Anti-poly(ADP-ribose)polymerase (PARP) drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes, and that both olaparib and niraparib act as PARP poisons at pharmacological concentrations. Therefore, we have proposed that PARP inhibitors should be evaluated based both on catalytic PARP inhibition and PARP-DNA trapping. Here, we evaluated the novel PARP inhibitor, BMN 673, and compared its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically-modified chicken DT40 and human cancer cell lines. Although BMN 673, olaparib and rucaparib are comparable at inhibiting PARP catalytic activity, BMN 673 is ~100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as single agent than olaparib, while olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient. BMN 673 is also ~100-fold more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sufonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor tested to date with the highest efficiency at trapping PARP-DNA complexes.
PARP inhibitor; poly(ADP-ribosyl)ation; PARP-DNA complex; chemotherapy; homologous recombination
The expression of a novel cardiac glucose transporter, SGLT1, is increased in glycogen storage cardiomyopathy secondary to mutations in PRKAG2. We sought to determine the role of SGLT1 in the pathogenesis of PRKAG2 cardiomyopathy and its role in cardiac structure and function.
Methods and Results
Transgenic mice with cardiomyocyte‐specific overexpression of human T400N mutant PRKAG2 cDNA (TGT400N) and transgenic mice with cardiomyocyte‐specific RNA interference knockdown of SGLT1 (TGSGLT1‐DOWN) were crossed to produce double‐transgenic mice (TGT400N/TGSGLT1‐DOWN). Tet‐off transgenic mice conditionally overexpressing cardiac SGLT1 in the absence of doxycycline were also constructed (TGSGLT‐ON). Relative to TGT400N mice, TGT400N/TGSGLT1‐DOWN mice exhibited decreases in cardiac SGLT1 expression (63% decrease, P<0.05), heart/body weight ratio, markers of cardiac hypertrophy, and cardiac glycogen content. TGT400N/TGSGLT1‐DOWN mice had less left ventricular dilation at age 12 weeks compared to TGT400N mice. Relative to wildtype (WT) mice, TGSGLT1‐ON mice exhibited increases in heart/body weight ratio, glycogen content, and markers of cardiac hypertrophy at ages 10 and 20 weeks. TGSGLT1‐ON mice had increased myocyte size and interstitial fibrosis, and progressive left ventricular dysfunction. When SGLT1 was suppressed after 10 weeks of overexpression (TGSGLT1‐ON/OFF), there was a reduction in cardiac hypertrophy and improvement in left ventricular failure.
Cardiac knockdown of SGLT1 in a murine model of PRKAG2 cardiomyopathy attenuates the disease phenotype, implicating SGLT1 in the pathogenesis. Overexpression of SGLT1 causes pathologic cardiac hypertrophy and left ventricular failure that is reversible. This is the first report of cardiomyocyte‐specific transgenic knockdown of a target gene.
cardiomyopathy; glucose; heart failure; hypertrophy
Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3′-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1–PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.
Small molecule inhibitors of polyADP-ribose polymerase (PARP) are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single strand breaks. However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study we demonstrate that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired single-strand breaks caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with MK-4827 > olaparib (AZD-2281) ≫ veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with pre-established deletions in specific DNA repair genes. This analysis revealed that, in addition to its function in homologous recombination, PARP also functions in post-replication repair and the Fanconi anemia pathway, and that polymerase β and FEN1 were critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.
PARP inhibitor; poly(ADP-ribosyl)ation; PARP-DNA complex; chemotherapy; homologous recombination
In order to asses performance of the LDA in describing physisorption on graphene, adsorptions of TCNE, TCNQ, TNF, TTF, and DMPD as well as four benzene derivatives on C54H18 and C110H30 were explored with a variety of DFTs such as MPWB1K, M06-2X, PBE-D and LSDA. Although it is well known that the LDA considerably overestimate non-covalent interaction, the LSDA predicted adsorption energies except for TCNE on C110H30 are systematically lower than those from the M06-2X by 0.4–3.2 kcal/mol, and they are more significantly lower than those from the PBE-D for all the molecules by 3–6 kcal/mol. However, the LSDA adsorption energy sequence is consistent with that from the PBE-D, TNF~TCNQ>TCNE~DMPD>TTF. Moreover, the domain interaction between the electron donor and acceptor molecules with graphene through cooperative π···π, C-H···π and N-H···π were visualized with sign(λ2)×ρ, and the relationships between the binding energy with London force, molecular electronegativity, and frontier orbital level were extensively discussed.
graphene; DFT; physisorption; electron donor and acceptor
Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.
