Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca2+ concentration ([Ca2+]i) elevation induced by infection can cause cell death, but [Ca2+]i changes and high [Ca2+]i-induced death of macrophages due to infection of Leptospira have not been previously reported.2+-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca2+]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca2+]i elevation was caused by both extracellular Ca2+ influx through the purinergic receptor, P2X7, and Ca2+ release from the endoplasmic reticulum, as seen by suppression of [Ca2+]i elevation when receptor-gated calcium channels were blocked or P2X7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3), which in turn induces intracellular Ca2+ release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S-1. Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca2+]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca2+]i. Moreover, PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca2+]i increases induced apoptosis and necrosis of macrophages, while mild [Ca2+]i elevation only caused apoptosis.We first used a CaL. interrogans infection induced [Ca2+]i elevation through extracellular Ca2+ influx and intracellular Ca2+ release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca2+]i elevation.This study demonstrated that  Leptospira species is a world-spread zoonotic infectious disease Leptospirosis caused by pathogenic Leptospira species Leptospira infection remain poorly understood.Many animals serve as the natural hosts of pathogenic Leptospira. However, unlike many other bacterial pathogens, only macrophages can kill the phagocytosed leptospires in individuals who do not have specific antibodies against LeptospiraLeptospira species Macrophages and neutrophils play an important role in innate immunity against infection through phagocytosis of microbial pathogens including 2+), an important intracellular messenger with multiple physiological functions, is increased when cells are infected with some bacterial pathogens Helicobacter pylori or Campylobacter jejuni cause an elevation of intracellular free Ca2+ concentration ([Ca2+]i) through Ca2+ release and/or influx mechanisms in gastric mucous epithelial cells or in intestinal epithelial cells during infection 2+]i in macrophages caused by infection with Listeria monocytogenes or Brucella abortus are involved in bacterial invasion and escape from phagocytotic vesicles for intracellular replication 2+]i can induce cell death, in which a mild [Ca2+]i increases typically stimulates cell apoptosis while a high [Ca2+]i change results in cell necrosis L. interrogans, a predominant pathogenic Leptospira species, could be phagocytized by human or mouse macrophages, but infection induces macrophage apoptosis and necrosis 2+]i in L. interrogans-infected macrophages and the role of high [Ca2+]i in Leptospira-induced macrophage death had not been investigated yet.Infection results from interaction between microbial pathogens and hosts 2) to produce inositol-1,4,5-trisphosphate (IP3) and DAG. IP3 can bind to the IP3-receptor (IP3R) on endoplasmic reticulum to trigger release of Ca2+ from the endoplasmic reticulum into cytosol, resulting in[Ca2+]i elevation Phospholipase (PL) is a group of enzymes in eukaryotes and prokaryotes that can be classified into the PLA, PLC, PLD and sphingonyelinase (SMsae) subfamilies according to the specificity of their substrates and diversity of products L. monocytogenes, few bacterial PLC have been well characterized L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai Leptospira-induced [Ca2+]i elevation in macrophage death have not been determined yet.Until now, except for a PI-PLC produced by 2+]i in macrophages during infection with L. interrogans strain Lai, the dependence of [Ca2+]i on Ca2+ influx through membrane calcium channels or release from endoplasmic reticulum, and the correlation between [Ca2+]i changes and macrophage apoptosis and necrosis. Subsequently, we characterized biochemically the PI-PLC or PC-PLC enzymatic activity of the proteins expressed by the three leptospiral PLC-domain-containing genes and demonstrated the function of leptospiral PI-PLC in [Ca2+]i elevation and macrophage death during infection. The results of this study identify a novel PI-PLC of L. interrogans and show its the role in the [Ca2+]i elevation and death of infected macrophages.In the present study, we investigated the changes of i) (494 nm excitation wavelength and 516 nm emission wavelength) according to the manufacturer’s protocol. In the detection procedure, the images of leptospire-cell co-cultures incubated for 15, 30, 45, 60, 90 and 120 min were analyzed. The change of [Ca2+]i in the leptospire-infected J774A.1 or THP-1 cells was calculated as the following formula: the fluorescence intensity in 500 cells infected with the spirochete for different times (Fx)/the fluorescence intensity in the same number of cells before infection (F0) ×100% J774A.1 or THP-1 cells  during infection with L. interrogans strain Lai (107), the two macrophage monolayers were pre-treated with 2 mM EGTA (Sigma), 100 µM BAPTA/AM (Sigma), 1.2 mM neomycin (Sigma), 20 µM SKF96365 (Sigma), 100 µM verapamil (Sigma) or 10 µM mibefradil (Sigma) for 30 min at 37°C 2+]i detection were the same as described above. In this assay, the normal J774A.1 or THP-1 cells before infection, the chelator or blocker-untreated, and P2X7-depleted J774A.1 or THP-1 cells infected with the spirochete were used as the controls.EGTA is an extracellular Ca5 cells per well) were infected with the ΔLB361 or CΔLB361 mutant or wild-type L. interrogans strain Lai (107) for a 120-min contiguous incubation at 37°C. To further determine the roles of cellular PI-PLC or L-PI-PLC in [Ca2+]i elevation of macrophages during infection with the spirochete, J774A.1 or THP-1 cell monolayers were pre-treated with 2 mM extracellular Ca2+ chelator EGTA (Sigma) 3 production blocker neomycin (Sigma) 7). The subsequent experimental steps for [Ca2+]i detection were the same as described above. In this detection, the normal J774A.1 or THP-1 cells before infection were used as the controls.J774A.1 or THP-1 cell monolayers (105 cells per well) were infected with the ΔLB361 or CΔLB361 mutant or wild-type L. interrogans strain Lai (107) for 0.5, 1 or 2 h. After trypsinization, washing with PBS and centrifugation, the harvested cell pellets were suspended in annexin-binding buffer. The cell suspensions were treated with both Alexa Fluor® 488-annexin V and propidium iodide (PI) dyes in a Vybrant® Apoptosis Assay Kit (Invitrogen) for 15 min at room temperature. The stained cells were detected using a flow cytometer  to distinguish the cells in early apoptotic cells (annexin V+/PĪ) from post-apoptosis/necrosis (annexin V+/PI+). In addition, the J774A.1 or THP-1 cells (105 per well), which were pretreated with 100 µM intracellular free Ca2+ chelator BAPTA/AM, 2 mM extracellular Ca2+ chelator EGTA, 2 mM EGTA plus 10 µM mammalian cell PI-PLC inhibitor U73122 or 1.2 mM IP3 production blocker neomycin 7) for the indicated times. Cell death was detected by flow cytometry as above. In the detection, the normal J774A.1 or THP-1 cells before infection as well as P2X7-depleted J774A.1 or THP-1 cells before or after infection with the spirochete were used as the controls.J774A.1 or THP-1 cell monolayers (104 cells per well) were infected with the ΔLB361 or CΔLB361 mutant or wild-type L. interrogans strain Lai (106) for 0.5, 1 or 2 h. After removal of supernatants and trypsinization, the J774A.1 and THP-1 cells were collected by a 500×g centrifugation for 15 min at 4°C, followed by lysis with 0.05% NaTDC-PBS J774A.1 or THP-1 cell monolayers  to remove leptospires 2+ influx on [Ca2+]i, J774A.1 and THP-1 cells were pretreated with 2 mM extracellular Ca2+ chelator EGTA (Sigma) at 37°C for 30 min 2+]i or apoptosis of the macrophages at different times was detected by laser confocal microscopy or flow cytometry as described above. In the detections, the normal J774A.1 or THP-1 cells without transfection, and wild-type pCMV-Tag2C-transfected or chpI-gene-transfected J774A.1 or THP-1 cells were used as the negative controls. In addition, the pCMV-Tag2CLB361-transfected J774A.1 or THP-1 cells pretreated with 1.2 mM IP3 production blocker neomycin were also used as the controls to further determine the function of the LB361 gene product.To avoid the influence of extracellular Cap<0.05.Data from a minimum of three experiments were averaged and presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test were used to determine significant differences. Statistical significance was defined as Leptospira strains was examined as a way to predict the potential role of the genes in leptospiral pathogenicity. The PCR and sequencing data confirmed that all the seven tested pathogenic L. interrogans strains but not the two non-pathogenic L. biflexa strains belonging to different serogroups and serovars