PK!u68|8|refs.MYD?Gossen, M. Bujard, H.1995ZEfficacy of tetracycline-controlled gene expression is influenced by cell type: commentary213-6; discussion 216-7 Biotechniques192Animal Cercopithecus aethiops Gene Expression Regulation/*DRUG EFFECTS Genetic Vectors Hamsters Hela Cells Human Kidney Tetracycline/*PHARMACOLOGY Trans-Activation (Genetics)/DRUG EFFECTS Vero Cells?.Baron, U. Freundlieb, S. Gossen, M. Bujard, H.1995QCo-regulation of two gene activities by tetracycline via a bidirectional promoter3605-6Nucleic Acids Res2317Cloning, Molecular/*METHODS Cytomegalovirus/GENETICS Gene Expression Regulation Genetic Vectors Hela Cells Human Operator Regions (Genetics) Promoter Regions (Genetics) Repressor Proteins/GENETICS Support, Non-U.S. Gov't Tetracycline/*PHARMACOLOGY Tetracycline Resistance Transfection? 1Gossen, M. Bonin, A. L. Freundlieb, S. Bujard, H.1994=Inducible gene expression systems for higher eukaryotic cells516-20Curr Opin Biotechnol55Animal Animals, Transgenic Escherichia coli/GENETICS Eukaryotic Cells Gene Expression Gene Expression Regulation Human Operon Plants, Transgenic Tetracycline Resistance/GENETICS Transcription, GeneticOf the wide variety of regulatory systems that have been developed to control gene expression in higher eukaryotes, those utilizing elements from prokaryotic systems presently achieve the highest specificity. In particular, systems based on the repressor/operator elements of an Escherichia coli tetracycline resistance operon appear broadly applicable. During the past year, the usefulness of these systems has been demonstrated not only at the cellular level, but also at the organismal level (i.e. in transgenic animals and plants).? EGossen, M. Freundlieb, S. Bender, G. Muller, G. Hillen, W. Bujard, H.1995>Transcriptional activation by tetracyclines in mammalian cells1766-9Science2685218]Amino Acid Sequence Doxycycline/*PHARMACOLOGY Genes, Reporter Hela Cells Human Molecular Sequence Data Operator Regions (Genetics) Recombinant Fusion Proteins/GENETICS Repressor Proteins/CHEMISTRY/*GENETICS Simplexvirus Support, Non-U.S. Gov't Trans-Activation (Genetics)/*DRUG EFFECTS Trans-Activators/*GENETICS Transfection Viral Proteins/GENETICSA transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.? _Furth, P. A. St Onge, L. Boger, H. Gruss, P. Gossen, M. Kistner, A. Bujard, H. Hennighausen, L.1994\Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter9302-6Proc Natl Acad Sci U S A9120-beta-Galactosidase/ANALYSIS/BIOSYNTHESIS Animal Base Sequence Cytomegalovirus/GENETICS DNA Insertion Elements DNA Primers Embryo/METABOLISM Enhancer Elements (Genetics) Escherichia coli/GENETICS Gene Expression Gestational Age Human Luciferase/ANALYSIS/BIOSYNTHESIS Mice Mice, Transgenic Molecular Sequence Data Muscles/METABOLISM Operon Organ Specificity Polymerase Chain Reaction Promoter Regions (Genetics)/DRUG EFFECTS Simplexvirus/GENETICS Support, Non-U.S. Gov't Tetracycline/*PHARMACOLOGY Time Factors Tongue/METABOLISM Trans-Activators/*BIOSYNTHESISPromoters whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in vivo. We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice. In this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1; see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence. Transgenic mice were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer. Whereas little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA in double-transgenic mice induced expression of the reporter genes up to several thousand-fold. This induction was abrogated to basal levels upon administration of tetracycline. These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control of transgene expression is required.? @Fruh, K. Gossen, M. Wang, K. Bujard, H. Peterson, P. A. Yang, Y.1994Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex3236-44Embo J1314Amino Acid Sequence Animal Antigen Presentation Cells, Cultured Comparative Study Cysteine Proteinases/CHEMISTRY/DRUG EFFECTS/GENETICS/*METABOLISM Gene Expression Regulation, Enzymologic Interferon Type II/PHARMACOLOGY Major Histocompatibility Complex/*PHYSIOLOGY Mice Mice, Inbred BALB C Molecular Sequence Data Multienzyme Complexes/CHEMISTRY/DRUG EFFECTS/GENETICS/*METABOLISM Peptide Peptidohydrolases/GENETICS/*METABOLISM Proteins/GENETICS/*METABOLISM Recombinant Proteins/METABOLISM Sequence Homology, Amino Acid TransfectioniThe degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.?6Weinmann, P. Gossen, M. Hillen, W. Bujard, H. Gatz, C.1994XA chimeric transactivator allows tetracycline-responsive gene expression in whole plants559-69Plant J54{Antiporters/GENETICS Base Sequence Chimera Dna DNA Insertion Elements Gene Expression Regulation/*DRUG EFFECTS Glucuronidase/GENETICS Molecular Sequence Data Plants, Transgenic Promoter Regions (Genetics) Repressor Proteins/GENETICS Simplexvirus/GENETICS Support, Non-U.S. Gov't Tetracycline/*PHARMACOLOGY Trans-Activators Transformation, Genetic TATA Box Viral Proteins/GENETICSThe chimeric transcriptional activator tTA, a fusion between the Tn10 encoded Tet repressor and the activation domain of the Herpes simplex virion protein VP16, was stably expressed in transgenic tobacco plants. It stimulates transcription of the beta-glucuronidase (gus) gene from an artificial promoter consisting of 7 tet operators and a TATA-box. Tetracycline, which interferes with binding of tTA to operator DNA, reduces gus expression over several orders of magnitude. This stringency of regulation suggests that the system can be used to construct transgenic plants encoding a potentially lethal gene product. Furthermore, the specific and fast inactivation of tTA allows study of the stability of RNAs and proteins.p?/Resnitzky, D. Gossen, M. Bujard, H. Reed, S. I.1994dAcceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system1669-79 Mol Cell Biol143Animal Cell Cycle Cell Line Culture Media Cyclins/*PHYSIOLOGY In Vitro Oncogene Proteins/*PHYSIOLOGY Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. TransfectionConditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence. ?"Gossen, M. Bonin, A. L. Bujard, H.1993VControl of gene activity in higher eukaryotic cells by prokaryotic regulatory elements471-5Trends Biochem Sci1812Animal Escherichia coli/GENETICS Gene Expression Regulation/*PHYSIOLOGY Hela Cells Human Lac Operon/GENETICS Repressor Proteins/*PHYSIOLOGY Saccharomyces cerevisiae/GENETICS Tetracycline Resistance/GENETICS Transcription FactorslRegulated gene expression systems for the study of gene function in prokaryotes and yeast have been available for several years. However, comparable systems in higher organisms are more complex and problematic. Recently, regulatory proteins from distant species have been used to establish highly specific control systems in eukaryotic cells. This is possible because their action can be modulated by effectors that are inert to the physiology of the organism or cell and therefore do not elicit pleiotropic effects. Such monospecific regulatory circuits open up new possibilities for the study of gene function in vivo.?Gossen, M. Bujard, H.1993mAnhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells4411-2Nucleic Acids Res2118.Animal Antiporters/METABOLISM Dictyostelium/GENETICS Gene Expression Regulation/*DRUG EFFECTS Hela Cells Human Luciferase/GENETICS Repressor Proteins/METABOLISM Saccharomyces cerevisiae/GENETICS Schizosaccharomyces/GENETICS Support, Non-U.S. Gov't Tetracycline/*PHARMACOLOGY Tetracyclines/*PHARMACOLOGYM?Gossen, M. Bujard, H.1992XTight control of gene expression in mammalian cells by tetracycline-responsive promoters5547-51Proc Natl Acad Sci U S A8912Bacterial Proteins/METABOLISM Base Sequence Escherichia coli/GENETICS Gene Expression/DRUG EFFECTS Hela Cells Human Kinetics Luciferase/GENETICS/METABOLISM Molecular Sequence Data Operon Promoter Regions (Genetics)/*DRUG EFFECTS Recombinant Fusion Proteins/METABOLISM Repressor Proteins/GENETICS/METABOLISM Restriction Mapping Simplexvirus/GENETICS Support, Non-U.S. Gov't Tetracycline/*PHARMACOLOGY Tetracycline Resistance/*GENETICS Trans-Activation (Genetics) Trans-Activators/*METABOLISM Transcription, Genetic/DRUG EFFECTS TransfectionxControl elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way. |?jYang, Y. Vanin, E. F. Whitt, M. A. Fornerod, M. Zwart, R. Schneiderman, R. D. Grosveld, G. Nienhuis, A. W.1995Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein1203-13 Hum Gene Ther69Animal Cell Line Culture Media Genetic Vectors/*BIOSYNTHESIS/GENETICS Herpes Simplex Virus Protein Vmw65/GENETICS Mice Moloney Leukemia Virus/*GENETICS Promoter Regions (Genetics) Repressor Proteins/GENETICS Steroids/PHARMACOLOGY Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Tetracycline Resistance/GENETICS Transfection Vesicular Stomatitis-Indiana Virus/*CHEMISTRY Viral Envelope Proteins/*GENETICSMurine leukemia viruses (MuLV) have been adapted for use as gene transfer vectors for experimental and human gene therapy applications. Their utility for these purposes has been circumscribed by the limited host range and relatively low titer of available producer clones. Pseudotyping of MuLV particles with the vesicular stomatitis virus envelope protein (VSV-G), expressed transiently in cells producing MuLV Gag and Pol proteins, has yielded vector preparations with a broader host range that can be concentrated by ultracentrifugation. We have explored the use of steroid-inducible and tetracycline-modulated promoter systems (necessary because the VSV-G protein is toxic to cells when constitutively expressed) to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MuLV particles. A packaging cell line and producer clones capable of expressing a chimeric transcription factor, composed of the tetracycline repressor (tetR) and the VP16 trans-activating sequences of herpes simplex virus VP16 gene and containing the VSV-G coding sequences linked to a minimal promoter having seven tandem copies of the tetracycline responsive operator (tetO), exhibited high levels of VSV-G protein expression when cultured in the absence of tetracycline. Vector particles, produced at titers of 10(5)-10(6) infectious colony forming units per ml (cfu/ml), could be concentrated effectively by ultracentrifugation yielding vector preparations having a titer of 10(9) cfu/ml. These cell lines grew normally when VSV-G protein expression was repressed by tetracycline. Such producer clones hold promise for future human gene therapy applications.L?