Inhibitors of topoisomerase I (Top1) that result in stalled Top1 cleavage complexes (Top1cc) are commonly employed against cancer. Combination chemotherapy with DNA repair inhibitors can potentially improve response to these widely used chemotherapeutics. One line of inquiry focuses on inhibitors of tyrosyl-DNA phosphodiesterase 1 (Tdp1), a repair enzyme for Top1cc. Tdp1 catalyzes the hydrolysis of DNA adducts covalently linked to the 3′-phosphate of DNA, including Top1-derived peptides and also 3′-phosphoglycolates. Tdp1 inhibitors should synergize not only with Top1-targeting drugs (camptothecins, indenoisoquinolines), but also with bleomycin, topoisomerase II (Top2) inhibitors (etoposide, doxorubicin) and DNA alkylating agents. Here, we summarize the structural-activity relationship obtained from the reported Tdp1 inhibitors. Better understanding of Top1cc repair in vivo coupled with detailed structural studies on Tdp1-inhibitor interaction will be crucial in guiding the rational design of Tdp1 inhibitors.
Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, either containing a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex is required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here we showed that MutS bound to a 30-bp heteroduplex containing an unpaired-T with a binding affinity ≈ 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and non-specific binding affinity, with the later marked by a larger drop, further increasing MutS specificity by ≈ 3-fold. Kinetic studies showed that the difference in MutS KD for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a non-cooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.
MMR; MutS specificity; binding cooperativity; binding kinetics; nucleotide cofactors
OBJECTIVES: Animal inhalation studies and theoretical models suggest that the pattern of formation of benzene metabolites changes as exposure to benzene increases. To determine if this occurs in humans, benzene metabolites in urine samples collected as part of a cross sectional study of occupationally exposed workers in Shanghai, China were measured. METHODS: With organic vapour monitoring badges, 38 subjects were monitored during their full workshift for inhalation exposure to benzene. The benzene urinary metabolites phenol, catechol, hydroquinone, and muconic acid were measured with an isotope dilution gas chromatography mass spectroscopy assay and strongly correlated with concentrations of benzene air. For the subgroup of workers (n = 27) with urinary phenol > 50 ng/g creatinine (above which phenol is considered to be a specific indicator of exposure to benzene), concentrations of each of the four metabolites were calculated as a ratio of the sum of the concentrations of all four metabolites (total metabolites) and were compared in workers exposed to > 25 ppm v < or = 25 ppm. RESULTS: The median, 8 hour time weighted average exposure to benzene was 25 ppm. Relative to the lower exposed workers, the ratio of phenol and catechol to total metabolites increased by 6.0% (p = 0.04) and 22.2% (p = 0.007), respectively, in the more highly exposed workers. By contrast, the ratio of hydroquinone and muconic acid to total metabolites decreased by 18.8% (p = 0.04) and 26.7% (p = 0.006), respectively. Similar patterns were found when metabolite ratios were analysed as a function of internal benzene dose (defined as total urinary benzene metabolites), although catechol showed a more complex, quadratic relation with increasing dose. CONCLUSIONS: These results, which are consistent with previous animal studies, show that the relative production of benzene metabolites is a function of exposure level. If the toxic benzene metabolites are assumed to be derived from hydroquinone, ring opened products, or both, these results suggests that the risk for adverse health outcomes due to exposure to benzene may have a supralinear relation with external dose, and that linear extrapolation of the toxic effects of benzene in highly exposed workers to lower levels of exposure may underestimate risk.
