RESUMEN
Growth factor receptor tyrosine kinases (RTK) have been implicated in tumor growth, metastasis and angiogenesis, and are thus considered promising targets for therapeutic intervention in malignant diseases. We present a novel drug discovery strategy to find inhibitors of RTKs based on comparative screening of compound libraries employing functional cellular assays. Cell lines stably expressing HER2 and the receptors for hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) have been established. All cell lines are based on FDC-P1, a murine myeloid progenitor cell line which allows a direct comparison of results obtained in primary screens. In addition, the same cell lines are suitable for compound optimization and for animal studies. Using this strategy we report the identification of promising lead candidates for further drug development which are highly selective, non-cytotoxic and cell permeable with potencies in the low micromolar range.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/enzimología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismoRESUMEN
The neurokinin receptor family is known to modulate phospholipase C activity. In order to find new compounds modulating the activity of these receptors we have developed a cellular screening system that measures the biological activity of receptors coupled to the IP3/DAG signal transduction pathway via the transcriptional activation of a reporter gene. For the establishment of neurokinin test cell lines the reporter cell line A20, stably transformed with the luciferase gene under the control of a promoter containing TPA response elements (TRE), which did not respond to neurokinin agonists, was used. Stable test cell lines were developed by transfecting the reporter cell line A20 with the genes for the human neurokinin receptors NK1, NK2 or NK3, respectively. In these cell lines, expression of luciferase was inducible by substance P, neurokinin A and neurokinin B, respectively. The order of potency of the three neurokinins substance P, neurokinin A and neurokinin B was consistent with published data and results from ligand binding studies performed with the NK1 and NK2 test cell lines. The agonistic effect of the neurokinins could be inhibited in a dose-dependent manner by simultaneous addition of neurokininspecific antagonists like the non-peptide antagonists CP-99,994 and SR 48968.
Asunto(s)
Bioensayo/métodos , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/metabolismo , Receptores de Neuroquinina-3/metabolismo , Línea Celular , Diglicéridos/metabolismo , Genes Reporteros , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Luciferasas/genética , Ensayo de Unión Radioligante , Receptores de Neuroquinina-1/análisis , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-2/análisis , Receptores de Neuroquinina-2/genética , Receptores de Neuroquinina-3/análisis , Receptores de Neuroquinina-3/genética , Transducción de Señal , Taquicininas/metabolismo , Taquicininas/farmacología , TransfecciónRESUMEN
Cytogenetic findings in 45 cases of peripheral T-cell lymphomas (PTL) diagnosed according to the updated Kiel classification are reported. Recurrent numerical chromosome aberrations comprised -X, -Y, -13, +X, +3, +5 and +7. Recurrent structural aberrations included t/del(1)(p31-32), t(2;5)(p23;q35), dup(5)(q23q31-32), t/dup(6q), t/del(6q), trisomy 7q, and trisomy 8q, mostly due to i(8)(q10), and changes in 14q11 and 14q32.1, mostly due to inv(14)(q11q32.1), t/del(13)(q14), t(6;7)(q13;q13), and t(13;17)(q11-13;p11). All deletions in 6q involved band 6q21 and all partial trisomies of 7q led to an amplification of band 7q21. Further studies are needed to ascertain whether these cytogenetic findings in PTL are of clinical and prognostic significance.
