Replication of the 2,6-Diamino-4-hydroxy-<i>N</i>⁵-(methyl)-formamidopyrimidine (MeFapy-dGuo) Adduct by Eukaryotic DNA Polymerases

Christov, Plamen P.,Yamanaka, Kinrin,Choi, Jeong-Yun,Takata, Kei-ichi,Wood, Richard D.,Guengerich, F. Peter,Lloyd, R. Stephen,Rizzo, Carmelo J. American Chemical Society 2012 Chemical research in toxicology Vol.25 No.8

<P><I>N</I><SUP>6</SUP>-(2-Deoxy-<SMALL>d</SMALL>-<I>erythro</I>-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-<I>N</I>-methylformamidopyrimidine (MeFapy-dGuo) has been identified as a stable DNA adduct that arises from the reaction of DNA with a variety of methylating agents. Since this lesion persists in DNA and may contribute to the overall mutagenesis from electrophilic methylating agents, the MeFapy-dGuo lesion was incorporated into oligonucleotides, and its replication bypass was examined in vitro with a panel of eukaryotic high fidelity (hPols α, β, and δ/PCNA) and translesion (hPols η, κ, ι, Rev1, ν, and yPol ζ) polymerases to address its miscoding potential. The MeFapy-dGuo was found to be a strong block to the high fidelity polymerases at either the insertion or the extension step. Efficient translesion synthesis was observed for hPols η and κ, and the combined activities of hRev1 and yPol ζ. The nucleotide sequences of the extension products were determined by mass spectrometry. The error-free extension product was the most abundant product observed for each polymerase. Misreplication products, which included misinsertion of Thy, Gua, and Ade opposite the MeFapy-dGuo lesion, as well as an interesting one-nucleotide deletion product, were observed when hPols η and κ were employed; these events accounted for 8–29% of the total extension products observed. The distribution and abundance of the misreplication products were dependent on the polymerases and local sequence context of the lesion. Collectively, these data suggest that although MeFapy-dGuo adducts represent a relatively minor proportion of the total alkylated lesions, their miscoding potentials could significantly contribute to genomic instability.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/crtoec/2012/crtoec.2012.25.issue-8/tx300113e/production/images/medium/tx-2012-00113e_0001.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/tx300113e'>ACS Electronic Supporting Info</A></P>

Basis of Miscoding of the DNA Adduct <i>N</i>²,3-Ethenoguanine by Human Y-family DNA Polymerases

Zhao, Linlin,Pence, Matthew G.,Christov, Plamen P.,Wawrzak, Zdzislaw,Choi, Jeong-Yun,Rizzo, Carmelo J.,Egli, Martin,Guengerich, F. Peter American Society for Biochemistry and Molecular Bi 2012 The Journal of biological chemistry Vol.287 No.42

<P><I>N</I><SUP>2</SUP>,3-Ethenoguanine (<I>N</I><SUP>2</SUP>,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (<I>e.g.</I> lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-<I>N</I><SUP>2</SUP>,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of <I>N</I><SUP>2</SUP>,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through <I>N</I><SUP>2</SUP>,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite <I>N</I><SUP>2</SUP>,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with <I>N</I><SUP>2</SUP>,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the <I>N</I><SUP>2</SUP>,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the <I>N</I><SUP>2</SUP>,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of <I>N</I><SUP>2</SUP>,3-ϵG.</P>

Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma

Radogna, F,Cerella, C,Gaigneaux, A,Christov, C,Dicato, M,Diederich, M Macmillan Publishers Limited 2016 Oncogene Vol.35 No.29

<P>A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.</P>

Recursive Model Free Controller: Application to Friction Compensation and Trajectory Tracking

Haoping Wang,Christian Vasseur,Yang Tian,Vladan Koncar,Nicolaï Christov 제어·로봇·시스템학회 2011 International Journal of Control, Automation, and Vol.9 No.6

In this paper friction compensation and trajectory tracking scheme is proposed for an X-Y robot using a Recursive Model Free Controller (RMFC). This controller is based on the theory of piecewise continuous systems which are a particular class of hybrid systems with autonomous switchings and controlled impulses. RMFC uses only the robot position measurements and does not require knowledge of electromechanical system parameters. The proposed control scheme is validated on a real time X-Y robot system.

