Induction Of DNA Damage Response Research Articles

Securin Enhances the Anti-Cancer Effects of 6-Methoxy-3-(3′,4′,5′-Trimethoxy-Benzoyl)-1H-Indole (BPR0L075) in Human Colorectal Cancer Cells

BPR0L075 [6-methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1H-indole] is a novel anti-microtubule drug with anti-tumor and anti-angiogenic activities in vitro and in vivo. Securin is required for genome stability, and is expressed abundantly in most cancer cells, promoting cell proliferation and tumorigenesis. In this study, we found that BPR0L075 efficiently induced cell death of HCT116 human colorectal cancer cells that have higher expression levels of securin. The cytotoxicity of BPR0L075 was attenuated in isogenic securin-null HCT116 cells. BPR0L075 induced DNA damage response, G2/M arrest, and activation of the spindle assembly checkpoint in HCT116 cells. Interestingly, BPR0L075 induced phosphorylation of securin. BPR0L075 withdrawal resulted in degradation of securin, mitotic exit, and mitotic catastrophe, which were attenuated in securin-null cells. Inhibition of cdc2 decreased securin phosphorylation, G2/M arrest and cell death induced by BPR0L075. Moreover, BPR0L075 caused cell death through a caspase-independent mechanism and activation of JNK and p38 MAPK pathways. These findings provided evidence for the first time that BPR0L075 treatment is beneficial for the treatment of human colorectal tumors with higher levels of securin. Thus, we suggest that the expression levels of securin may be a predictive factor for application in anti-cancer therapy with BPR0L075 in human cancer cells.

Thailandepsins are new small molecule class I HDAC inhibitors with potent cytotoxic activity in ovarian cancer cells: a preclinical study of epigenetic ovarian cancer therapy

BackgroundNew treatment strategies are emerging to target DNA damage response pathways in ovarian cancer. Our group has previously shown that the class I biased HDAC inhibitor romidepsin (FK228) induces DNA damage response and has potent cytotoxic effects in ovarian cancer cells. Here, we investigated newly discovered HDAC inhibitors, thailandepsin A (TDP-A) and thailandepsin B (TDP-B), to determine the effects on cell viability, apoptosis and DNA damage response in ovarian cancer cells.MethodsFK228, TDP-A and TDP-B were tested in five ovarian cancer cell lines. Cellular viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Immunofluorescence assays were used to assess activated caspase 3. Western blots were performed to detect protein expression of PARP cleavage, pH2AX, P-glycoprotein and tubulin acetylation.ResultsTreatment with TDPs decreased cell viability at nanonomolar concentrations in four of the five ovarian cancer cell lines studied. Similar to FK228, both TDP compounds exerted minimal effects on NCI/ADR-RES ovarian cancer cells. Across the four cell lines sensitive to the TDPs, TDP-B consistently had a greater inhibitory effect than TDP-A on cell viability. TDP-B also had relatively greater effects on promoting cell apoptosis and induction of pH2AX (a mark of DNA damage response), than TDP-A. These antitumor effects of TDP-B were of similar magnitude to those induced by an equal concentration of FK228. Similar to FK228, the nanomolar concentrations of the TDPs had little effect on tubulin acetylation (a mark of class II HDAC6 inhibition).ConclusionsThe new small molecule HDAC inhibitors TDP-A and TDP-B are FK228 analogues that suppress cell viability and induce apoptosis at nanomolar drug concentrations. TDP-B showed the most similarity to the biological activity of FK228 with greater cytotoxic effects than TDP-A in vitro. Our results indicate that FK228-like small molecule class I HDAC-biased HDAC inhibitors have therapeutic potential for ovarian cancer.

