MDMX (MDM4) oncogene is amplified or overexpressed in a significant percentage of human tumors. MDMX is thought to function as an oncoprotein by binding p53 tumor suppressor protein to inhibit p53-mediated transcription, and by complexing with MDM2 oncoprotein to promote MDM2-mediated degradation of p53. However, down-regulation or loss of functional MDMX has also been observed in a variety of human tumors that are mutated for p53, often correlating with more aggressive cancers and a worse patient prognosis. We have previously reported that endogenous levels of MdmX can suppress proliferation and promote pseudo-bipolar mitosis in primary and tumor cells derived from p53-deficient mice, and that MdmX-p53 double deficient mice succumb to spontaneously formed tumors more rapidly than p53-deficient mice. These results suggest that the MdmX oncoprotein may act as a tumor-suppressor in cancers with compromised p53 function. By using orthotopic transplantation and lung colonization assays in mice we now establish a p53-independent anti-oncogenic role for MdmX in tumor progression. We also demonstrate that the roles of MdmX in genome stability and in proliferation are two distinct functions encoded by the separate MdmX protein domains. The central Zn-finger domain suppresses multipolar mitosis and chromosome loss, whereas the carboxy-terminal RING domain suppresses proliferation of p53-deficient cells. Furthermore, we determine that it is the maintenance of genome stability that underlies MdmX role in suppression of tumorigenesis in hyperploid p53 mutant tumors. Our results offer a rationale for the increased metastatic potential of p53 mutant human cancers with aberrant MdmX function and provide a caveat for the application of anti-MdmX treatment of tumors with compromised p53 activity.The  The level of p53 activity in a cell is tightly regulated by the MDM2 and MDMX (MDM4) oncoproteins. These MDM homologs bind to the amino-terminal p53 transactivation domain and inhibit p53 regulation of heterologous gene expression. MDM2 and MDMX heterodimerize to facilitate MDM2-mediated ubiquitination and degradation of p53.MDM gene amplification and/or overexpression have been observed in a variety of human cancers, including leukemia and lymphoma, breast cancer, glioblastoma, soft tissue sarcoma, osteosarcoma and retinoblastoma.11 As most tumors with amplified copy numbers of MDM genes retain wild-type p53,12 the increased level of MDM2 or MDMX proteins is thought to promote oncogenesis by inhibiting p53 activity in these cells. Experiments involving Mdm2 or MdmX overexpression in cells or mice also suggest that MDM oncoproteins may have p53-independent roles in regulating cell growth and tumorigenesis17 although the precise contributions of these p53-independent, proto-oncogenic effects in human cancers remains uncertain.MDM2 and MDMX also regulate p53-mediated tumor suppression. MdmX and p53 succumb to spontaneous tumorigenesis faster than mice deleted solely for p53,18 suggesting p53-independent anti-oncogenic function for MdmX in mice. In contrast, deletion of Mdm2 fails to alter the tumorigenic potential of mice lacking functional p53.19 Down-regulation or loss of functional MDMX protein has also been associated with more aggressive or advanced osteosarcomas, soft tissue sarcomas, thyroid and prostate carcinomas, and chronic myelogenous leukemia.24 In addition, an alternatively spliced MDMX variant is often found in high-grade glioblastomas, papillary thyroid carcinomas, soft tissue sarcomas and osteosarcomas.24 In both human tumors25 and in mouse model with targeted MdmX internal deletion26 this altered splicing reduces the level of full-length (FL) MdmX transcript and generates a novel transcript encoding a severely truncated, unstable MdmX protein. The increase in short to FL transcript ratio in osteosarcomas correlates with reduced MDMX protein levels, faster metastatic progression and greatly reduced patient survival.20 Lower MDMX protein levels in many osteosarcoma or breast cancer cell lines and in soft tissue sarcomas correlate with compromised p53 function.20 Although it is likely that p53-mutant tumor cells have lost the selective pressure to maintain high levels of functional MDMX, it is unclear why loss of functional MDMX in these cells correlates with a more aggressive cancer.We have previously shown that mice co-deleted for both MdmX is also deleted, and that MdmX/p53-double-null cells have increased incidence of multipolar mitosis and reduced cell ploidy compared with p53-null cells.18 These findings suggest a p53-independent role for MdmX in suppression of proliferation and in maintenance of genome stability in hyperploid mouse cells. In the present study, we use human tumor cells in mouse orthotopic transplantation and lung colonization assays to explore the relevance of these p53-independent effects of MdmX in tumorigenesis. We provide the evidence that MdmX suppresses tumor progression and metastases in these mouse models of human cancer. Furthermore, we find the inhibition of cell proliferation and maintenance of genome stability to be separable MdmX functions encoded by different MdmX protein domains. We demonstrate that the ability of MdmX Zn-finger domain to suppress multipolar mitosis and large-scale ploidy reduction in p53-mutant cells underlies the role of MdmX in tumor suppression. We discuss the implications of our findings on cancer treatment strategies and on current models of genome instability and cancer progression.We previously observed that p53-deficient mouse embryo fibroblasts (MEFs) and p53-deficient mouse tumor cells proliferate faster when 27 that polyploid cells undergoing multipolar mitosis can indeed generate more than two daughter cells but many of the resulting progeny dies during one or two subsequent divisions. Time-lapse video microscopy analyses now revealed that only 21% of all multipolar mitosis results in multipolar division and 71% of such progeny died or arrested during the 69 h of filming. A majority (79%) of multipolar mitosis produced only two viable daughter cells  domain, a central acidic domain, a zinc-finger (ZnF) domain of unknown function and a carboxy-terminal RING domain critical for the interaction of MdmX with Mdm2. To characterize the ability of MdmX to suppress cell proliferation and multipolar mitosis, we utilized mouse MdmX expression constructs encoding either the FL, or mutant MdmX lacking carboxy-terminal RING domain (dRING), central zinc-finger domain (dZnF) or both  in transfected cells  are similar to the endogenous MdmX levels in p53 null mouse tumor cells (13.7±4.5) and in wild-type MEFs (11.07±4.6). Proliferation assays revealed that exogenous FL MdmX and dZnF mutant suppress proliferation of MdmX/p53-deficient cells . In contSince MdmX interacts with Mdm2 and forms heterodimer through its RING domain, we explored whether the p53-independent effect of RING domain on cell proliferation requires Mdm2. Tumor cells derived from Mdm2/MdmX/p53-triple deficient (TKO) mice were transduced with FL or MdmX deletion constructs . Cell proliferation assay  reveal t18 To identify the domain(s) in MdmX protein responsible for promoting genome stability, we analyzed the DNA content and chromosome number of mouse DKO tumor cells transduced with MdmX expression constructs. Propidium iodide staining and metaphase spread analysis indicate that the expression of FL MdmX in DKO cells increased DNA content and chromosome numbers to the levels seen in tumor cells from p53-null mice retaining endogenous MdmX  measured in the same cells. We first confirmed that MdmX also inhibits chromosome loss  and multin vivo experiment, cells were transplanted into the mammary fat pad of immune-compromised NSG (NOD-scid IL2ry null) mice and tumors were harvested and measured 16 days later. Tumors developed in all animals, but were much smaller in mice implanted with MdmX-FL and MdmX-dRING-containing cells than in mice with mock-control or MdmX-dZnF cells  and anti-oncogenic capabilities (in tumors mutated for p53). Here, we establish that the carboxy-terminal RING domain of MdmX prolongs the duration of cell cycle in p53-deficient cells, thereby suppressing cell proliferation. We also determine that MdmX-mediated suppression of multipolar mitosis correlates with increased genome stability in hyperploid mouse and human tumor cells. This p53-independent function of MdmX maps to the central Zn-finger domain of the MdmX protein that matches the consensus sequence for the Ran binding protein 2 (RanBP2) type zinc fingers . The Zn-fingers domain in RanBP2 nucleoporin facilitates its binding to the small GTPase Ran protein30 which is associated with nuclear envelop assembly and mitotic spindle morphogenesis.32 It can be speculated that MdmX demonstrates its centrosome clustering function as one of the downstream targets for Run in spindle formation. As such, MdmX might counteract the centrosome clustering inhibitory activity of previously identified Ran target, nuclear mitotic apparatus protein (Nu-MA).34 Both MdmX and Mdm2 proteins have conserved RanBP2 zinc finger-like consensus sequences. However, there is only 43% identity in amino acid sequence in Zn-finger domain between the two proteins that may account for the difference in their role in p53-independent tumorigenesis.in vivo imaging to measure metastatic activity, we determined that MdmX-mediated genome stabilization underlies the ability of MdmX to reduce tumorigenic potential of cells mutated for p53. Although we cannot formally exclude a role for MdmX-mediated inhibition of cell proliferation in tumor progression, it is noteworthy that exogenous MdmX fails to alter proliferative capacity of MB231 cells in vitro, yet it clearly suppresses tumor formation and metastasis in vivo. It suggests that the tumorigenic potential of hyperploid, p53-mutant cells is not driven by a proliferative advantage but rather by the genomic instability imparted by loss of MdmX.Increased cell proliferation and chromosomal instability are hallmarks of tumorigenesis. Since we determined that the suppression of proliferation and genome instability by MdmX are independently regulated, we were able to examine relative contributions of these activities to MdmX-mediated suppression of tumorigenesis. Utilizing standard orthotopic transplantation assays to measure tumor growth, and lung colonization by 35 Furthermore, polyploidy associated with increased genetic instability promotes aneuploidy and accelerated tumorigenesis in human cancers.37 Pan-cancer analyses of somatic copy number alteration revealed that the whole-genome doubling occurred in 37% of cancers with average estimated ploidy of 3.31, suggesting the large-scale genome loss being associated with whole-genome doubling.38 However, experiments with long-term culturing of tetraploid cells deriving from diploid cancer progenitors39 revealed that, on per chromosome basis, tetraploidization by itself does not trigger additional instability. Dewhurst et al.39 suggest that a tetraploid genome increases cell tolerance to segregation errors and aneuploidy. In agreement with this, our data provide direct evidence that it is not the presence of a large genome per se but rather the ability of these hyperploids to tolerate a greater