Unrepaired oxidative DNA damage induces an ATR/ATM apoptotic-like response in quiescent fission yeast

Programmed cell death is a term which refers to a genetic decision of self-killing or suicide of a cell. Programmed cell death is not restricted to multicellular organisms and was described in a wide range of unicellular eukaryotes, indicating phylogenetically conserved functions, that participate in an adaptive response to cellular stress. Here we review and discuss our observations recently published in the EMBO Journal1, that non-dividing fission yeast, Schizosaccharomyces pombe, exhibits a DNA damage response leading to cell death. We found that Tdp1 protects quiescent S. pombe cells against oxidative DNA damage. Tdp1 is a well-conserved tyrosyl-DNA phosphodiesterase required for single-strand break DNA repair, the mutation of Tdp1 is responsible for the recessively inherited syndrome spinocerebellar ataxia with axonal neuropathy (SCAN1) in humans. We found that tdp1 mutant yeast cells grow, as well as the wild-type cells, during the vegetative state, but progressively die in the quiescent state. We showed that, in the absence of Tdp1, the accumulation of unrepaired oxidative DNA damage triggers a genetic response, leading to checkpoint-dependent (ATM/ATR) nuclear DNA degradation, reminiscent of apoptosis. Our results indicate that the reactive oxygen species (ROS) produced during mitochondrial respiration are the main DNA damaging agents in the physiological quiescent state.     

Spinocerebellar ataxia with axonal neuropathy: consequence of a Tdp1 recessive neomorphic mutation?

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) cleaves the phosphodiester bond between a covalently stalled topoisomerase I (Topo I) and the 3' end of DNA. Stalling of Topo I at DNA strand breaks is induced by endogenous DNA damage and the Topo I-specific anticancer drug camptothecin (CPT). The H493R mutation of Tdp1 causes the neurodegenerative disorder spinocerebellar ataxia with axonal neuropathy (SCAN1). Contrary to the hypothesis that SCAN1 arises from catalytically inactive Tdp1, Tdp1-/- mice are indistinguishable from wild-type mice, physically, histologically, behaviorally, and electrophysiologically. However, compared to wild-type mice, Tdp1-/- mice are hypersensitive to CPT and bleomycin but not to etoposide. Consistent with earlier in vitro studies, we show that the H493R Tdp1 mutant protein retains residual activity and becomes covalently trapped on the DNA after CPT treatment of SCAN1 cells. This result provides a direct demonstration that Tdp1 repairs Topo I covalent lesions in vivo and suggests that SCAN1 arises from the recessive neomorphic mutation H493R. This is a novel mechanism for disease since neomorphic mutations are generally dominant.     

A tyrosyl DNA phosphodiesterase 1 from kinetoplastid parasite Leishmania donovani (LdTdp1) capable of removing topo I–DNA covalent complexes

Tyrosyl DNA phosphodiesterase 1 (Tdp1) is a member of phospholipase D superfamily, which cleaves a broad range of 3′‐DNA adducts, the best characterized of which is the phosphodiester bond formed between DNA and topoisomerase IB. This study describes cloning and functional characterization of the enzyme, termed as LdTdp1 in the kinetoplastid parasite Leishmania donovani. Sequence analysis confirmed conservation of the active site motifs typical for all Tdp1 proteins. LdTdp1 activity was detected in the parasite nucleus as well as in the kinetoplast. The enzyme harbours a nuclear localization signal at its C‐terminus. Overexpression of the active enzyme protected the parasites against topoisomerase IB inhibitor camptothecin (CPT) and oxidative agent H₂O₂‐mediated cytotoxicity and its downregulation rendered the parasites hypersensitive to CPT. Trapping of mutant LdTdp1 on DNA takes place following CPT treatment in L. donovani cells. The expression level and associated activity of LdTdp1 were found to be higher in CPT‐resistant L. donovani parasites. Altogether, this is the first report of Tdp1 from the kinetoplastid parasite L. donovani, which actively participates in topoisomerase I‐mediated DNA damage repair process and thereby counteracts the cytotoxic effect of topoisomerase I inhibitors.     

TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo

Defective Tyrosyl-DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single-strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1-/- mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1-/- mice display an inability to rapidly repair DNA SSBs associated with Top1-DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age-dependent and progressive cerebellar atrophy. Tdp1-/- mice treated with topotecan, a drug that increases levels of Top1-DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1-associated DNA strand breaks.     

In vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation

A homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1) has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive neurodegenerative disease. However, it is uncertain how the H493R mutation elicits the specific pathologies of SCAN1. To address this question, and to further elucidate the role of TDP1 in repair of DNA end modifications and general physiology, we generated a Tdp1 knockout mouse and carried out detailed behavioral analyses as well as characterization of repair deficiencies in extracts of embryo fibroblasts from these animals. While Tdp1^?/? mice appear phenotypically normal, extracts from Tdp1^?/? fibroblasts exhibited deficiencies in processing 3′-phosphotyrosyl single-strand breaks and 3′-phosphoglycolate double-strand breaks (DSBs), but not 3′-phosphoglycolate single-strand breaks. Supplementing Tdp1^?/? extracts with H493R TDP1 partially restored processing of 3′-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on phosphoglycolate (PG) termini on 3′ overhangs of double-strand breaks; these remained completely unprocessed. Altogether, these results suggest that for 3′-phosphoglycolate overhang lesions, the SCAN1 mutation confers loss of function, while for 3′-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate.     

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3′-end-blocking modifications, possesses DNA and RNA 3′-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3′-end of DNA have been used to prevent 3′-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3′-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3′-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3′-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.     

XRCC1-mediated repair of strand breaks independent of PNKP binding

Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3′-phosphate and 5′-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3′-OH and 5′-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage. To evaluate this documented repair process, a phosphorylation mutant of XRCC1, designed to eliminate PNKP binding, was stably expressed in Xrcc1^−/− mouse fibroblast cells. Analysis of PNKP-GFP accumulation at micro-irradiation induced damage confirmed that the XRCC1 phosphorylation mutant failed to support efficient PNKP recruitment, whereas there was rapid recruitment in cells expressing wild-type XRCC1. Recruitment of additional fluorescently-tagged repair factors PARP-1-YFP, GFF-XRCC1, PNKP-GFP and Tdp1-GFP to micro-irradiation induced damage was assessed in wild-type XRCC1-expressing cells. PARP-1-YFP recruitment was best fit to two exponentials, whereas kinetics for the other proteins were fit to a single exponential. The similar half-times of recruitment suggest that XRCC1 may be recruited with other proteins possibly as a pre-formed complex. Xrcc1^−/− cells are hypersensitive to the DNA-protein cross-link inducing agent camptothecin (CPT) and the DNA oxidative agent H₂O₂ due in part to compromised PNKP-mediated repair. However, cells expressing the PNKP interaction mutant of XRCC1 demonstrated marked reversal of CPT hypersensitivity. This reversal represents XRCC1-dependent repair in the absence of the phosphorylation-dependent PNKP recruitment and suggests either an XRCC1-independent mechanism of PNKP recruitment or a functional back-up pathway for cleaning of blocked DNA ends.     

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His^nuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (His^gab), which resolves the Tdp1-DNA linkage. A His^nuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His^gab to Arg. However, here we report that expression of the yeast His^nucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His^gab mutants, including H432N and the SCAN1-related H432R. Moreover, the His^nucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His^nucPhe mutant was catalytically inactive and suppressed His^gab mutant-induced toxicity. These data suggest that the activity of another nucleophile when His^nuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His^nuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.     

Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 A crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H(432)R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H(432)N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H(432) mutants were recessive to wild-type Tdp1. Thus, yeast H(432) acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts.     

Polynucleotide kinase/phosphatase, Pnk1, is involved in base excision repair in Schizosaccharomyces pombe

We previously reported that Schizosaccharomyces pombe pnk1 cells are more sensitive than wild-type cells to γ-radiation and camptothecin, indicating that Pnk1 is required for DNA repair. Here, we report that pnk1pku70 and pnk1rhp51 double mutants are more sensitive to γ-radiation than single mutants, from which we infer that Pnk1's primary role is independent of either homologous recombination or non-homologous end joining mechanisms. We also report that pnk1 cells are more sensitive than wild-type cells to oxidizing and alkylating agents, suggesting that Pnk1 is involved in base excision repair. Mutational analysis of Pnk1 revealed that the DNA 3'-phosphatase activity is necessary for repair of DNA damage, whereas the 5'-kinase activity is dispensable. A role for Pnk1 in base excision repair is supported by genetic analyses which revealed that pnk1apn2 is synthetically lethal, suggesting that Pnk1 and Apn2 may function in parallel pathways essential for the repair of endogenous DNA damage. Furthermore, the nth1pnk1apn2 and tdp1pnk1apn2 triple mutants are viable, implying that single-strand breaks with 3'-blocked termini produced by Nth1 and Tdp1 contribute to synthetic lethality. We also examined the sensitivity to methyl methanesulfonate of all single and double mutant combinations of nth1, apn2, tdp1 and pnk1. Together, our results support a model where Tdp1 and Pnk1 act in concert in an Apn2-independent base excision repair pathway to repair 3'-blocked termini produced by Nth1; and they also provide evidence that Pnk1 has additional roles in base excision repair.     