possessed the LA0543 and LB361 genes with high sequence identities, while the LA2250 gene was only detectable in genomic DNA from L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai . The IP3 fluorescence polarization assay confirmed that only the recombinant protein of the LB361 gene had PI-PLC activity to hydrolyze PIP2 into IP3  observed at the 2 h of post-infection  was secreted into the supernatants of co-cultures and cytosol of macrophages  hydrolyzes PIP2 into IP3 in macrophages.PI-PLC hydrolyzes PIProphages . The datL. interrogans strain Lai can cause [Ca2+]i-dependent death of macrophages, we measured the [Ca2+]i and apoptosis/necrosis of the LB361 gene-transfected J774A.1 or THP-1 cells. The results showed that the LB361 gene-transfected J774A.1 or THP-1 cells displayed a detectable increase in [Ca2+]i compared to the two normal or wild-type pCMV-Tag2C-transfected macrophages, but the IP3 production blocker neomycin inhibited the [Ca2+]i elevation  can increase due to extracellular Ca2+ influx through membrane calcium channels or intracellular Ca2+ release from endoplasmic reticulum 2+ release occurs when the IP3-receptor (IP3R) on endoplasmic reticulum is ligated with cytoplasmic IP32X4 and P2X7 receptor-gated calcium channels 2X7 protein. In the present study, we demonstrated that pretreatment with EGTA, an extracellular Ca2+ chelator, or neomycin, a blocker of IP3 production 2+]i elevation in the J774A.1 and THP-1 cells during infection with L. interrogans strain Lai. Moreover, the receptor-gated calcium channel blocker (SKF96365), but not the voltage-gated Ca2+ channel blockers, could inhibit the Leptospira-induced [Ca2+]i elevation in the two macrophages, while the depletion of P2X7 also prevented the [Ca2+]i elevation during infection. The data imply that L. interrogans causes the increase in [Ca2+]i in the infected macrophages due to extracellular Ca2+ influx through P2X7 calcium channel and intracellular Ca2+ release from endoplasmic reticulum.Intracellular free Ca2+]i after hydrolysis of PIP2 into IP3, which binds to IP3R to trigger Ca2+ release from endoplasmic reticulum into cytosol L. monocytogenes has been well characterized L. interrogans strain Lai, only the product of the LB361 gene (L-PI-PLC) displayed PI-PLC activity. More importantly, the increased expression and external secretion of L-PI-PLC were confirmed during infection of J774A.1 and THP-1 cells with the spirochete. Moreoever, all the seven tested L. interrogans strains belonging to different serogroups and serovars, but not the non-pathogenic L. interrogans strains, possess the LB361 gene. These data suggest that the product of the LB361 gene is a PI-PLC that is required by L. interrogans during infection.PI-PLC can increase cellular i and IP3 levels in the two macrophages during infection with the spirochete. These data imply that the cellular PI-PLCs also play a role in [Ca2+]i elevation in macrophages during infection with L. interrogans.The host cell PI-PLC during infection with some bacterial pathogens, such as Leptospira-specific antibodies Leptospira-induced macrophage death is important for the ability of the spirochetes to survive in infected hosts 2+]i induces cell apoptosis through a mitochondrion-dependent apoptotic pathway, while large [Ca2+]i increases cause cell necrosis through activation of Ca2+-dependent phospholipases and proteinases L. interrogans strain Lai caused both apoptosis and necrosis. However, blockage of extracellular Ca2+ influx and/or intracellular Ca2+ release favored apoptosis over necrosis. In addition, the LB361 gene-transfected macrophages only exhibited cell apoptosis, which may be due to L-PI-PLC causing a mild increase in [Ca2+]i. Taken together, our data show that infection with L. interrogans induced an increase in [Ca2+]i and [Ca2+]i-dependent apoptosis and necrosis of macrophages, and the LB361 gene product is a novel leptospiral PI-PLC that contributes to [Ca2+]i-dependent macrophage death.Infection-induced macrophage death is viewed as a common strategy used by different pathogens to evade the host immune response Figure S1Strategy for generation of ΔLB361 and CΔLB361 mutants. See Materials S1 for details.(TIF)Click here for additional data file.Figure S2Amplification and expression of LA0543, LA2250 and LB361 genes. (A). Amplification of LA0543, LA2250 and LB361 genes in different leptospiral strains. Lane M: DNA marker. Lane 1: blank controls. Lanes 2 to 8: amplicoms of the LA0543 gene (1320 bp) and LB361 gene (384 bp) from pathogenic L. interrogans serovar Lai strain Lai, serovar Grippotyphosa strain Lin-6, serovar Autumnalis strain Lin-4, serovar Pomona strain Luo, serovar Hebdomadis strain 56069, serovar Australis strain 65-9 and serovar Canicola strain Lin, respectively, but only L. interrogans strain Lai provided an amplicom (918 bp) of LA2250 gene, Lanes 9 and 10: no amplification products of the LA0543, LA2250 and LB361 genes from non-pathogenic L. biflexa serovar Patoc strain Patoc-1 and serovar Adamana strain CH-11. (B). Expression of LA0543, LA2250 and LB361 genes of L. interrogans strain Lai and purification of recombinant proteins. Lane M: protein marker. Lane 1: blank control of wild-type pET42a-transformed E. coli BL21DE3. Lanes 2 to 4: the recombinant proteins expressed by LA0543, LA2250 and LB361 genes, respectively. Lanes 5 to 7: the purified recombinant proteins of LA0543, LA2250 and LB361 genes by Ni-NTA affinity chromatography, respectively.(TIF)Click here for additional data file.Figure S3Confirmation of ΔLB361 and CΔLB361 mutants by PCR and sequencing. (A). PCR results for identification of the ΔLB361 mutant. Lane M: DNA marker. Lane 1: blank control. Lane 2: amplicon (2668 bp) of the 5′arm-kan-3′arm (2428 bp) plus two extending regions (120 bp each) from the ΔLB361 mutant. Lane 3: amplicon (1981 bp) of the 5′arm-LB361-3′arm (1741 bp) plus two extending regions (120 bp each) form wild-type L. interrogans strain Lai. (B). PCR results for identification of the CΔLB361 mutant. Lane M: DNA marker. Lane 1: blank control. Lane 2: amplicon (3228 bp) of the 5′arm-LB361-spc-3′arm segment (2988 bp) plus two extending regions (120 bp each) from the CΔLB361 mutant. Lane 3: amplicon (2668 bp) of the 5′arm-kan-3′arm (2428 bp) plus two extending regions (120 bp each) from the ΔLB361 mutant. Lane 4: amplicon (1981 bp) of the 5′arm-LB361-3′arm (1741 bp) plus two extending regions (120 bp each) form wild-type L. interrogans strain Lai. (C). Schematic diagram of sequencing result of the ΔLB361 mutant. The positions of PCR primers used are marked below. (D). Schematic diagram of sequencing result of the CΔLB361 mutant. The positions of PCR primers used are marked below.(TIF)Click here for additional data file.Figure S4Confirmation of ΔLB361 and CΔLB361 leptospiral mutants and LB361 or chpI2X7-depleted macrophages. gene-transfected and P (A). Expression of LB361 gene in the ΔLB361 and CΔLB361 mutants determined by Western Blot assay. Lane 1: the protein expressed by LB361 gene in wild-type L. interrogans strain Lai. Lane 2: no LB361 gene-encoding protein detectable in the ΔLB361 mutant. Lane 3: the protein expressed by LB361 gene in the CΔLB361 mutant. Lane 4: blank control. (B). Expression of the LB361 gene in the LB361 gene-transfected macrophages determined by Western Blot assay. Lane 1 or 3: the protein expressed by LB361 gene in the LB361 gene-transfected J774A.1 or THP-1 cells. Lane 2 or 4: no LB361 gene-encoding protein detectable in the normal J774A.1or THP-1 cells without transfection. Lane 5: blank control. (C). Expression of ChpI protein in the chpI gene-transfected macrophages determined by Western Blot assay. Lane 1 or 3: the expressed ChpI protein in the chpI gene-transfected J774A.1 or THP-1 cells. Lane 2 or 4: no ChpI protein detectable in the normal J774A.1or THP-1 cells without transfection. Lane 5: blank control. (D). Absence of P2X7 protein in the P2X7-depleted macrophages determined by Western Blot assay. Lane 1 or 3: no P2X7 protein detectable in the P2X7-depleted J774A.1 or THP-1 cells. Lane 2 or 4: the P2X7 protein expressed by the normal J774A.1 or THP-1 cells without transfection. Lane 5: blank control. (E). Expression of the LB361 gene product in the LB361 gene-transfected J774A.1 or THP-1 cells, determined by laser confocal microscopy. The small green spots correspond to the protein expreesed by the LB361 gene in the transfected J774A.1 or THP-1 cells. The large blue plaques correspond to the cell nucleus. The images at “0″ h indicate the results of laser confocal microscopic examination of normal J774A.1 or THP-1 cells before LB361 gene transfection.(TIF)Click here for additional data file.Table S1Sequences of the primers used in this study.(DOC)Click here for additional data file.Materials S1Detection and expression of LA0543, LA2250 and LB361 genes, and generation and identification of LB361 gene deletion and transfection.(DOC)Click here for additional data file.


Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.Our results further confirm that HDACs are  Acute myeloid leukemia (AML) accounts for one-fourth of acute leukemia in children, but is responsible for more than half of the leukemia deaths in this patient population de novo AML and demonstrated highly synergistic antileukemic activities of combined cytarabine/VPA in all of the cell lines and diagnostic blast samples, especially those with t. Our mechanistic studies revealed that cooperative induction of DNA damage by the two agents and induction of Bim by VPA underlay the observed synergistic antileukemic activities of this drug combination. Indeed, our results strongly suggested that HDACs are promising therapeutic targets for pediatric AML, however, which of the HDAC family members are involved in the synergy between cytarabine and VPA is not clear.Despite the well-characterized molecular and cellular effects of HDACIs, single-agent activity of this class of drugs has been modest. Therefore, there is an urgent need for developing rationally designed drug combinations including HDACIs. In our previous study HDACs comprise a large group of proteins divided into four classes based on their homologies to yeast HDACs, their subcellular localizations and their enzymatic activities HDACs control gene expression through chromatin modification. Recent studies have shown that exposure to HDACIs “resensitizes” AML cells to signals for differentiation and/or apoptosis, making HDACIs particularly promising agents for AML therapy In this study, we used 4 pediatric AML cell lines to identify HDAC family members which are involved in cytarabine sensitivities, and to select the optimal HDACIs that were most efficacious against pediatric AML when combined with cytarabine. We demonstrated that HDACs 1 and 6 are critical for cytarabine-induced apoptosis and suggest that pan-HDACIs, which simultaneously inhibit HDACs 1 and 6, may have the greatest potential for enhancing cytarabine activities in pediatric AMLs. Our results further support the use of HDACIs in the treatment of childhood AML.PXD101, LBH-589 and MGCD0103 were purchased from Selleck Chemicals . SAHA and MS-275 were purchased from United States Biological , and from ChemieTek , respectively. Cytarabine  and valproic acid (VPA) were purchased from Sigma Chemical Company .2/95% air.The THP-1, Kasumi-1, and MV4-11 pediatric AML cell lines were obtained from American Type Culture Collection . The CMS pediatric AML cell line was a gift from Dr A. Fuse . The parental and the engineered sublines were cultured in RPMI 1640 with 10-20% heat-inactivated fetal bovine serum  and 2 mM L-glutamine plus 100 U/mL penicillin and 100 µg/mL streptomycin in a 37°C humidified atmosphere containing 5% COTHP-1 cells were treated with various concentrations of HDACIs for up to 48 h and lysed in Cell Lysis Buffer  on ice for 2 hours. After centrifugation , 500 µg supernatant fraction (cell lysate) was incubated with 2 µg rabbit IgG, anti-HDAC1, anti-HDAC3 , anti-HDAC2  or 1000 µg supernatant fraction was incubated with 2 µg anti-HDAC8  overnight at 4°C, followed by incubation with 30 µL of Protein A/G® Dynabeads  for 3 hours at 4°C. The beads were washed three times with ice cold PBS and resuspended in HDAC Assay Buffer  for measuring HDAC enzymatic activities using the CycLex® HDACs Deacetylase Fluorometric Assay kit , or heated at 95°C for 5 min in 30 µl loading buffer for Western blotting.Soluble protein extracts prepared by sonication in hypotonic buffer , containing 1% SDS and proteolytic inhibitors, or immunoprecipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes  and immunoblotted with anti-HDAC1 (#2062), -HDAC2 (#2540), -HDAC3 (#2632), -HDAC4 (#2072), -HDAC5 (#2082), -HDAC7 (#2882) , -HDAC6 , -HDAC8 (H6412), -HDAC10 (H3412), -HDAC11 (H4539), –acetyl (ac)-tubulin (T7451) , -HDAC9 , -ac-Histone H4, -ac-Histone H3, -ac-Histone H4, -Histone H4, or –beta-actin antibodies , as described previously In vitro HDACI cytotoxicities of pediatric AML cell lines were measured by using MTT  assays, as previously described 50 values were calculated as drug concentrations necessary to inhibit 50% proliferation compared to untreated control cells. The data for the cell lines are presented as mean values ± standard errors from at least 3 independent experiments.HDAC1, HDAC2, HDAC3, HDAC4, and HDAC6 shRNA lentivirus clones were purchased from the RNAi Consortium (Sigma-Aldrich) and used to infect THP-1 cells. After selection with puromycin, a pool of infected cells was expanded and tested for HDAC1, HDAC2, HDAC3, HDAC4, or HDAC6 expression by Western blotting . A pool of cells from the negative control transduction was used as the negative control (designated NTC-shRNA).Bcl2L11 (encodes Bim) were quantitated using Taqman probes , and a LightCycler real-time PCR machine , based on the manufacturer's instructions. Real-time PCR results were expressed as mean values from 3 independent experiments using the same cDNA preparations and were normalized to GAPDH.Total RNA isolation, cDNA preparation and purification were as previously reported THP-1 cells were treated with HDACIs or cytarabine alone or in combination for up to 48 h. The cells were harvested, vigorously pipetted and triplicate samples taken to determine baseline and drug-induced apoptosis using the Apoptosis Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) Kit , as previously described Differences in cell apoptosis between cytarabine- and HDACI-treated  and untreated cells or between HDAC shRNA knockdown clones and NTC cells were compared using the paired t-test. Statistical analyses were performed with GraphPad Prism 4.0.de novo AML. Interestingly, Kasumi-1 and MV4-11 sublines were substantially more sensitive to VPA and showed greater responses to combined cytarabine/VPA, compared to THP-1 and CMS cells In our previous study, we demonstrated that VPA can enhance cytarabine-induced apoptosis in different subtypes of pediatric AML cells, including 4 cell lines  and 9 diagnostic blasts from children with To begin to address this important question, we first determined the protein levels for class I, II, and IV HDACs in the 4 pediatric AML cell lines  used in our previous study 50s for the HDACIs and inversely correlated with the responses to combined VPA/cytarabine among the cell lines Besides VPA  of the above HDACIs  with diverse substrate specificities to treat THP-1 cells, characterized by high level expression of both class I and II HDACs. Interestingly, treatments of THP-1 cells with MS-275 resulted in the highest levels of acetylation of both histones H3 and H4, compared to VPA and SAHA  in THP-1 cells treated with IChe cells . Intereshe cells . It is ihe cells  inverselhe cells , suggesthe cells , suggestTo further define the roles of the remainder of classes I and II HDACs in cytarabine sensitivities in pediatric AML, lentiviral shRNA knockdown of HDACs 1, 2, 3 (class I), 4 (representative of class IIa), and 6 (representative of class IIb) was performed in THP-1 cells. As shown in Bcl2L11 transcripts (encode Bim) , suggesting that a transcriptional mechanism may be responsible for the increased BimEL levels. Surprisingly, down-regulation of HDACs 2, 3, and 4 also resulted in increased levels for Bcl2L11 transcripts accompanying unchanged BimEL protein , VPA (inhibits classes I and IIa HDACs), MS-275, MGCD0103 (class I selective-HDACIs)], all at l trials  [24], [2l trials . IP folll trials . This wal trials . Essentil trials . These rEfforts were undertaken to better understand the molecular mechanisms which underlie the anti-leukemic effects of the aforementioned HDACIs. Reports from our own group and others demonstrated that HDACIs can induce DNA damage max concentrations  with different substrate specificities to further narrow down the HDAC isoforms likely to be involved in augmenting cytarabine sensitivity. Results from these studies suggested that HDAC8 is unlikely to be involved in cytarabine-induced apoptosis in THP-1 cells since none of the HDACI treatments resulted in significant enzyme inhibition, although they all enhanced cytarabine-induced apoptosis.Results from our shRNA knockdown studies unequivocally demonstrated that inhibition of HDACs 1 and 6 was pivotal for sensitizing pediatric AML cells to cytarabine. This could, at least partly, be mediated by Bim, a BH3-only pro-apoptotic protein. Bim was classified as an “activator” in view of its purported ability to act directly and to activate Bax and Bak in vitro treatments of pediatric AML cells with both class I selective- and pan-HDACIs. At clinically achievable concentrations, only the drugs (LBH-589 and PXD101) which simultaneously inhibited both HDACs 1 and 6 showed the best antileukemic activities and significantly enhanced cytarabine-induced apoptosis. Again, our mechanistic studies suggest that induction of DNA damage and Bim is critical for the activities of LBH-589 and PXD101 and their combinations with cytarabine.It has been a long-standing debate as to whether isoform specific- or pan-HDACIs result in better anti-cancer activities max concentrations for the HDACIs to combine with cytarabine to prove the concept. However, Cmax or the maximally tolerated doses of these HDACIs may not be the optimal doses for combination therapy with cytarabine. Detailed preclinical studies will be needed to establish the optimal scheduling and dosing for the combinational therapies involving cytarabine plus LBH-589 or PXD101 for treating pediatric AML.Altogether, our results not only confirmed that HDACs are promising therapeutic targets for pediatric AML, but also identified HDACs 1 and 6 as the most relevant drug targets. Accordingly, treating pediatric patients with pan-HDACIs may be more beneficial than HDAC isoform-specific drugs. Our study provides compelling rationale for the combination of cytarabine and HDACIs in pediatric AML clinical trials. It also provides a strong molecular basis for selecting the optimal HDACIs to combine with cytarabine. Since many biological features of AML are shared by adults and children, our results should also apply to the treatment of adult AML patients, as well. It is extremely important to note that we used CTable S1max and t1/2 values of HDACIs determined by phase I clinical trialsC(DOC)Click here for additional data file.


HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain ‘super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common ‘core' transcriptional program, which is  Although AML is highly heterogeneous at the genetic and biological level, unifying themes are evident.4 These include aberrant transcription and epigenetic dysfunction, which provide a potential basis for therapeutic intervention.5 In particular, the dynamic plasticity of the epigenome lends itself well to therapeutic manipulation. As an exemplar of this principle, we and others have recently shown the efficacy and mechanism of action of protein–protein interaction inhibitors of the bromodomain and extra-terminal (BET) family of epigenetic readers in animal models of mixed lineage leukemia (MLL)-rearranged leukemias,7 multiple myeloma8 and non-Hodgkins lymphoma.9 However, the efficacy and potential mechanism(s) of action of BET inhibitors in other forms of AML are largely unknown.Acute myeloid leukemia (AML) is an aggressive hematological malignancy where <30% of all patients are long-term survivors.NPM1) gene.10 NPM1 is a pleiotropic protein with roles in processes as diverse as ribosome biogenesis, chaperoning histones and centrosome duplication. The functional integrity of NPM1 is dependent on its ability to shuttle between the nucleus and cytoplasm, and this ability is severely compromised in NPM1 mutated AML.11 The mutations (termed NPM1c mutations) uniformly alter one or both critical tryptophan residues in the C-terminus of the protein, which prevent proper folding12 and destroys a nucleolar localization signal. In addition, the most common mutation 11 also generates an aberrant extra nuclear export signal. Although NPM1c mutations are heterozygous, hetro/homodimerization with wild-type (WT) NPM1 results in cytoplasmic mislocalization of both mutant and WT protein. This alteration in subcellular location perturbs normal NPM1 function, including the mislocalization and stabilization of critical proteins such as the TP53 regulator p14ARF,14 and leads to transformation. This process also generates a distinct transcriptional signature in NPM1c AML that facilitates the generation of leukemia.16 However, the nature of aberrant transcriptional regulation in NPM1c AML remains obscure.One of the most common mutations in AML, occurring in 35% of cases, involves the nucleophosmin  supplemented with 10–15% fetal calf serum (FCS). OCI-AML3 were grown in 80% alpha-MEM+20% FCS. All growth media were 1% penicillin/streptomycin. Murine progenitors retrovirally transformed with MOZ-TIF2 or NUP98-HOXA9 were grown in RPMI-1640 medium supplemented with 20% FCS+10 ng/ml of IL3. Primary human leukemia cells were also grown in RPMI-1640 medium supplemented with 20% FCS in the presence of 10 ng/ml of IL3, 10 ng/ml IL6 and 50 ng/ml SCF. Cells were incubated at 37 °C and 5% CO27 The following primer pairs were used in the chromatin immunoprecipitation analysis.Gene expression analysis and chromatin immunoprecipitation followed by downstream analysis with next-generation sequencing or RT-PCR were performed as previously described.The following primer pairs were then used in the gene expression analysis.in vivo studies. Ten million NPM1c AML cells from each leukemia were intravenously injected into 6–8-week-old sublethally irradiated (300 cGy) NOD-SCID mice, as previously described.6 Treatment with I-BET at 15 mg/kg was commenced on day 11 and mice were inspected twice daily. This dosing schedule maintained plasma levels of the compound above the in vitro IC50 , and all data analyzed with FlowJo software .Cell apoptosis and cell cycle analysis were performed as previously describedPeripheral blood or bone marrow containing >80% blasts, was obtained from patients following consent and under full ethical approval at each involved institute.The following antibodies were used in ChIP assays, western blotting assays and for immunoprecipitation: anti-H3 , anti-BRD4 ; anti-GFP  and Rabbit polyclonal IgG . Antibodies used for immunofluorescence were anti-BRD4  and Alex Fluor-488-conjugated IgG  immunofluorescence 1:250.Haematopoietic cells were washed once in 1 × phosphate buffered saline before cytocentrifugation onto polylysine coated microscope slides. Cells were fixed with buffered 4% paraformaldehyde, and following stepwise incubation with primary and then secondary fluorescent antibody (see antibodies) cells were stained with Hoechst 33258 (Sigma-Aldrich) and mounted with Vectashield mounting medium . Confocal laser images were captured with an Olympus Fluoview FV1000 microscope equipped with a 40 × oil lens . Image processing was carried out using PHOTOSHOP .6 before hybridization to Illumina Human HT12 v4 BeadChips . Gene expression data were processed using the lumi28 package in R. Probes were filtered to remove those where the detection P-value (representing the confidence that expression is above the background of the negative control probe) was greater than 0.01 in all samples. Expression data were transformed using variance stabilization,29 then normalized using quantile normalization. Comparisons between the dimethyl sulfoxide- and BET-inhibitor-treated samples for all three cell lines  were performed using the R package limma. Genes with a false discovery rate below 5% and a fold-change greater than two were considered significant. Agglomerative hierarchical clustering of the most downregulated genes was performed in R using complete linkage on the pairwaise Euclidean distance between gene signatures, and clusters were identified based on a cutoff of 10.RNA from OCI-AML3 was extracted after 6 h of treatment with I-BET151 and processed as described30 with default parameters. Only reads mapped with a mapping quality score >10 were retained, and multiple reads mapping to identical genomic loci were removed to limit potential PCR bias. Reads were extended along their strand to the estimated fragment size of 300 bp. Peaks were called using Model-based analysis of ChIP-seq31 with standard parameters. The distribution of reads about the transcriptional start site of all protein-coding genes >1 kb, as well as lincRNAs, miRNAs, snoRNAs and snRNAs, was calculated using the Repitools package in R.Sequenced reads were mapped to the reference human genome (hg19) using the Burrows–Wheeler aligner17 As there was an exceptionally strong correlation between MED1 and BRD4 binding intensity at enhancer regions in MM1.S cells, we estimated the number of ChIP-seq reads mapped to these enhancer regions for BRD4 binding in the OCI-AML3 cell line following treatment with dimethyl sulfoxide and I-BET, respectively. For both the treatments, reads per million were estimated and enhancer regions were ranked according to increasing BRD4 signal. Genes (N=6725) that were within 50 kb were assigned to the enhancer regions. Only a few genes were assigned to more than one enhancer.Enhancer and super-enhancer regions were defined as per Young and colleagues, based upon ChIP-seq enrichment for the mediator complex member MED1, the histone mark H3K27Ac and distance from promoters as defined by H3K4Me3 in the MMI.S cell line.in vivo  in growth and colony formation assays . Althougin vivo . Similarll lines , and I-Bll lines . Taken tBCL2 and C-MYC, leading to the induction of apoptosis and cell cycle arrest.6 On the basis of our demonstration of similar cellular phenotypic consequences following I-BET treatment in sensitive AML cell lines, we hypothesized that downregulation of a similar transcriptional program may have mediated these findings. To test this hypothesis, we analyzed the changes in gene expression following 6 h of I-BET treatment (before significant changes in cell cycle and apoptosis are evident) in the sensitive cell lines OCI-AML3 and SKM1, containing NPM1c and EZH2 Y641C mutations, respectively. Similarly to the MV4;11- and Molm13 MLL-rearranged cell lines, only a relatively small number of genes demonstrated a significant alteration in expression in either cell line, with the majority of genes unchanged  of the genes present within the core transcriptional program contain super-enhancers and BRD4 binding to these regions is dramatically decreased following just 6 h of inhibition with I-BET151  cells, relocation of NPM1c into the nucleus with LMB phenocopied treatment with I-BET in downregulating expression of both BCL2 and C-MYC .nd C-MYC . In contnd C-MYC . In OCI-se genes . Howeverse genes . To assese genes . Howeverse genes .NPM1 alleles) and expressed GFP-NPM1c in a proportion of these cells, allowing us to mechanistically test our hypothesis in an isogenic background  and DNMT3A mutations (as occurs in the OCI-AML3 cell line) is common and confers a relatively poor prognosis.21 Therefore, to further assess the efficacy of I-BET against NPM1c AML in cooperation with a number of other mutations, we utilized leukemia cells from an elegant, recently described murine model. In this model, NPM1c drives leukemia in cooperation with a range of other mutations generated through transposon-based insertional mutagenesis leading to unique tumors in each case with identified cooperating mutations in addition to NPM1c . Leukemia cells from all six independent tumors tested demonstrated sensitivity to I-BET in both liquid culture  and colony formation assays , mimicking the presentation of patients with AML in the clinic. Across all tumors, a significant survival advantage (P=0.01) was demonstrated for mice treated with I-BET in comparison with vehicle-treated controls (P=0.008). Althougn weight .Finally, we tested the efficacy of I-BET to inhibit clonal growth of samples from NPM1c AML patients. The majority (four out of five) of these samples also carried FLT3-ITD mutations . As is sin vitro following I-BET151 treatment, a specific inhibitor of BET proteins that we have previously shown to be of low toxicity in preclinical studies.23 These data suggest the clinical utility of BET inhibition across a number of AML subtypes. Of note, the related BET inhibitor I-BET76224 is currently in clinical trials for NUT midline carcinoma, an aggressive epithelial carcinoma characterized by genomic rearrangement of BRD4 or BRD3 . Our findings with I-BET151 and those with another inhibitor of BET proteins, JQ1, in AML7 now provide the basis for early phase clinical trials in these often fatal malignancies.AML remains an unmet medical need. Novel therapies are therefore urgently required, particularly therapeutics with toxicities acceptable for the increasingly elderly population who present with AML. In this report, we demonstrate that a wide range of disparate AML subtypes are sensitive to growth inhibition BCL2, C-MYC and IRF8. Although MLL fusion and NPM1c AML subtypes are associated with the aberrant expression of HOXA genes,25 the common transcription program and the profound in vitro and in vivo effects of BET inhibitors in these leukemias are independent of downregulation of the HOXA genes. Interestingly, a number of the common genes derived from our analyses in AML cell lines contain super-enhancers that are exquisitely sensitive to BRD4 inhibition by I-BET151. Notably, many of the genes downregulated in the human AML cell lines were also downregulated following I-BET treatment in multiple primary AML samples from patients with disparate genotypes. In addition, our 26-gene signature could classify AML into distinct molecular subgroups raising the possibility that this core transcriptional signature can provide biomarkers to predict sensitivity to BET inhibition and may also be used prospectively to monitor patient response to BET inhibition in real time. We propose that this gene set comprises a ‘core' BET-responsive transcriptional program abnormally regulated in AML, and that abrogation of this program underpins the apoptosis and cell cycle arrest that are uniform upon I-BET therapy.Our findings also demonstrate that BET proteins are transcriptional regulators of genes critically required for leukemogenesis. We have previously defined a set of genes in MLL-rearranged AML cell lines whose expression is altered following treatment with I-BET, but it was unknown whether a similar transcriptional program was