&Eldredge, E. R. Chiao, P. J. Lu, K. P.1995VUse of tetracycline operator system to regulate oncogene expression in mammalian cells481-91Methods Enzymol254Animal Bacterial Proteins/*BIOSYNTHESIS Base Sequence Cell Line Cells, Cultured Escherichia coli/GENETICS Gene Expression Regulation/DRUG EFFECTS Genetic Vectors Hela Cells Human Mammals Molecular Sequence Data Oncogene Proteins/*BIOSYNTHESIS Oncogenes Reading Frames Recombinant Proteins/BIOSYNTHESIS Repressor Proteins/*BIOSYNTHESIS Tetracycline/PHARMACOLOGY Transcription, Genetic Translation, Genetic?APaulus, W. Baur, I. Boyce, F. M. Breakefield, X. O. Reeves, S. A.1996dSelf-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells62-7J Virol701Animal Base Sequence Cell Line Cell Line, Transformed Gene Expression Regulation, Viral/*DRUG EFFECTS Gene Transfer Genetic Vectors/DRUG EFFECTS/*GENETICS Luciferase/METABOLISM Mammals Mice Molecular Sequence Data Promoter Regions (Genetics) Retroviridae/DRUG EFFECTS/GROWTH & DEVELOPMENT/*GENETICS Support, Non-U.S. Gov't Tetracycline/*PHARMACOLOGY Trans-Activators Tumor Cells, Cultured 3T3 Cells"Retroviral vectors that contain the tetracycline-inducible (Tet) system were developed. The two components of the Tet system were organized within the vectors in a manner that stringently maintains tetracycline-dependent regulation. Regulated expression of an indicator gene inserted into the retroviral vectors was examined in several different cell types. In infected NIH 3T3 cells, levels of induction in the absence of tetracycline were observed to be as much as 336-fold higher than levels in the presence of tetracycline, which were extremely low. Tetracycline-dependent regulation was observed in all other transduced cell types and ranged from 24- to 127-fold. The generation of retroviral vectors containing regulatory elements that allow for the regulated expression of heterologous genes and that have the ability to infect virtually all dividing target cells should greatly facilitate the biochemical and genetic examination of a broad range of genes. Moreover, these inducible retroviral vectors should prove useful in gene therapy applications.?;Dhawan, J. Rando, T. A. Elson, S. L. Bujard, H. Blau, H. M.1995`Tetracycline-regulated gene expression following direct gene transfer into mouse skeletal muscle233-40Somat Cell Mol Genet214nbeta-Galactosidase/ANALYSIS/BIOSYNTHESIS Animal Gene Expression Regulation/DRUG EFFECTS/*PHYSIOLOGY Gene Transfer Luciferase/ANALYSIS/BIOSYNTHESIS Male Mice Mice, Inbred C3H Muscle, Skeletal/*METABOLISM Plasmids Recombinant Proteins/ANALYSIS/BIOSYNTHESIS Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/*PHARMACOLOGY Tetracycline Resistance/GENETICSFor most experimental and therapeutic applications of gene transfer, regulation of the timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammalian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it is dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally administered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-controlled expression of genes in vivo, suggesting applications to developmental studies and gene therapy. f?KPrecious, B. Young, D. F. Bermingham, A. Fearns, R. Ryan, M. Randall, R. E.1995Inducible expression of the P, V, and NP genes of the paramyxovirus simian virus 5 in cell lines and an examination of NP-P and NP-V interactions8001-10J Virol6912Animal Base Sequence Blotting, Western Capsid/*BIOSYNTHESIS/ISOLATION & PURIFICATION/METABOLISM Cell Line Cloning, Molecular Comparative Study DNA Primers Gene Expression Regulation, Viral Genes, Viral Mammals Molecular Sequence Data Paramyxovirus/*GENETICS/PHYSIOLOGY Phosphoproteins/*BIOSYNTHESIS/ISOLATION & PURIFICATION/METABOLISM Phosphorylation Polymerase Chain Reaction Protein Binding Restriction Mapping Support, Non-U.S. Gov't Trans-Activators/METABOLISM Viral Proteins/*BIOSYNTHESIS/ISOLATION & PURIFICATION/METABOLISM=The P, V, and NP genes of the paramyxovirus simian virus 5 (SV5) were cloned such that their expression was regulated by the tetracycline-controlled transactivator (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992), and mammalian cell lines that inducibly expressed individually the P, V, or NP protein or coexpressed the P plus NP or V plus NP proteins were isolated. A plasmid that expresses the tetracycline-controlled transactivator linked, via the foot-and-mouth disease virus 2A cleavage peptide sequence, to the neomycin aminoglycoside phosphotransferase gene was constructed. Cells were cotransfected with this plasmid, and the appropriate responder plasmids and clonies were selected on the basis of their resistance to Geneticin (via the neomycin aminoglycoside phosphotransferase gene). The properties of these cell lines, in terms of the induction of the P, V, and NP genes, are described in detail. Both the P and V proteins were phosphorylated when expressed alone. In immunoprecipitation studies using a monoclonal antibody that recognizes both the P and V proteins, a nonphosphorylated host cell protein with an estimated molecular weight of 150,000 was coprecipitated with V but not P. Immunofluorescence data demonstrated that when expressed separately, the P protein had a diffuse cytoplasmic distribution, but the related V protein had both a nuclear and cytoplasmic distribution. The NP protein had a granular cytoplasmic distribution, giving rise to punctate and granular fluorescence. Coexpression of the NP and P proteins resulted in the accumulation of large cytoplasmic inclusion aggregates, similar to those visualized at late times in SV5-infected cells. Coexpression of V with NP led to a partial redistribution of the NP protein in that the NP protein had both a diffuse cytoplasmic and nuclear distribution in the presence of V, but no NP-V aggregates or inclusion bodies were visualized. Direct binding studies also revealed that NP bound to both P and V. For SV5, these studies suggest that V may have a role in keeping NP soluble prior to encapsidation.?Yin, D. X. Schimke, R. T.1995{BCL-2 expression delays drug-induced apoptosis but does not increase clonogenic survival after drug treatment in HeLa cells4922-8 Cancer Res5521Antineoplastic Agents, Phytogenic/*PHARMACOLOGY Aphidicolin/*PHARMACOLOGY Apoptosis/*DRUG EFFECTS/*PHYSIOLOGY Cell Death/DRUG EFFECTS/PHYSIOLOGY Cell Survival/DRUG EFFECTS/PHYSIOLOGY Clone Cells Demecolcine/*PHARMACOLOGY Enzyme Inhibitors/*PHARMACOLOGY Gene Expression Hela Cells Human Protein Synthesis Inhibitors/PHARMACOLOGY Proto-Oncogene Proteins/GENETICS/*PHYSIOLOGY Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGYcApoptosis is a major form of cell death induced by chemotherapeutic drugs. Overexpression of the proto-oncogene bcl-2 can prevent apoptosis in various types of cells. We have constructed a HeLa S3 cell line in which the expression of bcl-2 can be controlled by the concentration of tetracycline in the medium. Using this system, we show that apoptosis induced by various cytostatic treatments could be delayed by the overexpression of bcl-2, as assayed by vital dye exclusion, apoptotic nuclei morphology, DNA histogram shift, and DNA fragmentation. Quantitative analysis revealed a hyperbolic curve when protection from apoptosis was plotted against the amount of Bcl-2. When cells were treated with aphidicolin for 12, 24, or 36 h and then replated in fresh media to assay for colony formation, the majority of cells that did not show apoptotic morphology at the time of drug removal failed to form colonies. Furthermore, Bcl-2 did not increase colony formation after 12-36 h of aphidicolin treatment. Therefore, with aphidicolin treatment, cells were committed to the death program upstream of the point of Bcl-2 action.I?jEnglert, C. Hou, X. Maheswaran, S. Bennett, P. Ngwu, C. Re, G. G. Garvin, A. J. Rosner, M. R. Haber, D. A.1995VWT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis4662-75Embo J1419&Alternative Splicing Animal Animals, Newborn Apoptosis/*PHYSIOLOGY Base Sequence DNA-Binding Proteins/GENETICS/METABOLISM/*PHYSIOLOGY DNA, Neoplasm/METABOLISM Genes, Wilms' Tumor/*GENETICS Human Kidney/EMBRYOLOGY/METABOLISM Molecular Sequence Data Osteosarcoma Promoter Regions (Genetics)/GENETICS Protein p53/PHYSIOLOGY Rats Receptors, Epidermal Growth Factor-Urogastrone/*BIOSYNTHESIS Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Transcription Factors/GENETICS/METABOLISM/*PHYSIOLOGY Transcription, Genetic Transfection Tumor Cells, Cultured(The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.g?-Gjetting, T. Lukas, J. Bartek, J. Strauss, M.1995Regulated expression of the retinoblastoma susceptibility gene in mammary carcinoma cells restores cyclin D1 expression and G1-phase control441-6Biol Chem Hoppe Seyler3767Breast Neoplasms/*GENETICS/PATHOLOGY Cyclins/*GENETICS Gene Expression Regulation, Neoplastic Genes, Retinoblastoma G1 Phase Human Oncogene Proteins/*GENETICS Tumor Cells, CulturedThe product of the retinoblastoma susceptibility tumour suppressor gene, pRb, is a negative regulator of cell proliferation. In order to investigate the interaction between pRb and the cell cycle machinery in more detail, a functional Rb gene was reintroduced into the Rb-deficient human mammary carcinoma cell line Bt549. Since constitutive high level expression of Rb turned out to be difficult to maintain, the tetracycline-dependent gene expression system was used. A number of clones was generated which all showed low level expression in the noninduced state. Considerable induction rates were obtained. The low level of noninduced Rb expression was sufficient to induce the expression of cyclin D1 the level of which was not further increased by upregulation of Rb expression. Concomittantly, an increase in cell doubling time was observed due to retardation of the cell cycle in the G1-phase. The data suggest that limiting amounts of cyclin D1 determine, at least partly, the extent of growth-repressing properties of pRb. The inducible system allows for maintenance of Rb-reconstituted cells at a low level of expression and for their use in the investigation of downstream functions of pRb.?9Chen, Y. Q. Cipriano, S. C. Arenkiel, J. M. Miller, F. R.1995Tumor suppression by p21WAF14536-9 Cancer Res5520Animal Cell Adhesion Cell Division Cyclins/*PHYSIOLOGY Genes, Suppressor, Tumor Human In Vitro Mice Mice, Nude Neoplasm Transplantation Neoplasms, Experimental/*GENETICS/PATHOLOGY Support, Non-U.S. Gov't Transfection Tumor Cells, Cultured/PATHOLOGYThe p21WAF1 gene encodes a cyclin-dependent kinase inhibitor and mediates tumor suppressor gene p53-induced cell cycle arrest. To directly test whether p21WAF1 can act as a tumor suppressor, we have expressed the p21WAF1 cDNA in several human tumor cell lines using a tetracycline-inducible system. Overexpression of p21WAF1 suppresses proliferation and soft agar growth of tumor cells in vitro, as well as tumorigenicity in vivo. Our data provide direct evidence for the tumor-suppressive activity of p21WAF1. @?*Wu, Z. Xie, Y. Bucher, N. L. Farmer, S. R.1995lConditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis2350-63 Genes Dev9194Adipocytes/CYTOLOGY/*METABOLISM Animal Base Sequence Cell Differentiation/GENETICS Cell Fractionation Dexamethasone/PHARMACOLOGY Dna/metabolism DNA Probes DNA-Binding Proteins/*GENETICS/METABOLISM Gene Expression Regulation, Developmental Insulin/PHARMACOLOGY Mice Molecular Sequence Data Nuclear Proteins/*GENETICS/METABOLISM Receptors, Cytoplasmic and Nuclear/*GENETICS Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Transcription Factors/*GENETICS/METABOLISM 1-Methyl-3-isobutylxanthine/PHARMACOLOGY 3T3 Cells 5,8,11,14-Eicosatetraynoic Acid/PHARMACOLOGY9Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.%?5Agarwal, M. L. Agarwal, A. Taylor, W. R. Stark, G. R.1995wp53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts8493-7Proc Natl Acad Sci U S A9218Cell Cycle/*GENETICS Cell Division/*GENETICS Cell Line Cyclins/METABOLISM Fibroblasts/CYTOLOGY/METABOLISM Gene Expression Regulation Genes, p53 Human Support, Non-U.S. Gov'tIncreased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.Z?!OKirchhoff, S. Koster, M. Wirth, M. Schaper, F. Gossen, M. Bujard, H. Hauser, H.1995gIdentification of mammalian cell clones exhibiting highly regulated expression from inducible promoters219-20 Trends Genet116hAlkaline Phosphatase/ANALYSIS/BIOSYNTHESIS/GENETICS Animal Clone Cells/*METABOLISM DNA-Binding Proteins/GENETICS Gene Expression Regulation/*DRUG EFFECTS Genes, Reporter Mammals Membranes, Artificial Phosphoproteins/GENETICS Polyvinyls Promoter Regions (Genetics)/*DRUG EFFECTS Recombinant Proteins/*BIOSYNTHESIS/GENETICS Tetracycline/PHARMACOLOGY TransfectionV?"