range of genetic imbalances leading to the evolution of aggressive growth characteristics. We propose that MdmX exercises its tumor suppressive activity in hyperploid cells with compromised p53 by preventing multipolar mitosis and stabilizing the large genome of these cells, thus preventing chromosome loss. Loss of MdmX may trigger large-scale ploidy reductions (only tolerated in polyploid cells), reducing the ploidy to near-diploid levels until a new cycle of polyploidization occurs within the cell. Such a scenario of non-synchronized ‘genomic breathing' in populations of tumor cells might easily contribute to tumor heterogeneity, and is concordant with the model of parallel evolution of different subclones within the same tumor.41 Interestingly, overexpression of MdmX was recently shown to impair DNA damage response and promote chromosome and chromatid breaks in mouse embryo fibroblasts in vitro.42 In contrast to those aberrations that occur at the level of individual chromosome(s), the genomic instability imparted by the loss of MdmX that we refer to represents the large-scale genome loss restricted to the polyploid cells that can lead to aneuploidy and tumorigenesis. These two quantitatively and mechanistically distinct MdmX-mediated processes are not mutually exclusive and are likely dependent on cellular context.Computational quantification of somatic DNA alterations in human cancers reveals that tetraploidy generated by whole-genome doubling events occurs frequently during tumorigenesis.43 it is believed that the centrosome clustering represents major route for hyperploid cancer cells with amplified centrosomes to escape multipolar mitosis and survive. Not surprisingly, identification and the inhibition of proteins involved in centrosome clustering has become an attractive anticancer strategy.45 However, our experiments reveal that the majority of multipolar mitosis generates viable daughter cells with increased levels of aneuploidy and increased tumorigenic potential. Thus, our findings offer a caveat to proposed cancer treatment strategies that aim to promote multipolar mitosis. Furthermore, our data indicate that the loss of MdmX in p53-mutant cells promotes genome instability and cancer progression. As suggested by recent studies demonstrating the failure of anti-MdmX strategy in tumors expressing hypomorphic p53 mutant,46 our data indicate that cancer treatments centered on MdmX inhibition should be employed only in cases where tumor retains functional p53.Our findings also suggest that genome stabilization by MdmX is dependent upon the clustering of supernumerary centrosomes and the promotion of pseudo-bipolar mitosis. Although centrosome amplification and clustering is not limited to cancer cells,MdmX+/−, p53+/− mice and MdmX+/−, p53−/− mice. Mouse tumor cells were isolated from thymic tumors of MdmX−/−, p53−/− mice or p53−/− mice and genotyped by PCR. The MdmX mouse model used in this study previously obtained by gene-trapping47 does not generate a detectable MdmX protein.6 Mouse cells were grown at 37 °C with 5% CO2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin.MEFs were isolated from 13.5-day-old embryos deriving from crosses between 48 MB231 cells were grown at 37 °C with 5% CO2 in DMEM with penicillin, streptomycin and 10% serum. For MB157 cells media was supplemented with 15% serum. Cell line MB231 has missense mutation in exon 8 resulting in mutant p53 protein49 and it is tumorigenic in nude mice.50 MB157 cells are p53-deficient due to the deletion in TP53 exon 4 resulting in the absence of p53 protein.28 MB157 cells have hardly detectable levels of MDMX protein.51 All cells were tested and were free of mycoplasma.Human tumor cell lines MDA-MB157 (obtained from ATCC) and MDA-MB231  were established from metastatic pleural effusion of human breast carcinomas.MdmX complementary DNA (cDNA) was placed under the transcriptional control of a conjugate promoter bearing a cytomegaloviral enhancer and chicken-B actin promoter sequences (pCAGGS). Construction of MdmX expression plasmids with MdmX FL and MdmX deletion mutants MdmX-dRING (amino acids 1–444), and MdmX-dZnF-dRING (amino acids 1–300) was described previously.52 Those plasmids were generous gift from Steven J. Berberich (Wright State University). Deletion mutant MdmX-dZnF (d304-323) was generated from the wild-type MdmX cDNA by site-directed mutagenesis and confirmed by DNA sequencing .For the transfection experiments, murine Following tumor dissection, tumors were minced in high glucose DMEM supplemented with antibiotics and collagenase (2mg/ml), incubated at 37°C for 3 h with shaking, washed with PBS containing 5% FBS, resuspended in DMEM with antibiotics and 2% FBS and plated on collagen-coated plates. Cells were maintained in 2% FBS DMEM/F12 with antibiotics. Fibroblasts were removed by differential trypsinization. Once cells reached growth crises, cells were fed with low glucose 10% FBS DMEM.Cells were grown in DMEM with no antibiotics until 70–80% confluent. Linearized MdmX-expression constructs were co-transfected with PGK promoter-puromycin drug selection marker using Lipofectamine 2000  for transfection of mouse and MDA-MB231 human cells or XtremeGene-9  for MDA-MB157 human cells. Cells were split 24 h after transfection and selection for puromycin resistance started 24 h later. Pools of puromycin-resistant stably transfected clones were recovered for further analyses.Total mRNA was isolated using RNeasy kit . For the first-strand cDNA synthesis SuperScript III First-Strand Synthesis System (Invitrogen) was used according to manufacturer's instructions. Quantitative PCR reaction was performed with SYBR Select Master Mix (Invitrogen) using the ABI-9300 PCR machine. The MdmX mRNA data were normalized to actin and the level in Mock control was assigned a value 1. Primers used for quantitative PCR are as follows:Mouse MdmX forward, 5′-TCTCGCACAGGATCACACTATGGA-3′;Mouse MdmX reverse, 5′-TCATCTGCTCTGGAGTCTCTGCAT-3′;Mouse Actin forward, 5′-TCCTGTGGCATCCATGAAACT-3′Mouse Actin reverse, 5′-GAAGCACTTGCGGTGCACGAT-3′Human MdmX forward, 5′-GCAAGAAATTTAACTCTCCAAGCAA-3′Human mdmX reverse, 5′-CTTTGAACAATCTGAATACCAATCCTT-3′Human Actin forward, 5′-GGACTTCGAGCAAGAGATGG-3′;Human Actin reverse, 5′-AGCACTGTGTTGGCGTACAG-3′.53 and incubated at 37 °C. Individual cells were observed under the Zeiss Universal or Leica DMEXE microscope equipped with phase-contrast optics using × 10 objectives. Images were recorded with Orca ER, Orca100  Retiga EX and/or Retiga EXi cameras , acquired every 3 minutes with C-imaging software and exported as a QuickTime movies (100% of CinePak compression mode). The duration of cell cycle was defined as the time from anaphase onset in mother cell manifested as the chromosome disjunction and/or sudden start of cell elongation till the anaphase onset in daughter cells.Cells were plated on coverslips 24 h before filming in DMEM media with 10% serum, penicillin, streptomycin and 12.5 mM Hepes. Coverslips with attached cells were assembled into the sealed chambersCells were grown on glass coverslips, fixed in methanol and stained for microtubules with mouse monoclonal anti-α-tubulin  and for centrosomes with rabbit polyclonal anti-γ-tubulin  primary antibodies. Alexa488 and Alexa594  were used as secondary antibodies. DNA was stained with 4′-6-diamidino-2-phenylindole. Immunofluorescence images were recorded using a CCD camera attached to an epifluorescence Zeiss Axioplan 2 microscope . Single images were acquired using Metamorph Imaging Software .Actively proliferating cells were incubated with Colcemid  for 90 min, harvested and incubated in hypotonic solution (0.075 M KCl) for 15 min at 37 °C and fixed with methanol-acetic acid. Drops of cell suspension were placed on microscope slides, dried, 4′-6-diamidino-2-phenylindole stained and analyzed by fluorescent microscopy.6) in 200 μl were injected into the mammary fat pad of 4 weeks old NSG mice, five animals per group. Tumor growth was monitored externally and animals were killed 16 days after injection. Tumors were harvested and tumor volume was calculated: total volume=½ (length x width2).54 All animal work in this manuscript was approved by the Institutional Animal Care and Use Committee of UMMS.MDA-MB231 cells stably transduced with FL or MdmX deletion mutants were resuspended in PBS with 30% Matrigel . Cells  at 7 × 106 cells/ml. Cells were sorted for positive M-cherry expression using FACSAria II cell sorter in Core Flow Cytometry facility at UMMS and propagated under standard conditions.MDA-MB231 cells stable transduced with FL or MdmX deletion mutants in DMEM media without antibiotic with Polybrene , 60–70% confluent, were infected with FUW based retroviral vector encoding dual M-cherry-Luciferase reporter . Cells were incubated at 37 °C for 48 h and the expression of M-cherry was confirmed by fluorescent microscopy. Cells were propagated, harvested with Accumax (Innovative Cell Technologies) and resuspended in sorting buffer  was dissolved in DPBS (15 mg/ml), filter-sterilized and injected intraperitoneally (150 mg/kg body weight). Animals were anesthetized with isoflurane for 5 min and imaged from both dorsal and ventral position by Xenogen IVIS 100 Imaging System, Caliper Life Sciences in Optical Imaging Core Facility at UMMS. Mice were monitored for luciferase activity every other day for the first week and then weekly for the rest of the experiment. Bioluminescence images of upper dorsal region corresponding to the lung position were quantified using Living Image 2.60.1 software. The values for photon flux were normalized to those obtained at day 1.For tail vein injection, cells were resuspended in PBS at 1 × 10t-test was used to compare the mean between two experimental conditions with the significance level 0.05 and confidence interval 95%. Number of samples and P-values for specific group of experiments are provided in figure legends. The normality of data were tested by the stem and leaf plot or by Kolmogorov–Smirnov test (for time-lapse video microscopy data). For animal studies sample size was estimated using Mead's resource equation with the degrees of freedom between 10 and 20. No blinding or randomization methods were used.The two-sample two-sided unpaired


