And Rad3-related Protein Research Articles

Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression.

Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.

WWOX modulates the ATR-mediated DNA damage checkpoint response.

For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability.

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos.

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.

IFN-γ Induces Mimic Extracellular Trap Cell Death in Lung Epithelial Cells Through Autophagy-Regulated DNA Damage.

Treatment of interferon-γ (IFN-γ) causes cell growth inhibition and cytotoxicity in lung epithelial malignancies. Regarding the induction of autophagy related to IFN-γ signaling, this study investigated the link between autophagy and IFN-γ cytotoxicity. In A549 human lung cancer cells, IFN-γ treatment induced concurrent apoptotic and nonapoptotic events. Unexpectedly, the nonapoptotic cells present mimic extracellular trap cell death (ETosis), which was regulated by caspase-3 and by autophagy induction through immunity-related GTPase family M protein 1 and activating transcription factor 6. Furthermore, IFN-γ signaling controlled mimic ETosis through a mechanism involving an autophagy- and Fas-associated protein with death domain-controlled caspase-8/-3 activation. Following caspase-mediated lamin degradation, IFN-γ caused DNA damage-associated ataxia telangiectasia and Rad3-related protein (ATR)/ataxia telangiectasia mutated (ATM)-regulated mimic ETosis. Upon ATR/ATM signaling, peptidyl arginine deiminase 4 (PAD4)-mediated histone 3 citrullination promoted mimic ETosis. Such IFN-γ-induced effects were defective in PC14PE6/AS2 human lung cancer cells, which were unsusceptible to IFN-γ-induced autophagy. Due to autophagy-based caspase cascade activation, IFN-γ triggers unconventional caspase-mediated DNA damage, followed by ATR/ATM-regulated PAD4-mediated histone citrullination during mimic ETosis in lung epithelial malignancy.

A physical association between the human mutY homolog (hMYH) and DNA topoisomerase II-binding protein 1 (hTopBP1) regulates Chk1-induced cell cycle arrest in HEK293 cells.

BackgroundHuman DNA topoisomerase II-binding protein 1 (hTopBP1) plays an important role in DNA replication and the DNA damage checkpoint pathway. The human mutY homolog (hMYH) is a base excision repair DNA glycosylase that excises adenines or 2-hydroxyadenines that are mispaired with guanine or 7,8-dihydro-8-oxoguanine (8-oxoG). hTopBP1 and hMYH were involved in ATR-mediated Chk1 activation, moreover, both of them were associated with ATR and hRad9 which known as checkpoint-involved proteins. Therefore, we investigated whether hTopBP1 interacted with hMYH, and what the function of their interaction is.ResultsWe documented the interaction between hTopBP1 and hMYH and showed that this interaction increased in a hydroxyurea-dependent manner. We also mapped the hMYH-interacting region of hTopBP1 (residues 444–991). In addition, we investigated several cell cycle-related proteins and found that co-knockdown of hTopBP1 and hMYH significantly diminished cell cycle arrest due to compromised checkpoint kinase 1 (Chk1) activation. Moreover, we observed that hMYH was essential for the accumulation of hTopBP1 on damaged DNA, where hTopBP1 interacts with hRad9, a component of the Rad9-Hus1-Rad1 complex. The accumulation of hTopBP1 on chromatin and its subsequent interaction with hRad9 lead to cell cycle arrest, a process mediated by Chk1 phosphorylation and ataxia telangiectasia and Rad3-related protein (ATR) activation.ConclusionsOur results suggested that hMYH is necessary for the accumulation of hTopBP1 to DNA damage lesion to induce the association of hTopBP1 with 9-1-1 and that the interaction between hMYH and hTopBP1 is essential for Chk1 activation. Therefore, we suggest that the interaction between hMYH and hTopBP1 is crucial for activation of the ATR-mediated cell cycle checkpoint.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0042-x) contains supplementary material, which is available to authorized users.

Abstract A34: MC1R signaling reduces UV mutagenesis by ATR-mediated recruitment of XPA to photolesions

Abstract The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic Gs-coupled transmembrane receptor activated by binding to its high-affinity ligand, α-melanocyte stimulating hormone (MSH). Besides influencing pigment phenotype and mediating adaptive tanning, MC1R signaling is intricately linked with genome maintenance and DNA repair. Individuals harboring loss-of-function MC1R polymorphisms are UV sensitive and melanoma-prone. We have determined that MC1R-mediated cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435 which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing UV mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA and elevates UV-induced mutagenesis. PKA-mediated ATR phosphorylation on Ser435 appears to induce a DNA repair-specific function of ATR independent of Chk1 phosphorylation and cell cycle arrest. Importantly, MC1R signaling defects and sub-optimal DNA repair are overcome by forskolin-mediated cAMP induction in melanocytes and in whole skin. Our findings mechanistically link cAMP-PKA signaling to NER and illustrate the potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals. Citation Format: Stuart Gordon Jarrett, Erin M. Wolf Horrell, Perry A. Christian, Jillian C. Vanover, Mary C. Boulanger, Yue Zou, John August D'Orazio. MC1R signaling reduces UV mutagenesis by ATR-mediated recruitment of XPA to photolesions. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A34.