Abstract 1958: T-oligo mechanism of action and delivery with an alpha-helical peptide

Abstract When telomeres are critically short, the telomere overhang is exposed which induces cell senescence or apoptosis. T-oligo, an oligonucleotide homologous to the telomere overhang, induces DNA damage responses similar to those induced after exposure of the telomere overhang, and is an effective, cancer-specific therapy both in vitro and in vivo. However, T-oligo's mechanism of action is unclear, and as an oligonucleotide, its inherent instability severely limits its clinical use. Hence, our studies focus on its mechanism of action, stabilization and improved delivery. To understand T-oligo's mechanism of action, we studied the role of the telomere specific poly(ADP-ribose) polymerase, tankyrase-1, in T-oligo induced DNA damage responses. In normal cells, tankyrase-1 PARsylates TRF1 which causes disruption of the shelterin complex and exposure of the T-loop. This exposure activates telomerase resulting in elongation of telomeres leading to possible cell immortality. This mechanism has made tankyrase-1 inhibition a potential therapy for telomerase inhibitor resistant cancers where tankyrase-1 is found to be upregulated. Since several studies suggest that tankyrase-1 and T-oligo may have important roles in p53 mediated DNA damage responses to critically shortened telomeres, we measured total and phospho-p53 in MU melanoma cells after treatment with tankyrase-1 inhibitors by immunoblot analysis. We found that cells treated with T-oligo and tankyrase-1 inhibitors, 3AB and XAV939, exhibited a 4 and 2 fold down regulation of phospho-p53, and 2.5 and 2 fold down regulation of total p53, respectively, compared to T-oligo alone which increased phospho-p53 and total p53 by 4 and 2 fold, respectively. Furthermore, XAV939, when given with T-oligo, completely abrogated T-oligo induced inhibition of cell proliferation, indicating that tankyrase-1 may play a role in T-oligo induced DNA damage responses. To improve T-oligo's delivery, we combined T-oligo with a nano-sized cationic α-helical polypeptide (PVBLG150-8) which stabilizes the negatively charged T-oligo. We complexed and delivered T-oligo with PVBLG150-8 into H358 lung cancer and AN melanoma cells. We found that in vitro delivery of the T-oligo and peptide (0.025-.25 mg/ml) complex inhibited AN melanoma cell growth in a dose responsive manner from 4.5-8.3 fold compared to untreated controls, while T-oligo alone inhibited growth by only 3 fold. Similarly, when H358 cells were given T-oligo complexed with PVBLG150-8 (0.0125-.25 mg/ml), their growth was inhibited in a dose dependent manner from 3.3-6.6 fold, compared to T-oligo alone (2 fold). Our studies indicate that T-oligo has great potential as an anti-cancer therapy, and due to its unique mechanism of action, it could be used in patients with upregulated tankyrase-1 activity. Additionally, T-oligo delivery and efficacy can be greatly improved when complexed with a cationic α-helical polypeptide. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1958. doi:1538-7445.AM2012-1958

Silencing thioredoxin induces liver cancer cell senescence under hypoxia

Although thioredoxin 1 (TXN) has pleiotropic cellular functions as a redox-sensitive protein, very little is known about its role in tumor survival and growth under hypoxia. MHCC97H hepatocellular carcinoma cells have a high metastatic potential and high thioredoxin expression levels compared with their parent cell line, MHCC97. Thus, we used this cell line to explore the functional connections between TXN and hypoxia. MHCC97H cells were cultured under normoxia and hypoxia for specific periods after nucleofection with TXN siRNA or control siRNA. We assessed the β-phenylethyl isothiocyanate (PEITC) sensitivity of the cells, cell proliferation, cell cycle and senescence, and DNA damage response by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, colony formation assays, flow cytometry, β-galactosidase staining, western blotting, and immunohistochemistry. β-phenylethyl isothiocyanate treatment shifted reduced TXN to oxidized TXN in MHCC97H cells. Although silencing of TXN via siRNA had no effect on the PEITC sensitivity of the cells, it suppressed cell proliferation and colony formation under both normoxia and hypoxia. Under hypoxia, silencing TXN did not induce apoptosis but induced DNA damage response and cellular senescence. High TXN levels in MHCC97H cells protect them from DNA damage and cellular senescence under hypoxia. Targeting TXN might enhance the chemotherapeutic effects of some DNA-damaging agents against hepatocellular carcinoma.

DNA-Damage-Induced Differentiation in Hematopoietic Stem Cells

Aging of hematopoietic stem cells (HSCs) is accompanied by diminished functional potential. Wang et al. now provide evidence for an HSC-specific differentiation checkpoint mediated by the transcription factor BATF, which limits self-renewal of HSCs in response to the accumulation of DNA damage.