A large cohort of 74,828 benzene-exposed and 35,805 nonexposed workers employed between 1972 and 1987 in 12 cities in China was followed to determine mortality from all causes. Benzene-exposed study subjects were employed in a variety of occupations including coating applications, and rubber, chemical, and shoe production. Mortality was slightly increased among workers with greater cumulative exposure to benzene (ptrend < 0.05), but this excess was largely due to cancer deaths (ptrend < 0.01). Deaths due to lymphatic and hematopoietic malignancies (ptrend = 0.01) and lung cancer (ptrend = 0.01) increased with increasing cumulative exposure to benzene. Investigations continue to relate benzene exposure to specific lymphatic and hematopoietic malignancies and other causes of death.
An expanded cohort study of 74,828 benzene-exposed and 35,805 unexposed workers were followed during 1972 to 1987, based on a previous study in 12 cities in China. A small increase was observed in total cancer mortality among benzene-exposed compared with unexposed workers (relative risk [RR] = 1.2). Statistically significant excesses were noted for leukemia (RR = 2.3), malignant lymphoma (RR = 4.5), and lung cancer (RR = 1.4). When risks were evaluated by leukemia subtype, only acute myelogenous leukemia was significantly elevated (RR = 3.1), although nonsignificant excesses were also noted for chronic myelogenous leukemia (RR = 2.6) and acute lymphocytic leukemia (RR = 2.3). A significant excess was also found for aplastic anemia.
Previous occupational cohort studies of benzene-exposed workers have for the most part used only death certificates to validate diagnoses of workers developing leukemia and other hematopoietic and lymphoproliferative malignancies and related disorders (HLD). In a follow-up study of 74,828 benzene-exposed workers and a comparison group of 35,805 nonexposed workers from 12 cities in China, we sought to characterized clinicopathologically and to confirm diagnoses of all cases of HLD. Using medical records, laboratory hematology results, and histopathology, U.S. and Chinese expert hematopathologists, blinded to exposure status, carried out a detailed review using standardized evaluation forms. Key among the findings were a notable diversity of malignant and nonneoplastic hematopoietic and lymphoproliferative disorders, documentation of excess myelodysplastic syndromes among benzene workers, and widespread dyspoiesis involving all hematopoietic cell lines. As sophisticated clinicopathologic characterization and corresponding classification schemes for HLD become increasingly widespread, it is recommended that future epidemiologic investigations of benzene workers incorporate similarly detailed morphologic evaluation. In extending follow-up of this cohort of young workers, we will continue to use all available clinical, laboratory hematology, and pathology data as well as cytogenetic and biochemical markers to characterized various HLD outcomes. These careful surveillance mechanisms should also provide additional insight into carcinogenic mechanisms of benzene and allow comparison of the molecular pathogenesis of HLD induced by benzene versus chemotherapy, radiation, or other exposure.
We present a validation study of a quantitative retrospective exposure assessment method used in a follow-up study of workers exposed to benzene. Assessment of exposure to benzene was carried out in 672 factories in 12 cities in China. Historical exposure data were collected for 3179 unique job titles. The basic unit for exposure assessment was a factory/work unit/job title combination over seven periods between 1949 and 1987. A total of 18,435 exposure estimates was developed, using all available historical information, including 8477 monitoring data. Overall, 38% of the estimates were based on benzene monitoring data. The highest time-weighted average exposures were observed for the rubber industry (30.7 ppm) and for rubber glue applicators (52.6 ppm). Because of its recognized link with benzene exposure, the association between a clinical diagnosis of benzene poisoning and benzene exposure was evaluated to validate the assessment method that we used in the cohort study. Our confidence in the assessment method is supported by the observation of a strong positive trend between benzene poisoning and various measures, especially recent intensity of exposure to benzene.