Fe65 Ser228 is phosphorylated by ATM/ATR and inhibits Fe65-APP-mediated gene transcription.

Fe65 binds the amyloid precursor protein (APP) and regulates the secretase-mediated processing of APP into several proteolytic fragments, including amyloid β-peptides (Aβ) and APP intracellular domain (AICD). Aβ accumulation in neural plaques is a pathological feature of Alzheimer's disease (AD) and AICD has important roles in the regulation of gene transcription (in complex with Fe65). It is therefore important to understand how Fe65 is regulated and how this contributes to the function and/or processing of APP. Studies have also implicated Fe65 in the cellular DNA damage response with knockout mice showing increased DNA strand breaks and Fe65 demonstrating a gel mobility shift after DNA damage, consistent with protein phosphorylation. In the present study, we identified Fe65 Ser(228) as a novel target of the ATM (ataxia telangiectasia mutated) and ATR (ataxia-telangiectasia- and Rad3-related protein) protein kinases, in a reaction that occurred independently of APP. Neither phosphorylation nor mutation of Ser(228) affected the Fe65-APP complex, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Finally, mutation of Ser(228) to alanine (thus blocking phosphorylation) caused a significant increase in Fe65-APP transcriptional activity, whereas phosphomimetic mutants (S(228)D and S(228)E) showed decreased transcriptional activity. These studies identify a novel phosphorylation site within Fe65 and a novel regulatory mechanism for the transcriptional activity of the Fe65-APP complex.

Suppression of ATR reverses the cisplatin resistance in ovarian cancer SKOV3 cells

To explore the effect of ataxia telangiectasia mutated and RAD3 related protein (ATR) expression and ATR kinase activity on the sensitivity to cisplatin in ovarian cancer SKOV3 cells. SiRNA targeting ATR was transfected into SKOV3 cells for 48 h to reduce the ATR protein level, and ATR kinase inhibitor VE-821 was used for 12 h to inhibit the ATR pathway activity. The alteration of cell viability was examined by CCk-8 assay. Expression levels of ATR, p-ATR and γ-H2AX proteins were detected by Western blot. The DNA double strand breaks (DSB) marker γ-H2AX and homologous recombination repair key protein RAD51 and their co-localization in the cells were examined under the confocal microscope. The status of DNA double strand breaks (DSB) in single cells was visualized by alkaline comet assay. Finally, the cell cycle distribution was assessed using flow cytometry. DDP caused evident DNA double strands breaks and activated ATR kinase pathway. ATR-siRNA notably reduced ATR protein level, the 48 h IC(50) value of DDP was 72.12 µmol/L and 41.25 µmol/L, respectively, in the NC-siRNA and ATR-siRNA groups (P < 0.05). Confocal microscopic assay presented decreased recruitment of RAD51 at the DSB loci and comet assay showed enhanced DSB in the cells after ATR knocking down. After the inhibition of ATR kinase by VE-821, the 48 h IC(50) value of DDP was 75.32 µmol/L and 45.64 µmol/L, respectively, in the DMSO and VE-821 groups (P < 0.05 for both), confocal microscopic assay demonstrated reduced RAD51 recruitment, and comet assay showed increased DSB in cells after ATR kinase inhibition. Flow cytometry showed that percentage of cells distributed in G(0)/G(1), S and G(2)/M phases was 71.2%, 13.4% and 15.4%, repectively, after 40 µmol/L DDP treatment for 24 h. Compared with that of control group (G(0)/G(1): 54.2%, S: 21.3% and G(2)/M: 24.4%), DDP induced G(0)/G(1) phase arrest. DDP intervention resulted in the cell cycle status (G(0)/G(1): 43.2%, S: 20.4%, G(2)/M: 36.4%) in the ATR-siRNA group and (G(0)/G(1): 40.2%, S: 22.5%, G(2)/M: 37.3%) in the VE-821 group, indicating that the inhibition of ATR or ATR kinase could abrogate the effect of G(0)/G(1) phase arrest induced by DDP. Suppression of ATR can affect the homologous recombination repair in ovarian cancer cells, leading to accumulation of DNA double strand breaks in the cell nuclei as well as reduction of DDP-caused G(0)/G(1) phase arrest, finally enhances the sensitivity to cisplatin in the ovarian cancer SKOV3 cells.