Two sides of the Myc-induced DNA damage response: from tumor suppression to tumor maintenance

Activation of oncogenes is generally associated with the induction of DNA damage response (DDR) signaling, which acts as a barrier to tumor progression. In this review we will present an overview of the DDR associated with oncogenic activation of Myc, with special focus on two opposite and paradoxical aspects of this response: (1) the role of the Myc-induced DDR in tumor suppression; (2) its role in dampening Myc-induced replication stress, thereby protecting the viability of prospective cancer cells. These opposing effects on cancer progression are controlled by two different branches of DDR signaling, respectively ATM/CHK2 and ATR/CHK1. Indeed, while ATM activity constitutes a barrier to malignant transformation, full activation of ATR and CHK1 is essential for tumor maintenance, providing important opportunities for therapeutic intervention. Thus, the Myc-induced DDR acts as a double-edged sword in tumor progression.

Pteridium aquilinum and Its Ptaquiloside Toxin Induce DNA Damage Response in Gastric Epithelial Cells, a Link With Gastric Carcinogenesis

The multifactorial origin of gastric cancer encompasses environmental factors mainly associated with diet. Pteridium aquilinum-bracken fern-is the only higher plant known to cause cancer in animals. Its carcinogenic toxin, ptaquiloside, has been identified in milk of cows and groundwater. Humans can be directly exposed by consumption of the plant, contaminated water or milk, and spore inhalation. Epidemiological studies have shown an association between bracken exposure and gastric cancer. In the present work, the genotoxicity of P. aquilinum and ptaquiloside, including DNA damaging effects and DNA damage response, was characterized in human gastric epithelial cells and in a mouse model. In vitro, the highest doses of P. aquilinum extracts (40 mg/ml) and ptaquiloside (60 μg/ml) decreased cell viability and induced apoptosis. γH2AX and P53-binding protein 1 analysis indicated induction of DNA strand breaks in treated cells. P53 level also increased after exposure, associated with ATR-Chk1 signaling pathway activation. The involvement of ptaquiloside in the DNA damage activity of P. aquilinum was confirmed by deregulation of the expression of a panel of genes related to DNA damage signaling pathways and DNA repair, in response to purified ptaquiloside. Oral administration of P. aquilinum extracts to mice increased gastric cell proliferation and led to frameshift events in intron 2 of the P53 gene. Our data demonstrate the direct DNA damaging and mutagenic effects of P. aquilinum. These results are in agreement with the carcinogenic properties attributed to this fern and its ptaquiloside toxin and support their role in promoting gastric carcinogenesis.

The association of DNA damage response and nucleotide level modulation with the antibacterial mechanism of the anti-folate drug Trimethoprim

BackgroundTrimethoprim is a widely prescribed antibiotic for a variety of bacterial infections. It belongs to a class of anti-metabolites - antifolates - which includes drugs used against malarial parasites and in cancer therapy. However, spread of bacterial resistance to the drug has severely hampered its clinical use and has necessitated further investigations into its mechanism of action and treatment regimen. Trimethoprim selectively starves bacterial cells for tetrahydrofolate, a vital cofactor necessary for the synthesis of several metabolites. The outcome (bacteriostatic or bactericidal) of such starvation, however, depends on the availability of folate-dependent metabolites in the growth medium. To characterize this dependency, we investigated in detail the regulatory and structural components of Escherichia coli cellular response to trimethoprim in controlled growth and supplementation conditions.ResultsWe surveyed transcriptional responses to trimethoprim treatment during bacteriostatic and bactericidal conditions and analyzed associated gene sets/pathways. Concurrent starvation of all folate dependent metabolites caused growth arrest, and this was accompanied by induction of general stress and stringent responses. Three gene sets were significantly associated with the bactericidal effect of TMP in different media including LB: genes of the SOS regulon, genes of the pyrimidine nucleotide biosynthetic pathway and members of the multiple antibiotic resistance (mar) regulon controlled by the MarR repressor. However, the SOS response was identified as the only universal transcriptional signature associated with the loss of viability by direct thymine starvation or by folate stress. We also used genome-wide gene knock-out screen to uncover means of sensitization of bacteria to the drug. We observed that among a number of candidate genes and pathways, the effect of knock-outs in the deoxyribose nucleotide salvage pathway, encoded by the deoCABD operon and under the control of the DeoR repressor, was most informative.ConclusionTranscriptional induction of DNA damage response is an essential feature of the bactericidal effect of trimethoprim. Either the observation of the transcriptional response or DNA damage itself, or both, is made possible by thymine starvation when other folate-dependent metabolites are not limited. The effect of DNA damage by the drug takes place prior to its bactericidal effect, at the beginning of the lag stage of the treatment. Mutations in the deoxyribose nucleotide salvage pathway can affect duration of the lag as well as the rate of killing. This information can be used to postulate certain mechanistic differences between direct thymine starvation in thymidylate synthase deficient mutants and thymine starvation by anti-folate inhibitors.