required in other sensitive AML subtypes. When similar experiments were performed in other AML cell lines, an obvious overlap with the MLL program was evident. This overlap contains 26 genes that are consistently downregulated and includes the critical regulators of myelopoiesis and leukemogenesis BCL2 and C-MYC through a reduction in BRD4 recruitment to chromatin. Restoration of nuclear NPM1 following LMB treatment closely phenocopies the inhibitory effects of I-BET on transcription and BRD4 chromatin binding (Our studies also shed direct light on one of the prevailing mysteries in AML biology: the mechanisms of transcriptional dysregulation in NPM1c AML. We have previously demonstrated that WT NPM1 and BRD4 interact and further demonstrate this interaction in primary AML cells with WT NPM1. Our data predict that NPM1c mutations relieve an inhibitory interaction with BRD4 through cytosolic dislocation of NPM1c and WT NPM1, allowing BRD4 to activate aberrant transcription in NPM1c AML . In supp binding .in vitro sensitivity of AML with NPM1c mutations. NPM1c AML represents one of the single largest AML subgroups, comprising around 35% of all cases, and has a variable prognosis. This prognosis is largely dependent upon the presence or absence of other ‘cooperating' mutations, particularly the FLT3-ITD mutation. Importantly, our findings demonstrate that NPM1c AML cells are uniformly sensitive to I-BET inhibition regardless of the nature of the cooperating mutations (20 Of particular clinical relevance, around 15% of cases of AML harbor both an NPM1c mutation and a FLT3-ITD, and these patients have a relatively poor prognosis.20 Here, we demonstrate sensitivity to I-BET in both primary human AML cells and a mouse model that carry both an NPM1c mutation and an activating mutation of FLT3, suggesting significant clinical utility in this poor-risk subgroup.We and others have previously demonstrated the preclinical efficacy of BET inhibition in MLL-rearranged leukemias, and here we report a similar utations . These iTaken together, our data greatly inform the pathogenesis of NPM1c AML and provide compelling evidence supporting clinical trials of BET inhibitors across multiple AML subtypes. Moreover, our data identify potential predictive biomarkers of sensitivity and response to inform these studies. Novel, nontoxic, targeted therapies are desperately required in AML, and we eagerly await results of trials of BET inhibition in this aggressive hematological malignancy.


Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB) has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC) and highly compacted, transcriptionally inert, heterochromatin (HC), although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs) are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM), is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair. In vivo approaches have also demonstrated that the operation of these pathways and the choice between them, which appears to be determined by DNA end resection, is highly influenced by the chromatin environment surrounding the DSB. Of relevance to this review, the response to DSBs located within regions of heterochromatin (HC-DSBs) is substantially distinct to that which arises at DSBs formed in euchromatin (EC-DSBs). Additionally, there is increasing evidence that a significant role of the DNA damage response kinase, ataxia telangiectasia mutated (ATM), is to modify the chromatin environment in the DSB vicinity. Whilst these modifications can include changes in phosphorylation, ubiquitylation, SUMOylation and methylation, many of which enhance chromatin relaxation, there is, paradoxically, evidence that proteins promoting chromatin compaction, such as KAP-1, HP1 and HDACs, can become relocalized and/or recruited to DSBs [A broad range of studies including biochemical, structural and biophysical approaches have provided substantial detail and insight into the two most significant pathways of DNA double strand break (DSB) repair, namely DNA non-homologous end-joining (NHEJ) and homologous recombination (HR). Cell biology approaches have revealed that  to DSBs . Thus, dThe DDR to DSB formation encompasses repair pathways and a signal transduction response, which causes significant changes to the chromatin environment at a DSB. DNA non-homologous end-joining (NHEJ), which involves the Ku70-Ku80 heterodimer, the DNA dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, DNA ligase IV and XLF, represents the major DSB repair pathway in mammalian cells ,6. HomolHML and HMR loci, which are largely deacetylated and heterochromatic, does not occur in contrast to efficient spreading into euchromatic sequences [A range of studies have demonstrated that chromatin undergoes rapid decondensation at DSBs, leading to a more open configuration –23. Howeequences . Supportequences . A furthequences . Relatedequences . Thus, wequences ,28. A moequences . Finallyequences . FurtherThe use of carbon ion trajectory irradiation has shown convincingly that DSB-containing regions of DNA migrate to the periphery of HC regions . In Droset al., γH2AX modifications originating from an HO-induced DSB penetrate avidly into EC but not HC [Whilst some observations described above may be attributed to DSB movement within HC, there is distinct evidence that HC is refractory to IRIF expansion; for example, in the studies of Kim t not HC . Furthert not HC ,33. For t not HC ,34. Sucht not HC . HoweverIn our own studies with DSBs induced by gamma or X-rays, we have not, even at early times post IR, detected γH2AX foci within the core of densely DAPI-staining regions. Indeed, we have observed differential kinetics of γH2AX foci formation at EC versus HC regions, consistent with the notion that IRIF predominately form on the periphery of HC ,33. HoweWhile there remains some uncertainty about the relationship between DDR signaling and HC, there is strong evidence that HC poses a barrier to DSB repair. Most strikingly, the loss of ATM protein kinase activity confers a DSB repair defect and the persisting DSBs localize to the HC periphery, suggesting that ATM signaling is necessary to facilitate repair of HC-DSBs . This isC-terminal region [In addition to the requirement for ATM for HC-DSB repair, there is also a requirement for downstream components of ATM signaling including γH2AX, MRE11, NBS1, MDC1, RNF8, RNF168 and 53BP1 ,37. In cl region ,39. The l region . These fl region ,41. Rathl region ,42,43.In addition to distinct genetic requirements for HC versus EC-DSB repair, the repair of these two classes of DSBs occurs with markedly different kinetics ,41. For i.e., it is required for the repair of HC-DSBs and its requirement can be overcome by siRNA-mediated knockdown of a number of HC component proteins [As stated above, 53BP1 shows a similar phenotype to ATM for DSB repair in G0/G1 phase—proteins . 53BP1 rproteins ,48. Impoproteins ,49. Thusproteins ,48.versus KAP-1 signal above the pan-nuclear background staining. In this way, pKAP-1 foci were shown to be 53BP1-dependent (in contrast to pan nuclear pKAP-1), to form at only a subset of DSBs (HC-DSBs) and for the loss of these foci (taken to represent DSB repair) to be both 53BP1 and ATM-dependent. Thus, the evidence strongly suggests that HC-DSB repair requires pKAP-1 foci formation, whilst pan-nuclear pKAP-1 is not sufficient to promote HC-DSB repair.Two distinct modes of KAP-1 S824 phosphorylation (pKAP-1) have been delineated, the basis of which may derive from the different mechanisms of ATM retention ,38,41. AA somewhat distinct finding is that KAP-1 as well as all three isoforms of HP1 are rapidly and transiently recruited to DSBs induced by laser microirradiation. KAP-1 and HP1α recruitment occurs via a p150CAF-1-dependent process whilst HP1α recruitment is KAP-1 dependent [The HP1β isoform of HP1 is also known to undergo a dynamic modification during the DDR. Indeed, HP1β is phosphorylated at T51 by Casein Kinase 2 (CK2) in response to DSB formation, triggering its rapid mobilisation from H3K9me3 sites to which it is bound (via its chromodomain) in undamaged cells . The relThe studies discussed above suggest that ATM triggers localized HC relaxation to promote DSB repair. Interestingly, KAP-1 phosphorylation results in pan nuclear chromatin relaxation as monitored by sensitivity to micrococcal nuclease . HoweverN-terminal RING finger [C-terminal region. SUMOylation of KAP-1 is known to mediate interactions with the multi-subunit nucleosome remodeling and deacetylase (NuRD) complex as well as with the histone methyltransferase, SETDB1. The interaction between SUMOylated KAP-1 and NuRD occurs via the ATP-dependent chromatin remodeling subunit, CHD3  [C-terminus, which, as its name implies, interacts with SUMO1-modified KAP-1. One possible model is that phosphorylation at S824 on KAP-1 might inhibit SUMOylation at the nearby residues, K804/K779/K554. However, although a previous finding was supportive of such a model [C-terminal region of KAP-1 produces a SIM-like domain within KAP-1, which can interfere with the KAP-1SUMO1:CHD3SIM interaction and causes CHD3 dispersal from the DSB site , potentially via an auto-SUMOylation mechanism involving KAP-1’s G finger . The S82 CHD3.1) ,54. Unli a model . The modDSB site . This exAs mentioned above, DSBs that form within HC rapidly become localized to the HC periphery. Although IRIF expansion occurs slightly slower at these DSBs, at later times post IR their size is similar to those at EC regions, largely due to the expansion of γH2AX foci into the surrounding EC . Howeveri.e., there appears to be a defined threshold of DDR signaling required to activate checkpoint arrest [Cell cycle checkpoint arrest represents a significant consequence of the DDR. Such checkpoints include the G1/S, intra-S and G2/M checkpoint. The G2/M checkpoint is interesting in this context since a range of studies have shown that low doses of ionizing radiation fail to efficiently activate checkpoint arrest—t arrest ,58. Simit arrest . A similAnalysis of the DNA damage response in the past few years has revealed, somewhat unexpectedly, that the chromatin undergoes enormous changes following exposure to DNA damaging agents. The majority of these changes arise at the DSB site but pan-nuclear chromatin alterations have also been described. Perhaps surprisingly, these changes appear to effect both relaxation and compaction of chromatin, depending on the situation or time post damage. In this review, we have focused on those changes that take place when a DSB arises in HC-DNA. Although ATM signaling, which is known to exert a massive impact on chromatin in the DSB vicinity, appears to lie at the heart of the changes observed, some responses appear to be ATM-independent. However, critically, ATM is required for the repair of HC-DSBs. Further, a diminution of HC compaction, as observed in human disorders with HC-relaxation, leads to hypersensitive signaling and checkpoint arrest. Despite this, there remains only limited direct evidence that HC relaxation occurs at the level of a single DSB. One reason for this is that the magnitude of relaxation may be modest, either due to the nature of the relaxation or its extent, rendering it difficult to observe except by sophisticated and sensitive methods. Further innovative approaches are required to determine how the magnitude of HC compaction is modified at HC-DSBs. In this article, we have divided chromatin simply into “EC” or “HC”; however, chromatin is in fact composed of a highly diverse mosaic of chromatin states, with many shades of grey between the classic definition of transcriptionally active, open EC and compacted, silent HC. Specific questions that we feel need to be addressed are understanding the finer details of how chromatin compaction and specific epigenetic DNA and histone modifications combine to regulate DNA end resection and the outcome of repair, the magnitude of DDR signaling and its impact of cell cycle checkpoints. If HC DSBs more readily undergo DNA end resection, then it is important to determine its cellular consequence and whether HC affects the fidelity of the repair process and the likelihood of translocations.