Gli1 Protein Regulates the S-phase Checkpoint in Tumor Cells via Bid Protein, and Its Inhibition Sensitizes to DNA Topoisomerase 1 Inhibitors

Aberrant expression of hedgehog molecules, particularly Gli1, is common in cancers of many tissues and is responsible for their aggressive behavior and chemoresistance. Here we demonstrate a novel and tumor-specific role for aberrant Gli1 in the regulation of the S-phase checkpoint that suppresses replication stress and resistance to chemotherapy. Inhibition of Gli1 in tumor cells induced replication stress-mediated DNA damage response, attenuated their clonogenic potential, abrogated camptothecin (CPT)-induced Chk1 phosphorylation, and potentiated its cytotoxicity. However, in normal fibroblasts, Gli1 siRNAs showed no significant changes in CPT-induced Chk1 phosphorylation. Further analysis of ataxia telangiectasia and Rad3-related protein (ATR)/Chk1 signaling cascade genes in tumor cells revealed an unexpected mechanism whereby Gli1 regulates ATR-mediated Chk1 phosphorylation by transcriptional regulation of the BH3-only protein Bid. Consistent with its role in DNA damage response, Bid down-regulation in tumor cells abolished CPT-induced Chk1 phosphorylation and sensitized them to CPT. Correspondingly, Gli1 inhibition affected the expression of Bid and the association of replication protein A (RPA) with the ATR- interacting protein (ATRIP)-ATR complex, and this compromised the S-phase checkpoint. Conversely, complementation of Bid in Gli1-deficient cells restored CPT-induced Chk1 phosphorylation. An in silico analysis of the Bid promoter identified a putative Gli1 binding site, and further studies using luciferase reporter assays confirmed Gli1-dependent promoter activity. Collectively, our studies established a novel connection between aberrant Gli1 and Bid in the survival of tumor cells and their response to chemotherapy, at least in part, by regulating the S-phase checkpoint. Importantly, our data suggest a novel drug combination of Gli1 and Top1 inhibitors as an effective therapeutic strategy in treating tumors that expresses Gli1.

HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability.

CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.

Abstract 1637: Discovery of ATR kinase inhibitors from natural products

Abstract Inhibition of the DNA damage checkpoint and repair functions is a promising approach to improve chemosensitivity. Ataxia telangiectasia and Rad3-related protein (ATR) is an important checkpoint kianse responsible for organizing both normal and stressful DNA replication. In previous, we discovered a natural compound protoapigenone (WYC02) and its synthetic derivate WYC0209 are able to inhibit ATR-mediated Chk1 phosphorylation, impair the G2/M checkpoint, and improve cancer sensitivity to cisplatin both in vitro and in vivo. Although all evidences convince ATR signaling is disrupted by WYC compounds, the effect of compounds on ATR kinase activity is still unknown. In this study, we further provide evidences to prove that WYC02 and WYC0209 are undoubted ATR inhibitors by using of in vitro kinase assay. The data showed WYC0209 inhibits ATR kinase activity at least 4 times greater than WYC02, and enhances MDA-MB-231cells sensitivity to DNA replication-affecting agents such as doxorubicin, mitomycin C, and etoposide, but has little or no effect to mitosis-affecting microtubule inhibitor palcitaxol. The result of this study proposes a strategy to improve most of current chemotherapeutics in which aim to selective disrupting DNA replication in fast growing cancers. Citation Format: Hui-Chun Wang, Alan Yueh-Luen Lee, Chin-Chung Wu, Yang-Chang Wu. Discovery of ATR kinase inhibitors from natural products. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1637. doi:10.1158/1538-7445.AM2014-1637

Constitutive Photomorphogensis Protein1 (COP1) mediated p53 pathway and its oncogenic role