Abstract B72: Assessing specific biological mechanisms of drug resistance in adult and pediatric AML.

Abstract Background: Acute myeloid leukemias (AML) non-responsive to induction chemotherapy generally have poor prognoses. Understanding the biological mechanisms of drug resistance, or sensitivity, specific to each individual AML could inform biologically-based treatment selection in the salvage setting. The current study was undertaken to identify resistance mechanisms to commonly used AML drugs. Methods: Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Herein we use SCNP to investigate biological mechanisms of in vitro resistance to chemotherapeutics relevant to AML treatment (Ara-C/Daunorubicin, Gemtuzumab Ozogamicin, Decitabine, Azacitidine and Clofarabine) by assessing in a quantitative fashion the induction of DNA Damage Responses (as measured by phospho-Chk2 and phospho-H2A.X levels) and apoptosis (as measured by cleaved PARP levels and loss of membrane integrity) in 6 pediatric and 6 elderly AML samples. In addition, SCF and FLT3L induced survival signaling (as measured by phospho-Akt and phospho-Erk levels) were simultaneously assessed. Results: Treatment with Ara-C/Dauno and GO revealed cross-resistance among AML samples and was associated with elevated SCF induced PI3K/Erk survival signaling. In vitro treatment of AML samples with the different genotoxic agents (Ara-C/Dauno, GO or CLO), resulted in three distinct profiles of DNA Damage and apoptosis responses: A) Samples with induced DNA Damage Response and apoptosis, B) Samples with induced DNA Damage response but no detectable apoptosis and C) Samples lacking significant induced DNA Damage response and apoptosis. In the latter group the presence of drug efflux activity in some samples, as measured by sensitization with an MDR1 (MultiDrug Resistance) inhibitor, may explain the observation. Of note, for each sample the drug response varied with the specific agent tested. Differential sensitivity among individual patient samples was also observed between treatment with two hypomethylating agents (Decitabine and Azacitidine). Additionally, distinct kinetics of DNA Damage and apoptosis were observed among drugs: genotoxic agents (CLO, Ara/Dauno, GO) induced DNA Damage prior to induction of apoptosis while hypomethylating agents (Decitabine and Azaciditine) induced DNA Damage coincident with induction of apoptosis. Conclusions: Using SCNP to dissect in vitro response to clinically used AML drugs reveals: 1) heterogeneity of individual patient biology characteristic of AML, 2) distinct resistance mechanisms present in unique samples and 3) mechanistic differences between distinct classes of agents. Studies are ongoing to assess the clinical relevance of these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B72.

PML contributes to p53-independent p21 up-regulation in gamma-irradiation induced DNA damage responses

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is a critical cell cycle regulator which translocates into the nucleus to participate in DNA repair during DNA damage responses. In the present study, we showed that the tumor suppressor, promyelocytic leukemia protein (PML) contributes to the up-regulation of p21 in a p53-independent pathway. Knock-down of PML in p53-null H1299 and HCT 116 (p53−/−) tumor cells by specific siRNA resulted in down-regulation of p21 protein expression, inhibition of γ-irradiation-induced p21 up-regulation, and a decrease in p21 protein half-life. In PML knockdown H1299 cells, the down-regulation of p21 protein expression was reversed by MG132 treatment indicating that the proteasomal degradation of p21 protein was increased. Thus, PML positively regulates p21 expression by inhibiting proteasome-mediated proteolysis. Knockdown of PML decreased the repair of γ-irradiation-induced double strand breaks (DSBs) as indicated by the delayed disappearance of γ-H2AX foci and a decreased association between p21 and proliferating cell nuclear antigen (PCNA). Over-expression of p21 significantly restored the delayed DSB repair function. Taken together, these data provide evidence for a p53-independent functional relationship between PML and p21 in γ-irradiation-induced DNA damage responses, and identify PML as a positive post-translational regulator of p21 in p53-deficient tumor cells.