Asunto(s)
Aberraciones Cromosómicas , Linfoma de Células T Periférico/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Cariotipificación , Masculino , Persona de Mediana Edad , RecurrenciaRESUMEN
Cytogenetic studies on lymph node and skin biopsy specimens and peripheral blood in 104 patients with peripheral T-cell lymphomas (PTL) were compared with histopathologic diagnoses made according to the updated Kiel classification. Low-grade lymphomas presented normal metaphases more frequently than high-grade ones (P < .0001). This difference remained significant if cases with greater than 10% and greater than 50% normal metaphases in unstimulated cultures and in cultures stimulated by different mitogens were compared. On the other hand, high-grade lymphomas more often showed aberrant clones (P < .05), triploid to tetraploid clones (P < .0001), and complex clones with more than four chromosome changes (P < .01). Low-grade PTL showed consistent cytogenetic features. Clones with both inv(14)(q11q32.1) and trisomy 8q, mostly caused by i(8q)(q10), were found in all cases of T-cell chronic lymphocytic leukemia (T-CLL) and T-cell prolymphocytic leukemia (T-PLL). Trisomy 3 was observed only in angioimmunoblastic lymphadenopathy with dysproteinemia (AILD)-type PTL, T-zone lymphoma, and lymphoepithelioid lymphoma. Moreover, the proportion of normal metaphases in these PTL was higher than in the other low-grade PTL (P < .01). On the contrary, T-CLL, T-PLL, and cutaneous T-cell lymphomas (CTCL) showed complex clones (P < .0001), duplications in 6p (P < .01), deletions in 6q (P < .01), trisomy 8q (P < .00001), inv(14) (P < .00001), and monosomy 13 or changes of 13q14 (P < .001) more frequently than the other low-grade PTL. Trisomy 5 and + X predominated in AILD-type PTL. A cytogenetic feature characteristic of AILD-type PTL and CTCL was unrelated clones, which were found in 15% of AILD-type PTL and 17% of CTCL. The only chromosome aberration restricted to a certain high-grade PTL was t(2;5)(p23;q35) in large-cell anaplastic lymphoma. Deletions in 6q, total or partial trisomies of 7q, and monosomy 13 or changes of 13q14 turned out to be significantly more frequent in high-grade than in low-grade lymphomas (P < .01, P < .01, and P < .05, respectively). In summary, the cytogenetic findings in our series of 104 PTL enabled us to distinguish not only between low-grade and high-grade lymphomas but also between various entities of PTL. Thus, the cytogenetic findings paralleled the histopathologic diagnoses made according to the updated Kiel classification.
Asunto(s)
Aberraciones Cromosómicas , Linfoma no Hodgkin/genética , Linfoma de Células T Periférico/genética , Humanos , Linfadenopatía Inmunoblástica/genética , Linfoma no Hodgkin/ultraestructura , Linfoma Cutáneo de Células T/genética , Linfoma de Células T Periférico/ultraestructuraRESUMEN
Cytogenetic studies were performed in 21 cases of Hodgkin's disease. Fourteen cases revealed chromosomally aberrant clones which could be fully described in 12 cases. Two cases showed different unrelated clones and five cases only single cell aberrations. Recurrent breakpoints were 1p13/21 (six cases), 7q32/34 (five cases), 2p16/21 and 19p13 (four cases each), 4q25/28, 6q15/21 and 12q22/23 (three cases each). In two cases, a translocation between band 19q13 and band 14q11 or 14q32 was found. This finding may indicate that an unknown oncogene in 19p13 is activated by juxtaposition next to a T-cell receptor or immunoglobulin gene in 14q11 or 14q32, respectively. In eight cases each, total or partial monosomy 4 or 6 was present suggesting that tumor suppressor genes in 4q or 6q play a role in tumor development in Hodgkin's disease. Moreover, the aberrant clones lacked the Y-chromosome in men and the second X-chromosome in women in eight out of nine and in two out of three cases, respectively. Although different cell populations, especially T cells, showed mitotic activity in unstimulated short term culture, combined immunophenotyping and karyotyping unequivocally demonstrated that CD30 and CD15 positive Hodgkin and Sternberg-Reed cells represented the chromosomally aberrant clones.