Recent measurements of gene expression show a lot of randomness between cells. Steven Frank proposes a theory in which robustness protects against the effects of randomness and so allows relaxed regulatory control and greater randomness to evolve. Gene expression varies widely in cells with the same genotype and environment  This essay is part of the Challenges Series: highlighting fundamental, unifying challenges in biology.Some phenotypes arise by averaging the inputs from many components. For example, a cell may capture an external signal and store the information internally. If the half-life of the internally stored information is sufficiently long, then a cell can average the inputs over a period of time Frank I have emphasized that combining component inputs increases the robustness of the system by reducing the consequences of component variability. I now turn the problem around. How does an increase in system robustness affect the variability of components?n independent inputs, each input having a variance of σ2, the variance of the average is σ2/n. If we double the number of inputs, then the system can tolerate a doubling of the variance of each component and still achieve the same variability in output.Suppose that multiple component inputs are averaged to obtain a system output. The more independent component inputs, the less each component input affects the average. Therefore, as the number of inputs increases, the system can tolerate greater component variability to achieve the same level of output variability If we assume that natural selection is acting on the system outputs—the phenotypes—then increasing the number of inputs weakens the selective force acting on the variability of each component. As selection on the reliability of the components weakens, we may expect greater component variability. In general, any evolutionary change that increases the robustness of system output tends to reduce the selective pressure acting on the reliability and variability of the individual components of the system This idea leads to a prediction: the more protections a system has against the variability of the underlying components, the more stochastic variability there will be in the underlying components. The logic is so simple and inevitable that it must, in a sense, be true. But a true theory is not necessarily an interesting or important biological theory. The force may be weak and of little consequence. To study the problem empirically, we may compare a phenotype that arises by aggregation of few components versus a phenotype that arises by aggregation of many components. Microbes often provide good model systems in which to test simple theories about natural selection.Bacteria often secrete enzymes to digest complex extracellular resources Several other microbial functions also happen extracellularly, such as quorum sensing and binding of free iron and vitamins by secreted molecules In multicellular organisms, some functions arise at the aggregate tissue level, whereas other functions depend on individual cellular expression. Cellular variability should be relatively high when function is at the aggregate tissue level compared to cases in which function is at the cellular level. Within cells, functional expression may arise by aggregation over the expression of several different gene products or pathways that effectively provide the same function. Redundant gene products or pathways are widely observed. Variability in redundant components is relatively weakly constrained and therefore is expected to be high.Aggregation reduces the sensitivity of overall function with respect to the performance of each component. Thus natural selection may often favor a less costly, lower performance design of component traits If one focused solely on the design and performance of the components over time, it would appear as if they were becoming increasingly maladapted. That apparent maladaptation arises because natural selection operates only on overall function rather than on the design and reliability of individual components. Clear interpretations of design in physiology and systems biology require consideration of components in relation to overall system function. Improving design at the system level can actually favor degradation of performance at the component level.It can be difficult to test the effects of a complex evolutionary sequence on the design of traits. Microbes may provide a good place to start. Consider a clonal microbial population faced with the challenge of digesting complex extracellular food sources. Initially, the population may be placed in a situation with low extracellular diffusion, so that each cell tends to be near only the digested products caused by its own enzymatic secretions . Here, tThen a period of selection follows in a well-mixed environment. With mixture, each cell is near the digested products caused by the overall enzymatic secretions of the population . The funAfter a period of evolution in the well-mixed environment, the performance of individual cells may be compared to the cells derived from the initial period of selection in the low-diffusion environment. The prediction is that variability in expression will be greater in cells derived from the well-mixed environment. In addition, the overall performance and reliability of the cells from the well-mixed environment may be degraded relative to the cells from the low-diffusion environment.To test for reduced costs of enzyme production in cells from the well-mixed environment, one may supplement the environment so that no benefit is derived from extracellular digestion but extracellular enzymatic secretion continues to be stimulated. In direct competition, the cells from the well-mixed environment may have an advantage relative to the cells from the spatially structured environment, because the well-mixed environment may have favored a reduction in the costs associated with the secretory traits.There are, of course, many complexities of experimental design and interpretation in this scenario. For example, genetic variability may introduce competition between different genotypes, adding additional selective pressures with respect to the level at which function arises With regard to the challenge of explaining patterns of stochastic variation in gene expression, the processes of aggregation and robustness provide intriguing hypotheses. Increased aggregation reduces the consequence of variability at the component level. Reduced selective pressure on the components may often lead to increased stochastic fluctuations of those components. Particular genes almost always play a component role within a larger functional system. The system structure by which component genes aggregate to determine function may be an important factor determining the amount of stochastic variation in gene expression.