$Resnitzky, D. Hengst, L. Reed, S. I.1995{Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip14347-52 Mol Cell Biol158Animal Cell Line Cyclins/*METABOLISM G1 Phase/*PHYSIOLOGY Human Microtubule-Associated Proteins/*METABOLISM Phosphorylation Protein Kinases/*METABOLISM Rats Retinoblastoma Protein/METABOLISM S Phase/*PHYSIOLOGY Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S.pWe have created fibroblast cell lines that express cyclin A under the control of a tetracycline-repressible promoter. When stimulated to reenter the cell cycle after serum withdrawal, these cells were advanced prematurely into S phase by induction of cyclin A. In an asynchronous population, induction of cyclin A caused a decrease in the percentage of cells in G1. These results demonstrate that expression of cyclin A is rate limiting for the G1-to-S transition and suggest that cyclin A can function as a G1 cyclin. Although the level of exogenous cyclin A was constant throughout the cell cycle, its associated kinase activity increased as cells approached S phase. Low kinase activity in early G1 was found to correlate with the presence of p27Kip1 in cyclin A-associated complexes, while high kinase activity in late G1 was correlated with its absence. These results suggest that a function of p27Kip1 in G1 is to prevent premature activation of cyclin A-associated kinase. Cyclin A expression in early G1 led to phosphorylation of the product of the retinoblastoma susceptibility gene (pRb). Thus, cyclin A expression can be rate limiting for pRb phosphorylation, implicating pRb as a physiological substrate of the cyclin A-dependent kinase. Taken together, these results demonstrate that deregulated expression of cyclin A can perturb the normal regulation of the G1-to-S transition. ?#:Shockett, P. Difilippantonio, M. Hellman, N. Schatz, D. G.1995A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice6522-6Proc Natl Acad Sci U S A9214Animal Blotting, Western Cells, Cultured DNA Nucleotidyltransferases/BIOSYNTHESIS/METABOLISM Gene Expression Regulation/*DRUG EFFECTS Herpes Simplex Virus Protein Vmw65/BIOSYNTHESIS Mice Mice, Transgenic Open Reading Frames Plasmids Recombinant Fusion Proteins/BIOSYNTHESIS Repressor Proteins/BIOSYNTHESIS Restriction Mapping RNA, Messenger/ANALYSIS/BIOSYNTHESIS Sequence Deletion Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/*PHARMACOLOGY Trans-Activators/BIOSYNTHESIS Transfection 3T3 CellsA system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining the tetracycline repressor and the transcriptional activation domain of VP16 [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs. constitutive, 18%). In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.|?%Cayrol, C. Flemington, E. K.1995Identification of cellular target genes of the Epstein-Barr virus transactivator Zta: activation of transforming growth factor beta igh3 (TGF-beta igh3) and TGF-beta 14206-12J Virol697|Base Sequence DNA-Binding Proteins/GENETICS/*PHYSIOLOGY Gene Expression Regulation/DRUG EFFECTS Hela Cells Herpesvirus 4, Human/*GENETICS Human Molecular Sequence Data Promoter Regions (Genetics) Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Trans-Activators/GENETICS/*PHYSIOLOGY Transforming Growth Factor beta/*GENETICS Viral Proteins/*PHYSIOLOGYThe lytic switch transactivator Zta initiates the ordered cascade of Epstein-Barr virus gene expression that culminates in virus production. Zta is a sequence-specific DNA-binding protein that transactivates early viral promotes via cis-acting sequences. Activation of some of these genes is mediated through binding to consensus AP-1 promoter elements. This observation suggests that Zta may also regulate the expression of cellular genes. While many targets of Zta have been identified in the Epstein-Barr virus genome, putative host cell targets remain largely unknown. To address this issue, a tetracycline-regulated Zta expression system was generated, and differential hybridization screening was used to isolate Zta-responsive cellular genes. The major target identified by this analysis is a gene encoding a fasciclin-like secreted factor, transforming growth factor beta igh3 (TGF-beta igh3), that was originally identified as a gene that is responsive to the potent immunosuppressor TGF-beta 1. Northern (RNA) blot analysis demonstrated that induction of Zta expression results in a 10-fold increase in TGF-beta igh3 mRNA levels. Zta was also found to increase TGF-beta 1 mRNA levels as well as the amount of active TGF-beta 1 secreted into the medium. Interestingly, alpha 1-collagen IV, which has been shown to potentiate the effects of TGF-beta 1, is also a cellular target of Zta. These results suggest that Zta could play a role in modulating the host cell environment through activating the expression of secreted factors.?&]Fruh, K. Ahn, K. Djaballah, H. Sempe, P. van Endert, P. M. Tampe, R. Peterson, P. A. Yang, Y.1995BA viral inhibitor of peptide transporters for antigen presentation415-8Nature3756530Amino Acid Sequence Animal Antigen Presentation ABC Transporters/*ANTAGONISTS & INHIBITORS/METABOLISM Biological Transport/DRUG EFFECTS Endoplasmic Reticulum/METABOLISM Hela Cells Histocompatibility Antigens Class I/METABOLISM Human Immediate-Early Proteins/GENETICS/*PHYSIOLOGY Interferon Type II/PHARMACOLOGY Mice Molecular Sequence Data Precipitin Tests Recombinant Proteins/GENETICS/METABOLISM Simplexvirus/*IMMUNOLOGY/PHYSIOLOGY Tetracyclines/PHARMACOLOGYCytotoxic T lymphocytes lyse target cells after T-cell-receptor-mediated recognition of class I major histocompatibility complex molecules presenting peptides. Antigenic peptides are generated in the cytoplasm by proteasomes and translocated into the lumen of the endoplasmic reticulum (ER) by peptide transporters (TAP). Herpes simplex virus (HSV) expresses a cytoplasmic protein, ICP47, which seems to interfere with such immune surveillance by mediating retention of 'empty' class I molecules in the ER. By expressing ICP47 in HeLa cells under an inducible promoter, we show that ICP47 efficiently inhibits peptide transport across the ER membrane such that nascent class I molecules fail to acquire antigenic peptides. This inhibition was overcome by transfecting murine TAP. Further, we demonstrate that ICP47 colocalizes and physically associates with TAP within the cell. Inhibition of peptide translocation by a viral protein indicates a previously undocumented potential mechanism for viral immune evasion. ?'Miller, K. Rizzino, A.1995JThe function of inducible promoter systems in F9 embryonal carcinoma cells144-50 Exp Cell Res2181Actins/*BIOSYNTHESIS Animal Carcinoma, Embryonal Cell Differentiation Cell Line Chloramphenicol Acetyltransferase/BIOSYNTHESIS Enhancer Elements (Genetics) Gene Expression Hela Cells Human Mammary Tumor Viruses, Mouse Mice Plasmids Promoter Regions (Genetics) Rats Receptors, Glucocorticoid/*BIOSYNTHESIS Recombinant Fusion Proteins/BIOSYNTHESIS Recombinant Proteins/BIOSYNTHESIS Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Transfection Tumor Cells, CulturediEmbryonal carcinoma (EC) cells represent an important model for studying the regulation of cellular differentiation during embryonic development and tumor formation. The differentiation of EC cells is associated with changes in the expression of a number of cellular genes, some of which have been implicated directly in the regulation of differentiation. To facilitate further studies of the possible roles of cellular gene products during the differentiation of EC cells, we have used transient transfection assays to compare the function of three promoter systems that direct the conditional expression of recombinant gene constructs. One system employs the mouse mammary tumor virus (MMTV) promoter, which is induced by glucocorticoid hormones. The other two systems are based on chimeric transactivator proteins consisting of the bacterial lac repressor or tet repressor, respectively, fused with a viral transactivation domain. The chimeric proteins function in mammalian cells as sequence-specific activators of transcription that are regulated by either lactose analogs or tetracycline. Transient transfections of mouse F9 EC cells and their differentiated cells with an MMTV promoter-reporter gene construct and a second plasmid encoding the rat glucocorticoid receptor resulted in a dramatic induction of reporter gene expression by glucocorticoid hormone of approximately 200-fold. The conditional expression system based on the tetracycline-responsive transactivator exhibited a similar range of reporter gene expression in response to tetracycline. In contrast, the system based on the lac repressor exhibited a much more limited range of conditional reporter gene expression in our studies. These findings and others discussed in this report suggest that the tetracycline-responsive promoter system may be useful for the conditional expression of recombinant gene constructs in F9 EC cells. Furthermore, data are presented indicating that the human beta-actin promoter should be suitable for stable expression of conditional transactivators, such as the tetracycline-responsive transactivator, in F9 cells before and after differentiation. L?)<Efrat, S. Fusco-DeMane, D. Lemberg, H. al Emran, O. Wang, X.1995Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene3576-80Proc Natl Acad Sci U S A928uAnimal Antigens, Viral, Tumor/BIOSYNTHESIS/*GENETICS Blood Glucose/ANALYSIS Cell Division/DRUG EFFECTS Cell Line Cell Transformation, Neoplastic/*GENETICS Diabetes Mellitus, Experimental/METABOLISM Dose-Response Relationship, Drug Gene Expression Regulation, Neoplastic/*DRUG EFFECTS Herpes Simplex Virus Protein Vmw65/GENETICS Insulinoma/*GENETICS Islets of Langerhans/CYTOLOGY/DRUG EFFECTS/PATHOLOGY Mice Mice, Transgenic Neoplasms, Experimental/GENETICS Pancreatic Neoplasms/*GENETICS Polyomavirus macacae/GENETICS Repressor Proteins/GENETICS Support, Non-U.S. Gov't Tetracycline/PHARMACOLOGY Tetracycline Resistance/*GENETICSConditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.\?,QSopher, B. L. Fukuchi, K. Smith, A. C. Leppig, K. A. Furlong, C. E. Martin, G. M.1994wCytotoxicity mediated by conditional expression of a carboxyl-terminal derivative of the beta-amyloid precursor protein207-17Brain Res Mol Brain Res261-2Alzheimer's Disease/METABOLISM Amyloid beta-Protein Precursor/*BIOSYNTHESIS Brain/METABOLISM Bucladesine/PHARMACOLOGY Cell Line Cell Survival/DRUG EFFECTS Flow Cytometry Gene Expression/DRUG EFFECTS Human Neuroblastoma Promoter Regions (Genetics) Recombinant Fusion Proteins/BIOSYNTHESIS Restriction Mapping Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Transfection Tumor Cells, CulturedThe beta amyloid peptide which accumulates within the brains of patients with Alzheimer's disease (AD) is proteolytically derived from a precursor protein (beta PP). We established and characterized four stably transformed human neuroblastoma cell lines which conditionally expressed a partial beta PP fusion protein (amino-17 residues+carboxyl-99 residues; S beta C). Conditional expression of S beta C was achieved using a tetracycline-responsive promoter system. Expression of this fusion protein in one of the cell lines resulted in pronounced cytotoxicity. Addition of n6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate and/or fetal bovine serum to the culture medium of this cell line further elevated the level of S beta C expression and enhanced the associated cytotoxicity. Conditioned medium, acquired from cells expressing S beta C, was not cytotoxic. These findings suggest that modulation of beta PP expression and/or metabolism can have cytotoxic consequences. This is the first report of cytotoxic effects mediated by conditional expression of a beta PP derivative. This immortal cell line provides a unique opportunity to screen for complementary DNAs which suppress this toxicity. Such cDNAs could help elucidate the processes underlying S beta C mediated cytotoxicity which in turn could further our understanding of the pathogenesis of AD and could also provide additional candidate genes for various forms of familial AD.=?