Asunto(s)
Enfermedad de Hodgkin/genética , Adulto , Anciano , Femenino , Enfermedad de Hodgkin/inmunología , Humanos , Inmunofenotipificación , Cariotipificación , Masculino , Persona de Mediana EdadRESUMEN
A large number of G-protein coupled receptors are known to modulate adenylyl cyclase activity. In order to find new compounds modulating the activity of specific receptor subtypes we developed a cellular screening system that measures the biological activity of drugs acting on receptors rather than merely their binding characteristics. The activity of the receptor coupling to the cAMP signal transduction pathway was measured via transcriptional activation of a reporter gene. A chinese hamster ovary cell line was stably transformed with a reporter plasmid containing the firefly luciferase gene under the transcriptional control of multiple cAMP responsive elements (CRE). This CRE reporter cell line exhibited 20 to 30-fold induction of luciferase activity upon stimulation of adenylyl cyclase with forskolin, but did not respond to dopamine agonists. Stable test cell lines were developed by transfecting reporter cell lines with human dopamine D1 and D5 receptor genes, respectively. Treatment of these test cell lines with dopamine receptor agonists and antagonists modulated the luciferase expression in a dose-dependent manner. The rank of potency of dopamine receptor agonists and antagonists was in agreement with reported data obtained from binding studies. The non-isotopic assay can be performed in microtiter plate format and is far less work intensive than the determination of adenylyl cyclase activity by direct cAMP measurement. This technology could also be utilized for discovery of new classes of compounds, e.g. allosteric effectors or non competitive ligands.
Asunto(s)
AMP Cíclico/fisiología , Luciferasas/genética , Receptores de Dopamina D1/fisiología , Receptores Dopaminérgicos/fisiología , Animales , Secuencia de Bases , Células CHO , Cricetinae , Humanos , Luciferasas/análisis , Datos de Secuencia Molecular , Plásmidos/genética , Receptores de Dopamina D5 , Proteínas Recombinantes , Secuencias Reguladoras de Ácidos Nucleicos , Transfección/métodosRESUMEN
A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, linked to the phospholipase C second messenger system. The human adenocarcinoma cell line A549 was transformed with the Photinus pyralis luciferase gene under the control of the ICAM-1 gene 5'regulatory region and, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the phospholipase C signal transduction pathway. In this NK2 receptor test cell line, expression of luciferase was inducible by neurokinin A and other NK2-specific agonists. The order of potency of the three neurokinins substance P, neurokinin A and neuromedin K was consistent with published data and results from ligand binding studies performed with the same NK2 test cell line. The agonistic effect of neurokinin A could be inhibited in a dose-dependent manner by simultaneous addition of NK2-specific antagonists or protein kinase C-inhibitors. Similarly, a stable test cell line expressing the human serotonin 2 receptor was established. Agonist-induced luciferase expression in this cell line was abolished in the presence of 5-HT2-specific antagonists. These cellular assay systems can be employed for the identification of competitive, non-competitive and allosteric modulators of the NK2 and the 5-HT2 receptor, and they represent prototypes for analogous test cell lines for other phospholipase C-coupled receptors.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Neuroquinina-2/análisis , Receptores de Serotonina/análisis , Bioensayo/métodos , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Molécula 1 de Adhesión Intercelular/genética , Luciferasas/genética , Neuroquinina A/farmacología , Neuroquinina B/farmacología , Fosfatidilinositoles/metabolismo , Sistemas de Mensajero Secundario , Sustancia P/farmacología , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
The numerous biological activities of tumor necrosis factor (TNF) appear mediated by two types of receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular mass. To test TR55 for its individual role in signaling across the membrane, a cDNA coding for the human TR55 was stably expressed in murine 70Z/3 pre-B cells, which lack binding sites for, and proved nonresponsive to human TNF. The transfected TR55 showed high affinity ligand binding and active internalization. It is demonstrated that the TNF signaling cascade, i.e. stimulation of protein kinase C, sphingomyelinase, and phospholipase A2, production of the second messengers diacylglycerol and ceramide, can occur completely through exclusive binding of TNF to TR55. The p55 TNF-binding site functions as an autonomous TNF receptor that mediates key signal transduction pathways, which may control the majority of TNF actions.