Separation of Recombination and SOS Response in Escherichia coli RecA Suggests LexA Interaction Sites

RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET) analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root.

Activation of the aryl hydrocarbon receptor is the major toxic mode of action of an organic extract of a reference urban dust particulate matter mixture: The role of polycyclic aromatic hydrocarbons

Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo- p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.

Homeodomain transcription factor and tumor suppressor Prep1 is required to maintain genomic stability

Prep1 is a homeodomain transcription factor that is essential in embryonic development and functions in the adult as a tumor suppressor. We show here that Prep1 is involved in maintaining genomic stability and preventing neoplastic transformation. Hypomorphic homozygous Prep1(i/i) fetal liver cells and mouse embryonic fibroblasts (MEFs) exhibit increased basal DNA damage and normal DNA damage response after γ-irradiation compared with WT. Cytogenetic analysis shows the presence of numerous chromosomal aberrations and aneuploidy in very early-passage Prep1(i/i) MEFs. In human fibroblasts, acute Prep1 down-regulation by siRNA induces DNA damage response, like in Prep1(i/i) MEFs, together with an increase in heterochromatin-associated modifications: rapid increase of histone methylation and decreased transcription of satellite DNA. Ectopic expression of Prep1 rescues DNA damage and heterochromatin methylation. Inhibition of Suv39 activity blocks the chromatin but not the DNA damage phenotype. Finally, Prep1 deficiency facilitates cell immortalization, escape from oncogene-induced senescence, and H-Ras(V12)-dependent transformation. Importantly, the latter can be partially rescued by restoration of Prep1 level. The results show that the tumor suppressor role of Prep1 is associated with the maintenance of genomic stability.

Telomere dysfunction and cell cycle checkpoints in hematopoietic stem cell aging

Stem cells are believed to be closely associated with tissue degeneration during aging. Studies of human genetic diseases and gene-targeted animal models have provided evidence that functional decline of telomeres and deregulation of cell cycle checkpoints contribute to the aging process of tissue stem cells. Telomere dysfunction can induce DNA damage response via key cell cycle checkpoints, leading to cellular senescence or apoptosis depending on the tissue type and developmental stage of a specific stem cell compartment. Telomerase mutation and telomere shortening have been observed in a variety of hematological disorders, such as dyskeratosis congenital, aplastic anemia, myelodysplastic syndromes and leukemia, in which the hematopoietic stem cells (HSC) are a major target during the pathogenesis. Moreover, telomere dysfunction is able to induce both cell-intrinsic checkpoints and environmental factors limiting the self-renewal capacity and differentiation potential of HSCs. Crucial components in the cascade of DNA damage response, including ataxia telangiectasia mutated, CHK2, p53, p21 and p16/p19(ARF), play important roles in HSC maintenance and self-renewal in the scenarios of both sufficient telomere reserve and dysfunctional telomere. Therefore, a further understanding of the molecular mechanisms underlying HSC aging may help identity new therapeutic targets for stem cell-based regenerative medicine.

Microarray analysis of global gene expression in Caenorhabditis elegans exposed to potassium dichromate

Hexavalent chromium [Cr (VI)] can be considered as carcinogen heavy metal to human population. Especially, potassium dichromate [K2Cr2O7; Cr (VI)] induces DNA damage response and oxidative stress. It also causes inhibition of protective process including DNA repair as well as apoptosis. However, genomic responses to Cr (VI) exposure in Caenorhabditis elegans (C. elegans) have not been performed yet. As an alternative animal model, C. elegans is well known model for genetical studies including mRNA expression (etc.) and also serves as a living biomonitor in ecotoxicological field. Moreover, recognition of chromium biomarkers for the toxicity assessment is of prime importance. In present study, we have investigated the changes of gene expression in C. elegans in response to potassium dichromate exposure using microarray consisting of 22K nematode-specific oligonucleotide probes. We have found 28 genes as a Cr (VI) responsive genes that were differentially expressed (>2 fold) following 24 hours exposure to potassium dichromate. In addition, using the comparative toxicogenomics database, we have deduced molecular targets including cyclin B, alpha-B crystalline, G-protein coupled receptor kinase, nucleobindin, U2AF splicing factor, and SR protein (splicing factor) in response to potassium dichromate toxicity in C. elegans. In consistency with previous studies in human, it has also found that alteration in expression level of these genes contributes to particular syndromes including cataract, prostatic neoplasms, liver disease and neurotoxicity syndromes. In conclusion, our investigation would be able to present precise route to figure out important biomarkers in response to potassium dichromate in the field of ecotoxicology.