-]Van Meir, E. G. Polverini, P. J. Chazin, V. R. Su Huang, H. J. de Tribolet, N. Cavenee, W. K.1994hRelease of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells171-6 Nat Genet82Animal Brain Neoplasms/*PATHOLOGY Cell Movement Cornea/BLOOD SUPPLY Disease Progression Endothelium, Vascular/PATHOLOGY Female Gene Expression Regulation, Neoplastic Glioblastoma/*PATHOLOGY Human Neoplasm Proteins/*BIOSYNTHESIS/GENETICS/PHYSIOLOGY Neovascularization/PHYSIOPATHOLOGY Protein p53/BIOSYNTHESIS/GENETICS/*PHYSIOLOGY Proteins/*BIOSYNTHESIS/PHARMACOLOGY Rats Rats, Inbred F344 Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tumor Cells, CulturedqThe earliest genetic alteration in human astrocytoma progression is mutation of the p53 tumour suppressor gene, while one of the earliest phenotypic changes is the stimulation of neovascularization. Here, we tested the role of p53 in the angiogenic process by introducing a tetracycline-regulated wild type p53 gene into null glioblastoma cells. The parental cells expressed strong angiogenic activity while upon induction of wild type, but not mutant, p53 expression, the cells secreted a factor able to neutralize the angiogenicity of the factors produced by the parental cells as well as of basic fibroblast growth factor. ?./Bergman, M. Joukov, V. Virtanen, I. Alitalo, K.1995Overexpressed Csk tyrosine kinase is localized in focal adhesions, causes reorganization of alpha v beta 5 integrin, and interferes with HeLa cell spreading711-22 Mol Cell Biol152Cell Adhesion Cell Adhesion Molecules/METABOLISM Cytoskeletal Proteins/METABOLISM Gene Expression Gene Expression Regulation/DRUG EFFECTS Hela Cells Human Insulin Receptor Protein-Tyrosine Kinase/METABOLISM Integrins/*METABOLISM Kinetics Phenotype Phosphoproteins/METABOLISM Protein-Tyrosine Kinase/BIOSYNTHESIS/*METABOLISM Proto-Oncogene Protein pp60(c-src)/METABOLISM Sequence Deletion Support, Non-U.S. Gov't Tetracycline/PHARMACOLOGY TransfectionThe C-terminal Src kinase p50csk phosphorylates Src family tyrosine kinases and down-regulates their activity in vitro. To gain insight into the cellular functions of this potentially antioncogenic enzyme, we have overexpressed the csk cDNA by using an inducible promoter in HeLa cells. Despite some differences in basal Src activity in the clones analyzed, Src activity was not significantly suppressed, while the amount of p50csk and Csk activity increased at least 10-fold during 3 days of induction. Immunofluorescence for the induced p50csk was localized in the cytoplasm and distinctly in focal adhesions, in which the amount of phosphotyrosine containing proteins was also increased. Point and deletion mutagenesis experiments showed that localization in focal adhesions was dependent on the SH2 and SH3 domains of Csk but not on its catalytic activity. Csk formed a complex with the focal adhesion protein paxillin in cells, and its SH2 domain was shown to interact with pp125FAK and paxillin in vitro. After Csk induction, the cells became spherical and more loosely attached to the culture substratum, and the alpha v beta 5 integrin complex (vitronectin receptor) of focal adhesions was redistributed to a novel type of structure consisting of punctate plaques on the ventral cell surface. These phenotypic changes occurred in several clones analyzed and were totally reversible when Csk was switched off, but they did not occur in cells overexpressing the catalytically inactive Csk R-222 mutant or luciferase. Our results thus show that a fraction of cellular Csk is targeted to focal adhesions via its SH2 and SH3 domains, probably interacting with tyrosyl-phosphorylated focal adhesion proteins. They also suggest that Csk is involved in the regulation of integrins controlling cell attachment and shape.?/Passman, R. S. Fishman, G. I.1994ERegulated expression of foreign genes in vivo after germline transfer2421-5 J Clin Invest946 Animal DNA-Binding Proteins/*GENETICS Gene Expression Regulation/DRUG EFFECTS Heart/*GROWTH & DEVELOPMENT Mice Mice, Transgenic Myosin/*GENETICS Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Tissue Distribution Trans-Activators/*GENETICS)Tight transcriptional control of foreign genes introduced into the germline of transgenic mice would be of great experimental value in studies of gene function. To develop a system in which the spatial and temporal expression of candidate genes implicated in cardiac development or function could be tightly controlled in vivo, we have generated transgenic mice expressing a tetracycline-controlled transactivator (tTA) under the control of a rat alpha myosin heavy chain promoter (MHC alpha-tTA mice), as well as mice harboring a candidate target gene implicated in the control of differentiation, Id1 (tet-Id1 mice). No expression of the target transgene was detected in any tissues of hemizygous tet-Id1 mice. Genetic crosses with MHC alpha-tTA mice resulted in transactivation of the Id1 transgene, but expression was restricted to heart, where tTA was expressed. Furthermore, transactivation of the target gene was tightly and reversibly controlled by systemic therapy with tetracycline, both in utero and postnatally. These studies demonstrate the feasibility of such a binary approach for tightly controlling the timing and extent of expression of transgenes in vivo. This approach should be generally useful for the ectopic expression of candidate genes in selected tissues during delineated developmental stages.?0Shan, B. Lee, W. H.1994LDeregulated expression of E2F-1 induces S-phase entry and leads to apoptosis8166-73 Mol Cell Biol1412Animal Apoptosis Cell Cycle Cell Line DNA Damage Gene Expression In Vitro Rats RNA, Messenger/GENETICS S Phase Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Transcription Factors/*METABOLISME2F-1, the first gene product identified among a family of E2F transcription factors, is thought to play a critical role in G1/S progression of the cell cycle. Transcriptional activities of E2F are modulated during the cell cycle, mainly by the formation of complexes between E2F and several key regulators of cell cycle such as the retinoblastoma protein and related proteins. To further understand the roles of E2F in the cell cycle progression, we have overexpressed exogenous E2F-1 by using a tetracycline-controlled expression system. We have found that the induced expression of E2F-1 in Rat-2 fibroblasts promotes S-phase entry and subsequently leads to apoptosis. The apoptosis occurs in an E2F-1 dose-dependent manner. Cells resistant to the induction of apoptosis have lost the ability to express exogenous E2F-1. Cells growing in low serum are more sensitive to the E2F-1-mediated cell death. Overexpression of E2F-1 mutants that impair DNA binding or transactivation does not alter cell cycle progression or induce apoptosis. These results define a novel pathway to apoptosis and demonstrate that premature S-phase entry is associated with apoptotic cell death. R?1/Damke, H. Baba, T. Warnock, D. E. Schmid, S. L.1994RInduction of mutant dynamin specifically blocks endocytic coated vesicle formation915-34 J Cell Biol1274$Animal Base Sequence Blotting, Western Cell Membrane/METABOLISM Clathrin/ANALYSIS/BIOSYNTHESIS Coated Pits, Cell-Membrane/*PHYSIOLOGY Comparative Study Drosophila/GENETICS DNA, Complementary Endocytosis Enzyme Induction Fluorescent Antibody Technique GTP Phosphohydrolase/ANALYSIS/*BIOSYNTHESIS/METABOLISM Hela Cells Human Kinetics Mammals Molecular Sequence Data Mutagenesis Oligoribonucleotides Protein Processing, Post-Translational Receptors, Transferrin/BIOSYNTHESIS/*METABOLISM Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. TransfectionnDynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.?24Buckbinder, L. Talbott, R. Seizinger, B. R. Kley, N.1994ZGene regulation by temperature-sensitive p53 mutants: identification of p53 response genes10640-4Proc Natl Acad Sci U S A9122Apoptosis Base Sequence Binding Sites Blotting, Northern Blotting, Western Cell Line DNA, Neoplasm/METABOLISM Gene Expression Regulation, Neoplastic/DRUG EFFECTS Gene Library Genes, p53 Human Kinetics Luciferase/BIOSYNTHESIS Molecular Sequence Data Mutagenesis Mutation Nuclear Proteins/ISOLATION & PURIFICATION/METABOLISM Osteosarcoma Plasmids Protein p53/GENETICS/*METABOLISM RNA, Messenger/ISOLATION & PURIFICATION/METABOLISM Temperature Tetracycline/PHARMACOLOGY Transcription, Genetic Tumor Cells, Cultured,The ability of the p53 protein to act as a sequence-specific transcriptional activator suggests that genes induced by p53 may encode critical mediators of p53 tumor suppression. Using a tetracycline-regulated p53 expression system and cDNA library subtraction procedure, we identified several p53-induced gene transcripts in human Saos-2 osteosarcoma cells that are novel on the basis of their size, regulation, and low abundance. Wild-type p53-dependent induction of these transcripts was observed in cells that are growth arrested by p53, as well as in cells that undergo apoptosis upon expression of an inducible wild-type p53 transgene. These results show that p53 activates the expression of numerous response genes and suggest that multiple effectors may play a role in mediating cellular functions of p53.?4 Barinaga, M.1994iKnockout mice: round two [news; comment] [published erratum appears in Science 1994 Aug 12;265(5174):855]26-8Science2655168?Animal Antiporters/GENETICS DNA Nucleotidyltransferases/GENETICS/METABOLISM DNA Polymerase I/GENETICS/METABOLISM Gene Deletion Gene Expression Regulation Genetic Engineering/*METHODS Mice Mice, Knockout/*GENETICS Mice, Transgenic Repressor Proteins/GENETICS Stem Cells T-Lymphocytes/ENZYMOLOGY Tetracycline/PHARMACOLOGY ?5,Fishman, G. I. Kaplan, M. L. Buttrick, P. M.19946Tetracycline-regulated cardiac gene expression in vivo1864-8 J Clin Invest934Animal Female Gene Expression Regulation/*DRUG EFFECTS Gene Therapy Myocardium/*METABOLISM Rats Rats, Wistar Support, U.S. Gov't, P.H.S. Tetracycline/*PHARMACOLOGY Trans-Activation (Genetics)Tight regulation of foreign genes expressed in vivo would facilitate studies of many biologic processes and would be useful for gene transfer-based therapies. To test the ability of a tetracycline-regulated gene expression system to function in vivo, we directly injected chimeric tet repressor-VP16 transactivator expression plasmids and luciferase target genes into the hearts of adult rats. Cardiac luciferase activity increased over two orders of magnitude in response to small changes in input tetracycline-controlled transactivator DNA. Transactivation was repressed to background levels by subtherapeutic concentrations of tetracycline in a dose-dependent manner. Target gene expression could be rapidly and reversibly controlled by manipulating antibiotic administration. This system may be particularly useful for in vivo studies of gene function or gene therapies where the timing or extent of expression are critical variables.?6(Haase, S. B. Heinzel, S. S. Calos, M. P.1994ZTranscription inhibits the replication of autonomously replicating plasmids in human cells2516-24 Mol Cell Biol144Actins/GENETICS Cell Line Chromosomes, Human Cytomegalovirus/*GENETICS Dna/biosynthesis DNA Replication/DRUG EFFECTS DNA, Viral/BIOSYNTHESIS Enhancer Elements (Genetics) Genes, Immediate-Early Human Kidney Plasmids/*METABOLISM Promoter Regions (Genetics)/DRUG EFFECTS Restriction Mapping Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Terminator Regions (Genetics) Tetracycline/PHARMACOLOGY Transcription, GeneticThis study addresses the effect of transcription on replication, using a system based on autonomously replicating plasmids in human cells. We added transcriptional elements from the human cytomegalovirus promoter/enhancer and the human beta-actin promoter to autonomously replicating plasmids based on human sequences and found that the