Asunto(s)
Receptores de Superficie Celular/fisiología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Ceramidas/metabolismo , Diglicéridos/metabolismo , Elementos de Facilitación Genéticos , Duplicado del Terminal Largo de VIH , VIH-1/genética , Humanos , Cinética , Células L , Ratones , Datos de Secuencia Molecular , Peso Molecular , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/metabolismo , Ensayo de Unión Radioligante , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral , Sistemas de Mensajero Secundario/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Soluble forms of receptors for tumor necrosis factor (TNF) might be important for regulating the actions of TNF. Site-directed in vitro mutagenesis was employed to examine the processing of the p55 tumor necrosis factor receptor chain (TNF-R55) into soluble TNF-binding protein (TNF-R55-BP). An Asn/Val sequence close to the transmembrane region in TNF-R55 was indicated as a putative cleavage site for proteolytic processing. By mutagenesis, Asn and Val were replaced with Gly and Ala, respectively. Expression in Chinese hamster ovary (CHO) cells resulted in identical binding of ligand to mutated receptors as compared to receptors not subjected to mutagenesis. Turnover rates of receptors as judged by disappearance of TNF binding capacity after inhibition of de novo protein synthesis and downregulation in response to incubation with phorbol esters were also identical between wild-type and mutated receptors. However, mutations of the cleavage site resulted in a decreased spontaneous and phorbol ester-induced release of soluble receptor (TNF-R55-BP) which was almost abolished when both Asn and Val were mutated. Our results clearly demonstrate the importance of an Asn/Val sequence for proteolytic processing of the TNF-R55 into soluble TNF-R55-BP and indicate that phorbol ester-induced downregulation of the TNF-R55 may be dissociated from proteolytic cleavage of the receptor.
Asunto(s)
Receptores de Superficie Celular/genética , Animales , Secuencia de Bases , Unión Competitiva , Células CHO , Cricetinae , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/química , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes/biosíntesis , SolubilidadRESUMEN
Clones encoding the entire coding and 3' untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping. DNA blot analysis and sequence analysis. The coding region and the 3' untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.
Asunto(s)
Cromosomas Humanos Par 12 , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Factor de Necrosis Tumoral alfa , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Humanos , Células Híbridas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores del Factor de Necrosis Tumoral , Mapeo RestrictivoAsunto(s)
Proteínas Asociadas a Microtúbulos , Secuencia de Aminoácidos , Animales , Fenómenos Químicos , Química Física , Metabolismo Energético , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/metabolismo , Datos de Secuencia MolecularRESUMEN
Tumor necrosis factor-alpha (TNF-alpha), a protein released by activated macrophages, is involved in a wide variety of human diseases including septic shock, cachexia, and chronic inflammation. TNF binding protein (TNF-BP), a glycoprotein with high affinity to TNF-alpha isolated from urine, acts as an inhibitor of TNF-alpha by competing with the cell-surface TNF receptor. We report here the partial amino acid sequencing of human TNF-BP as well as the isolation, sequence, and expression of cDNA clones encoding a human and rat TNF receptor. The calculated Mr of the mature human and rat TNF receptor chains is 47,526 and 48,072, respectively. The extracellular ligand binding domain represents the soluble TNF-BP which is released by proteolytic cleavage. TNF-BP contains 24 cysteine residues and three potential N-glycosylation sites and shows sequence homology to the extracellular portions of TNF-R p80 chain and nerve growth factor receptor. Transfection of the human TNF receptor cDNA into mammalian cells resulted in increased binding capacity for TNF-alpha and increased reactivity with a monoclonal antibody directed against the human TNF receptor chain p60. When a stop codon was introduced into the cDNA at the site corresponding to the carboxyl terminus of TNF-BP, transfected cells secreted a protein that reacted with antibodies raised against natural TNF-BP.
Asunto(s)
Expresión Génica , Receptores de Superficie Celular/genética , Factor de Necrosis Tumoral alfa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Plásmidos , Poli A/análisis , Reacción en Cadena de la Polimerasa , ARN/análisis , ARN Mensajero , Ratas , Receptores de Factor de Crecimiento Nervioso , Receptores del Factor de Necrosis Tumoral , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A case of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) with four cytogenetically different cell clones (49,XX,+5,+19,+21/47,XX,+X/46,XX,inv (14)(q11q32)/45,X,-X) is reported. To our knowledge, this is the first case of AILD with an inv(14)(q11q32), thus probably involving the T-cell receptor alpha-chain gene. The cytogenetic findings are discussed with respect to the possible progression of AILD to malignant lymphoma.