Requirement of DDX39 DEAD box RNA helicase for genome integrity and telomere protection

Human chromosome ends associate with shelterin, a six-protein complex that protects telomeric DNA from being recognized as sites of DNA damage. The shelterin subunit TRF2 has been implicated in the protection of chromosome ends by facilitating their organization into the protective capping structure and by associating with several accessory proteins involved in various DNA transactions. Here we describe the characterization of DDX39 DEAD-box RNA helicase as a novel TRF2-interacting protein. DDX39 directly interacts with the telomeric repeat binding factor homology domain of TRF2 via the FXLXP motif (where X is any amino acid). DDX39 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT but has no effect on telomerase activity. Whereas overexpression of DDX39 in telomerase-positive human cancer cells led to progressive telomere elongation, depletion of endogenous DDX39 by small hairpin RNA (shRNA) resulted in telomere shortening. Furthermore, depletion of DDX39 induced DNA-damage response foci at internal genome as well as telomeres as evidenced by telomere dysfunction-induced foci. Some of the metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an aberrant telomere structure. Our findings suggest that DDX39, in addition to its role in mRNA splicing and nuclear export, is required for global genome integrity as well as telomere protection and represents a new pathway for telomere maintenance by modulating telomere length homeostasis.

Abstract 2636: Mechanisms of therapy-induced senescence with ABT-737

Abstract Aberrant expression of the antiapoptotic Bcl-2 family of proteins in human cancer is correlated with poor outcomes after standard chemotherapy. ABT-737 is a cell permeable Bcl-2 family antagonist that is capable of binding to Bcl-2, Bcl-xL, and Bcl-w. ABT-737 functions by displacing BH3 proteins, such as Bim, from these proteins activating Bax and Bak to induce apoptotic cell death. Resistance to ABT-737 appears to be mediated by the elevated expression of Mcl-1 or Bfl-1/A1 which are not capable of binding this compound. Since renal, prostate and lung carcinomas, exhibit elevated levels of endogenous Mcl-1 many of these tumor cell lines are resistant to killing by this agent. Gene chip analysis in order to understand anticancer action of ABT-737 in human cancer cells reveals an intrigue data that ABT-737 regulates key genes involving cytokines and chemokines associated with senescence phenotype. Because senescence is considered to be a key mechanism that protects against cancer development, we investigated the mechanism of action for ABT-737 in human cancer cells where this compound is incapable of inducing apoptotic cell death, inhibit growth of tumor cells by the induction of cell senescence. Here, we identify a novel pathway involving the induction of DNA damage response by caspase-3 cleavage that normally induces apoptosis, which instead leads to promotion of cellular senescence. Treatment of renal, lung and prostate cancer cell lines with ABT-737, but not an inactive enantiomer, caused the induction of senescence-associated β-galactosidase, and additional changes associated with the senescence phenotype including increases in specific gene transcription, secretion of IL-6 and IL-8, and stimulation of the activity of C/EBPβ. ABT-737 treatment of these cell lines induced activation of the DNA damage response pathway as demonstrated by increases in γ-H2AX and phosphorylation of both the ATM and Chk2 protein kinases. Decreasing ATM levels with shRNA blocked the senescent phenotype suggesting that this pathway was critical for the induction of ABT-737-mediated senescence. DNA damage induced by ABT-737 treatment was caused by low level activation of the caspase cascade, which was blocked by decreasing caspase-3 levels with shRNA. ABT-737-induced senescence appeared to be p53-dependent, since the overexpression of dominant-negative p53 inhibited the ABT-737-induced increases in p21 protein levels, β-galactosidase, and IL-6 transcription. These findings suggest that in multiple cancer cell types where ABT-737 is incapable of inducing sufficient DNA damage to activate apoptosis, it instead inhibits cancer cell growth by inducing senescence, a phenotype that is associated with major changes in gene transcription and protein secretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2636. doi:10.1158/1538-7445.AM2011-2636