transcriptional elements inhibited plasmid replication. Furthermore, conditional inhibition of plasmid replication was demonstrated by using a tetracycline-responsive promoter. We found that replication activity of plasmids carrying this promoter was inversely correlated with promoter activity. Replication activity was partially restored on plasmids when a transcriptional termination sequence was placed directly downstream of the promoter element. Transcriptional activity of the promoters and the efficacy of the terminator sequence were confirmed by using steady-state RNA analysis. These experiments suggest that transcription inhibits DNA replication on these plasmids and that the degree of inhibition is dependent on transcription strength. The possible significance of these results for chromosomal DNA replication are discussed.W?7<Wimmel, A. Lucibello, F. C. Sewing, A. Adolph, S. Muller, R.1994dInducible acceleration of G1 progression through tetracycline-regulated expression of human cyclin E995-7Oncogene93Cyclins/BIOSYNTHESIS/GENETICS/*PHYSIOLOGY Gene Expression Regulation/DRUG EFFECTS G1 Phase Hela Cells Human Support, Non-U.S. Gov't Tetracycline/PHARMACOLOGYCyclin E is a cell cycle-regulated protein that activates the cdc2-related protein kinases cdk2 shortly before S-phase entry. In order to analyse the biological role of cyclin E, we have generated HeLa cells that allow the conditional expression of ectopic human cyclin E. In these cells, a cyclin E cDNA is under the control of a bacterial tetracycline repressor-VP16 activator hybrid protein. In the absence of tetracycline, the endogenous gene becomes activated and leads to the synthesis of elevated levels of cyclin E. Concomitant with this increase in cyclin E expression we show by a combined time-lapse video recording/5-bromo-deoxyuridine labelling procedure a significant acceleration of G1 transition by approximately 1.5 hours. This observation is consistent with the idea that cyclin E is a rate-limiting factor with respect to the control of G1-->S transition. The experimental system described here should also prove useful to address the function of cyclin E in further detail.?8RBoldin, M. P. Varfolomeev, E. E. Pancer, Z. Mett, I. L. Camonis, J. H. Wallach, D.1995vA novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain7795-8 J Biol Chem27014Amino Acid Sequence Animal Antigens, Surface/GENETICS/*METABOLISM Apoptosis/*GENETICS Carrier Proteins/GENETICS/*METABOLISM Cloning, Molecular DNA, Complementary Hela Cells Human Mice Molecular Sequence Data Proteins/*METABOLISM Sequence Homology, Amino Acid Support, Non-U.S. Gov'tSignaling for cell death by Fas/APO1 occurs via a distinct region in its intracellular domain. This region contains a conserved sequence motif, the death domain motif, that is also found in the intracellular domains of the p55 tumor necrosis factor receptor and the low affinity nerve growth factor receptor, as well as in the regulatory domain of the ankyrins. A novel protein that specifically binds to the death domain of Fas/APO1 but not to Fas/APO1 molecules with a loss of function point mutation occurring in lprcg mice was cloned by a two-hybrid screen of a HeLa cells' cDNA library. The cloned protein itself contains a death domain motif, and this region binds to the death domain of Fas/APO1, while the region upstream to the death domain prompts self-association of the protein. Induced expression of the protein results in ligand-independent triggering of cytotoxicity, suggesting that it is involved in cell death induction by Fas/APO1.7?9Lu, K. P. Hunter, T.1995<Evidence for a NIMA-like mitotic pathway in vertebrate cells413-24Cell813BAmino Acid Sequence Animal Aspergillus nidulans/GENETICS Calmodulin-Dependent Protein Kinases Cell Compartmentation Cell Cycle/*PHYSIOLOGY Cell Nucleus/PHYSIOLOGY Chromatin/PHYSIOLOGY Female Gene Expression Regulation G2 Phase/PHYSIOLOGY Hela Cells Human Mitosis/PHYSIOLOGY Molecular Sequence Data Mutation Oocytes/CYTOLOGY Protein p34cdc2/METABOLISM Protein-Serine-Threonine Kinases/GENETICS/*METABOLISM Proto-Oncogene Proteins c-mos Recombinant Proteins/METABOLISM Species Specificity Structure-Activity Relationship Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. XenopusnNIMA is essential for entry into mitosis in Aspergillus nidulans. To examine whether there is a NIMA-like pathway in other eukaryotic cell cycles, we expressed NIMA and its dominant negative mutants in two different eukaryotic systems. In Xenopus oocytes, NIMA induced germinal vesicle breakdown without activating Mos, CDC2, or MAP kinase. In HeLa cells, NIMA induced premature mitotic events without activating CDC2, whereas the mutants caused a specific G2 arrest but did not block mutant CDC2T14AY15F-induced premature entry into mitosis. A sequence essential for both these phenotypes was mapped to a region of approximately 100 amino acids lying just after the catalytic domain of NIMA that shows a significant similarity to protein interaction domains in other proteins. These results provide evidence for the existence of a NIMA-like mitotic pathway in vertebrate cells.?:5Damke, H. Baba, T. van der Bliek, A. M. Schmid, S. L.1995mClathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin69-80 J Cell Biol1311Clathrin/*PHYSIOLOGY Coated Pits, Cell-Membrane/PHYSIOLOGY Endocytosis/PHYSIOLOGY GTP Phosphohydrolase/*GENETICS Hela Cells/CYTOLOGY/PHYSIOLOGY Human Microtubules/*GENETICS Pinocytosis/*PHYSIOLOGY Point Mutation/*PHYSIOLOGY Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. TemperatureA stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.?;&Dirks, R. W. Daniel, K. C. Raap, A. K.1995URNAs radiate from gene to cytoplasm as revealed by fluorescence in situ hybridization2565-72 J Cell Sci108Pt 7)Animal Cell Line Cell Nucleus/METABOLISM/ULTRASTRUCTURE Cytomegalovirus/*GENETICS/METABOLISM Gene Expression Genes, Immediate-Early Genes, Viral Herpesvirus 4, Human/*GENETICS/METABOLISM Human In Situ Hybridization, Fluorescence/METHODS Luciferase/BIOSYNTHESIS Rats Recombinant Proteins/BIOSYNTHESIS RNA, Messenger/ANALYSIS/*BIOSYNTHESIS Transcription, Genetic Viral Proteins/ANALYSIS/*BIOSYNTHESISBGenes for Epstein-Barr virus, human cytomegalovirus immediate early antigen and luciferase are abundantly transcribed in Namalwa, rat 9G and X1 cells, respectively. The EBV transcripts and HCMV-IE transcripts are extensively spliced, while in the luciferase transcript only a small intron sequence has to be spliced out. EBV transcripts are hardly localized in the cytoplasm while the luciferase and HCMV-IE transcripts are present in the cytoplasm and translated into proteins. We have correlated these characteristics with nuclear RNA distribution patterns as seen by fluorescence in situ hybridization. Transcripts of the HCMV-IE transcription unit were shown to be present in a main nuclear signal in the form of a track or elongated dot and as small nuclear RNA signals that radiate from this site towards the cytoplasm. A similar distribution pattern of small RNA signals was observed for transcripts of the luciferase gene, whereas the main nuclear signal was always observed as a dot and never as a track or elongated dot. In Namalwa cells, EBV transcripts were only present as track-like signals. The results suggest that when the extent for splicing is high, unspliced or partially spliced mRNAs begin to occupy elongated dot or track-like domains in the vicinity of the gene. When the extent of splicing is low, splicing is completed co-transcriptionally, leading to a bright dot-like signal. The presence of small nuclear spots in addition to the main signal correlates with cytoplasmic mRNA expression. The small spots most likely represent, therefore, mRNAs in transport to the cytoplasm.?<Resnitzky, D. Reed, S. I.1995LDifferent roles for cyclins D1 and E in regulation of the G1-to-S transition3463-9 Mol Cell Biol157{Animal Clone Cells Cyclins/GENETICS/*METABOLISM Fibroblasts/CYTOLOGY Gene Expression Regulation G1 Phase/*PHYSIOLOGY Immunoblotting Models, Biological Oncogene Proteins/GENETICS/*METABOLISM Phosphorylation Precipitin Tests Rats Recombinant Proteins/METABOLISM Retinoblastoma Protein/*METABOLISM S Phase/*PHYSIOLOGY Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Time FactorsEctopic expression of cyclins D1 and E was previously shown to accelerate the G1/S-phase transition, indicating that both classes of G1 cyclin control an event(s) that is rate limiting for entry into S phase. In order to determine whether cyclins D1 and E control the same or two different rate-limiting events, we have created cell lines that express both cyclins in an inducible manner. We show here that ectopic expression of both cyclins E and D1 in the same cell has an additive effect on shortening of the G1 interval relative to expression of any single cyclin. In order to further explore the molecular basis for G1 cyclin action, we used cell lines capable of expressing cyclin D1, E, or both prematurely and measured the effect of cyclin expression in early G1 on phosphorylation of the retinoblastoma susceptibility gene product (pRb). We show here that while premature expression of either cyclin alone advances the G1/S-phase transition to the same extent, premature expression of cyclin D1 leads to immediate appearance of hyperphosphorylated pRb, while premature expression of cyclin E does not. Ectopic expression of both cyclins E and D1 in the same cell has an additive effect on shortening of the G1 interval, while the effect on pRb phosphorylation is similar to the effect of cyclin D1 alone. These results suggest that cyclins E and D1 control two different events, both rate limiting for the G1/S-phase transition, and that pRb phosphorylation might be the rate-limiting event controlled by cyclin D1.?>CSternsdorf, T. Guldner, H. H. Szostecki, C. Grotzinger, T. Will, H.1995{Two nuclear dot-associated proteins, PML and Sp100, are often co-autoimmunogenic in patients with primary biliary cirrhosis257-68Scand J Immunol422Autoantibodies/BLOOD/*IMMUNOLOGY Cell Line, Transformed Escherichia coli/GENETICS/METABOLISM Gene Transfer Human Liver Cirrhosis, Biliary/*IMMUNOLOGY Nuclear Proteins/IMMUNOLOGY Support, Non-U.S. Gov't Transcription Factors/GENETICS/*IMMUNOLOGYThe nucleoproteins Sp100 and PML, the first an autoantigen predominant in patients with primary biliary cirrhosis (PBC) and the second a transformation and cell growth suppressing protein aberrantly expressed in promyelocytic leukaemia cells, were recently shown to colocalize in dot-like nuclear domains. Here we analysed whether PML, like Sp100, is also an autoantigen in patients with PBC and other autoimmune diseases, and wether both proteins interact directly. Testing sera from autoimmune patients using an immunoprecipitation assay with radiolabelled PML and an immunofluorescence assay based on a cell line overexpressing PML, autoantibodies (Aabs) against PML were found in the majority o anti-Sp100 Aab positive patients. Only very few patients with PBC or other autoimmune diseases contained anti-PML or anti-Sp100 Aabs exclusively. In contrast to Sp100, immunoreactivity of recombinant PML in immunoblots was only weak and was directed to one region. This suggests that anti-PML Aabs recognize fewer and preferentially conformation-dependent epitopes. In an immunoprecipitation assay using in vitro synthesized Sp100 and PML proteins and Abs to recombinant proteins, no direct interaction was observed. Taken together, these data indicate that Aabs against PML are as highly prevalent and specific for patients with PBC as those against Sp100. The colocalization of these autoantigens and the frequent co-occurrence of the corresponding Aabs might reflect an association of both proteins mediated by one or several other proteins.