We have reviewed the COP1 mediated tumor suppressor protein p53 pathway and its oncogenic role. COP1 is a negative regulator of p53 and acts as a pivotal controller of p53-Akt death-live switch (Protein kinase B). In presence of p53, COP1 is overexpressed in breast, ovarian, gastric cancers, even without MDM2 (Mouse double minute-2) amplification. Following DNA damage, COP1 is phosphorylated instantly by ATM (Ataxia telangiectasia mutated) and degraded by 14-3-3σ following nuclear export and enhancing ubiquitination. In ATM lacking cell, other kinases, i.e. ATR (ataxia telangiectasia and Rad3-related protein), Jun kinases and DNA-PK (DNA-dependent protein kinase) cause COP1 & CSN3 (COP9 signalosome complex subunit-3) phosphorylation and initiate COP1’s down regulation. Although, it has been previously found that co-knockout of MDM2 and COP1 enhance p53’s half life by eight fold, the reason is still unknown. Additionally, while interacting with p53, COP1 upregulate MDM2’s E3 ubiquitin ligase, Akt, CSN6 (COP9 signalosome 6) activity and inhibit 14-3-3σ’s negative regulation on MDM2 and COP1 itself. Conclusively, there persists an amplification loop among COP1, MDM2, Akt and 14-3-3σ to regulate p53’s stability and activity. However, the role of another tumor suppressor PTEN (phosphatase and tensin homologue) is yet to be discovered. This study provides insight on the molecular genetic pathways related to cancer and might be helpful for therapeutic inventions.

RETRACTED: PKA-Mediated Phosphorylation of ATR Promotes Recruitment of XPA to UV-Induced DNA Damage

The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals.

Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation.

In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb). Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length) and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis.

UV-Induced Nuclear Import of XPA Is Mediated by Importin-α4 in An ATR-Dependent Manner

Xeroderma pigmentosum Group A (XPA) is a crucial factor in mammalian nucleotide excision repair (NER) and nuclear import of XPA from the cytoplasm for NER is regulated in cellular DNA damage responses in S-phase. In this study, experiments were carried out to determine the transport mechanisms that are responsible for the UV (ultraviolet)-induced nuclear import of XPA. We found that, in addition to the nuclear localization signal (NLS) of XPA, importin-α4 or/and importin-α7 are required for the XPA nuclear import. Further investigation indicated that, importin-α4 and importin-α7 directly interacted with XPA in cells. Interestingly, the binding of importin-α4 to XPA was dependent on UV-irradiation, while the binding of importin-α7 was not, suggesting a role for importin-α7 in nuclear translocation of XPA in the absence of DNA damage, perhaps with specificity to certain non-S-phases of the cell-cycle. Consistent with the previous report of a dependence of UV-induced XPA nuclear import on ataxia telangiectasia and Rad3-related protein (ATR) in S-phase, knockdown of ATR reduced the amount of XPA interacting with importin-α4. In contrast, the GTPase XPA binding protein 1 (XAB1), previously proposed to be required for XPA nuclear import, showed no effect on the nuclear import of XPA in our siRNA knockdown analysis. In conclusion, our results suggest that upon DNA damage transport adaptor importin-α4 imports XPA into the nucleus in an ATR-dependent manner, while XAB1 has no role in this process. In addition, these findings reveal a potential new therapeutic target for the sensitization of cancer cells to chemotherapy.

Curcumin suppresses multiple DNA damage response pathways and has potency as a sensitizer to PARP inhibitor

Inhibitors of poly(ADP-ribose) polymerase (PARP) are promising anticancer drugs, particularly for the treatment of tumors deficient in the DNA damage response (DDR). However, it is challenging to design effective therapeutic strategies for use of these compounds against cancers without DDR deficiencies. In this context, combination therapies in which PARP inhibitors are used alongside DDR inhibitors have elicited a great deal of interest. Curcumin, a component of turmeric (Curcuma longa), has been tested in clinical studies for its chemosensitizing potential; however, the mechanisms of chemosensitization by curcumin have not been fully elucidated. This study demonstrates that curcumin suppresses three major DDR pathways: non-homologous end joining (NHEJ), homologous recombination (HR) and the DNA damage checkpoint. Curcumin suppresses the histone acetylation at DNA double-strand break (DSB) sites by inhibiting histone acetyltransferase activity, thereby reducing recruitment of the key NHEJ factor KU70/KU80 to DSB sites. Curcumin also suppresses HR by reducing expression of the BRCA1 gene, which regulates HR, by impairing histone acetylation at the BRCA1 promoter. Curcumin also inhibits ataxia telangiectasia and Rad3-related protein (ATR) kinase (IC50 in vitro = 493 nM), resulting in impaired activation of ATR-CHK1 signaling, which is necessary for HR and the DNA damage checkpoint pathway. Thus, curcumin suppresses three DDR pathways by inhibiting histone acetyltransferases and ATR. Concordantly, curcumin sensitizes cancer cells to PARP inhibitors by enhancing apoptosis and mitotic catastrophe via inhibition of both the DNA damage checkpoint and DSB repair. Our results indicate that curcumin is a promising sensitizer for PARP inhibitor-based therapy.