Asunto(s)
Inversión Cromosómica , Cromosomas Humanos Par 14 , Linfadenopatía Inmunoblástica/genética , Bandeo Cromosómico , Femenino , Humanos , Linfadenopatía Inmunoblástica/patología , Cariotipificación , Ganglios Linfáticos/ultraestructura , Persona de Mediana EdadRESUMEN
The inv(14)(q11q32) has been reported to be a specific chromosome aberration in T-cell chronic lymphocytic leukemia (T-CLL) and different types of T-cell lymphomas. Here, the first case of B-CLL with a clonal inv(14)(q11q32) in a phytohemagglutinin-stimulated bone marrow culture is presented. In a subsequent study 9 months later, tetradecanoyl-o-phorbol-13-acetate stimulated blood and bone marrow cultures revealed a clone with a t(1;11)(p13.2;q23.1) and an aberrant chromosome 17.
Asunto(s)
Inversión Cromosómica , Cromosomas Humanos Par 14 , Leucemia Linfocítica Crónica de Células B/genética , Anciano , Bandeo Cromosómico , Humanos , Cariotipificación , MasculinoRESUMEN
Tau proteins consist of a family of proteins, heterogeneous in size, which associate with microtubules in vivo and are induced during neurite outgrowth. In humans, tau is one of the major components of the pathognomonic neurofibrillary tangles in Alzheimer's disease brain. Screening of a cDNA library prepared from bovine brain led to the isolation of several cDNA clones encoding tau proteins with different N termini and differing by insertions or deletions, suggesting differential splicing of the tau transcripts. One of the N-terminal domains and the repeated C-terminal domain of the encoded tau proteins are recognized by polyclonal antibodies to bovine tau. The bovine tau proteins are highly homologous to murine and human tau, especially within the repeated C-terminal domain. Compared with murine and human tau, bovine tau contains the insertion of three longer segments, one of which is an additional characteristic repeat. Portions of tau proteins generated by in vitro translation were used to show that these repeats represent tubulin-binding domains, two of which are sufficient to bind to microtubules assembled from purified tubulin in the presence of taxol.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Clonación Molecular , ADN/genética , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/genética , Proteínas tauRESUMEN
Tau, a major class of microtubule-associated proteins, consists of a family of proteins that are heterogeneous in molecular weight. The presence of internal deletions in previously described cDNA clones for murine and bovine tau suggested that alternative splicing of transcripts could account for the protein size heterogeneity. Analysis of the exon-intron structure of the bovine tau gene provided sequence information necessary to detect new variants of tau transcripts by in vitro amplification techniques. The variant transcripts found corresponded to mRNA species missing one or more exons, which suggested that by skipping various exons during mRNA splicing, a family of proteins is generated. Four major tau protein isoforms isolated from bovine brain were identified by comparison with translation products of cDNA constructs and the use of antisera raised against synthetic peptides. These studies provide reagents and a basis for analyzing potentially altered forms of tau proteins in brains of patients with Alzheimer's disease.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Empalme del ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN/genética , Exones , Intrones , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Mapeo Restrictivo , Transcripción Genética , Proteínas tauRESUMEN
Using a human interferon-alpha (IFN-alpha) cDNA probe, several recombinant phages containing type I IFN genes were isolated from a canine genomic library. One of these phages contains two complete CaIFN-alpha genes with identical coding sequences, and a second one a slightly different IFN-alpha gene. The IFN-alpha protein sequences contain six cysteine residues as well as two or three potential N-glycosylation sites. Expression of mature CaIFN-alpha 1 in E. coli results in antiviral activity on dog cells. Genomic analysis using an equine IFN-omega probe and DNA sequencing suggests the deletion of IFN-omega genes from canine genome.