>?@5Reynisdottir, I. Polyak, K. Iavarone, A. Massague, J.1995]Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta1831-45 Genes Dev915Animal Carrier Proteins/METABOLISM Cell Cycle/DRUG EFFECTS/*PHYSIOLOGY Cells, Cultured Cyclin-Dependent Kinases/*ANTAGONISTS & INHIBITORS/*METABOLISM Cyclins/METABOLISM Epithelium/CYTOLOGY/GROWTH & DEVELOPMENT Human Microtubule-Associated Proteins/METABOLISM Mink Models, Biological Phosphorylation Protein Binding Protein-Serine-Threonine Kinases/METABOLISM Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Time Factors Transforming Growth Factor beta/*PHARMACOLOGYG1 progression in mammalian cells requires the activity of the cyclin D-dependent kinases Cdk4 and/or Cdk6 and the cyclin E-dependent kinase Cdk2. Proliferating Mv1Lu mink lung epithelial cells and human keratinocytes contain high levels of the universal Cdk inhibitor p27Kip1 distributed in complexes with Cdk2, Cdk4, and Cdk6. Addition of the antimitogenic cytokine transforming growth factor-beta (TGF-beta) elevates expression of the Cdk4/6-specific inhibitor p15Ink4B and induces the release of p27 from Cdk4 and Cdk6. In Mv1Lu cells, this release of p27 coincides with increased binding of p27 to Cdk2. Recombinant p15 inhibits p27 binding to Cdk4 in vitro, and p15 overexpression induces the transfer of p27 from Cdk4 to Cdk2 in vivo, suggesting that the release of Cdk4-bound p27 in TGF-beta-treated cells is caused by the surge in p15 levels. In keratinocytes, TGF-beta increases not only p15 but also p21Cip1, which binds to Cdk2. These events correlate with Cdk2 inhibition and cell cycle arrest and occur without a loss of G1 Cdk components. The results suggest that TGF-beta induces G1 arrest in these two epithelial cell types by inhibiting various cyclin-Cdk kinases through the cooperative action of an Ink4 Cdk inhibitor and a Cip/Kip Cdk inhibitor. Subsequent to cell cycle arrest, Cdk2 and Cdk4 levels decline as part of a second set of events that may represent a program of cell adaptation to the quiescent state.P?A@Maheswaran, S. Englert, C. Bennett, P. Heinrich, G. Haber, D. A.1995GThe WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis2143-56 Genes Dev917Animal Apoptosis Base Sequence Cell Line Cell Line, Transformed Dna/metabolism DNA-Binding Proteins/GENETICS/METABOLISM/*PHYSIOLOGY Genes, p53 Genes, Wilms' Tumor Human Molecular Sequence Data Nephroblastoma/METABOLISM Oncogene Proteins, Viral/PHYSIOLOGY Protein p53/GENETICS/METABOLISM/*PHYSIOLOGY Rats Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Trans-Activation (Genetics) Transcription Factors/GENETICS/METABOLISM/*PHYSIOLOGY Transcription, Genetic Transfection Tumor Cells, Cultured Zinc FingersThe Wilms' tumor-suppressor gene product WT1 coimmunoprecipitates with p53 from baby rat kidney (BRK) cells and Wilms' tumor specimens, and expression of WT1 in BRK cells is associated with increased levels of endogenous wild-type p53 protein. To study the effect of WT1 on p53 function, we cotransfected expression constructs into Saos-2 cells, an osteosarcoma cell line without endogenous expression of either gene. Expression of WT1 resulted in increased steady-state levels of p53, attributable to a prolongation in protein half-life, and associated with protection against papillomavirus E6-mediated degradation of p53. This effect mapped to zinc fingers 1 and 2 of WT1 and was not observed with the closely related EGR1 protein. The stabilized p53 demonstrated enhanced binding to its target DNA sequence and increased trans-activation of a promoter containing this RGC site, but reduced transcriptional repression of a TATA-containing promoter lacking this site. Expression of WT1 inhibited p53-mediated apoptosis triggered by UV irradiation or by expression of temperature-sensitive p53 in the wild-type conformation, but did not affect p53-mediated cell cycle arrest. We conclude that WT1 protein can stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis. This effect may contribute to the elevated levels of wild-type p53 protein that are observed in Wilms' tumors.?C>Schaack, J. Guo, X. Ho, W. Y. Karlok, M. Chen, C. Ornelles, D.1995OAdenovirus type 5 precursor terminal protein-expressing 293 and HeLa cell lines4079-85J Virol697Adenoviruses, Human/*METABOLISM Base Sequence Cell Division Cell Line Cytopathogenic Effect, Viral Hela Cells Human Molecular Sequence Data Phosphoproteins/*BIOSYNTHESIS/GENETICS Protein Precursors/*BIOSYNTHESIS/GENETICS Support, U.S. Gov't, P.H.S. Transfection HeLa and 293 cell lines that express biologically active adenovirus type 5 precursor terminal protein (pTP) have been made. The amount of pTP synthesized in these cell lines ranges from barely detectable to greater than that observed in cells infected with the wild-type virus. The pTP-expressing cell lines permit the growth of a temperature-sensitive terminal protein mutant virus sub100r at the nonpermissive temperature. A higher percentage of the stably transfected 293 cell lines expressed terminal protein, and generally at considerably higher levels, than did the HeLa cell lines. While 293 cells appeared to tolerate pTP better than did HeLa cells, high-level pTP expression in 293 cells led to a significantly reduced growth rate. The 293-pTP cell lines produce infectious virus after transfection with purified viral DNA and form plaques when overlaid with Noble agar after infection at low multiplicity. These cell lines offer promise for the production of adenoviruses lacking pTP expression and therefore completely defective for replication.?DdBoldin, M. P. Mett, I. L. Varfolomeev, E. E. Chumakov, I. Shemer-Avni, Y. Camonis, J. H. Wallach, D.1995Self-association of the "death domains" of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects387-91 J Biol Chem2701Antigens, Surface/*METABOLISM Apoptosis Cell Survival Hela Cells Human Interleukin-8/GENETICS Receptors, Tumor Necrosis Factor/*METABOLISM Signal Transduction Support, Non-U.S. Gov't Transcription, GeneticSignaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H.J. and Mett, I. (1994) Cytokine 6, 556; Song, H.Y., Dunbar, J.D., and Bonner, D.B. (1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell death, as well as for some other effects (the "death domain", specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.?EgSchulze, A. Zerfass, K. Spitkovsky, D. Middendorp, S. Berges, J. Helin, K. Jansen-Durr, P. Henglein, B.1995UCell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site11264-8Proc Natl Acad Sci U S A9224Animal Base Sequence Carrier Proteins/PHYSIOLOGY Cell Cycle Cyclin-Dependent Kinases/PHYSIOLOGY Cyclins/*GENETICS/*PHYSIOLOGY DNA-Binding Proteins/METABOLISM Gene Expression Mice Molecular Sequence Data Oligodeoxyribonucleotides/CHEMISTRY Oncogene Proteins/*PHYSIOLOGY Promoter Regions (Genetics) RNA, Messenger/GENETICS Support, Non-U.S. Gov't Trans-Activation (Genetics) Transcription Factors/*METABOLISM Transcription, Genetic 3T3 CellsCyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.?H<Damke, H. Gossen, M. Freundlieb, S. Bujard, H. Schmid, S. L.1995rTightly regulated and inducible expression of dominant interfering dynamin mutant in stably transformed HeLa cells209-20Methods Enzymol257Animal Cell Line, Transformed Cell Survival/DRUG EFFECTS Cloning, Molecular/METHODS Endocytosis Escherichia coli Gene Expression/DRUG EFFECTS Gene Expression Regulation, Neoplastic GTP Phosphohydrolase/*BIOSYNTHESIS Hela Cells Human Kinetics Mammals Mutagenesis Receptors, Transferrin/BIOSYNTHESIS/METABOLISM Recombinant Proteins/BIOSYNTHESIS Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Transferrin/METABOLISM?U;Orimoto, K. Tsuchiya, H. Kobayashi, T. Matsuda, T. Hino, O.1996iSuppression of the neoplastic phenotype by replacement of the Tsc2 gene in Eker rat renal carcinoma cells70-5Biochem Biophys Res Commun2191Animal Blotting, Northern Blotting, Southern Cell Cycle Cell Division Chloramphenicol Acetyltransferase/BIOSYNTHESIS Genes, Dominant Genes, Suppressor, Tumor Kidney Neoplasms/*GENETICS Kinetics Mutation Phenotype Rats Rats, Mutant Strains Recombinant Proteins/BIOSYNTHESIS Repressor Proteins/*BIOSYNTHESIS/*GENETICS Support, Non-U.S. Gov't Transfection Tuberous Sclerosis/GENETICS Tumor Cells, Cultured$The hereditary renal carcinoma (RC) in the rat, originally reported by R. Eker in 1954, is an excellent example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. Recently, we have identified a germline mutation of the sclerosis (Tsc2) gene in the Eker rat (Nature Genetics 9, 70-74, 1995), suggested to be a novel tumor suppressor gene, fitting Knudson's two-hit hypothesis. In this study, the effect of wild-type Tsc2 gene expression in Eker RC cells was tested using a tetracycline-responsive promoter system. Transfection and expression of a exogenous Tsc2 gene affected both cell morphology and growth rate. This demonstration of suppression of the neoplastic phenotype provides direct evidence for an essential role of the Tsc2 gene in tumorigenesis.I?V,Zeidler, M. Gatz, C. Hartmann, E. Hughes, J.1996QTetracycline-regulated reporter gene expression in the moss Physcomitrella patens199-205Plant Mol Biol301@Antibiotics, Tetracycline/*PHARMACOLOGY Chimeric Proteins/BIOSYNTHESIS Gene Expression Regulation, Plant/*GENETICS Genes, Reporter Glucuronidase/*BIOSYNTHESIS/GENETICS Models, Genetic Mosses/*GENETICS Plants, Transgenic Repressor Proteins/GENETICS Simplexvirus/GENETICS Tetracycline/*PHARMACOLOGY Viral Proteins/GENETICSJAs ancestors of higher plants, mosses offer advantages as simple model organisms in studying complex processes such as development and signal transduction. Overexpression of transgenes after genetic transformation is a powerful technique in such studies. To establish a controllable expression system for this experimental approach we expressed a chimeric protein consisting of the Tn1O-encoded Tet repressor and the activation domain of Herpes simplex virion protein 16 in the moss Physcomitrella patens. We showed that this protein activates transcription from a suitable target promoter (Top 1O) containing seven operators upstream of a TATA box. In media containing very low levels of tetracycline (1 mg/l), expression levels of a beta-glucuronidase (GUS) reporter gene dropped to <1% of that in the absence of tetracycline. This regulation is due to interference of tetracycline with the DNA binding activity of the Tet repressor portion of the chimeric transcriptional activator. Stable transformants grown for three weeks on tetracycline-containing media showed negligible GUS activity, whereas GUS was expressed strongly within 24 h of transfer to tetracycline-free media. Potent and stringently regulated expression of other, physiologically active genes is thus readily available in the moss system using the convenient ToplO expression system.?WGil, P. Green, P. J.1996Multiple regions of the Arabidopsis SAUR-AC1 gene control transcript abundance: the 3' untranslated region functions as an mRNA instability determinant1678-86Embo J157qArabidopsis/*GENETICS/METABOLISM Auxins/PHARMACOLOGY Drug Stability Gene Expression Regulation, Plant/DRUG EFFECTS Genes, Plant Genes, Reporter Plant Proteins/*GENETICS Plants, Transgenic Promoter Regions (Genetics) RNA, Messenger/*GENETICS/*METABOLISM RNA, Plant/*GENETICS/*METABOLISM Support, Non-U.S. Gov't Support, U.S. Gov't, Non-P.H.S. Tobacco/GENETICS/METABOLISMThe small-auxin-up-RNA (SAUR) transcripts are rapidly induced by auxin and are among the most short-lived mRNAs in higher plants. In this study, we investigate the regulation of SAUR-AC1, a well characterized SAUR gene of Arabidopsis. Be examining the expression of chimeric genes in transgenic tobacco, we demonstrate that the promoter region of SAUR-AC1 mediates auxin induction. Sequences downstream of the promoter region were found to limit mRNA accumulation in a manner that was independent of auxin treatment. Both the coding region and the 3' untranslated region (UTR) of SAUR-AC1 independently contribute to this limitation. Effects on mRNA stability were assayed using chimeric genes under the control of the tetracycline-repressible Top10 promoter. mRNA half-life analysis following tetracycline treatment showed that the SAUR-AC1 coding region does not contain elements that decrease mRNA stability. In contrast, the 3' UTR was found to act as a potent mRNA instability determinant. This finding and the general utility of the Top10 system should provide the means to elucidate mRNA decay pathways that are potentially novel and specific for certain unstable transcripts.?XYin, D. X. Schimke, R. T.1996Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment3394-8Proc Natl Acad Sci U S A9386Aphidicolin/PHARMACOLOGY Apoptosis/DRUG EFFECTS/*GENETICS Cell Cycle/DRUG EFFECTS/GENETICS Gene Amplification/DRUG EFFECTS Gene Expression/DRUG EFFECTS Hela Cells Human Models, Genetic Proto-Oncogene Proteins/*GENETICS Tetracycline/PHARMACOLOGY Tetrahydrofolate Dehydrogenase/GENETICS Trimetrexate/PHARMACOLOGYTo study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation. ?Z7Lukas, J. Petersen, B. O. Holm, K. Bartek, J. Helin, K.1996uDeregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression1047-57 Mol Cell Biol163Animal Carrier Proteins/GENETICS/*METABOLISM Cell Cycle/GENETICS Cell Line Fibroblasts/CYTOLOGY/METABOLISM Gene Expression Regulation Rats S Phase Support, Non-U.S. Gov't Transcription Factors/GENETICS/*METABOLISMThe E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated ED2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rate fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody to cyclin D1. The p16INK4-induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S-phase entry, and overcomes a p16-mediated G1 block, and since E12F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p126-cyclin D-pRB pathway. Furthermore, our date suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.?\`Zwijsen, R. M. Klompmaker, R. Wientjens, E. B. Kristel, P. M. van der Burg, B. Michalides, R. J.1996XCyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit2554-60 Mol Cell Biol166Breast Neoplasms/*PATHOLOGY Cell Cycle/PHYSIOLOGY Cell Division/PHYSIOLOGY Culture Media Cyclins/GENETICS/*PHYSIOLOGY Female Gene Expression/DRUG EFFECTS Human Kinetics Oncogene Proteins/GENETICS/*PHYSIOLOGY Phosphorylation Retinoblastoma Protein/METABOLISM Support, Non-U.S. Gov't Tetracycline/PHARMACOLOGY Trans-Activation (Genetics)/DRUG EFFECTS Transfection Tumor Cells, CulturedpCyclin D1 controls G1-associated processes, including G0-to-G1 and G1-to-S transitions. This study demonstrates a novel aspect of cyclin D1 as a regulator of the transition between G1 and G0. Overexpression of cyclin D1 in MCF7 breast tumor cells resulted in a continued proliferation under low-serum conditions, whereas nonoverexpressing cells ceased to grow. This difference in growth was due to a reduced exit from G1 to G0 in cyclin D1-overexpressing cells. Our data therefore suggest a model in which cyclin D1 overexpression in tumor cells is responsible for hyperproliferation under growth factor-deprived conditions. ?^$Hofmann, A. Nolan, G. P. Blau, H. M.1996lRapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette [see comments]5185-90Proc Natl Acad Sci U S A9311Sbeta-Galactosidase/*BIOSYNTHESIS Animal Cells, Cultured Cytomegalovirus/DRUG EFFECTS/*GENETICS Enhancer Elements (Genetics) Flow Cytometry Gene Expression Regulation/*DRUG EFFECTS Herpes Simplex Virus Protein Vmw65/BIOSYNTHESIS/METABOLISM Human Kinetics Mice Mice, Inbred C57BL Moloney Leukemia Virus Muscles Promoter Regions (Genetics) Recombinant Fusion Proteins/BIOSYNTHESIS/METABOLISM Repetitive Sequences, Nucleic Acid Repressor Proteins/BIOSYNTHESIS/*METABOLISM Retroviridae Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/*PHARMACOLOGY Transcription, Genetic TransfectionWe describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.?_Shockett, P. E. Schatz, D. G.1996VDiverse strategies for tetracycline-regulated inducible gene expression [see comments]5173-6Proc Natl Acad Sci U S A9311beta-Galactosidase/BIOSYNTHESIS Animal Cytomegalovirus/DRUG EFFECTS/ENZYMOLOGY/GENETICS Escherichia coli/DRUG EFFECTS/GENETICS Gene Expression Regulation/*DRUG EFFECTS Human Repressor Proteins/BIOSYNTHESIS/METABOLISM Tetracycline/*PHARMACOLOGY ?arBargou, R. C. Wagener, C. Bommert, K. Arnold, W. Daniel, P. T. Mapara, M. Y. Grinstein, E. Royer, H. D. Dorken, B.1996Blocking the transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in SCID mice1205-13 J Exp Med1833=Animal Apoptosis Base Sequence Breast Neoplasms/*GENETICS/*PATHOLOGY Cell Cycle Cell Division Cell Line Chloramphenicol Acetyltransferase/BIOSYNTHESIS DNA Primers DNA Replication Female Fibroblasts Gene Expression Regulation Genes, myc Genes, Dominant Human Kinetics Mice Molecular Sequence Data Neoplasm Transplantation Polymerase Chain Reaction Promoter Regions (Genetics) Proto-Oncogene Proteins c-myc/BIOSYNTHESIS Recombinant Proteins/BIOSYNTHESIS RNA, Messenger/ANALYSIS/BIOSYNTHESIS Transcription Factors/ANTAGONISTS & INHIBITORS/BIOSYNTHESIS/*METABOLISM TransfectionThe transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.m?b,Hoshimaru, M. Ray, J. Sah, D. W. Gage, F. H.1996Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene1518-23Proc Natl Acad Sci U S A934Animal Base Sequence Cell Cycle Cell Differentiation/DRUG EFFECTS Cell Line, Transformed Cytomegalovirus/GENETICS Fibroblast Growth Factor, Basic/PHARMACOLOGY Gene Expression Regulation/DRUG EFFECTS Genes, myc Genes, Synthetic Genes, Viral Genetic Vectors Hippocampus/CYTOLOGY Molecular Sequence Data Moloney Sarcoma Virus/GENETICS Neurites/ULTRASTRUCTURE Neurons/*CYTOLOGY Oncogene Protein p55(v-myc)/BIOSYNTHESIS/PHYSIOLOGY Promoter Regions (Genetics) Rats Recombinant Fusion Proteins/BIOSYNTHESIS Repetitive Sequences, Nucleic Acid Simplexvirus/GENETICS Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Trans-Activation (Genetics)A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.d?c#Beauparlant, P. Lin, R. Hiscott, J.1996]The role of the C-terminal domain of I kappa B alpha in protein degradation and stabilization10690-6 J Biol Chem27118)Animal Cell Line Dna/metabolism DNA-Binding Proteins/CHEMISTRY/*METABOLISM Gene Expression Regulation/DRUG EFFECTS Human Hydrolysis Mice NF-kappa B/ANTAGONISTS & INHIBITORS/METABOLISM Protease Inhibitors/PHARMACOLOGY Proteins/*METABOLISM Support, Non-U.S. Gov't Tetracycline/PHARMACOLOGY 3T3 CellszIn the present study, the role of the I kappa B alpha C terminus in NF-kappa B/I kappa B alpha regulation was examined in NIH 3T3 cells engineered to inducibly express wild type or mutated human I kappa B alpha proteins under the control of the tetracycline responsive promoter. Deletion studies demonstrated that the last C-terminal 30 amino acids (amino acids (aa) 288 to aa 317, deleted in I kappa B alpha delta 3), including most of the PEST domain, were dispensable for I kappa B alpha function. However, deletions from aa 261 to 317 or aa 269 to 317 (I kappa B alpha delta 1 and I kappa B alpha delta 2 respectively), lacked the ability to dissociate NF-kappa B/DNA complexes in vitro and were unable to inhibit NF-kappa B dependent transcription. Moreover, I kappa B alpha delta 1 and I kappa B alpha delta 2 mutants were resistant to inducer-mediated degradation. Analysis of I kappa B alpha deletions in the presence of protein synthesis inhibitors revealed that, independently of stimulation, I kappa B alpha delta 1 and I kappa B alpha delta 2 had a half-life four times shorter than wild type I kappa B alpha and the interaction of I kappa B alpha delta 1 and I kappa B alpha delta 2 with p65 was dramatically decreased in vivo as measured by co-immunoprecipitation. Interestingly, protease inhibitors which blocked inducer-mediated degradation of I kappa B alpha also stabilized the turnover of I kappa B alpha delta 1 and I kappa B alpha delta 2. Based on these studies, we propose that in the absence of stimulation, the C-terminal domain between aa 269 and 287 may play a role to protect I kappa B alpha from a constitutive protease activity. ?d:Lin, R. Beauparlant, P. Makris, C. Meloche, S. Hiscott, J.1996uPhosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability1401-9 Mol Cell Biol164Base Sequence Binding Sites DNA-Binding Proteins/GENETICS/*METABOLISM Enzyme Stability Human Immunoblotting Molecular Sequence Data NF-kappa B/ANTAGONISTS & INHIBITORS/GENETICS/*METABOLISM Phosphorylation Protein-Serine-Threonine Kinases/GENETICS/*METABOLISM Proto-Oncogene Proteins/GENETICS/METABOLISM Recombinant Fusion Proteins/GENETICS/METABOLISM Sequence Deletion Sequence Homology Support, Non-U.S. Gov'tThe NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.o?fBargou, R. C. Wagener, C. Bommert, K. Mapara, M. Y. Daniel, P. T. Arnold, W. Dietel, M. Guski, H. Feller, A. Royer, H. D. Dorken, B.1996Overexpression of the death-promoting gene bax-alpha which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice [see comments]2651-9 J Clin Invest9711Animal Antigens, CD95/PHYSIOLOGY Apoptosis Base Sequence Breast/METABOLISM Breast Neoplasms/*GENETICS/*PATHOLOGY Cell Division Cell Line Comparative Study DNA Primers DNA, Neoplasm/BIOSYNTHESIS Epithelium/METABOLISM Female Gene Expression Regulation, Neoplastic Human Mice Mice, SCID Molecular Sequence Data Polymerase Chain Reaction Proto-Oncogene Proteins/*BIOSYNTHESIS Recombinant Proteins/BIOSYNTHESIS Reference Values RNA, Messenger/ANALYSIS/BIOSYNTHESIS Transcription, Genetic Transfection Tumor Cells, CulturedWe have studied the expression of members of the bcl-2 family in human breast cancer. The expression pattern of these genes in breast cancer tissue samples was compared with the expression pattern in normal breast epithelium. No marked difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and cancer tissue. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined so far. Of interest, downregulation of bax-alpha was found in different histological subtypes. Furthermore, we transfected bax-alpha into breast cancer cell lines under the control of a tetracycline-dependent expression system. We were able to demonstrate for the first time that induction of bax expression in breast cancer cell lines restores sensitivity towards both serum starvation and APO-I/Fas-triggered apoptosis and significantly reduces tumor growth in SCID mice. Therefore, we propose that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the bcl-2 gene family.?hYue, Z. Shatkin, A. J.1996xRegulated, stable expression and nuclear presence of retrovirus double-stranded RNA-binding protein sigma3 in HeLa cells3497-501J Virol706Amino Acid Sequence Cell Nucleus/*CHEMISTRY Hela Cells Human Molecular Sequence Data Reoviridae/*CHEMISTRY/PHYSIOLOGY RNA-Binding Proteins/*ANALYSIS RNA, Double-Stranded/*METABOLISM Viral Proteins/*ANALYSIS/PHYSIOLOGYuReovirus genome segment S4 codes for polypeptide sigma3, a major outer capsid component of virions and a double-stranded RNA (dsRNA)-binding protein implicated in viral cytopathogenesis. We have constructed a stable HeLa cell line (S4tTA) that produces functional sigma3 under tetracycline transactivator control. In the absence of tetracycline, S4tTA cells synthesized stable dsRNA-binding sigma3 that accumulated in the nucleus as well as in the cytoplasm. However, in induced S4tTA cells also expressing reovirus outer shell polypeptide mu1/mu1C, migration of sigma3 into the nucleus was blocked, probably as a result of formation of a complex with mu1/mu1C which was exclusively in the cytoplasm. Mutant analyses indicated a correlation between dsRNA-binding activity and nuclear entry of sigma3, suggesting an additional role(s) for this capsid protein in virus-cell interactions.+?iLanger, S. J. Schaack, J.1996a293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein172-9Virology2211UAdenoviruses, Human/*GROWTH & DEVELOPMENT Base Sequence Cell Line, Transformed Cell Separation DNA Primers Gene Expression Human Molecular Sequence Data Phosphoproteins/*BIOSYNTHESIS/GENETICS Protein Precursors/*BIOSYNTHESIS/GENETICS Recombinant Proteins/BIOSYNTHESIS/GENETICS Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Transfection!293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein (pTP) under the control of a tetracycline-dependent promoter were constructed. To construct the cell lines expressing pTP, 293 cells were stably transfected with a plasmid encoding the tetracycline repressor/VP16 transactivator protein (tTA) using selection with hygromycin. Cell lines that expressed high levels of tTA activity were then stably transfected with plasmids in which pTP expression is directed by the tTA-dependent promoter from either a cDNA or a modified genomic construct using selection with G418. Cell lines that expressed high, inducible levels of pTP efficiently complemented a temperature-sensitive pTP mutant virus for growth and plaque formation at the nonpermissive temperature.?j6Yu, H. Rabson, A. B. Kaul, M. Ron, Y. Dougherty, J. P.1996BInducible human immunodeficiency virus type 1 packaging cell lines4530-7J Virol707Cell Line/*VIROLOGY Cell Line, Transformed Cell Transformation, Viral Defective Viruses/GENETICS/PHYSIOLOGY Gene Expression Regulation, Viral Gene Products, env/GENETICS Genetic Vectors Hela Cells Human Hiv-1/genetics/*physiology Support, U.S. Gov't, P.H.S. Virus ReplicationPackaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. ?khKnaus, P. I. Lindemann, D. DeCoteau, J. F. Perlman, R. Yankelev, H. Hille, M. Kadin, M. E. Lodish, H. F.1996A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma3480-9 Mol Cell Biol167OAmino Acid Sequence Animal Carcinoma, Hepatocellular Cell Division/DRUG EFFECTS Cell Line Cercopithecus aethiops Genes, Dominant Human Liver Neoplasms Lymphoma, T-Cell, Cutaneous/*GENETICS/PATHOLOGY Molecular Sequence Data Point Mutation Receptors, Transforming Growth Factor beta/*BIOSYNTHESIS/CHEMISTRY/*GENETICS Recombinant Proteins/BIOSYNTHESIS/CHEMISTRY Sequence Homology, Amino Acid Signal Transduction Skin/PATHOLOGY Skin Neoplasms/*GENETICS/PATHOLOGY Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Transfection Transforming Growth Factor beta/*PHARMACOLOGY Tumor Cells, Cultured5In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.*?l-Iida, A. Chen, S. T. Friedmann, T. Yee, J. K.1996eInducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system6054-9J Virol709ICell Line Chloramphenicol Acetyltransferase/BIOSYNTHESIS Estrogens/PHARMACOLOGY Fibrosarcoma Gene Expression Regulation, Viral/DRUG EFFECTS Genetic Vectors Human Kidney Moloney Leukemia Virus/GENETICS Promoter Regions (Genetics) Receptors, Estrogen/BIOSYNTHESIS Recombinant Fusion Proteins/BIOSYNTHESIS Retroviridae Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Tetracycline/*PHARMACOLOGY Tetracycline Resistance/*GENETICS Trans-Activators/METABOLISM Transfection Tumor Cells, Cultured Vesicular Stomatitis-Indiana Virus/*GENETICS/METABOLISM Viral Envelope Proteins/*BIOSYNTHESISThe ability to regulate gene expression via exogenous stimuli will facilitate the study of gene functions in mammalian cells. In the present study, we modified the tetracycline-controlled inducible system by the addition of the ligand-binding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator. A single retroviral vector can transduce both the transactivator gene and the gene of interest controlled by the tTA-inducible promoter into mammalian cells. We show that cell lines expressing the transactivator can readily be established and that expression of the gene of interest depends on the removal of tetracycline and the addition of estrogen. By using this system, cell lines with inducible expression of the G protein of vesicular stomatitis virus, a potentially toxic gene product, were established. The combination of a powerful inducible system and retrovirus-mediated gene transfer can not only be used to study gene function but may also be applied in the future to clinical trials in human gene therapy.?m(Zhang, Q. X. Davis, I. D. Baldwin, G. S.1996Controlled overexpression of selected domains of the P85 subunit of phosphatidylinositol 3-kinase reverts v-Ha-Ras transformation207-14Biochim Biophys Acta13123src Homology Domains Animal Base Sequence Binding Sites Cell Division/DRUG EFFECTS Cell Transformation, Neoplastic DNA Primers Gene Expression Genes, ras Macromolecular Systems Mice Molecular Sequence Data Oncogene Protein p21(ras)/BIOSYNTHESIS Peptide Fragments/*BIOSYNTHESIS Phosphotransferases (Alcohol Group Acceptor)/*BIOSYNTHESIS Platelet-Derived Growth Factor/PHARMACOLOGY Recombinant Proteins/BIOSYNTHESIS/PHARMACOLOGY Retroviridae Support, Non-U.S. Gov't Tetracycline/PHARMACOLOGY Transfection 3T3 CellsSelected domains of the regulatory p85 subunit of phosphatidylinositol 3-kinase have been expressed under the control of the tetracycline transactivator in NIH 3T3 fibroblasts transformed by the v-Ha-Ras oncogene. The domains expressed were the SH3 domain, the BCR homology domain, the region between the two SH2 domains which contains the p110 binding site (the inter SH2 (IS) domain), and the C-terminal (CT) domain (containing both SH2 domains and the IS domain). The levels of IS or SH3 domain expressed in the presence of tetracycline were sufficient to reverse the transforming effects of v-Ha-Ras, and no further inhibition of proliferation was observed when expression was increased 7-fold by removal of tetracycline. In contrast inhibition of proliferation by the CT domain was observed only when the level of expression was increased 5-fold by removal of tetracycline. Overexpression of the BCR domain of p85 had no effect on v-Ha-Ras transformation. Expression of the IS domain disrupted the interaction of the p85 regulatory subunit with the p110 catalytic subunit. These results indicate that the association of the p85 subunit of PI 3-kinase with the p110 subunit is necessary for v-Ha-Ras-induced transformation in NIH 3T3 cells. ?n[Wang, J. Han, W. Zborowska, E. Liang, J. Wang, X. Willson, J. K. V. Sun, L. Brattain, M. G.1996Reduced expression of transforming growth factor beta type I receptor contributes to the malignancy of human colon carcinoma cells17366-71 J Biol Chem27129{Animal Antigens, CD/BIOSYNTHESIS Cell Division/DRUG EFFECTS Cell Line Cloning, Molecular Colonic Neoplasms/METABOLISM/PATHOLOGY DNA Replication Fibronectins/BIOSYNTHESIS Human Mice Protein-Serine-Threonine Kinases/BIOSYNTHESIS/*PHYSIOLOGY Rats Receptors, Transforming Growth Factor beta/BIOSYNTHESIS/*PHYSIOLOGY Recombinant Proteins/BIOSYNTHESIS/METABOLISM/PHARMACOLOGY RNA, Messenger/BIOSYNTHESIS Support, Non-U.S. Gov't Support, U.S. Gov't, Non-P.H.S. Support, U.S. Gov't, P.H.S. Tetracycline/PHARMACOLOGY Transcription, Genetic/DRUG EFFECTS Transfection Transforming Growth Factor beta/METABOLISM/*PHARMACOLOGY Tumor Cells, CulturedTransforming growth factor beta (TGFbeta) type I (RI) and type II (RII) receptors are essential for TGFbeta signal transduction. A human colon carcinoma cell line, designated GEO, is marginally responsive to TGFbeta and expresses a low level of RI mRNA relative to colon carcinoma cells, which are highly responsive to TGFbeta. Hence, the role of RI as a limiting factor for TGFbeta sensitivity and the contribution of low RI levels to the malignant phenotype of GEO cells were examined. Stable transfection of a tetracycline-regulatable rat RI cDNA increased TGFbeta1 binding to RI and resulted in increased growth inhibition by exogenous TGFbeta1. In contrast, although stable transfection of an RII expression vector into the same GEO cells increased TGFbeta1 binding to RII, growth inhibition by exogenous TGFbeta1 was not altered. This indicated that the low level of RI is a limiting factor for the growth-inhibitory effects of TGFbeta in GEO cells. RI-transfected cells were growth-arrested at a lower saturation density than GEO control cells. They also showed reduced growth and clonogenicity in plating efficiency and soft agarose assays, whereas RII-transfected cells did not show any differences from the NEO control cells in these assays. Tetracycline repressed RI expression in transfected cells and reversed the reduction in plating efficiency of RI-transfected clones, confirming that growth effects were due to increased RI expression in transfected cells. TGFbeta1 neutralizing antibody stimulated the proliferation of RI-transfected cells but had little effect on GEO control cells, indicating that increased autocrine-negative TGFbeta activity also resulted from increased RI expression. Tumorigenicity in athymic nude mice was significantly delayed in RI-transfected cells. These results indicate that low RI expression can be a limiting factor for response to exogenous TGFbeta, as well as TGFbeta autocrine-negative activity, and that reduction of RI expression can contribute to malignant progression.?oDNeering, S. J. Hardy, S. F. Minamoto, D. Spratt, S. K. Jordan, C. T.1996WTransduction of primitive human hematopoietic cells with recombinant adenovirus vectors1147-55Blood884Adenovirus Infections, Human/GENETICS Adenoviruses, Human/*GENETICS Antigens, CD34/ANALYSIS Base Sequence Bone Marrow/CYTOLOGY Cell Survival Cells, Cultured Colony-Forming Units Assay DNA Primers/CHEMISTRY Fetal Blood/CYTOLOGY Gene Expression Regulation, Viral Gene Transfer Genetic Vectors Hematopoiesis Hematopoietic Stem Cells Human Interphase Molecular Sequence Data Transduction, GeneticxWe have examined the ability of recombinant adenoviral vectors to transduce human hematopoietic cells. Our findings indicate that adenovirus readily infects a large proportion of CD34+ cells. Using adenovirus vectors that transduce either a lacZ or an alkaline phosphatase reporter gene, we observed up to 45% of total CD34+ cells infected. Upon more detailed analysis, we observed comparable levels of transduction for CD34+/CD38- cells and for CD34+ cells in G(zero) phase of the cell cycle. Importantly, exposure to adenovirus resulted in negligible levels of toxicity as assayed by propidium iodide staining and colony-forming ability. Using adenovirus vectors, we also describe a model system for regulated gene expression in early hematopoietic tissues. CD34+ cells were simultaneously infected with two viruses, one carrying a TetR/VP16 transactivator (tTA) and the second carrying a tTA-dependent lacZ reporter gene. Using this approach, beta-gal expression was only observed upon coinfection with the transactivator vector. In addition, as shown previously (Gossen and Bujard, Proc Natl Acad Sci USA 89:5547, 1992), tetracycline was able to inhibit tTA mediated induction, thereby providing an effective means to regulate expression of the reporter gene. We conclude that recombinant adenovirus is an effective vehicle for transiently expressing genes in primitive human hematopoietic cells. ?p Kitamura, M.1996{Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus7387-91Proc Natl Acad Sci U S A9314`beta-Galactosidase/BIOSYNTHESIS Animal Cells, Cultured Gene Expression Regulation/DRUG EFFECTS Gene Transfer Genetic Engineering Glomerular Mesangium/CYTOLOGY/*METABOLISM Male Plasmids Promoter Regions (Genetics)/DRUG EFFECTS Rats Rats, Sprague-Dawley Support, Non-U.S. Gov't Tetracycline/PHARMACOLOGY Trans-Activators/METABOLISM Transcription, GeneticxUsing genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controll