Late Activation of Stress-activated Protein Kinases/c-Jun N-terminal Kinases Triggered by Cisplatin-induced DNA Damage in Repair-defective Cells

Although stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) are rapidly activated by genotoxins, the role of DNA damage in this response is not well defined. Here we show that the SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK (Thr-183/Tyr-185) correlates with the level of cisplatin-DNA adducts at late times (16-24 h) after drug treatment in both human and mouse cells. Transfection of platinated plasmid DNA also caused SAPK/JNK activation. A defect in transcription-coupled nucleotide excision repair resting on a mutation in Cockayne syndrome group B protein promoted the late SAPK/JNK activation following cisplatin exposure. Signaling to SAPK/JNK was accompanied by activation of Ataxia telangiectasia mutated- and Rad3-related kinase, replication protein A, and checkpoint kinases as well as by the formation of DNA double strand breaks (DSBs). Ionizing radiation-induced DSBs did not provoke SAPK/JNK activation, and inhibition of transcription also failed to provoke this response. Late activation of SAPK/JNK stimulated by cisplatin-induced DNA lesions was reduced in the absence of specific DNA repair proteins, such as xeroderma pigmentosum protein C, pointing to an essential function of individual repair factors in DNA damage signaling to SAPK/JNK. Collectively, the data indicate that late SAPK/JNK activation is triggered by non-repaired cisplatin adducts in transcribed genes and involves replication-associated events, DSBs, tyrosine kinases, Rho GTPases, and specific repair factors.

S1P lyase regulates DNA damage responses in vitro and in vivo

The injurious consequences of radiation to normal human cells and the acquired radiation resistance of cancer cells represent limitations to cancer radiotherapy. Radiation induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair and apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly, radiation activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in radiation resistant cancer cells. S1P lyase (SPL) catalyzes degradation of S1P in the final step of sphingolipid metabolism. We show that by reducing S1P and enhancing ASMase-mediated ceramide generation, SPL modulates the kinetics of DNA repair, the speed of recovery from G2 cell cycle arrest, and the extent of apoptosis after irradiation. In contrast, inhibiting SPL in vitro enhances DNA repair, and oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body irradiation. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.

Ionizing radiation-induced TAp63α phosphorylation at C-terminal S/TQ motifs requires the N-terminal transactivation (TA) domain

TAp63α, a homolog of p53 and one of six alternatively spliced p63 isoforms, is a critical mediator of the ionizing radiation (IR)-induced DNA damage response in female germ cells and also tumor suppression in somatic cells. The ΔNp63α isoform, lacking the N-terminal transactivation (TA) domain, is associated with oncogenic potential. The mechanism of p63 functional regulation is not well understood. TAp63α is phosphorylated by ionizing radiation (IR)-induced DNA damage and gene transactivation is likely to be involved. Based on information gleaned from studies on p53, we explored the possibility that TAp63α S/TQ sites may be phosphorylated by IR-induced DNA damage. Our findings show a wortmanin-sensitive kinase phosphorylates TAp63α at C-terminal Ser-Gln and Thr-Gln (S/TQ) sites but not N-terminal S/TQ sites. ΔNp63α, lacking the TA domain, and TAp63γ, lacking C-terminal domains, including S/TQ sites, fail to undergo IR-induced phosphorylation. We propose a model for TA domain-dependent C-terminal phosphorylation drawing from previously described self-inactivating intramolecular interaction between N-terminal TA domain and C-terminal Transactivation Inhibitory Domain (TID) of TAp63α. A specific topology adopted only by TAp63α, but not possible for ΔNp63α or TAp63γ, may lead to TAp63α-specific kinase recruitment, phosphorylation and self-inactivation release. TID-lacking TAp63γ, like p53, is constitutively active and thus may forgo phosphorylation-dependent activation. Thus, p53 is regulated by protein stabilization and TAp63α by protein activation but both appear to involve S/TQ phosphorylation. The difference in phosphorylation potential of TAp63α and ΔNp63α may in part help explain why the two similar isoforms have diametrically opposite tumor suppression and oncogene functions, respectively.