Both external and internal Ca2+ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca2+-buffering protein, is able to bind Ca2+ reversibly, it can serve as a mobile store of easily releasable Ca2+  in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca2+ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca2+ are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca2+ storage and mobilization during sexual reproduction of angiosperms.Calcium (Ca Recent work from our lab provided evidence that CRT is highly expressed and accumulated in the ER of Petunia cells during pistil transmitting tract maturation, the progamic phase and early embryogenesis  and ovules (Petunia and Haemanthus) were prepared according to standard protocols, stained with 0.1% methylene blue and observed by light microscopy. Then, selected tissue samples of styles and ovules were prepared for immunocytochemical and cytochemical (potassium antimonate precipitation) studies according to the protocols as described below. All experiments were repeated many times during several growing seasons with similar results.Commercial cultivars of v/v) formaldehyde and 0.25% (v/v) glutaraldehyde in phosphate-buffered saline  for 1 h at room temperature (slight vacuum infiltration) followed by overnight fixation at 4 °C. Fixed samples were dehydrated in graduated ethanol concentrations, embedded in LR Gold resin (Fluka) according to the standard protocol, and then semithin and ultrathin longitudinal or cross-sections were collected on microscope slides covered with Biobond  or Formvar-coated nickel grids. After blocking in 3–5% bovine serum albumin (Sigma-Aldrich), sections were incubated with a rabbit polyclonal antibody against maize CRT  or with 10–20 nm diameter gold-conjugated goat anti-rabbit IgG antibody . As a final step, DNA was stained with 2 μg/ml 4′, 6-diamidino2-phenylindole . In the controls, incubations with the CRT PAb were omitted. Images were acquired using an Olympus BX50 fluorescence microscope, Olympus Xc50 digital color camera, and CellB software (Olympus Soft Imaging Solutions gmbH); ultrathin sections were examined by transmission electron microscopy (Jeol EM 1010) at 80 kV.Immunofluorescence and immunogold localizations of CRT were performed according to protocols described previously . Signals were detected with the following secondary antibodies: 20 nm diameter gold-conjugated goat anti-rabbit IgG (for CRT) and 10 nm diameter gold-conjugated goat anti-mouse IgG , both from BBInternational. To quantitate the amount of CRT localized intra/extracellularly in different Petunia cells involved in pollen-pistil interactions, the average number of gold traces was determined in different cell sections of compartments/structures (min. 20) labeled with CRT PAb conjugated with immunogold secondary antibody. In the negative control, incubation with the primary antibodies was omitted. To verify if the CRT PAb specifically bound to protein epitopes, the immunolocalizaton was performed on ultrathin sections pretreated by incubation with a proteinase K solution  uranyl acetate and 0.4% (w/v) lead citrate solutions and examined by transmission electron microscopy as above. The specificity of maize CRT PAb in Petunia and Haemanthus was previously verified by immunoblotting  potassium antimonate, 2% (v/v) glutaraldehyde, and 2% (v/v) formaldehyde in 0.1 M phosphate buffer  for 4 h at room temperature, and then subsequently postfixed with 1% (v/v) osmium tetroxide (OsO4) in the same buffer-antimonate solution for 12 h at 4 °C. Next, samples were dehydrated in graduated ethanol concentrations and embedded in Poly/Bed 812 resin (Polysciences) according to the standard protocol. Ultrathin longitudinal or cross-sections were collected on copper grids, stained with 2.5% (w/v) uranyl acetate and 0.4% (w/v) lead citrate solutions, and examined by transmission electron microscopy as above. The presence of Ca2+ in the precipitates was confirmed previously using energy-dispersive X-ray microanalysis  were observed in the same localizations where CRT was found; there were the ER , as well as Ca2+ ppts , were also found in the cortical cytoplasm, the plasma membrane/wall attached patches and plasmodesmata. It should be noted that long ER cisternae commonly occurred in the peripheral cytoplasm of transmitting cells and were associated with intercellular connections. It has been also long recognized that plasmodesmata of higher plants contain a central strand of tightly compressed ER, called the rod or desmotubule, that creates the cytosolic sleeve providing continuity of cytoplasm between adjacent cells  are enriched at sink plasmodesmata. However, CRT was also found to be strongly expressed and accumulated near the neck region of closed channels in response to different stresses, irrespective of callose deposition , and/or in cell adhesion during the cell shape formation. Directional growth of the pollen tube in vivo involves both the tube cell adhesion to the style transmitting tract and diverse cell signaling pathways that regulate complicated pollen-pistil interactions . Other studies have demonstrated that a lily pollen-specific protein LP2 and two pistil-specific proteins, 120 kDa and PELPIII glycoproteins, were also located in the callosic cell wall of developing pollen and pollen tubes growing in situ  between nucellus cells surrounding the embryo sac and its preferential accumulation within the ER and the filiform apparatus of the synergids. Similar results were observed in 2+ levels in the synergid resulting in preferential accumulation of CRT and Ca2+ in the ER and the filiform apparatus (Lenartowski et al. 2+ is in the synergid (see review by Ge et al. 2+ is delivered from the extracellular space, the ER, or both. We postulate that CRT accumulated in the filiform apparatus may provide an exchangeable Ca2+ external store in the synergid and thus modulate the local concentration of cytoplasmic Ca2+ to prevent extra pollen tubes from entering the embryo sac. Generally, only one pollen tube enters each embryo sac in angiosperms, and precise regulation of the Ca2+ level in the penetrated ovule determines its receptivity. Upon pollen tube arrival at the Arabidopsis receptive synergid, Ca2+ oscillation begins at the micropylar pole and spreads toward the chalazal pole, and Ca2+ reaches a maximum level at pollen tube rupture Iwano et al. (Arabidopsis synergid cell is observed at a site distinct from the filiform apparatus (Lesherm et al. The filiform apparatus is a thickened cell wall of heterogeneous structure that involves fibrillar and amorphous fractions with numerous finger-like projections into the synergid cytoplasm (Płachno et al. o et al. . Even th


A detailed understanding of technological change as an evolutionary process is currently not well understood. To increase our understanding, we build upon theory from organizational ecology to develop a model of endogenous technological growth and determine to what extent the pattern of technological growth can be attributed to the structural or systemic characteristics of the technology itself. Through an empirical investigation of patent data in the biotechnology industry from 1976 to 2003, we find that a technology’s internal  ecological characteristics have a positive effect on its growth rate. The niche’s external characteristics of crowding and status have a negative effect on its growth rate. Hence, applying theory from organizational ecology increases our understanding of technological change as an evolutionary process. We discuss the implications of our findings for the study of technological growth and evolution, and suggest avenues for further research. Although the Schumpeterian  conceptiIn line with  and 8],,8], we aThe theoretical claim that this paper makes is as follows. To come to a better understanding of the process of technological growth, we argue that a systemic perspective towards technology is warranted, bringing in insights from organizational ecology. Furthermore, these technological growth patterns are to a large extent determined by structural characteristics of technology. The contribution of this paper is twofold. First, we extend the notion of the technological niche by adding internal diversity as a key structural feature. In so doing, we illustrate the importance of technological diversity in evolutionary and ecological models of technological growth. Second, we demonstrate that technology should effectively be studied as an ecological system, composed of interdependent and interacting technological components. To fully understand technological growth requires understanding the environmental dependencies and interactions between technological components.The structure of this chapter is as follows. We develop our theoretical model and associated hypotheses in Section 2. After developing our theory, we will test a series of specific hypotheses that follow from this ecological logic through an empirical analysis of patents and patent citations in one of the leading sets of technology developed in the 20th century : i.e., bThe concept of the niche was first developed by , and is We define the technological niche as the relational context of a technological component , embedded within a technological system . Hence, we view technology as a system that cuts across organizational boundaries , and absOur key dependent variable is growth of the technological component niche as reflected in entry by new inventions, coined component growth. Below, we will subsequently discuss the dimensions of the component niche central to our theory that we take as our independent variables, focusing on both a component niche’s internal  and external  features. Note that, given our application to component niches of biotechnology, we often refer to the short-cut component for component niche.Ecologists have observed a characteristic pattern of evolution of diverse organizational populations: initially, after a slow kick-off, population size–measured in terms of the number of organizations, or density–increases rapidly, and then stabilizes or even declines in numbers . IntriguLegitimation refers to “the standing as a taken-for-granted element in a social structure” , and is Density also has an obvious link with diffuse competition, which is defined as common dependence on the same resource pool. If density increases linearly, the number of potential competitive links increases exponentially . This imEven though the theory of density dependence has been primarily applied to organizational populations, and very successfully so, recent research illustrates that, due to its general nature, this argument can also be effectively applied in other settings, such as the birth and death rates of national laws , 27 and It is widely acknowledged that technologies need to be legitimized , 31. AccIdeas and innovations compete with one another for the attraction of resources and attention . That isHypothesis 1: There is an inverted U-shaped effect of component density on component growth.Over the years, density dependence theory has received considerable critique. This is mainly the result of the generality of the model. On the one hand, regarding the legitimation processes, opponents–mainly institutionalists–argue that legitimation is a multi-dimensional construct that cannot be adequately represented by a measure as crude as population density . This crThe competition leg of the theory has also been challenged. It is argued that populations are not fully homogeneous, and that segments of the population respond differently to (mainly) competitive processes . Indeed,One of the proposed extensions is to employ multi-level models, where processes of legitimation are allowed to operate more broadly than competitive processes . Here, wHypothesis 2: System density is positively associated with component growth.Because different population segments may well respond differently to processes of competition and legitimation, it is important to consider whether a population is subdivided into segments. In the context of our current study, three motives come to mind for considering such across-niche diversity. First, according to , there iSecond, diversity mitigates lock-in and provides flexibility in uncertain environments . BecauseThird, diversity is one of