Identification of Tyrosyl-DNA Phosphodiesterase as a Novel DNA Damage Repair Enzyme in Arabidopsis

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a key enzyme that hydrolyzes the phosphodiester bond between tyrosine of topoisomerase and 3′-phosphate of DNA and repairs topoisomerase-mediated DNA damage during chromosome metabolism. However, functional Tdp1 has only been described in yeast and human to date. In human, mutations of the Tdp1 gene are involved in the disease spinocerebellar ataxia with axonal neuropathy. In plants, we have identified the functional nuclear protein AtTDP, homolog to human Tdp1 from Arabidopsis (Arabidopsis thaliana). The recombinant AtTDP protein certainly hydrolyzes the 3′-phosphotyrosyl DNA substrates related to repairing in vivo topoisomerase I-DNA-induced damage. The loss-of-function AtTDP mutation displays developmental defects and dwarf phenotype in Arabidopsis. This phenotype is substantially caused by decreased cell numbers without any change of individual cell sizes. The tdp plants exhibit hypersensitivities to camptothecin, a potent topoisomerase I inhibitor, and show rigorous cell death in cotyledons and rosette leaves, suggesting the failure of DNA damage repair in tdp mutants. These results indicate that AtTDP plays a clear role in the repair of topoisomerase I-DNA complexes in Arabidopsis.In all living organisms, a variety of DNA damage is constantly caused by replication errors, UV light, ionizing radiation, DNA damage agents, etc. Once DNA damage has occurred, specific DNA repair proteins, such as AP endonuclease, RAD1 (for radiation sensitive), RAD9, RAD51, XRCC2 (for x-ray repair cross-complementing), Ku80 (XRCC6), and ligase, initiate to act through the repair pathways (Wood et al., 2001). Defects in DNA damage repair have evolved into cancer or genetic diseases in mammals and affect productivity or growth in plants (Tuteja et al., 2001; Wood et al., 2001).In the repair of DNA-protein cross-links, tyrosyl-DNA phosphodiesterase 1 (Tdp1) is known as a unique protein. Tdp1 was initially reported as an active enzyme in Saccharomyces cerevisiae that specifically removes the Tyr group from the covalent intermediate between the Tyr residue and the terminal 3′- phosphate of the oligonucleotide (Yang et al., 1996). Subsequently, the yeast TDP1 gene was identified and showed highly conserved sequences with other organisms, such as Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (Pouliot et al., 1999). The Tdp1 homologs of these species are members of the phospholipase D (PLD) superfamily (Pouliot et al., 1999; Interthal et al., 2001). Yeast Tdp1 is mainly studied concerning the topoisomerase I-repair pathway using double or triple mutants. The deletion mutations of yeast Tdp1 were shown lacking in the repair of DNA damage induced by a topoisomerase inhibitor, the anticancer drug camptothecin (CPT; Pouliot et al., 2001; Liu et al., 2002; Vance and Wilson, 2002). Tdp1 has been further implicated in multiple repair pathways, including the damage repair of topoisomerase II-DNA in yeast (Nitiss et al., 2006).In multicellular eukaryotes, the defect of human Tdp1 has resulted in the neurodisorder disease spinocerebellar ataxia with axonal neuropathy (SCAN1; Takashima et al., 2002). SCAN1 is a rare autosomal recessive neurodegenerative disease, and the patients present distal muscle weakness and peripheral neuropathy (Interthal et al., 2001; Takashima et al., 2002). SCAN1 is caused by a missense mutation (His-493Arg) in the Tdp1 catalytic site. As in yeast, the human Tdp1 protein plays a role in the repair of topoisomerase I-DNA complex lesions in SCAN1 cells (El-Khamisy et al., 2005; Miao et al., 2006). SCAN1 cells are hypersensitive to CPT (Interthal et al., 2005; Miao et al., 2006) and accumulate single-strand break and double-strand break DNAs by CPT (El-Khamisy et al., 2005).At present, although the functional analysis of Tdp1 has been widely conducted in yeast and human cell lines, its role in the overall growth and development of higher plants remains unknown. Here, we investigate the function of a novel Arabidopsis (Arabidopsis thaliana) TDP, a human and yeast Tdp1 homolog. The AtTDP protein shows the DNA damage-repairing activity and substrate specificities in biochemical assay. The dwarf phenotype of the Arabidopsis tdp mutant may be due to the reduced cell number caused by the accumulation of DNA damage and progressive cell death during Arabidopsis development.     

Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1^?/?/Aptx^?/? double knockout quiescent mouse astrocytes compared with Tdp1^?/? or Aptx^?/? single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1^?/? and Tdp1^?/?/Aptx^?/? double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1^?/?, Aptx^?/? or Tdp1^?/?/Aptx^?/? astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.     

Increased SOD1 association with chromatin,DNA damage,p53 activation,and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly⁹³ → Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.     

Down-regulation of statin,a nonproliferation-specific nuclear protein,and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G₁ phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G₁ phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley-Liss, Inc.     

The neutrophil elastase mutant affects viability and differentiation of the human monocytic THP‐1 cell

Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has been extensively investigated. Monocytes and neutrophils are derived from a common myeloid progenitor; therefore, ELA2 mutations can also influence monocyte development. These effects have not been well described. In this study, we used the human monocytic THP‐1, to carry the human wild‐type and G185R mutant ELA2 gene. Growth, death, differentiation and BiP expression were evaluated in the two stable cell lines and in the wild‐type THP‐1 cells. Exogenous wild‐type ELA2 markedly increased THP‐1 differentiation, whereas G185R ELA2 was incompetent to promote THP‐1 differentiation in response to all‐trans retinoic acid (ATRA). Indeed, during differentiation induced by ATRA, G185R cell line showed significant cell death. Also, up‐regulated BiP expression accompanied cell death in the G185R cells, suggesting that the overexpression of G185R elastase increases apoptosis through an unfolded protein response. The G185R cells treated with lithium chloride (LiCl; a Wnt signalling activator) displayed higher BiP expression but similar cell viability compared with THP1 and HNEwt/THP1 cells treated with LiCl. This suggested that Wnt signalling might increase cellular tolerance to endoplasmic reticulum stress, enabling mutant monocyte survival. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of novel inhibitors for the tyrosyl-DNA-phosphodiesterase 1 (Tdp1) mutant SCAN1 using virtual screening

Spinocerebellar ataxia syndrome with axonal neuropathy (SCAN1) is a debilitating neurological disease that is caused by the mutation the Tyrosyl-DNA phosphodiesterase 1 (TDP1) DNA repair enzyme. The crucial His493 in TDP1′s binding site is replaced with an arginine amino acid residue rendering the enzyme dysfunctional. A virtual screen was performed against the homology model of SCAN1 and seventeen compounds were identified and tested in a novel SCAN1 specific biochemical assay. Six compounds showed activity with IC₅₀ values between 3.5 and 25.1 µM. The most active ligand 5 (3.5 µM) is a dicoumarin followed by a close structural analogue 6 at 6.0 µM. A less potent series of β-carbolines (14 and 15) was found with potency in the mid-teens. According to molecular modelling an excellent fit for the active ligands into the binding pocket is predicted. To the best of our knowledge, data on inhibitors of the mutant form of TDP1 has not been reported previously. The virtual hits were also tested for wild type TDP1 activity and all six SCAN1 inhibitors are potent for the former, e.g., ligand 5 has a measured IC₅₀ at 99 nM.In the last decade, TDP1 is considered as a promising target for adjuvant therapy against cancer in combination with Topoisomerase 1 poisons. The active ligands are mostly non-toxic to cancer cell lines A-549, T98G and MCF-7 as well as the immortalized WI-38 human fetal lung cells. Furthermore, ligands 5 and 7, show promising synergy in conjunction with topotecan, a clinically used topoisomerase 1 anticancer drug. The active ligands 5, 7, 14 and 15 have a good balance of the physicochemical properties required for oral bioavailability making the excellent candidates for further development.