activity than either alone. Our studies identified two putative TPC subpopulations in GBM with distinct cell cycle dynamics, DNA damage response and cell division mode. Ongoing work will investigate the role of Plk1 activity in linking these divergent phenotypes. Understanding this phenotypic heterogeneity in adjacent TPC types will allow the design of treatment strategies that eradicate multiple putative TPC populations.

Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae

In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora ( Ravintsara aromatica) and Helichrysum italicum ( Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum > Coriandrum sativum > Artemisia herba alba > Cinnamomum camphora > Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine.

Coordinate alterations in the expression of BRCA1, BRCA2, p300, and Rad51 in response to genotoxic and other stresses in human prostate cancer cells.

BRCA1 and BRCA2 participate in cell cycle progression, apoptosis, and DNA repair pathways. The latter role may be mediated by interaction with DNA recombinase Rad51. The purpose of this study was to evaluate the effects of genotoxic and other cytotoxic agents on expression of DNA damage-response genes (BRCA1, BRCA2, p300, and Rad51) in human prostate cancer cells. Subconfluent proliferating cultures of Tsu-Prl or DU-145 cells were treated with various stressful agents and assayed 24 hr later for alterations in: 1) mRNA expression (by semiquantitative reverse transcription-PCR); 2) cell viability (by trypan blue dye exclusion); and 3) protein expression (by Western blotting). Of 26 agents screened, BRCA1 and BRCA2 mRNA reductions were observed in both cell lines after exposure to adriamycin (ADR), camptothecin (CPT), sodium selenite (SLN), and ultraviolet radiation (UV), while nitrogen mustard (HN2) caused mRNA reduction in DU-145 but not in Tsu-Prl. Inhibition of BRCA1/2 expression by ADR and HN2 was blocked by cycloheximide, suggesting that this requires new protein synthesis, while inhibition by CPT, SLN, and UV did not require protein synthesis. Reduction of p300 and Rad51 mRNA levels occurred in parallel with that of BRCA1/2, suggesting coordinate regulation of these genes. The ability of an agent to inhibit mRNA expression was not directly correlated with cytotoxicity. ADR, CPT, UV, and SLN also caused reduction of protein levels; but the kinetics of decreases in protein vs. mRNA differed. After ADR treatment, high molecular weight (Mr hyperphosphorylated) BRCA1 decreased more rapidly than the low Mr species. BRCA2 showed a more rapid decrease in protein than mRNA, while Rad51 showed the opposite. By 48 and 72 hr post-ADR, all four mRNAs and proteins were reduced to well below control levels, except for Rad51 protein, which was only moderately decreased. Selected DNA-damaging agents (ADR, CPT, and UV) and a reducing agent (SLN) inhibited BRCA1/2, p300, and Rad51 expression in prostate cancer cells, although decreases in mRNA vs. protein did not coincide. We postulate that temporal changes in relative protein levels affect different phases of the stress response, and that the ultimate downregulation of all four genes promotes prostate cancer survival.