the central principles in evolutionary economics, see , 41. JusHypothesis 3: Component diversity is positively associated with component growth.Even though processes of legitimation at the system level affect all components within the system, it is highly unlikely that system-level legitimation will affect all components equally. Furthermore, according to the institutional argument, component density is a proxy that cannot accurately capture the distribution of system-level legitimation among components. This means that we need another way to distinguish between the legitimation of individual components relative to the other components within the technological system. A well-known construct that measures legitimation at the individual member level is status, which is defined as a focal member’s ‘perceived’ quality in relation to the ‘perceived’ quality of other population members , 44.Status is an instance of endogenous system or population structuring that results from the interactions among members and stakeholders in a population. Akin to the importance of legitimation in the formative (or uncertain) stages of population development, status is mainly used by resource controllers to guide their decisions in uncertain environments. Due to the uncertainty, the quality of population members cannot be objectively determined. Therefore, resource controllers rely on status to guide their decisions . In the According to these studies, as the uncertain environment makes quality perceptions dependent on status, status becomes important in guiding the flow of resources in technological developments. As other organizations build upon the focal organization’s technology, a certain status is conferred to that focal organization’s technology . Here, aHypothesis 4: Component status is positively associated with component growth.In ecological studies, niche crowding is usually equated with competition, as it implies a similarity in resource requirements , 15, whiBecause inventions recombine technology from prior antecedent inventions, these prior or antecedent inventions constitute the building blocks of the focal inventions. The uniqueness of the invention’s building blocks determines the uniqueness of the invention itself . AggregaHowever, niche crowding can also contribute in a positive way to niche entry or growth, due to knowledge and reputation spillovers , 48, ecoFirst, technology can become a taken-for-granted means to accomplish an organizational objective, implying the existence of legitimation or reputation spillovers from (the use of) one technology to the (use of the) other. Furthermore, knowledge spillovers within technological development are well-documented. As the usage of technology increases, the documentation of technology also increases–for example, in patent documents, manuals, and books. As a result, the characteristics and behaviors of often-used technologies are better known.Second, economies of standardization or infrastructure sharing relate to the costs of transportation, communication, and ease of supply , which aHow can we accommodate for both a positive and negative effect of component crowding? The answer to this question lies in an analysis of the spatial pattern of crowding. The investigation of spatial patterns in the distribution of organisms is central in ecology, as they play an important role in population dynamics . AccordiHypothesis 5: Local crowding is negatively associated with component growth.Hypothesis 6: Global crowding is positively associated with component growth.We have argued under component status that a certain amount of legitimacy is transferred to a focal organization when another organization builds upon the focal organization’s technology. However, according to , such a Hypothesis 7a: The interaction term of local crowding and component status is negatively associated with component growth.Hypothesis 7b: The interaction term of global crowding and component status is not negatively associated with component growth.Patents and patent citations provide the core of the data that we will use to test our hypotheses. Patents and patent citations have been used extensively in the study of technological change and organizational innovation , 47, 55.Previous research has illustrated that the US patent system offers the most complete dataset for technological analysis, since the US is the world’s largest and most international marketplace . FurtherBiotechnology patents are registered in classes 435 and 800 of the USPC. The domain of biotechnology has an average of 57 per cent of self-citations, and can therefore be considered as highly autonomous and independent. Hence, biotechnology offers a setting highly suitable for an empirical investigation of the kind proposed here. The biotechnology domain contains 27 main sub-classes , as listed in Component growth, our dependent variable, is measured by the count of the number of patents that enter our focal components in a particular month in the period between 1976 and 2003. As we have repeated observations for the same components, our data form a time-series–cross-sectional panel. This panel is unbalanced, though, as not all components were in existence at the start of our time window.Component density is a count of the total number of patents (divided by 1000) in the focal component in the month prior to the date of measurement of our dependent variable. So, this measure represents the stock of patents contained in the focal component. System density is a count of the total number of patents (divided by 1000) within the domain of biotechnology  in the month prior to the date of measurement of our dependent variable. To avoid double counting, we subtract focal component density from system density.Focal Total crowding) as follows:COit refers to the overlap experienced by focal component i at time t, Aikt to the antecedent inventions of component i’s inventions from component k at time t, Ajkt to the antecedent inventions of component j’s inventions from component k at time t, J and K to the set of all components, ∩ to the intersection of two sets , |.| to the cardinality of a set , and t to the twelve months prior to the month of observation of our dependent variable. To make the number of non-focal components manageable, we have defined the non-focal components at the class level instead of the sub-class level. Strictly speaking, this implies that our non-focal components are actually composed of alternative technological systems.Component crowding refers to the extent to which the building blocks  of our focal components are crowded (or used) by other components. To provide for a baseline model to test our hypotheses regarding the distinction between local and global crowding, we calculate the aggregate measure of crowding . In this case, J refers to the set of focal components only, while K denotes the set of both focal and non-focal components. Global crowding measures the overlap of our focal components with the non-focal technological components. To calculate this measure, we again use (1), but now J refers to the set of all non-focal components, whilst K is the set of all components . An example of how our crowding measures are calculated is provided in For our measure of Component status is based upon the patent citations received by the focal component. Patent citations reveal system-wide perceptions of the relative importance of patented technologies  fi fifixed eived by , there idynamic vis-à-vis dynamic panel estimation issue. Because we include a lag of the dependent variable  in Third, we investigated the On the basis of our results, we can conclude that during our period of observation, biotechnology’s components during our period of observation. In this stage, legitimation processes dominate competitive processes within technological components. As a result, we only find a significant positive effect of component density on component growth. Furthermore, a dominant design or technological paradigm appears to be absent at the system level (biotechnology as a whole) at this stage, as we do not find any significant positive spillovers between components at the system level. Diversity plays a key role in the early stages of technological development within components. Studying the role of diversity more directly in the evolution of technologies could thus lead to a better understanding of processes of endogenous technological change. In developing such a theory of diversity dependence in technological populations, both centripetal and centrifugal forces would have to be taken on board . That isThe question of how technological changes come about endogenously has been left largely unanswered. One of the main reasons for this blind spot is that most studies view technology as a single component, without considering its multi-level nature. That is, these studies ignore the embeddedness of this component within a larger technological system, and thereby disregard the interdependence between components . Hence, First, in developing a systemic view on technological growth, we have assumed that our technological system is stable, with technological components behaving in reliable and predictable ways. In doing so, we have been able to demonstrate that biotechnology–or any other technology, for that matter–can effectively be studied as a technological system, composed of a set of interdependent and interacting technological components. However, by no means does this imply that we perceive technology as a stable system, with components behaving in reliable and predictable ways. Even though we acknowledge that some technologies could, at a certain point in time, be characterized by such a stable state, at this moment, biotechnology is most definitely not one of them. On the contrary, biotechnology can be characterized as a highly dynamic technology, with many components that are just being developed . ObviousSecond, an important limitation of our study is that we have abstracted from the role of the innovating organization. Our results clearly indicate that organizations play a major role in the growth process of a technological component. This signifies the importance of developing a co-evolutionary model, where both the evolution of technologies and the evolution of organizations are considered in unison. It is well-recognized that technologies and organizations/industries co-evolve , 80, 81.Moreover, at the level of an individual organization, we can relate the dynamics of technological components to the characteristics of an organization’s technological search behavior. Organizations search as members of a population , and by Third, another interesting avenue to consider are ecological models with spatial effects (cf. ). In ourFourth, another limitation is that our empirical setting is the domain of biotechnology. Studying this technological domain has the advantage that patents form a reliable indicator of processes of technological growth , 57, herFifth and finally, in this paper, we have applied insights from organizational ecology to develop a model of endogenous technological growth. This demonstrates how organizational ecology can contribute to the field of evolutionary economics . This isS1 Appendix(DOCX)Click here for additional data file.