A retrospective cohort study was carried out in 1982–1983 among 28,460 benzene-exposed workers (15,643 males, 12,817 females) from 233 factories and 28,257 control workers (16,621 males, 12,366 females) from 83 factories in 12 large cities in China. All-cause mortality was significantly higher among the exposed (265.46/100,000 person-years) than among the unexposed (139.06/100,000 person-years), as was mortality from all malignant neoplasms (123.21/100,000 versus 54.7/100,000, respectively). For certain cancers, increased mortality was noted among benzene-exposed males in comparison with that among unexposed males; the standardized mortality ratios (SMR) were elevated for leukemia (SMR = 5.74), lung cancer (SMR = 2.31), primary hepatocarcinoma (SMR = 1.12), and stomach cancer (SMR = 1.22). For females only leukemia occurred in excess among the exposed. Risk of leukemia rose as duration to exposure to benzene increased up to 15 years, and then declined with additional years of exposure. Leukemia occurred among some workers with as little as 6 to 10 ppm average exposure and 50 ppm-years (or possibly less) cumulative lifetime exposure (based on all available measurements for the exposed work units). Among the 30 leukemia cases identified in the exposed cohort, the proportion of subjects with acute lymphocytic leukemia was substantially lower and the proportion with acute nonlymphocytic leukemias was higher than in the general population. During 1972 to 1981, the annual incidence of leukemia ranged from 5.83 to 28.33 per 100,000 with higher rates occurring in the interval 1977 to 1981 than in the earlier years of the study period. Future studies should evaluate more precisely the relationship between exposure levels, job title, and development of leukemia among cases and noncases within the exposed cohort.
A method for rapidly determining t,t-muconic acid (MA) by high performance liquid chromatography was developed and successfully applied to urine samples from 152 workers exposed to benzene (64 men, 88 women) and 213 non-exposed controls (113 men, 100 women). The MA concentrations in urine correlated linearly with time weighted average benzene concentrations in the breath zone air of workers. A cross sectional balance study showed that about 2% of benzene inhaled is excreted into the urine as MA. The MA concentrations in the urine of the non-exposed was below the detection limit (less than 0.1 mg/l) in most cases, and the 95% lower confidence limit of MA for those exposed to benzene at 5 ppm (5.0 mg/l as a non-corrected value) was higher than the 97.5%-tile values for the non-exposed (1.4 mg/l). In practice, it was possible to separate those exposed to 6-7 ppm benzene from the non-exposed by means of urine analysis for MA. The urinary MA concentration was suppressed by coexposure to toluene.
Of a total of 528,729 workers exposed to benzene or benzene mixtures in China, 508,818 (96.23%) were examined. Altogether 2,676 cases of benzene poisoning were found, a prevalence of 0.15%. A higher prevalence of benzene poisoning was found in the cities of Hangjou, Hefei, Nanjing, Shenyang, and Xian. The geometric mean concentration of benzene in 50,255 workplaces was 18.1 mg/m3 but 64.6% of the workplaces had less than 40 mg/m3. There was a positive correlation between the prevalence of benzene poisoning and the concentration in shoemaking factories. The prevalence of benzene induced aplastic anaemia in shoemakers was about 5.8 times that occurring in the general population. The results of this investigation show the need for a practicable hygiene standard to prevent benzene poisoning.
A retrospective cohort study was conducted in 233 benzene factories and 83 control factories in 12 cities in China. The benzene cohort and the control cohort consisted of 28,460 benzene exposed workers (178,556 person-years in 1972-81) and 28,257 control workers (199,201 person-years). Thirty cases of leukaemia (25 dead and 5 alive) were detected in the former and four cases (all dead) in the latter. The leukaemia mortality rate was 14/100,000 person-years in the benzene cohort and 2/100,000 person-years in the control cohort; the standardized mortality ratio was 5.74 (p less than 0.01 by U test). The average latency of benzene leukaemia was 11.4 years. Most (76.6%) cases of benzene leukaemia were of the acute type. The mortality due to benzene leukaemia was high in organic synthesis plants followed by painting and rubber synthesis industries. The concentration of benzene to which patients with a leukaemia were exposed ranged from 10 to 1000 mg/m3 (mostly from 50 to 500 mg/m3). Of the 25 cases of leukaemia, seven had a history of chronic benzene poisoning before the leukaemia developed.