技术资料

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore,overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria,committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU,TMZ,and MMS,which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy. View Publication

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates,its activity in combination with other antiretrovirals,and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L,T215Y/F,plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V,Y115F,and M184V plus one other NAM) or stavudine (V75T/M,M41L,T215F/Y,and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold,respectively (these changes gave values comparable to or less than the corresponding values for zidovudine,lamivudine,abacavir,and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades,including A/G,B,C,D,A(E),D/F,F,and H. SPD754 showed additive effects in combination with other NRTIs,tenofovir,nevirapine,or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro,SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity. View Publication

OBJECTIVE: In vitro models of hematopoiesis used in investigative hematopathology and in safety studies on candidate drugs,involve clonogenic assays on colony-forming unit granulocyte macrophage (CFU-GM). These assays require live and unstained colonies to be counted. Most laboratories still rely on visual scoring,which is time-consuming and error-prone. As a consequence,automated scoring is highly desired. An algorithm that recognizes and scores CFU-GM colonies by data fusion has been developed. Some preliminary results are presented in this article. METHODS: CFU-GM assays were carried out on hematopoietic progenitors (human umbilical cord blood cells) grown in methylcellulose. Colony images were acquired by a digital camera and stored. RESULTS: The classifier was designed to process images of layers sampled from a three-dimensional (3D) domain and forming a stack. Structure and texture information was extracted from each image. Classifier training was based on a 3D colony model applied to the image stack. The number of scored colonies (assigned class) was required to match the count supplied by the human expert (class of belonging). The trained classifier was validated on one more stack and then applied to a stack with overlapping colonies. Scoring in distortion- and caustic-affected border areas was also successfully demonstrated. Because of hardware limitations,compact colonies in some cases were missed. CONCLUSIONS: The industry's scoring methods all rely on structure alone and process 2D data. Instead,the classifier here fuses data from a whole stack and is capable,in principle,of high-throughput screening. View Publication

Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis,even if initial gene transfer efficiency is low. Moreover,selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However,a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore,understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)-expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly,not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection,thus underscoring the potential value of MGMT P140K selection for clinical gene therapy. View Publication

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences (mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique� View Publication

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly,we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre,we designed a lentiviral vector that integrates into the host genome,expresses Cre in the target cell,and is subsequently deleted from the genome in a Cre-dependent manner. Thus,the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression,transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo. View Publication

In February 2013,zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported,a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population,there is interest in identifying other correlates of protection,such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly,but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here,we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells,obtained from HLA-typed study subjects,with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that,apart from recognition of individual H7N9 variant epitopes,CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus. View Publication

Down's syndrome (DS),caused by trisomy of human chromosome 21,is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons,and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally,we show that the FDA-approved antibiotic drug,minocycline,partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B,GFAP,inducible nitric oxide synthase,and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. View Publication

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-β[2-(di-n-butylamino)-6-naphthyl]butadienylquinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses,we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision,generating nearly identical values for AP duration (AP durations at 10%,50%,and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure,with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally,di-4-ANBDQBS allowed for moderate-throughput analyses,increasing throughput textgreater10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs. View Publication

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture,the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs,inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation,differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs,mainly as a consequence of increased replication. Furthermore,trisomy 12 increases the tumorigenicity of hPSCs in vivo,inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last,a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together,these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications. View Publication