We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca2+ rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP3 receptor mediated ER Ca2+ release, but decreased receptor-operated extracellular Ca2+ influx. Impaired Gq protein-coupled intracellular Ca2+ rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2α-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered Gq protein-coupled intracellular Ca2+ dynamics in cortical pyramidal neurons.PERK (EIF2AK3) is an ER-resident eIF2α kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates CaThe online version of this article (doi:10.1186/s13041-016-0268-5) contains supplementary material, which is available to authorized users. We first tested if PERKi impacts cholinergic activation induced [Ca2+]i rise by treating neurons with 250 μM carbachol, and found that 15 min 500 nM PERKi pretreatment significantly reduced carbachol-induced [Ca2+]i rise  from extracellular medium. To study PERKi’s effect on internal Ca2+ release, we measured [Ca2+]i rise upon carbachol treatment in the absence of extracellular Ca2+, to exclude any contribution from nicotinic acetylcholine receptor or receptor-operated Ca2+ channel (ROCC)-dependent Ca2+ influx. Cells were perfused with Ca2+-free bath for 100 sec before stimulation with 250 μM carbachol. Carbachol treatment in Ca2+-free bath triggered a transient and small [Ca2+]i increase due to Ca2+ release from intracellular stores, which was significantly higher in PERK-inhibited neurons  refers to cytosol Ca2+ influx mediated by cell membrane Ca2+ channels triggered by ER Ca2+ store depletion. Since ROCE and SOCE are two closely related processes, and store depletion is an integral component of ROCE, we next examined PERKi’s effect on SOCE in primary cortical neurons. As shown in Fig. 2+-containing bath, thapsigargin treatment only elicited a transient [Ca2+]i rise, which is the result of the combined effect of thapsigargin-induced ER Ca2+ release and SOCE, suggesting that thapsigargin stimulation alone did not significantly induce SOCE in primary neurons. To maximally activate SOCE, we followed a “Ca2+ re-addition” protocol [2+-free bath for 300 sec to fully deplete ER Ca2+ and activate store-operated Ca2+ channels (SOCC). Subsequent reintroduction of 2 mM Ca2+ into the bath elicited a sustained [Ca2+]i elevation, reflecting SOCC mediated Ca2+ influx. No difference was observed between PERK-inhibited neurons and DMSO controls -independent i mobilization could be mimicked by genetic ablation of Perk, primary cortical neurons from brain-specific Perk KO (BrPKO) mice were examined. BrPKO mice were generated by crossing Perk-floxed mice [Nestin-Cre mice strain [loxP-flanked Perk gene sequence in neurons and glial cells during embryonic stage [BrPKO mice at postnatal day 0  [q protein-coupled [Ca2+]i mobilization regulates working memory in several ways. First, the induced [Ca2+]i rise is known to activate several Ca2+-dependent protein enzymes, including the phosphatase calcineurin, and the kinases CaMKII and PKC, all of which have been shown to regulate working memory capacity i rise has direct effects on intrinsic neuronal excitability. It has been demonstrated that pharmacological activation of mGluR1 in prefrontal cortex pyramidal neurons triggers a biphasic electrical response-SK channel-dependent neuronal hyperpolarization followed by TRPC-dependent neuronal depolarization, and the amplitude of both are regulated by the extent of [Ca2+]i rise [q protein-coupled [Ca2+]i mobilization in pyramidal neurons.Does PERK’s regulation of G KO mice , and we  animals –48, and t (ICAN)  , which capacity . Secondlependent . Finally+]i rise , 51. TakPerk knockout mice [q protein-coupled Ca2+ dynamics in primary cortical neurons, together with the earlier finding that PERK regulates Ca2+ dynamics-dependent working memory [2+ dynamics.Considering PERK’s role in eIF2α-dependent protein synthesis and translational control, it has been hypothesized that PERK’s regulation over memory flexibility and mGluR1-dependent long-term depression is eIF2α-dependent , 9. Howeout mice , 9. Thusg memory , supportq protein-coupled Ca2+ dynamics in pyramidal neurons? We speculate that during early vertebrate evolution, PERK first played an eIF2α-dependent role in CNS. Given its localization on the ER, which is the major organelle for intracellular Ca2+ storage, and its regulation by ER/cytosolic Ca2+[2+ may have provided PERK the opportunity to evolve an additional function to regulate intracellular Ca2+ dynamics through mechanism independent of eIF2a and protein translation. The fact that PERK is activated by ER Ca2+ depletion [3R and a positive regulator of ROCC shown herein, fit well into this hypothesis: when ER Ca2+ stores are depleted under physiological responses such as activation of Gq protein-coupled receptor, PERK is activated due to Ca2+ dissociation from its regulatory domain in the ER, and it subsequently replenishes ER Ca2+ by inhibiting IP3R mediated ER Ca2+ release and activating ROCE (Fig. As an eIF2α kinase, how did PERK evolve to be a modulator of Golic Ca2+, 55, theOCE Fig. .Fig. 6Pr


PARP1 and LIG3) in acute myeloid leukemia (AML) patients and assessed their potential correlationwith the formation of chromosomal translocations. Chromosomal translocations are among the most common mutational events in cancer development, especiallyin hematologic malignancies. However, the precise molecular mechanism of these events is still not clear. It has beenrecently shown that alternative non-homologous end-joining , a newly described pathway for double-stranded DNAbreak repair, mediates the formation of chromosomal translocations. Here, we examined the expression levels of the maincomponents of alt-NHEJ (PARP1 and LIG3 at the transcript level in AML patients (n=78) and healthyindividuals (n=19). This experimental study used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify the expression levels of PARP1 was the only gene overexpressed in the AML group when compared with healthy individuals(P=0.0004), especially in the poor prognosis sub-group. Both genes were, however, found to be up-regulatedin AML patients with chromosomal translocations (P=0.04 and 0.0004 respectively). Moreover, patients withone isolated translocation showed an over-expression of only LIG3 (P=0.005), whereas those with two or moretranslocations over-expressed both LIG3 (P=0.002) and PARP1 (P=0.02).PARP1 and LIG3 expression and the rate of chromosomaltranslocations in AML patients provides a molecular context for further studies to investigate the causality of this association.The significant correlations observed between  One of the major chromosomal aberrations identified in cancer tissues, particularly in hematological malignancies, is balanced chromosomal translocation, which is thought to play a central role in initiating malignancy. In most cases, these chromosomal abnormalities substantially impact prognosis -3. ApproRecent findings have elucidated how balancedtranslocations are implicated in leukemia development, however, the question of how such translocations are generated in the first place has remained to be fully resolved. It is experimentally clear that these translocations mostly occur due to DNA double-strand breaks (DSBs) in distinct chromosomes . DSBs inTo maintain genomic integrity and protect cells from adverse consequences of these lesions, such as oncogenic transformation following DSB, two major repair pathways are induced, namely homologous recombination (HR), where sequence integrity is preserved, and non-homologous end-joining (NHEJ), which results in small-scale mutations . Data derived from cloning and sequencing junctions of chromosomal translocations in patients with leukemia have revealed that the breakpoints showed no consistent homologous sequences and often strongly showed repair signatures of NHEJ including small deletions and duplications, non-template insertions and micro-homologies. These observations indicate that these translocations predominantly arise by NHEJ and HR are not thought to play a significant role in chromosomal translocations , 2, 11. Recent studies have argued against the previous paradigm that NHEJ mediates translocations by demonstrating that chromosomal translocations are more common when cNHEJ components such as KU70 and DNA ligase IV are missing, suggesting that these cNHEJ components repress chromosomal translocations , 14. HoPARP1 and LIG3 in the accelerated rate of chromosomal translocation formation in AML patients. To investigate this possibility, we quantified their expression levels at the transcript level in subsets of AML patients. Our findings suggest that these molecular markers may be used as potential therapeutic targets for AML therapy. Since PARP1 and LIG3 are the main components of alt-NHEJ, we aimed to examine the potential role of 2daily for 3 days) combined with cytarabine (100 mg/m2) for a 7-day continuous intravenous infusion. AML-M3 patients received ATRA plus arsenic trioxide for several cycles. Moreover, peripheral blood samples from healthy individuals (n=19) were analyzed as the control group. Bone marrow (BM) specimens were collected from AML patients (n=78) admitted to the Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran, Iran. All patients gave informed written consent for sample collection. This experimental study was approved by the Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.REC.1395.2697). Non-M3 AML patients received standard induction chemotherapy consisting of 1 or 2 courses of daunorubicin (45 mg/mBone marrow samples were collected in EDTA containing tubes. The mononuclear cells (MNCs) were isolated within two hours of bone marrow sample collection using Ficoll-Paque  density-gradient centrifugation according to the manufacturer’s instructions. After washing twice using phosphate-buffered saline , the cell pellets were directly lysed in TriPure Isolation Reagent . Fresh bone marrow aspirate samples were directly cultured and harvested following standard cytogenetic methods. G-banding and FISH (if necessary) were undertaken on each bone marrow sample to detect all cytogenetic abnormalities in accordance with an in-house validated protocol. The chromosomal aberrations in this study were described according to the recommendations of the International System of Human Cytogenetic Nomenclature (ISCN 2013) . Total RNA was isolated from bone marrow MNCs using TriPure Isolation Reagent according to the manufacturer’s procedure . To determine the quantity and quality of RNA samples, absorbance at 260/280 nm wavelengths was measured by using the NanoDrop spectrophotometer. Moreover, 1 µl of each RNA sample was electrophoresed on a 1.5% agarose gel to observe rRNA bands corresponding to the 28S and 18S subunits, and to assess the integrity of RNA samples. The PrimeScript First Strand cDNA Synthesis kit  was used to reverse transcribe 1 µg of total RNA into complementary DNA (cDNA). The synthesized cDNA was stored at -20°C for further analysis. To assess the relative quantity of mRNA transcripts, qPCR was undertaken in a StepOnePlus Real-Time PCR System  by using the SYBR Green assay in duplicate. The cycling conditions were an initial denaturation step at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds and 60°C  for 30 seconds. Ultimately a melting curve was generated to ensure primer specificity for each target gene. A standard curve was also generated using a serial dilution (5-fold dilutions) of cDNA samples to determine the efficiency of quantitative polymerase chain reactions (qPCR). All reactions were conducted in a final volume of 20 µl comprising 10 µl qPCR Master Mix (Takara), 2 µl (200 ng/µl) of cDNA, 0.5 µl of each primer and 7 µl of ddH2O. Expression levels of all target genes were normalized with ABL1, a housekeeping gene recommended for such analysis by Europe Against Cancer Program, . The priPARP1 and LIG3. A P<0.05 was considered statistically significant. All statistical analyses were implemented in the Statistical Package for Social Sciences (SPSS) version 20 . Mann-Whitney U test was used to compare PARP1 and LIG3 expression levels between the healthy and AML patient groups. This test was also used to compare the two subgroups of AML patients  with respect to the expression levels of Based on the FAB classification, the number of patients in each category were 4 (5.1%) M0, 17 (21.8%) M1, 8 (10.3%) M2, 21 (26.9%) M3, 18 (23.1%) M4, 8 (10.3%) M5, 1 (1.3%) M6 and 1 (1.3%) MDS. Flow cytometric analysis demonstrated that 8 to 90 % (mean of 57.4%) of bone marrow mononuclear cells were immature blood cells. The cells were positive for the surface markers specific for AML subgroups including CD2, CD10, CD13, CD14, CD19, CD33, CD34, CD45, CD64, CD117 and HLA DRI3. All AML samples were negative for TdT . The control group comprised the healthy individuals aged from 20 to 66 years (median=35), of whom 50 % were male. The patients with AML  were between 14 and 76 years of age (median=39.5 years). The patient diagnosis was based on the revised French-American-British (FAB) classification. The clinic-pathological characteristics of patients are given in Table2. PARP1 in different cytogenetic risk-based subgroups of AML patients are summarized in Table 3. In contrast, no statistically significant difference was observed for LIG3 expression (P=0.08) in the overall comparison and also between AML subgroup with structural chromosomal aberrations and healthy controls (P=0.67). Interestingly, PARP1 was also significantly upregulated in the poor prognosis group when compared with the good or intermediate prognosis subgroup (P=0.01). By comparing the AML and control groups, PARP1 was significantly differentially expressed  . The norPARP1 and LIG3 expression with the presence of chromosomal translocations, we subgrouped AML patients based on presence of chromosomal translocations and compared the expression levels of PARP1 and LIG3 between the two subgroups. LIG3 and PARP1 were significantly up-regulated in the subgroup with chromosomal translocations   . We furtcompared  and consKU70, a component of the classical pathway, in bone marrow specimens of AML patients. KU70 was not significantly dysregulated in AML patients accordingto absence or presence of chromosomal translocations(P=0.08). To examine the involvement of the classical NHEJpathway in different chromosomal translocation-basedsubgroups, we analyzed the mRNA expression level of Wide spectra of hematologic malignancies display one or several balanced chromosomal translocations. In most cases, these aberrations have a major role in diagnosis and treatment of hematological disorders. Although the precise underlying molecular mechanism behind these translocations are yet unknown, recent studies have demonstrated that unlike the components of the cNHEJ pathway, alt-NHEJ factors such as CtIP, DNA ligase III and PARP1 are required for chromosomal translocation formation , 16, 18.PARP1 and LIG3 expression levels and formation of chromosomal translocations in AML patients. Given the overexpression of PARP1 and not LIG3 in the overall AML patient group, especially in patients with poor prognosis, suggests a potential role of PARP1 in the development of AML. This result, on its own, is particularly significant since several PARP inhibitors have been well-tolerated in clinical trials of breast and ovarian cancer and may therefore be potential candidates for AML therapy  may potentially induce genetic instability and complex cytogenetic abnormalities in myelodysplastic (MDS) patients . PournazFinally, many studies have reported karyotype instability and development of chromosomal translocations in patients with relapsed leukemia, especially those affected by AML -7. Kern PARP1 expression increases the rate of leukemogenic translocation formation. Accordingly, due to the availability and tolerability of PARP1 inhibitors, it is possible to reduce the risk of translocation developmentby using a concurrent treatment with PARP1 inhibitorsas a reasonable therapeutic option to reduce the risk of developing further translocations, hence avoiding the emergence of relapse and therapy resistance in AML patients. Second, our results highlight the importance of PARP1 and LIG3 overexpression as different risk markers of translocation formation. The findings presented here have clinical significance under three perspectives. First, it is possible that increased This association may be used for predicting risk of translocation formation and secondary leukemia in patients undergoing high risk therapy. Thirdly, given the correlation between the expression levels of PARP1 and LIG3 with formation of translocations and the possible occurrence of these chromosomal aberrations between diagnosis and the relapsing stage, we suggest measuring these two markers to predict the translocation formation and possibly to introduce them as novel therapeutic targets to prevent the relapsing phase in AML. However, further investigations are required to fully establish this association.


Our results showed that NaHS significantly increased the level of [Ca2+]i in rat medullary neurons in a concentration-dependent manner. L-Cysteine and SAM significantly raised the level of [Ca2+]i in the medullary neurons while HA and/or AOAA produced a reversal effect. In addition, L-cysteine and SAM significantly increased but HA and/or AOAA decreased the production of H2S in the cultured neurons. The [Ca2+]i elevation induced by H2S was significantly diminished by EGTA-Ca2+-free solutions, and this elevation was also reduced by nifedipine or nimodipine and mibefradil, suggesting the role of L-type and/or T-type Ca2+ channels. Moreover, the effect of H2S on [Ca2+]i level in neurons was significantly attenuated by BAPTA-AM and thapsigargin, suggesting the source of Ca2+. Therefore, we concluded that both exogenous and endogenous H2S elevates [Ca2+]i level in primarily cultured rat medullary neurons via both increasing calcium influx and mobilizing intracellular Ca2+ stores from ER.In the present study, we attempted to elucidate mechanisms for the regulation of intracellular calcium levels by H It was trocytes  and regutrocytes . Anotheractivity . It has um store . However2S exerts its cardiovascular effects by decreasing oxidative stress via inhibition of NADPH oxidase activity in the rostral ventrolateral medulla (RVLM) of SHRs i. Our study provides the evidence that H2S increases [Ca2+]i in neurons through several different mechanisms.We recently reported that H of SHRs . RVLM, w of SHRs –25. HoweDMEM/F-12, neurobasal medium, and B27 supplement were obtained from Gibco Invitrogen Corporation . Fura-2 AM and BAPTA-AM were obtained from Dojindo Molecular Technologies, Inc. Sodium hydrosulfide hydrate (NaHS), EGTA, thapsigargin (TG), nifedipine, nimodipine, and mibefradil were obtained from Sigma-Aldrich. Fura-2 AM and TG were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO did not exceed 0.1%. Anti-MAP-2 antibody produced in mouse, anti-MAP-2 antibody produced in rabbit, anti-CBS antibody produced in mouse, and anti-MST antibody produced in rabbit were obtained from Abcam. Anti-glutamate antibody produced from rabbit was obtained from Sigma-Aldrich. FITC goat anti-rabbit IgG (H+L) and Cy3 goat anti-mouse IgG (H+L) were obtained from Beyotime Biotechnology. Alexa Fluor® 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 594 goat anti-mouse IgG (H+L) were obtained from Invitrogen.2HPO4·12H2O, 0.06 KH2PO4, 0.35 NaHCO3, and 1 glucose, pH 7.2~7.4, and finally chopped. The tissues were then treated with 0.125% trypsin in D-hanks' solution for 10~12 min at 37°C and gently triturated using flame-polished Pasteur pipettes. Cell suspension was centrifuged for 8 min at 1000 ×g. Then the cell pellets were resuspended in Dulbecco's modified Eagle's medium (DMEM) and F-12 supplement (1 : 1) with 10% fetal bovine serum (FBS) before plating onto glass-bottomed dishes coated with poly-L-lysine (20 μg/mL for 12~24 h) and kept at 37°C in 5% CO2 incubator. After overnight incubation in DMEM, the medium was changed to neurobasal medium (Gibco) containing 15 mM glucose supplemented with 2% B27, 2 mM glutamine, 10 μg/mL penicillin, and 10 μg/mL streptomycin. The culture medium for medullary neurons was changed every 48 h. Microscopically, glial cells were not apparent in medullary neurons employing this protocol. The neurons were maintained for 7–10 days in primarily culture until used for calcium imaging.Primary cultures of medullary neurons were prepared from 14-day-old embryos of Sprague Dawley rats. The fetal rats were humanely taken out and killed by decapitation, and then brain slices containing the entire medullary were prepared. The dissected tissues were removed and transferred to D-hanks' solution containing (in g/L) 8.0 NaCl, 0.4 KCl, 0.134 NaμL D-hanks' solution was added to the cell dishes for confocal comicroscopy .The primarily cultured medullary neurons were washed three times with D-hanks' solution and then cells were fixed with 1 mL 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer  for 20 min. Afterward, cells were blocked with 1 mL 5% fetal bovine serum (FBS) for 30 min after being washed three times with D-hanks' solution. Then, cells were incubated with primary antibodies, namely, anti-MAP-2  and anti-glutamate , anti-MAP-2 antibody  and anti-CBS , anti-MAP-2 antibody  and anti-MST , and anti-MAP-2 antibody  and anti-CSE , for 1 h at 37°C, plus an additional 12 h at 4°C. On the next day, the cell were washed three times with D-hanks' solution and incubated with fluorescent secondary antibodies, namely, FITC goat anti-rabbit IgG (H+L) (1 : 100) and Cy3 goat anti-mouse IgG (H+L) (1 : 100) or Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1 : 500) and Alexa Fluor 594 goat anti-mouse IgG (H+L) (1 : 500), for 1 h at 37°C. Then cells were incubated with DAPI for 5 min at 37°C after being washed three times with D-hanks' solution. Finally, 500 2+]i, neurons were loaded with Ca2+-specific dye Fura-2 by incubating with 2.5 μM Fura-2/AM (Molecular Probes) in HBSS  at 37°C for 30 minutes and subsequently washed three times with HBSS to remove the excess extracellular Fura-2/AM. [Ca2+]i was expressed as the ratio (R) of emitted fluorescence corresponding to excitation wavelengths of 340 nm and 380 nm. R/R0 was applied to assess the change of [Ca2+]i level, in which R0 represents the fluorescent signal before drug treatment and R represents the signal after drug treatment.To determine the level of i induced by NaHS was determined in Ca2+-free HBSS containing the extracellular Ca2+ chelator EGTA (2 mM). In addition, separate cultures were treated with two selective L-type Ca2+ channel blockers, nifedipine (10 μM) and nimodipine (10 μM), and with the selective T-type Ca2+ channel inhibitor mibefradil (2 μM). To determine the involvement of intracellular Ca2+ stores in neurons, the cultures were treated with the intracellular Ca2+-chelating agent BAPTA-AM (50 μM) and thapsigargin (1 μM) with NaHS (200 μM) stimulation. Thapsigargin is known to release Ca2+ from the endoplasmic reticulum by inhibiting Ca2+ ATPase. All experiments were carried out at 37°C and were repeated 4~6 times using different batches of cells.To identify the source of Ca4 cells/well. Cells were cultured for 6~7 days at 37°C in 5% CO2 incubator. On the following day, the cultured neurons were treated for 30 min with different concentrations of NaHS. Afterward, each well of plates was added to 10 μL CCK-8 solution and then incubated at 37°C, 5% CO2 for 2 h. Subsequently, the cell viability was assessed by measuring the absorbance at 450 nm.Cell viability was analyzed by using Cell Counting Kit-8 . CCK-8 allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. Dojindo's highly water-soluble tetrazolium salt, WST-8, is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. The primary medullary neurons were cultured in 96-well plates at a cellular density of 0.5 × 102S levels in primarily cultured neurons were measured according to previously described methods with some modifications i in the medullary neurons in a concentration-dependent manner , SAM (a CBS activator), and HA and AOAA (two CBS inhibitors), respectively. The results showed that L-cysteine and SAM significantly raised the level of [Ca2+]i in the medullary neurons  or in normal HBSS. Similar to the data shown in Figures μM caused a robust increase in [Ca2+]i within 10 min after incubation of normal Ca2+ containing HBSS  and nimodipine (10 μM), two L-type Ca2+ channel blockers, significantly suppressed the effect of NaHS at 200 μM , also inhibited the elevation of [Ca2+]i level induced by NaHS  and thapsigargin (a sarco/endoplasmic reticulum Ca2+-ATPase blocker) with or without NaHS. The effect of NaHS on [Ca2+]i level in neurons was significantly attenuated by depletion of BAPTA-AM  and mibefradil (an antagonist of T-type Ca2+ channel), suggesting the role of L-type and/or T-type Ca2+ channels. Lastly, the effect of H2S on [Ca2+]i level in neurons was significantly attenuated by BAPTA-AM  and thapsigargin , suggesting the source of Ca2+.In the present study, we attempted to elucidate mechanisms for the regulation of intracellular calcium levels by H2S homeostasis in hypertension. Administration of H2S donors and precursors decreases mean blood pressure in various hypertensive models  i in SH-SY5Y neuronal cells by increasing Ca2+ influx via plasma membrane and in turn releasing calcium from intracellular calcium store [2S regulates calcium homeostasis in neurons via both increasing calcium influx and mobilizing calcium from ER [2S activates L-type Ca2+ channels in rat cerebellar granule neurons to increase calcium signals which were inhibited by nifedipine and nimodipine [2+ waves in astrocytes [2S also activates T-type Ca2+ channels in NG108-15 (neuroblastoma cell line) involved in neuronal differentiation [2+]i in the medullary neurons by H2S has not been reported. Our results showed that NaHS (50–300 μM) significantly increased the level of [Ca2+]i in rat medullary neurons in a concentration-dependent manner. On the other hand, L-cysteine and SAM significantly raised the level of [Ca2+]i in the medullary neurons while HA and/or AOAA produced a reversal effect. Meanwhile, HA or AOAA can no longer inhibit [Ca2+]i level in the presence of SAM. However, the stimulatory effect of SAM on [Ca2+]i was not affected by HA or AOAA. The possible reasons may be considered as follows: on the one hand, the affinity of SAM to CBS is much greater than that of HA or AOAA so that the stimulatory effect of SAM is dominating; on the other hand, the possibility that the increasing effects of L-cysteine and SAM on Ca2+ may be a H2S-independent manner is not ruled out and is needed to be addressed in the future. In addition, L-cysteine and SAM significantly increased the production of H2S in the cultured neurons and HA and/or AOAA decreased the production of H2S. These results suggested that both exogenous H2S and endogenous H2S increase the level of [Ca2+]i in the medullary neurons.Calcium plays an important role in regulating a great variety of neuronal processes such as release of neurotransmitters, synaptic plasticity, neuronal excitation, and gene transcription. It was reported that Hum store . Exertin from ER . In CNS,modipine  and in htrocytes . H2S alsntiation  and actintiation . The reg2+]i is controlled by Ca2+ channels in the membrane and intracellular Ca2+ stores [2+]i due to extracellular Ca2+ influx may be facilitated by voltage-gated channels, transmitter-gated Ca2+ permeant ion channels, transient receptor potential (TRP) ion channels, and Ca2+ pumps located in the plasma membrane i in NaHS-stimulated medullary neurons, we applied to the EGTA-Ca2+-free solution and BAPTA-AM to chelate the extracellular Ca2+ and intracellular Ca2+, respectively. The results showed that the Ca2+ elevation induced by H2S was significantly diminished by EGTA-Ca2+-free solutions and/or BAPTA-AM, suggesting that the elevation of [Ca2+]i level in medullary neurons partly involved both an influx of extracellular Ca2+ and the intracellular Ca2+ stores. That thapsigargin (a sarco/endoplasmic reticulum Ca2+-ATPase blocker) significantly attenuates the effect of H2S on [Ca2+]i level in neurons further suggests that NaHS releases calcium from intracellular Ca2+ stores.i level in primarily cultured rat medullary neurons via both increasing calcium influx and mobilizing intracellular Ca2+ stores from ER.In summary, the present study demonstrates that both exogenous H


Combination targeted therapy is commonly used to treat acute myeloid leukemia (AML) patients, particularly in refractory/relapse (RR) population. However, concerns have been raised regarding the safety and patient tolerance of combination chemotherapy. It is critical to choose the appropriate treatment for precision therapy. We performed genome-wide RNA profiling using RNA-Seq to compare the RR group and the complete remission (CR) group . The Hedgehog (Hh) and PI3K/AKT pathways were upregulated in the RR population, which was further confirmed by western blot and/or qPCR. Overexpression of GLI1 in AML cells led to increased AKT phosphorylation and decreased drug sensitivity, which was attenuated by GLI1 inhibition. By contrast, neither the expression of GLI1 nor apoptosis in response to Ara-C treatment of AML cells was significantly affected by PI3K inhibition. Furthermore, co-inhibition of GLI1 and PI3K induced apoptosis of hematopoietic stem/progenitor cells (HSPCs), which raised serious concerns about the side effects of this treatment. These results indicated that GLI1 inhibition alone, but not combined inhibition, is sufficient to enhance AML drug sensitivity, which provides a novel therapeutic strategy for AML treatment. Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. The high recurrence of AML reflects the incomplete eradication of leukemia stem cells by conventional chemotherapy and Ara-C combination chemotherapy, which are the most commonly used treatments. Approximately 20% of adult patients with AML fail to achieve remission with initial induction chemotherapy, and approximately 50% ultimately experience relapse after achieving complete remission124As the pathogenesis of AML involves regulation of different signalling pathways, the interaction and feedback among different signalling pathways are expected to be involved in the clinical treatment of AML. It is therefore critical to understand the interactions of these signalling pathways to provide a precise diagnosis for patients. Genomic approaches have identified somatic mutations in coding genes that are associated with patients’ prognosis. In transcriptome research, microarrays have been extensively used to identify a number of differentially expressed genes679et al. also demonstrated that a key transcription factor of the Hh pathway, GLI1, affects drug resistance by inducing glucuronidation, which could be reversed by a Hh inhibitorThe Hh pathway plays an important role in cell proliferation, differentiation, apoptosis and migration. It has been shown to cross-talk with other signalling pathways  and has been identified as a major target in the treatment of hematological malignancies1112Meanwhile, sustained activation of the PI3K/AKT pathway has been identified in different types of leukemia, and this effect promotes cell cycle progression, inhibits cell apoptosis and facilitates invasion and metastasis of cancer cells17182122However, whether there is cross-talk between the Hh and PI3K/AKT pathways in AML drug resistance/relapse and the potential risk of combined inhibition of these pathways in AML patients are still unknown. The possibility of using Hh and PI3K double inhibition in clinical AML treatment requires further study.By comparing the clinical samples of AML-RR and AML-CR patients using RNA-Seq, we showed that both the Hh and PI3K/AKT pathways were upregulated in the AML-RR group. Moreover, overexpression of GLI1 decreased drug sensitivity in AML cells, which could be reversed by a GLI1 inhibitor. In addition, GLI1 inhibition blocked the PI3K/AKT pathway, whereas PI3K inhibition had no effect on GLI1 expression or AML cell drug sensitivity. This study describes a novel mechanism of Hh and PI3K/AKT pathway interaction in chemo-resistance and relapse of AML patients, which provides guidance to develop an efficient and safe combination targeted therapy for AML-RR patients.10RPKM −2.5 to 3.5), with the majority of the genes ranging from 1 to 100 (log10RPKM 0 to 2). The majority of genes involved in the Hh pathway, such as GLI, SMO, SHH, and PTCH,  group and the complete remission (AML-CR) group  and 2. Rnd PTCH, , Table 3As previously noted, we also found that the majority of genes involved in the PI3K/AKT pathway, such as AKT1, AKT2, AKT3, and BTK , Table 3To explore the role of GLI1 and the relationship of Hh and the PI3K/AKT pathway in AML relapse and drug resistance, GLI1 was overexpressed in both HL60 and NB4 cells by a lentiviral vector carrying EGFP for selection. Seventy-two hours after infection, cells were sorted by FACS to establish new cell lines, which were named HL60/GLI1 and NB4/GLI1. The upregulation of GLI1 in HL60/GLI1 and NB4/GLI1 was also confirmed by both RT-PCR  and west50 of 4 μM for the HL60 cells , and Ara-C + LY294002 (20 μM) separately for 48 hours, and cell apoptosis was assessed using Annexin V assays . ConsistAs the morbidity and mortality of leukemia are increasing every year, developing effective and safe treatment strategies is of great urgency. In recent years, targeted therapy, which is considered to be one of the most promising treatments for AML, has been extensively researched. However, only a few targeted drugs have been successful in clinical practice, such as all trans-retinoic acid and imatinib2425in vitro study showed that inhibition of GLI1 depressed p-AKT activation and increased Ara-C sensitivity in leukemia cells, which is consistent with previous reports12152829In this study, by comparing gene expression profiles of clinical bone marrow samples from AML-CR and AML-RR patients using RNA-Seq technology, we found that the Hh and PI3K/AKT pathways were upregulated in the AML-RR group. Consistent with the results of RNA-Seq, RT-PCR and western blot analyses also showed upregulated expression of GLI1 and p-AKT in the AML-RR patients. An Other groups have shown that the AKT1 promoter possesses two GLI1 binding sites (BS1 and BS2) and proved the expression of AKT1 was regulated at the transcriptional level by GLI1Our study characterizes the Hh and PI3K/AKT interaction in AML drug sensitivity, which may provide a theoretical basis for further research of targeted therapy.2  or 20 μM LY294002  for 72 h prior to subsequent measurements.HL60 and NB4 cells were obtained from the Cell Resource Centre . The cell lines were maintained in RPMI-1640 medium  containing 10% foetal bovine serum (Corning Inc.) and 1% antibiotic solution of penicillin/streptomycin  in a 37 °C incubator with a humidified atmosphere of 5% CO2 . To over2 . To evalLiquid bone marrow samples from 42 patients between 18 and 64 years old, who were diagnosed with AML according to the 2008 WHO criteria and treated at Xiangya Hospital of Central South University, Hunan, China, were included in the AML group . For comRNA sample quality was analysed, and the cDNA libraries were synthesized and sequenced using BGI technology. In brief, the quality of the RNA samples was assessed by an Agilent Bioanalyzer (Agilent). cDNA libraries were generated using TruSeq RNA Sample Preparation (Illumina). Each library was sequenced using single-reads on a HiSeq2000/1000 (Illumina). Gene expression levels were measured in RPKM using Cufflinks336 cells using TRIzol reagent . Total RNA (500 ng) was reverse transcribed into first strand cDNA using a PrimeScriptTM RT Reagent Kit . cDNA was amplified in a total volume of 10 μl using SYBR qPCR Mix , and the amplification was performed by ABI StepOnePlus  with specific primers  membranes . The membranes were saturated in TBST containing 5% BSA (Bio Sharp Sigma A-4612) for an hour at room temperature and incubated with the primary antibodies overnight at 4 °C. After incubation with secondary antibody, the blots were then washed and detected with a ChemiDoc MP System . The antibodies were used as follows: mAb rabbit anti-GLI1 (C68H3) , mAb rabbit anti-phospho-AKT (Ser473) , mAb rabbit anti-AKT , anti-GAPDH , and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG .4 cells were seeded in 96-well culture plates in 100 μL of medium per well and treated with Ara-C at IC50  and GANT61/LY294002 (20 μm) for 24 h. Finally, 10 μl CCK8 solution was added to each well, and the cells were incubated further for 3 h at 37 °C. Cell viability was evaluated based on the absorbance at 450 nm compared with 630 nm. The growth inhibition rate =  × 100%.The inhibition effect of cell proliferation was measured by a Cell Counting Kit-8 assay  using different concentrations of Ara-C  according to the protocol provided by the manufacturer. Then, 2 × 106 cells were seeded in 6-well culture plates and treated with Ara-C at its IC50 and GANT61/LY294002 (20 μm). After 24 h of culture, cells were analysed by flow cytometry with an Annexin V-APC kit (Beyotime Institute) to determine the cell death rate.In each well, 1 × 10P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.All experiments were repeated independently at least three times, and the data were represented as the mean ± SD of the vehicle controls. All groups were compared using the Statistical Package for the Social Sciences (SPSS) version 20.0. The differences between the populations were assessed by analysis of covariance (ANCOVA). The diagrams were generated by GraphPad Prism 6 software. Significance was defined as *The experimental protocols and methods were performed in accordance with relevant guidelines and regulations. The informed consent was obtained from all the research subjects.How to cite this article: Liang, H. et al. Targeting the PI3K/AKT pathway via GLI1 inhibition enhanced the drug sensitivity of acute myeloid leukemia cells. Sci. Rep.7, 40361; doi: 10.1038/srep40361 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.