Tubulin-binding anticancer polysulfides induce cell death via mitotic arrest and autophagic interference in colorectal cancer

Yagdi Efe, Esma,Mazumder, Aloran,Lee, Jin-Young,Gaigneaux, Anthoula,Radogna, Flavia,Nasim, Muhammad Jawad,Christov, Christo,Jacob, Claus,Kim, Kyu-Won,Dicato, Mario,Chaimbault, Patrick,Cerella, Claudia Elsevier 2017 Cancer letters Vol.410 No.-

<P><B>Abstract</B></P> <P>Polysulfanes show chemopreventive effects against gastrointestinal tumors. We identified diallyl tetrasulfide and its derivative, dibenzyl tetrasulfide (DBTTS), to be mitotic inhibitors and apoptosis inducers. Here, we translate their application in colorectal cancer (CRC). MALDI-TOF-MS analysis identified both compounds as reversible tubulin binders, validated by <I>in cellulo</I> α-tubulin degradation. BRAF(V600E)-mutated HT-29 cells were resistant to DBTTS, as evidenced by mitotic arrest for 48 h prior to apoptosis induction compared to KRAS(G12V)-mutated SW480/620 cells, which committed to death earlier. The prolonged mitotic block correlated with autophagy impairment and p62 protein accumulation in HT-29 but not in SW480/620 cells, whereas siRNA-mediated p62 inhibition sensitized HT-29 cells to death. <I>In silico</I> analysis with 484 colorectal cancer patients associated higher p62 expression and reduced autophagic flux with greater overall survival. Accordingly, we hypothesized that DBTTS targets CRC survival/death through autophagy interference in cell types with differential autophagic capacities. We confirmed the therapeutic potential of DBTTS by the inhibition of spheroid and colony formation capacities in CRC cells, as well as in HT-29 zebrafish xenografts <I>in vivo</I>.</P>

The dialkyl resorcinol stemphol disrupts calcium homeostasis to trigger programmed immunogenic necrosis in cancer

Ji, Seungwon,Lee, Jin-Young,Schrö,r, Jan,Mazumder, Aloran,Jang, Dong Man,Chateauvieux, Sé,bastien,Schnekenburger, Michael,Hong, Che Ry,Christov, Christo,Kang, Hyoung Jin,Lee, Youngjo,Han, By Elsevier 2018 Cancer letters Vol.416 No.-

<P><B>Abstract</B></P> <P>Stemphol (STP) is a novel druggable phytotoxin triggering mixed apoptotic and non-apoptotic necrotic-like cell death in human acute myeloid leukemia (AML). Use of several chemical inhibitors highlighted that STP-induced non-canonical programmed cell death was Ca<SUP>2+</SUP>-dependent but independent of caspases, poly (ADP-ribose) polymerase-1, cathepsin, or calpains. Similar to thapsigargin, STP led to increased cytosolic Ca<SUP>2+</SUP> levels and computational docking confirmed binding of STP within the thapsigargin binding pocket of the sarco/endoplasmic reticulum (ER) Ca<SUP>2+</SUP>-ATPase (SERCA). Moreover, the inositol 1,4,5-trisphosphate receptor is implicated in STP-modulated cytosolic Ca<SUP>2+</SUP> accumulation leading to ER stress and mitochondrial swelling associated with collapsed cristae as observed by electron microscopy. Confocal fluorescent microscopy allowed identifying mitochondrial Ca<SUP>2+</SUP> overload as initiator of STP-induced cell death insensitive to necrostatin-1 or cycloheximide. Finally, we observed that STP-induced necrosis is dependent of mitochondrial permeability transition pore (mPTP) opening. Importantly, the translational immunogenic potential of STP was validated by HMGB1 release of STP-treated AML patient cells. STP reduced colony and <I>in vivo</I> tumor forming potential and impaired the development of AML patient-derived xenografts in zebrafish.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Stemphol induces cell death by disrupting calcium homeostasis. </LI> <LI> Stemphol induces necrosis by mediating mPTP opening. </LI> <LI> Stemphol triggers immunogenic cell death markers ER stress and HMGB1 release. </LI> <LI> Stemphol impairs development of leukemia patient-derived zebrafish xenografts. </LI> </UL> </P>