How is motion information that has been obtained through multiple viewing apertures integrated to form a global motion percept? We investigated the mechanisms of motion integration across apertures in two hemifields by presenting gratings through two rectangles  and recording the perceived direction of motion by human observers. To this end, we presented dual barber poles in conditions with various inter-component distances between the apertures and evaluated the degree to which the hemifield information was integrated by measuring the magnitude of the perceived barber pole illusion. Surprisingly, when the inter-component distance between the two apertures was short, the perceived direction of motion of the dual barber poles was similar to that of a single barber pole formed by the concatenation of the two component barber poles, indicating motion integration is achieved through a simple concatenation mechanism. We then presented dual barber poles in which the motion and contour properties of the two component barber poles differed to characterize the constraints underlying cross-hemifield integration. We found that integration is achieved only when phase, speed, wavelength, temporal frequency, and duty cycle are identical in the two barber poles, but can remain robust when the contrast of the two component barber poles differs substantially. We concluded that a motion stimulus presented in bilateral hemifields tends to be integrated to yield a global percept with a substantial tolerance for spatial distance and contrast difference. A common problem faced by the neural system is integrating spatially distributed information to infer a global percept. Researchers proposing Gestalt psychology observed that we tend to link multiple components together to create a global pattern percept ,2. HowevIntegration and segregation are two processes that are complementary to each other –6. For eWe hypothesized that the motion processing property provides a unique opportunity to qualify the integration properties. The aperture problem of motion processing is a well-studied example of the integration process –22. WhenGuilford found that a barber pole, constructed by the presentation of a drifting grating through a rectangular aperture, yields a visual motion illusion which can characterize how motion integration is processed in the human brain ,40–42. TIn the present study, we presented dual barber poles which consisted of two component barber poles placed symmetrically in two hemifields and then recorded the perceived direction of motion in human observers. By analyzing the strength of the barber pole illusion induced by the dual barber poles, we could gauge the strength of motion integration across the hemifields. We varied the distance between the component barber poles to characterize the spatial constraints of integration, and then we presented dual barber poles in which the motion and contour properties of the two component barber poles differed to characterize the motion and contour constraints underlying integration. The results indicated that motion information presented in the bilateral hemifields tended to be integrated to yield a global percept with a substantial tolerance of spatial distance and contrast difference between the two component barber poles.Five healthy naive subjects  participated in all the experiments in the present study. All subjects reported normal corrected vision and no history of central nervous system disease. The corrected vision of all subjects was assessed by the Snellen chart, and all subjects showed visual acuity equal to or better than 1.0. Written informed consent was obtained from each subject. All testing procedures were performed in compliance with the policies and procedures of the Chang Gung Medical Foundation Institutional Review Board. The Chang Gung Medical Foundation Institutional Review Board specifically approved this study. Subjects were paid for their participation.°, 135°, 225°, or 315°, which were ± 45° relative to the orientation of the long axis of the aperture. Unless otherwise specified in the following text, the wavelength (stripe-to-stripe distance) of the drifting square-wave gratings constituting the barber poles was 0.5°, their duty cycle 50%, their speed 10°/s, and their contrast 75% (computed using the Michelson formula). The luminance levels of the grating stripes were 4.5 cd/m2 and 31.7 cd/m2, respectively, against a background of 18.1 cd/m2. We used this wavelength and duty cycle because it yielded a clear percept of the barber pole illusion as determined in preliminary experiments and by other researchers [A barber pole was constructed by presenting a drifting square-wave grating within a rectangular aperture (example barber poles are shown in earchers . The str°: °). The rationale for choosing this aspect ratio is that the side-by-side concatenation of two component barber poles yields a single horizontal barber pole with an aspect ratio of 1.41:1 (width: height: = 2°: °), a design that could balance the effects between the component barber poles and the global barber pole ensemble. Unless otherwise specified, the phase of the two component barber poles was identical.Dual barber poles consist of two component barber poles symmetrically presented in bilateral visual hemifields, separated by an inter-component distance  with a resolution of 1024*768, a pixel size of 0.02°, and a refresh rate of 60 Hz. The software was Matlab  with Psychophysics Toolbox . On eachperceptual bias, which gauged the magnitude of the barber pole illusion, was expressed as the deviation of the perceived direction from the grating’s direction of motion and was defined as positive when it deviated horizontally. In other words, a positive perceptual bias indicated that the perceived direction was biased toward horizontal directions and a negative perceptual bias indicated that the perceived direction was biased toward vertical directions.The global probability as the proportion of trials where the subject reported the “global percept” by choosing the perceived global direction, instead of reporting the “individual percept” by only clicking the right mouse button.As mentioned in the task structure section, the subject could either perceive a global motion coherently moving at one velocity  or two motion velocities corresponding to the velocity of each of the two component barber poles . We then computed the The perceptual bias among repetitions was averaged using the vector average method and the data was represented as mean ± standard error of mean. The comparisons among conditions were analyzed using repeated-measures ANOVA.2 (We presented single barber poles with various aspect ratios and with a fixed aperture area to gauge the magnitude of the barber pole illusion in each subject. The three different aspect ratios (height/width) were 1:1.41 (1°/1.41°), 1:1 (1.19°/1.19°), and 1.41:1 (1.41°/1°) and their aperture area was fixed as 1.41(°)2 .p < 0.001).We presented dual barber poles with different inter-component distances to measure the degree to which the inter-component distance affected the strength of motion integration across the visual hemifields. The distance between the two component barber poles was 0°, 0.03°, 0.07°, 0.10°, 0.14°, 0.17°, 0.21°, 0.41°, 0.83°, or 1.66°  and then approached the perceived direction of the single vertical component barber pole measured in Experiment 1  in all conditions and in all subjects, indicating these dual barber poles always elicit a single global percept; the perceptual bias peaked when the inter-component distance was zero, yielding a single horizontal barber pole with a width-to-height aspect ratio of 1.41:1 . This finding was incompatible with their perceived directions to the dual barber poles (circle trace in p < 0.001). As can be seen in p > 0.05 when the width of the completed barber pole was 2° and 2.03°) and gradually shifted to that of the single component barber pole (dashed line) as the inter-component distance increased , suggesting that cross-hemifield integration follows the simple concatenation mechanism when the inter-component distance is small, and the strength of integration faded away when the two components are far apart.Perceptual bias to the completed barber poles, reflecting the filling-the-gap predictions, monotonically increased as the inter-component distance increased  the same. The parameters of the right component barber pole in this and the remaining experiments were identical to those delineated in Experiment 3. The wavelength of the left component barber pole was 0.33° (0.5°*2/3), 0.5°, or 0.75° (0.5°*3/2) and that of the right component barber pole was 0.5°. The speed, which is the product of wavelength and temporal frequency, changed proportionally as we altered the wavelength. In other words, the speed of the left component barber-pole was 2/3, 1, or 3/2 times of the speed of the right component barber poles (10°/s), respectively. The inter-component distance was 0°, 0.1°, 0.2°, or 0.8° . The othp = 0.001; interaction effect between the wavelength and inter-component distance, F = 11.3, p < 0.001). Post-hoc analysis showed that the perceptual bias decreased when the wavelength differed in the component barber poles and the inter-component distance was small (p < 0.05 when the inter-component distance was 0° and 0.1°) (example subject in p > 0.05 when the inter-component distance was 0.2° and 0.8°).The standard dual barber poles almost always elicit a global percept  and the dual barber poles, in which the wavelength and corresponding speed in the component barber poles differed, still tended to elicit a global percept  , respectively.p = 0.048; interaction effect between temporal frequency and inter-component distance, F = 4.31, p < 0.01). Post-hoc analysis showed that the perceptual bias decreased when the temporal frequency differed in the component barber poles and the inter-component distance was small (p < 0.05 when the inter-component distance was 0° and 0.1°) (example subject in p > 0.05 when the inter-component distance was 0.2° and 0.8°).Dual barber poles in which the temporal frequency and corresponding speed in the component barber poles differs tend to elicit a global percept   or temporal frequency (in Experiment 5) will change the speed proportionally. To determine whether the results in Experiments 4 and 5 could be simply accounted for by the differences in speed between the component barber poles, we varied the wavelength and temporal frequency in the left component barber pole while keeping the speed constant in all conditions. Specifically, the wavelength and temporal frequency of the left component barber pole was 0.33° (0.5°*2/3) & 30 Hz (20 Hz*3/2), 0.5° & 20 Hz, or 0.75° (0.5°*3/2) & 13.3 Hz (30 Hz*2/3) yielding a constant speed of 10°/s . The wavp = 0.02; the interaction effect between the temporal frequency and inter-component distance, F = 5.1, p < 0.01). Post-hoc analysis showed that the perceptual bias decreased when the temporal frequency and wavelength differed in the component barber poles and the inter-component distance was small (p < 0.05 when the inter-component distance was 0° and 0.1°) (example subject in p > 0.05 when the inter-component distance was 0.2° and 0.8°).Similar to those observed in Experiments 4 and 5, the dual barber poles whose wavelength and temporal frequency differed in the component barber poles still tended to elicit a global percept  (See the example subject in We then varied the duty cycle in the left component barber pole while keeping the other parameters the same to test whether a difference in the duty cycle between the component barber poles affects the strength of cross-hemifield integration. The duty cycle of the left component barber pole was 33.3% (50% *2/3), 50%, or 75% (50% *3/2) and that of the right component barber pole was 50% .p < 0.01; interaction effect between the duty cycle and inter-component distance, F = 3.1, p = 0.02). Post-hoc analysis showed that the perceptual bias decreased when the duty cycle differed in the component barber poles and the inter-component distance was small  (example subject in p > 0.05 when the inter-component distance was 0.8°).Dual barber poles in which the duty cycle differed in the two component barber poles tended to elicit a global percept   = 10.8, p < 0.001). Post-hoc analysis showed that the perceptual bias decreased when the phase differed in the component barber poles and the inter-component distance was small (p < 0.05 when the inter-component distance was 0° and 0.1°) (example subject in p > 0.05 when the inter-component distance was 0.2° and 0.8°).Dual barber poles in which the phase differed in the two component barber poles tended to elicit a global percept   = 0.7, p = 0.65),   on an occluding surface is perceived as moving vertically. This barber pole illusion greatly diminished when the gratings in the rectangular apertures were placed in front of the occlusion plane, indicating that motion integration across apertures occurs only when the grating plane is behind the occlusion plane . In the


Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets.Ki≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α2C-adrenoceptor (Ki = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α1A-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (KB = 3.7 µM). JDTic bound to the noradrenaline transporter (Ki = 54 nM), but only weakly inhibited transport (IC50 = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (Ki = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers.In binding assays, the three antagonists showed no detectable affinity (1A-adrenoceptors). This may contribute to GNTI's severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α In vitro, these compounds are potent and selective antagonists at the κ opioid receptor (κ-OR), with much lower potency at μ- (mu) and δ- (delta) OR Selective κ (kappa) opioid antagonists may have therapeutic potential against conditions such as depression and anxiety disorders in vivo differ dramatically from other opioid antagonists. Attainment of maximal κ opioid antagonism may be delayed by hours or days, compared to minutes for competitive antagonists like naloxone The pharmacodynamics of these compounds in vitroSurprisingly, despite the extremely protracted timecourse of κ antagonism, other effects of nor-BNI, GNTI and JDTic are of rapid onset and brief duration These effects are maximal when κ antagonism is submaximal (<2 hours), and undetectable when antagonism is maximal (24 hours) 50 = 570 mg/kg s.c.) While κ antagonism cannot plausibly account for these transient effects, the fact that some of them are caused by several of these compounds suggests that they may nonetheless be κ-OR-mediated. They might, for instance, result from inverse or biased agonism. However, effects seen after only one of these agents suggest the involvement of targets other than κ opioid receptors (κ-OR). Dramatically, we found that administration of a high dose of GNTI to mice (30 mg/kg i.p.) caused ataxia, convulsions and death within 18 minutes. By contrast, even at 100 mg/kg, nor-BNI and JDTic caused no convulsions or deaths 1 muscarinic receptors (M1-R) In vivo, M1 agonist McN-A-343 inhibited GNTI-induced scratching, consistent with the in vitro evidence for the involvement of this receptor Nor-BNI Ki>1 µM) for the N-methyl-d-aspartate (NMDA) receptor Ki = 780 nM) Less evidence is available on the affinities of nor-BNI and JDTic for non-opioid targets. Nor-BNI has been reported to show very low affinity , and JDTic for the opioid-like receptor NOP (Ki = 12 nM) and the noradrenaline (norepinephrine) transporter .As expected, nor-BNI, GNTI and JDTic bound selectively to κ- over μ- and δ-OR, albeit with lower selectivity (3 to 44-fold) than in some previous reports 3H]CGP-39,653 from the glutamate site 3H]-MDL-105,519 from the glycine site by dynA 3H]MK-801 from the phencyclidine (cation channel) site at 10 µM,. This does not exclude the possibility of binding to the glutamate and glycine sites. Note that in previous reports, binding to these sites was only detectable at nor-BNI concentrations above 1 µM, consistent with the negligible affinity observed here.Contrary to previous reports, we detected no binding by nor-BNI to NMDA receptors. However, this receptor's multiple binding sites complicate comparisons. Nor-BNI was reported to weakly displace 1A adrenoceptors (α1A-AR). We therefore measured functional activity in an intracellular calcium mobilization assay. Surprisingly, even at 30 µM GNTI neither activated α1A-AR nor inhibited activation by noradrenaline (norepinephrine). To reconcile this apparent discrepancy, we tested for allosteric enhancement of noradrenaline's effects.In initial tests, GNTI showed affinity for α1A-AR, but increased the maximal response to noradrenaline by up to 20% without affecting potency (50 of GNTI for this enhancement was 41 nM (95% CI = 24 to 72 nM). At the highest concentration of GNTI tested (30 µM), the potency of noradrenaline and the increase in maximal response were both reduced, suggesting that weak competitive antagonism occurs at this concentration.In the calcium mobilization assay, GNTI did not activate α potency . This su1A-AR-mediated calcium mobilization reportedly exist 1A-AR, selectively enhancing efficacy towards a PI-independent signalling pathway.By contrast, GNTI slightly reduced the maximal response to noradrenaline in an assay measuring hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI); weak competitive antagonism again occurred above 10 µM . This di125I]HEAT from α1A-AR only at concentrations approaching 10 µM . In a previous report 1-R at 1 µM, while we observed only 49% displacement at 10 µM. One potential reason for this small difference is our use of a different radioligand, [3H]3-quinuclidinyl benzilate (QNB); the previous report used [3H]N-methylscopolamine. Given the convergent evidence reported previously for the involvement of M1-R 1-R, and inhibited the effect of acetylcholine with very low potency: KB = 3.7 µM (95% CI = 3.2 to 4.3 µM). Given the consistently sub-nanomolar potency of GNTI at κ-OR in functional assays GNTI showed negligible affinity for all non-opioid targets (Ki = 54 nM versus [3H]nisoxetine). However, in a functional assay . This was not due to assay insensitivity, since the positive control nisoxetine exhibited high potency . The surprisingly low potency of JDTic suggests that it may bind with high affinity to an allosteric site, modulating the affinity of [3H]nisoxetine without affecting noradrenaline transport. Evidence for at least one affinity-modulating allosteric site in NET has been reported previously JDTic bound to the noradrenaline transporter with moderately high affinity . Nor-BNI showed negligible affinity, consistent with prior reports 2 = 7.1, 95% CI = 5.9 to 8.3, KB of 73 nM for a competitive antagonist . Maximally-effective concentrations of N/OFQ (>100 nM) were not affected even by extremely high concentrations of JDTic (30 µM); this saturable effect suggests negative allosteric modulation rather than competitive antagonism  JDTic bound with high affinity to NOP  is comparable to our estimate of the unbound brain concentration after a high dose of GNTI (5 nM after 39 mg/kg) 1-R, towards which the potency of GNTI is orders of magnitude lower.Allosteric enhancement of noradrenaline's actions at α1 antagonist is consistent with a previous report of weak binding to that receptor in vivo. In the prior report, an M1 agonist (McN-A-343) inhibited GNTI-induced scratching in mice, providing apparent in vivo confirmation that GNTI acts upon this receptor 1 antagonist had no effect. However, those results are difficult to interpret, for several reasons. Firstly, in earlier reports M1 agonists induced scratching, while antagonists inhibited it 1 agonist is paradoxical. Secondly, McN-A-343 was administered intrathecally (i.t.), while GNTI was injected s.c. 1-R. Indeed, GNTI induces scratching after s.c. but not i.t. administration 1 agonists show the opposite profile 1 receptors 1 receptors. Further exploration of this issue would benefit from the use of more selective M1 ligands, administered by the same route as GNTI.Our finding that GNTI acts as a weak M1 receptors may be involved in some transient effects of GNTI. We previously observed peak unbound GNTI concentrations in plasma of 2 µM at a dose of 10 mg/kg, and 8 µM at 39 mg/kg 1 antagonism, given KB = 3.7 µM. At these concentrations, detectable receptor occupancy would also be expected at five other peripheral targets, including M2-R . JDTic•2HCl•H2O: F. Ivy Carroll, Research Triangle Institute, NC. Naltrexone•HCl•2H2O: Tocris Bioscience (batch 5B/93329). Nor-BNI•2HCl•H2O: Tocris Bioscience (batches 8A/90732 and 9A/93084). Other compounds were purchased from Sigma-Aldrich .3H]N/OFQ in membranes prepared from HEK293T cells transiently transfected with human NOP as described previously Inhibition of the noradrenaline transporter (NET) was determined using fluorescent dye uptake  Caco-2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. The cells were trypsinized twice a week with trypsin/ethylenediaminetetraacetic acid (EDTA) (0.02% each) and the medium was changed twice a week. Cells were harvested with trypsin-EDTA and seeded onto a MultiScreen Caco-2 assay system  at a density of 10,000 cells per well. The culture medium was replaced every 48 h for the first 6 days and every 24 h thereafter, and after 21 days in culture, the Caco-2 monolayer was utilized for the permeability experiments. Trans-epithelial electrical resistance (TEER) of the monolayers was measured daily before and after the experiment using an epithelial volt-ohm meter . TEER values were greater than 1800 Ω for 21-day cultures.app, A→B) and basolateral to apical  permeability of compounds were measured at 120 min and at various compound concentrations (1–100 µM). Compounds were dissolved in Hanks' balanced salt solution  and sterile filtered. After 21 days of cell growth, the medium was removed from filter wells and from the receiver plate. The filter wells were filled with 75 µL of fresh HBSS buffer and the receiver plate with 250 µL per well of the same buffer. This procedure was repeated twice, and the plates were incubated at 37°C for 30 min. After incubation time, the HBSS buffer was removed and compound solutions added to the filter well (75 µL). HBSS without the compound was added to the receiver plate (250 µL). The plates were incubated at 37°C for 120 min. After incubation time, samples were removed from the apical (filter well) and basolateral (receiver plate) side of the monolayer and then were stored in a freezer (−20°C) pending analysis. Concentrations were calculated from UV absorbance at the following wavelengths (nm): 278 (nor-BNI); 285 (GNTI); 276 (JDTic); 228 . The apparent permeability (Papp), in nm sec−1, was calculated using the following equation:A is the volume (in mL) in the acceptor well; Area is the surface area of the membrane (0.11 cm2 of the well); time is the total transport time in seconds (7200 s); [compound]acceptor is the concentration of the compound measured by UV spectroscopy; [compound]initial is the initial compound concentration in the apical or basolateral wells.Apical to basolateral (PDataset S1Raw data for .(XML)Click here for additional data file.Dataset S2Raw data for .(XML)Click here for additional data file.Dataset S3Raw data for .(XML)Click here for additional data file.Dataset S4Raw data for .(XML)Click here for additional data file.Dataset S5Raw data for .(XML)Click here for additional data file.File S1Radioligand displacement curves.(PDF)Click here for additional data file.Table S1Binding affinities with standard error, radioligand, membrane type, and species.(XLS)Click here for additional data file.