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.

HDAC turnover, CtIP acetylation and dysregulated DNA damage signaling in colon cancer cells treated with sulforaphane and related dietary isothiocyanates

Histone deacetylases (HDACs) and acetyltransferases have important roles in the regulation of protein acetylation, chromatin dynamics and the DNA damage response. Here, we show in human colon cancer cells that dietary isothiocyanates (ITCs) inhibit HDAC activity and increase HDAC protein turnover with the potency proportional to alkyl chain length, i.e., AITC < sulforaphane (SFN) < 6-SFN < 9-SFN. Molecular docking studies provided insights into the interactions of ITC metabolites with HDAC3, implicating the allosteric site between HDAC3 and its co-repressor. ITCs induced DNA double-strand breaks and enhanced the phosphorylation of histone H2AX, ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase-2 (CHK2). Depending on the ITC and treatment conditions, phenotypic outcomes included cell growth arrest, autophagy and apoptosis. Coincident with the loss of HDAC3 and HDAC6, as well as SIRT6, ITCs enhanced the acetylation and subsequent degradation of critical repair proteins, such as CtIP, and this was recapitulated in HDAC knockdown experiments. Importantly, colon cancer cells were far more susceptible than non-cancer cells to ITC-induced DNA damage, which persisted in the former case but was scarcely detectable in non-cancer colonic epithelial cells under the same conditions. Future studies will address the mechanistic basis for dietary ITCs preferentially exploiting HDAC turnover mechanisms and faulty DNA repair pathways in colon cancer cells vs. normal cells.

Abstract 2447: Activation of Chk1 critical for Piperine induced cell cycle arrest and apoptosis in melanoma cells.

Abstract The National Cancer Institute estimates that about 76,250 new cases of melanomas will be diagnosed in 2012 in the United States leading to at least 9810 related deaths. Therefore, novel strategies to control and treat melanoma are of prime importance. In this study, we determined the cytotoxic effects of Piperine, a major constituent of black (Piper nigrum Linn) and long pepper (Piper longum Linn), in melanoma cell lines. Piperine treatment inhibited the growth of SKMEL-28 and B16-F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine was correlated with the down-regulation of cyclin D1 and induction of p21. The growth arrest was associated with DNA damage as indicated by phosphorylation of H2A.X at Ser139, activation of ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). The G1 arrest by piperine was also associated with the down-regulation of E2F1 and decreased phosphorylation of retinoblastoma protein (Rb), hence indicating the inhibition of the dynamic transcription complex. In addition, apoptosis induced by piperine was associated with down-regulation of XIAP and Bid (uncleaved) and cleavage of Caspase-3 and PARP. Pretreatment of SK-MEL-28 with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Silencing of Chk1 in SK-MEL28 cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine treatment showing the involvement of Chk1. Through immunofluorescence of Phospho Chk1 (Ser 256), we found significant activation of Chk1 in the cells that were treated with piperine hence, establishing the role of Chk1 activation. Furthermore, our results showed that piperine treatment caused ROS generation in melanoma cells. Blocking ROS generation by Tiron pre-treatment prevented the cells from piperine mediated cell cycle arrest. Moreover, western-blotting results showed that Tiron pre treatment prevented the activation of Chk1 hence, confirming the role played by Chk1 in piperine mediated cell cycle arrest. Taken together, our results demonstrate that piperine induced G1 phase cell cycle arrest by generating ROS and inducing DNA damage with activation of Chk1 being a critical event. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Neel M. Fofaria, Sanjay K. Srivastava. Activation of Chk1 critical for Piperine induced cell cycle arrest and apoptosis in melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2447. doi:10.1158/1538-7445.AM2013-2447

The nucleolus stress response is coupled to an ATR-Chk1–mediated G2 arrest

We report experiments on the connection between nucleolar stress and cell cycle progression, using HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator. Nucleolar stress elicited by brief exposure of cells to a low concentration of actinomycin D that selectively inhibits rRNA synthesis had no effect on traverse of G1 or S, but stalled cells in very late interphase. Additional experiments revealed that a switch occurs during a specific temporal window during nucleolar stress and that the subsequent cell cycle arrest is not triggered simply by the stress-induced decline in the synthesis of rRNA or by a ribosome starvation phenomenon. Further experiments revealed that this nucleolus stress-induced cell cycle arrest involves the action of a G2 checkpoint mediated by the ataxia telangiectasia and Rad3-related protein (ATR)-checkpoint kinase 1 (Chk1) pathway. Based on analysis of the cell cycle stages at which this nucleolar stress effect is put into action, to become manifest later, our results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint.