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1.
Cancer Gene Ther ; 16(2): 149-60, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18670452

ABSTRACT

Interferon-gamma-inducible protein 10 is a potent chemoattractant for natural killer cells and activated T lymphocytes. It also displays angiostatic properties and some antitumor activity. Tumor necrosis factor-alpha (TNF-alpha) is a powerful immunomodulating cytokine with demonstrated tumoricidal activity in various tumor models and the ability to induce strong immune responses. This prompted us to evaluate the antitumor effects of recombinant parvoviruses designed to deliver IP-10 or TNF-alpha into a glioblastoma. When Gl261 murine glioma cells were infected in vitro with an IP-10- or TNF-alpha-transducing parvoviral vector and were subcutaneously implanted in mice, tumor growth was significantly delayed. Complete tumor regression was observed when the glioma cells were coinfected with both the vectors, demonstrating synergistic antitumor activity. In an established in vivo glioma model, however, repeated simultaneous peritumoral injection of the IP-10- and TNF-alpha-delivering parvoviruses failed to improve the therapeutic effect as compared with the use of a single cytokine-delivering vector. In this tumor model, cytokine-mediated immunostimulation, rather than inhibition of vascularization, is likely responsible for the therapeutic efficacy.


Subject(s)
Chemokine CXCL10/metabolism , Chemokine CXCL10/therapeutic use , Glioblastoma/drug therapy , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/therapeutic use , Animals , Chemokine CXCL10/administration & dosage , Chemokine CXCL10/immunology , Dendritic Cells/cytology , Dendritic Cells/virology , Drug Synergism , Female , Genetic Vectors , Glioblastoma/blood supply , Glioblastoma/immunology , Glioblastoma/metabolism , Glioblastoma/virology , H-1 parvovirus/physiology , Humans , Immunocompetence , Mice , Mice, Inbred C57BL , Minute Virus of Mice/physiology , Necrosis/metabolism , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects , Tumor Necrosis Factor-alpha/administration & dosage , Tumor Necrosis Factor-alpha/genetics
2.
Cancer Gene Ther ; 15(4): 252-67, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18202715

ABSTRACT

Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 (AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants.


Subject(s)
Genetic Vectors , Parvovirus/genetics , Transgenes , Animals , Blotting, Western , Cell Line, Transformed , HeLa Cells , Humans , Rats
3.
J Gene Med ; 3(4): 326-37, 2001.
Article in English | MEDLINE | ID: mdl-11529662

ABSTRACT

BACKGROUND: The oncosuppressive properties of some autonomous parvoviruses such as H-1 virus, together with their low pathogenicity, make them attractive vectors for tumor-directed gene therapy. Indeed, it was recently shown that these viruses became endowed with an enhanced oncosuppressive activity after they had been engineered to deliver a recognized therapeutic transgene. This prompted us to use a parvoviral vector to analyse the antineoplastic capacity of MCP-3 (monocyte chemotactic protein-3), a CC chemokine which has a broad spectrum of target cells, and can thus be considered to be a promising candidate for cancer treatment. METHODS: We explored the use of a parvovirus H-1-based vector encoding human MCP-3 for its antitumor potential on human cervical carcinoma cells. HeLa cells were infected in vitro with the recombinant virus hH1/MCP-3 at a low multiplicity [1 replication unit (RU)/cell] and we investigated the effect of parvovirus-mediated MCP-3 transduction on tumor formation and growth upon implantation of HeLa cells in nude mice. RESULTS: Infection of HeLa cells with hH1/MCP-3 led to secretion of high levels of MCP-3 and to significant retardation of tumor growth in recipient mice, as compared with HeLa cells that were either buffer-treated or infected with a MCP-3-free vector. Tumors from hH1/MCP-3-infected HeLa cells were heavily infiltrated with activated macrophages and showed increased numbers of dendritic cells. In addition, activated natural killer (NK) cells were also recruited into MCP-3-transduced tumors. CONCLUSION: These observations indicate that parvovirus H-1-transduced MCP-3 is able to exert a significant antitumor activity which is mediated, at least in part, through macrophages and NK cells, under conditions in which activated T cells are lacking.


Subject(s)
Cytokines , Monocyte Chemoattractant Proteins/genetics , Parvovirus/genetics , Uterine Cervical Neoplasms/therapy , Animals , Chemokine CCL7 , Female , HeLa Cells , Humans , Mice , Mice, Nude , Monocyte Chemoattractant Proteins/pharmacokinetics , Monocyte Chemoattractant Proteins/therapeutic use , Plasmids , Recombinant Proteins/analysis , Transcription, Genetic , Transduction, Genetic , Transplantation, Heterologous
4.
Cancer Gene Ther ; 8(12): 958-65, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11781658

ABSTRACT

The oncotropic and oncolytic behaviors of certain autonomous rodent parvoviruses make them promising vectors for anticancer gene therapies. However, these parvoviruses are often not potent enough to kill all tumor cells equally well. With the aim of enhancing the intrinsic antitumor effect and the range of natural parvoviruses, a recombinant H1 parvovirus vector was constructed that produces the Apoptin protein, a tumor cell-specific, p53-independent, Bcl-2-insensitive apoptotic effector. We compared the apoptotic activity exerted by a recombinant hH1/Apoptin virus with that of a Green Fluorescent Protein (GFP)-transducing recombinant virus, hH1/GFP, in three human tumor cell lines differing in their susceptibility to wild-type parvovirus H1-induced killing. We found that in cells that were rather resistant to the basal cytotoxic effect of wild-type H1 or the GFP recombinant virus, a parvovirus that expressed Apoptin caused a pronounced, additional cytotoxic effect. In contrast to its enhanced cytotoxicity toward tumor cells, hH1/Apoptin virus was not more toxic to normal human fibroblasts than was the wild-type H1 virus. Taken together, these data indicate that enhancing the oncotropic behavior of wild-type H1 parvoviruses with the tumor-specific apoptotic potency of Apoptin should lead to an effective replicative parvoviral vector.


Subject(s)
Apoptosis , Capsid Proteins , Capsid/genetics , Genetic Therapy , Genetic Vectors , Parvovirus/genetics , Apoptosis/genetics , Capsid/pharmacology , HeLa Cells , Humans , Reassortant Viruses/genetics , Virus Replication/genetics
5.
Hum Gene Ther ; 11(4): 597-609, 2000 Mar 01.
Article in English | MEDLINE | ID: mdl-10724038

ABSTRACT

The possible use of recombinant autonomous parvoviruses as vectors to efficiently express therapeutic cytokines in human tumor cells was evaluated in vitro and in vivo. The parvovirus H1 was used to generate recombinant viruses (rH1) that carried transgenes encoding either human interleukin 2 (IL-2) or monocyte chemotactic protein 1 (MCP-1), in replacement of part of the capsid genes. Such rH11 viruses have been shown to retain in vitro the intrinsic oncotropic properties of the parental virus. On infection with the recombinant viruses at an input multiplicity of 1 replication unit (RU) per cell, HeLa cultures were induced to release 4-10 microg of cytokine per 10(6) cells over a period of 5 days. The expression of the rH1-transduced human cytokine/chemokine could also be detected in tumor material recovered from nude mice that had been subcutaneously engrafted with in vitro-infected HeLa cells. The formation of tumors from HeLa xenografts was reduced by 90% compared with wild-type or mock-infected cells as a result of cells preinfected with IL2-expressing virus at an input multiplicity as low as 1 RU per cell. Tumors arising from HeLa cells infected with transgene-free or MCP1-expressing vectors or with wild-type H1 virus were not rejected at this virus dose. Tumors infected with rH1/IL-2 virus displayed markers indicative of their infiltration with NK cells in which the cytocidal program was activated, whereas little NK activity was detected in wild-type virus or mock-infected tumors. Altogether, these data show that the IL-2 expressing H1 vector was a more potent antineoplastic agent than the parental virus, and point to the possible application of recombinant autonomous parvoviruses toward therapy of some human tumors.


Subject(s)
Cytokines/genetics , Gene Expression Regulation , Genetic Vectors , Neoplasms, Experimental/prevention & control , Parvovirus/genetics , Transduction, Genetic , Animals , Base Sequence , Cell Line , Cytotoxicity, Immunologic/genetics , DNA Primers , Female , Humans , Mice , Mice, Nude , Neoplasms, Experimental/immunology , Recombination, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Transgenes , Viral Nonstructural Proteins/genetics
6.
Hum Gene Ther ; 10(10): 1619-32, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10428207

ABSTRACT

The replication of viral genomes and the production of recombinant viral vectors from infectious molecular clones of parvoviruses MVMp and H1 were greatly improved by the introduction of a consensus NS-1 nick site at the junction between the left-hand viral terminus and the plasmid DNA. Progressive deletions of up to 1600 bp in the region encoding the structural genes as well as insertions of foreign DNA in replacement of those sequences did not appreciably affect the replication ability of the recombinant H1 virus genomes. In contrast, the incorporation of these genomes into recombinant particles appeared to depend on in cis-provided structural gene sequences. Indeed, the production of H1 viral vectors by cotransfection of recombinant clones and helper plasmids providing the structural proteins (VPs) in trans, drastically decreased when more than 800 bp was removed from the VP transcription unit. Furthermore, titers of viral vectors, in which most of the VP-coding region was replaced by an equivalent-length sequence consisting of reporter cDNA and stuffer DNA, were reduced more than 50 times in comparison with recombinant vectors in which stuffer DNA was not substituted for the residual VP sequence. In addition, viral vector production was restricted by the overall size of the genome, with a mere 6% increase in DNA length leading to an approximately 10 times lower encapsidation yield. Under conditions fulfilling the above-mentioned requirements for efficient packaging, titers of virus vectors from improved recombinant molecular DNA clones amounted to 5 x 10(7) infectious units per milliliter of crude extract. These titers should allow the assessment of the therapeutic effect of recombinant parvoviruses expressing small transgenes in laboratory animals.


Subject(s)
DNA, Viral , Genetic Vectors , Parvovirus , Capsid/genetics , Capsid Proteins , Cell Line, Transformed , Chemokine CCL2/genetics , DNA Replication , Gene Expression , HeLa Cells , Humans , Mutagenesis , Parvovirus/genetics , Parvovirus/physiology , Recombination, Genetic , Virus Replication
7.
J Virol ; 72(11): 8893-903, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9765434

ABSTRACT

The human promonocytic cell line U937 undergoes apoptosis upon treatment with tumor necrosis factor alpha (TNF-alpha). This cell line has previously been shown to be very sensitive to the lytic effect of the autonomous parvovirus H-1. Parvovirus infection leads to the activation of the CPP32 ICE-like cysteine protease which cleaves the enzyme poly(ADP-ribose)polymerase and induces morphologic changes that are characteristic of apoptosis in a way that is similar to TNF-alpha treatment. This effect is also observed when the U937 cells are infected with a recombinant H-1 virus which expresses the nonstructural (NS) proteins but in which the capsid genes are replaced by a reporter gene, indicating that the induction of apoptosis can be assigned to the cytotoxic nonstructural proteins in this cell system. The c-Myc protein, which is overexpressed in U937 cells, is rapidly downregulated during infection, in keeping with a possible role of this product in mediating the apoptotic cell death induced by H-1 virus infection. Interestingly, four clones (designated RU) derived from the U937 cell line and selected for their resistance to H-1 virus (J. A. Lopez-Guerrero et al., Blood 89:1642-1653, 1997) failed to decrease c-Myc expression upon treatment with differentiation agents and also resisted the induction of cell death after TNF-alpha treatment. Our data suggest that the RU clones have developed defense strategies against apoptosis, either by their failure to downregulate c-Myc and/or by activating antiapoptotic factors.


Subject(s)
Apoptosis/physiology , Parvovirus/pathogenicity , Tumor Necrosis Factor-alpha/physiology , Apoptosis/drug effects , Caspases/metabolism , Down-Regulation , Enzyme Activation , Genes, myc , Humans , Parvovirus/genetics , Poly(ADP-ribose) Polymerases/metabolism , Recombination, Genetic , Signal Transduction , Tumor Necrosis Factor-alpha/pharmacology , U937 Cells
8.
Blood ; 89(5): 1642-53, 1997 Mar 01.
Article in English | MEDLINE | ID: mdl-9057647

ABSTRACT

The human promonocytic cell line U937 is highly sensitive to the lytic effect of the autonomous parvovirus H-1. Rare cell variants that resisted H-1 virus infection could be isolated, of which four (RU1, RU2, RU3, and RU4) were further characterized. In contrast to parental cells, the RU clones sustained an abortive H-1 virus infection. Three of the clones showed a significant decrease in the accumulation levels of the c-Myc oncoprotein and in their capacity for forming tumors in immunodeficient mice. Surprisingly, all RU clones resisted the suppressing effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on c-myc oncogene expression and cell proliferation. In contrast, RU clones exhibited the TPA-induced changes in membrane surface antigens and nonspecific esterase activities that are characteristic of monocytic differentiation. Studies of the activation steady-state of RU cells demonstrated the constitutive production of significant amounts of nitric oxide (NO) and superoxide anion (O-.2). Inhibitors of NO and O-.2, production sensitized all RU cells to the killing effect of parvovirus H-1 and increased the production of infectious viral particles. These data argue for the participation of active oxygen species in macrophage defence mechanisms against parvovirus infection. Moreover, the use of parvovirus H-1 as a selective agent in a cell-colony formation assay allowed us to show that expression of defined markers of monocytic differentiation can be uncoupled from suppression of proliferation.


Subject(s)
Clone Cells/virology , Monocytes/virology , Parvoviridae Infections/virology , Parvovirus , Animals , Cell Differentiation , Humans , Mice , Monocytes/pathology , Virus Replication
9.
J Virol ; 70(3): 1331-9, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8627649

ABSTRACT

The P4 promoter of parvovirus minute virus of mice (MVMp) directs transcription of the genes coding for nonstructural proteins. The activity of promoter P4 is regulated by several cis-acting DNA elements. Among these, a promoter-proximal GC box was shown to be essential for P4 activity (J.K. Ahn, B.J. Gavin, G. Kumar, and D.C. Ward, J. Virol. 63:5425-5439, 1989). In this study, a motif homologous to an Ets transcription factor-binding site (EBS), located immediately upstream from the GC box, was found to be required for the full activity of promoter P4 in the ras-transformed rat fibroblast cell line FREJ4. In normal parental FR3T3 cells, the transcriptional function of P4 EBS was insignificant but could be restored by transient cell transfection with the c-Ha-ras oncogene. P4 EBS may thus contribute to the stimulation of promoter P4 in ras-transformed cells. Electrophoretic mobility shift assays using crude extracts from FREJ4 cells revealed the binding of a member(s) of the Ets family of transcription factors to the P4 EBS, as well as the interaction of two members of the Sp1 family, Sp1 and Sp3, with the adjacent GC box. When produced in Drosophila melanogaster SL2 cells, Ets-1 and Sp1 proteins acted synergistically to transactivate promoter P4 through their respective cognate sites.


Subject(s)
Gene Expression Regulation, Viral , Genes, ras , Minute Virus of Mice/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , Cell Line, Transformed , DNA, Viral , DNA-Binding Proteins/metabolism , Drosophila melanogaster/cytology , Mice , Molecular Sequence Data , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins c-ets , Rats , Rats, Inbred F344 , Recombinant Proteins/metabolism , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor , Transcriptional Activation
10.
Mol Cell Biol ; 12(3): 1218-25, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1545803

ABSTRACT

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.


Subject(s)
Adenylyl Cyclases/genetics , Flagella/enzymology , Trypanosoma brucei brucei/genetics , Variant Surface Glycoproteins, Trypanosoma/genetics , Adenylyl Cyclases/metabolism , Animals , Genetic Complementation Test , Immunoblotting , Microscopy, Electron , Multigene Family , Mutation , Saccharomyces cerevisiae/genetics , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/ultrastructure , Variant Surface Glycoproteins, Trypanosoma/metabolism
11.
Biochem Biophys Res Commun ; 180(3): 1460-8, 1991 Nov 14.
Article in English | MEDLINE | ID: mdl-1659418

ABSTRACT

The cDNA of RDC4, a putative receptor of the G protein-coupled receptor family, has been cloned by PCR methodology. The primary structure of this receptor showed homology with the serotonin 5-HT1A receptor. In this work, RDC4 mRNA has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 cDNA has been transfected transiently in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the transfected cos-7 cells using [3H]-LSD showed a pattern of drug affinities consistent with the known properties of a 5-HT1D receptor. RDC4 therefore codes for a 5-HT1D receptor which in the studied systems is positively coupled to adenylate cyclase.


Subject(s)
GTP-Binding Proteins/metabolism , Receptors, Serotonin/genetics , 8-Hydroxy-2-(di-n-propylamino)tetralin , Adrenal Cortex , Animals , Binding, Competitive , Cell Line , Cloning, Molecular , Cyclic AMP/metabolism , DNA/genetics , Kinetics , Lysergic Acid/metabolism , Polymerase Chain Reaction , RNA, Messenger/genetics , Receptors, Serotonin/metabolism , Recombinant Proteins/metabolism , Tetrahydronaphthalenes/pharmacology , Transfection
12.
J Clin Endocrinol Metab ; 73(4): 857-60, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1716261

ABSTRACT

The sequential epitopes on the human thyroperoxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease were investigated using a recombinant DNA technique. Previous studies led to the isolation of two overlapping cDNA clones that encode polypeptides of TPO (85 residues, C2; 100 residues, C21) recognized by sera from several patients with autoimmune disease that contained antimicrosomal autoantibodies. In this report the vector pUEX1 was used to clone and express small random fragments of TPO cDNA in Escherichia coli as a beta-galactosidase fusion protein. Colonies were screened with a serum from a patient with Hashimoto's thyroiditis, and immunoreactive peptides were identified by sequencing the corresponding DNA inserts. Two linear epitopes of human TPO (amino acids 590-622 and 710-722) were recognized by the autoantibodies. This confirmed our previous results and provide a more precise localization of the antigenic determinants involved. The same approach has been applied in an attempt to identify the binding site(s) for autoantibodies on the human TSH receptor. In contrast to the data obtained with TPO, sera from patients with blocking (from idiopathic myxoedema) or stimulating (from Graves' disease) activity did not recognize the linear TSH receptor peptide fragments generated in our libraries.


Subject(s)
Autoantibodies/immunology , Epitopes/genetics , Genetic Vectors , Iodide Peroxidase/genetics , Receptors, Thyrotropin/genetics , Amino Acid Sequence , DNA/genetics , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Graves Disease/blood , Graves Disease/genetics , Humans , Iodide Peroxidase/immunology , Molecular Sequence Data , Myxedema/blood , Myxedema/genetics , Plasmids , Receptors, Thyrotropin/immunology , Recombinant Proteins/immunology , Thyroiditis, Autoimmune/blood , Thyroiditis, Autoimmune/genetics
13.
J Clin Endocrinol Metab ; 68(6): 1091-6, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2470771

ABSTRACT

Previous studies carried out by screening a lambda gt11 human thyroid cDNA library with serum samples from selected patients with Hashimoto's thyroiditis and a polyclonal antibody to porcine thyroid peroxidase (TPO) confirmed, at the molecular level, that TPO is a major component of the thyroid microsomal antigen (M). That investigation led to the isolation of a clone (C2) which encodes an 85-amino acid segment of TPO and harbors a major epitope recognized by serum from several patients with autoimmune thyroid disease that contained anti-M autoantibodies (MAb). In this study, C2 antigen that was produced as a beta-galactosidase fusion protein was used to establish an enzyme-linked immunoabsorbent assay for the detection of anti-C2 autoantibodies (C2Ab). C2Ab then were assayed in 191 patients with different autoimmune and nonautoimmune thyroid disorders, and 50 patients with nonthyroidal autoimmune diseases. The results were compared with the titers of anti-TPO antibodies (TPOAb; as detected by monoclonal antibody-assisted RIA) and MAb (as detected by passive hemagglutination). Positive C2Ab was found in the serum of 85 of 136 (63%) patients whose serum contained TPOAb and/or MAb. A significant positive correlation was found between the levels of C2Ab and those of TPOAb (r = 0.76; P less than 0.001) or MAb (r = 0.69; P less than 0.001), which was independent of the type of underlying autoimmune thyroid disorder. Low levels of C2Ab also were found in 10 of 105 (9%) serum samples that did not contain TPOAb. Western blot analysis carried out on the latter samples showed that in 2 samples the apparent C2Ab reactivity was due to the presence of antibodies reacting with beta-galactosidase. In conclusion, we confirmed the validity of screening lambda gt11 cDNA human thyroid libraries to better characterize thyroid autoantigens and demonstrated the feasibility of using recombinant proteins to establish diagnostic assays for autoantibodies.


Subject(s)
Antibodies/analysis , DNA, Recombinant/analysis , Epitopes/analysis , Iodide Peroxidase/immunology , Thyroiditis, Autoimmune/enzymology , Antibodies, Monoclonal/analysis , Antigen-Antibody Complex/analysis , Cloning, Molecular , DNA/analysis , Enzyme-Linked Immunosorbent Assay , Humans , Immune Sera/analysis , Iodide Peroxidase/genetics , Recombinant Fusion Proteins , Thyroiditis, Autoimmune/genetics , Thyroiditis, Autoimmune/immunology
14.
Science ; 244(4904): 569-72, 1989 May 05.
Article in English | MEDLINE | ID: mdl-2541503

ABSTRACT

An approach based on the polymerase chain reaction has been devised to clone new members of the family of genes encoding guanosine triphosphate-binding protein (G protein)-coupled receptors. Degenerate primers corresponding to consensus sequences of the third and sixth transmembrane segments of available receptors were used to selectively amplify and clone members of this gene family from thyroid complementary DNA. Clones encoding three known receptors and four new putative receptors were obtained. Sequence comparisons established that the new genes belong to the G protein-coupled receptor family. Close structural similarity was observed between one of the putative receptors and the 5HT1a receptor. Two other molecules displayed common sequence characteristics, suggesting that they are members of a new subfamily of receptors with a very short nonglycosylated (extracellular) amino-terminal extension.


Subject(s)
Cloning, Molecular , GTP-Binding Proteins/metabolism , Gene Amplification , Receptors, Neurotransmitter/genetics , Amino Acid Sequence , Base Sequence , DNA/genetics , DNA-Directed DNA Polymerase , Humans , Molecular Sequence Data , Receptors, Adrenergic, alpha/genetics , Receptors, Adrenergic, beta/genetics , Receptors, Muscarinic/genetics , Receptors, Neurokinin-2 , Receptors, Serotonin/genetics , Sequence Homology, Nucleic Acid , Thyroid Gland/analysis , Transcription, Genetic
16.
Cytogenet Cell Genet ; 47(3): 170-2, 1988.
Article in English | MEDLINE | ID: mdl-3378456

ABSTRACT

A 2.0-kb thyroid peroxidase cDNA of human origin was used as probe for Southern blot hybridization of genomic DNA from human somatic cells and human-rodent somatic cell hybrids. The results showed that the gene coding for human thyroid peroxidase is located on chromosome. 2. Further analysis of hybrids derived from Burkitt lymphoma cells carrying a (2;8)(p12;q24) translocation revealed that the gene maps to the region 2pter----p12.


Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 2 , Iodide Peroxidase/genetics , Animals , Burkitt Lymphoma/genetics , Cricetinae , Cricetulus , DNA/genetics , Humans , Hybrid Cells , Mice , Nucleic Acid Hybridization , Tumor Cells, Cultured
17.
EMBO J ; 6(13): 4193-6, 1987 Dec 20.
Article in English | MEDLINE | ID: mdl-3443105

ABSTRACT

A lambda gt11 cDNA library was constructed from a normal human thyroid and screened with a rabbit anti-porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the 'microsomal antigen' (McAg). Comparison of the cDNA sequences from TPO clones and McAg clones provides definite proof that the McAg is TPO. A short segment of TPO was characterized as bearing a major epitope involved in autoimmunity. The primary structure of TPO was 42% homologous to myeloperoxidase (MPO). It contains, in addition, a C-terminal extension with a membrane anchor region contiguous to two domains encoded by modules belonging to the EGF and C4b gene families. The existence in TPO of still another domain presenting a significant homology with a putative heme-binding region of cytochrome C oxidase polypeptide I raises the possibility that a mitochondrial gene module has contributed a piece to the evolution of a typical nuclear mosaic gene.


Subject(s)
Autoantigens/genetics , Cell Nucleus/metabolism , DNA, Mitochondrial/genetics , Genes , Iodide Peroxidase/genetics , Mitochondria/enzymology , Thyroid Gland/enzymology , Amino Acid Sequence , Cloning, Molecular , DNA/genetics , DNA/isolation & purification , Humans , Molecular Sequence Data , Sequence Homology, Nucleic Acid
19.
Mutat Res ; 151(1): 9-14, 1985 Aug.
Article in English | MEDLINE | ID: mdl-2991761

ABSTRACT

UV irradiation of simian virus 40 (SV40)-transformed human and hamster cells induced them both to express a mutator phenotype and to produce SV40. The mutator could also be activated indirectly by transfecting unirradiated cells with UV-damaged calf thymus DNA. In contrast, UV-damaged exogenous DNA failed to rescue SV40 from unirradiated transformed cells. These results suggest that the expression of transforming viruses and of cellular mutator functions is regulated by at least partially independent mechanisms. Unlike the activation of a cellular mutator phenotype, the rescue of SV40 from virus-transformed mammalian cells by UV light might require that the integrated viral DNA and/or specific cellular sequences are directly damaged.


Subject(s)
Mutation/radiation effects , Simian virus 40/growth & development , Virus Replication/radiation effects , Animals , Cell Transformation, Viral , Cells, Cultured , Cricetinae , DNA/radiation effects , Humans , Kidney , Phenotype , Transfection , Ultraviolet Rays
20.
Mol Cell Biol ; 4(2): 324-8, 1984 Feb.
Article in English | MEDLINE | ID: mdl-6700594

ABSTRACT

Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.


Subject(s)
DNA/radiation effects , Mutation , Parvoviridae/genetics , Transfection , Animals , Cells, Cultured , DNA/biosynthesis , Humans , Phenotype , Rats , Ultraviolet Rays
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