RESUMO
A patient with a primary undifferentiated carcinoma of the nasopharynx manifested the clinical syndrome of inappropriate antidiuretic hormone secretion (SIADH). Immunohistochemical techniques demonstrated the presence of vasopressin, neurophysin, and their precursor (propressophysin) in the cancer cells. In situ hybridization additionally confirmed the expression of propressophysin messenger RNA in these cells. To the knowledge of the authors, this represents not only the first case of SIADH caused by carcinoma of the nasopharynx, but also the first report of pathologic confirmation of the syndrome with the use of both molecular and immunologic probes.
Assuntos
Carcinoma de Células Escamosas/complicações , Síndrome de Secreção Inadequada de HAD/etiologia , Neoplasias Nasofaríngeas/complicações , Ocitocina , Arginina Vasopressina/análise , Carcinoma de Células Escamosas/química , DNA de Neoplasias/análise , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/química , Neurofisinas/análise , Hibridização de Ácido Nucleico , Precursores de Proteínas/análise , Vasopressinas/análiseRESUMO
A monoclonal antibody (mAb L6) to a small-cell lung carcinoma surface antigen recognizes a common epitope of vasopressin-neurophysin and oxytocin-neurophysin in hypothalamic nuclei. We now report on the identification of a neurophysin-like precursor in human lung carcinoma (LX-1) cell membrane. mAb L6 immunoaffinity chromatography of solubilized membranes resulted in a single band of approximately 45 kDa. Western blot analysis demonstrated immunoreactivity of this band with mAb L6, anti-vasopressin, and an antibody to the vasopressin precursor, pro-pressophysin. N-terminal sequencing of this band demonstrated a 21-amino acid homology with the N terminus of human pro-pressophysin, and substitution of a Cys33 residue in the tumor antigen with Arg33. Absence of immunoreactivity with the antibodies described above in cytosolic extracts and culture medium suggests nonsecretion of processed or intact pro-pressophysin-like peptide. Northern analysis of LX-1 mRNA with a 30-mer to the C terminus of rat pro-pressophysin resulted in a band of approximately 1000 base pairs, 250 base pairs larger than hypothalamic message. In situ hybridization of LX-1 tumor-bearing nude rat brain with the same probe demonstrated specific hybridization in rat hypothalamus and xenografted tumor. These findings suggest expression of a pro-pressophysin-like protein in this tumor cell line that is preferentially targeted to the cell membrane.
Assuntos
Antígenos de Neoplasias/genética , Carcinoma de Células Pequenas/imunologia , Epitopos/análise , Neoplasias Pulmonares/imunologia , Neurofisinas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/isolamento & purificação , Northern Blotting , Linhagem Celular , Membrana Celular/imunologia , Cromatografia de Afinidade , Humanos , Dados de Sequência Molecular , Neurofisinas/genética , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Homologia de Sequência do Ácido NucleicoRESUMO
A monoclonal antibody (mAb L6) to a carcinoma surface antigen has previously been shown to recognize neurophysins (NP), proteins associated with oxytocin and vasopressin. L6-reactivity in rat hypothalamus was confined to magnocellular neuronal systems. No staining was detected in parvicellular suprachiasmatic or paraventricular systems. mAb L6 immunoprecipitated vasopressin-neurophysin only under reducing conditions, and detected it in Western blots only after gel-renaturation and electroblotting in basic buffer. These findings suggest L6-reactivity to NP is conformation-sensitive, and imply NP expression in a unique configurational form in hypothalamic parvicellular systems.
Assuntos
Anticorpos Monoclonais , Hipotálamo/citologia , Neurônios/citologia , Neurofisinas/análise , Animais , Hipotálamo/química , Immunoblotting , Imuno-Histoquímica , Masculino , Eminência Mediana/citologia , Peso Molecular , RatosRESUMO
Mouse monoclonal antibody (mAb) L6 identifies an antigen expressed on the cell surface of many different human carcinomas. While studying the binding activity of mAb L6 to intracerebral tumor xenografts of human lung carcinoma LX-1 cells in nude rats using immunohistological techniques, we observed that L6 can also bind to a cytoplasmic antigen expressed in the magnocellular component of the hypothalamo-neurohypophysial system. Double-labeling experiments with antisera to vasopressin and oxytocin confirmed the localization of L6 immunoreactivity within both peptide-containing cell groups. L6 immunoreactivity in Brattleboro rats (with genetic deletion in the vasopressin gene) was exclusively localized within oxytocin neurons. Oxytocin and vasopressin failed to block L6 staining which suggested that its target epitope resides within the neurophysin sequence, and this explanation was supported by the finding that adsorption of L6 with porcine neurophysin completely eliminated hypothalamic immunoreactivity. Western blot analysis of bovine neurophysin and human pituitary extracts identified L6-immunoreactive bands which corresponded to the position of neurophysin and pro-pressophysin, confirming that L6 immunoreactivity in hypothalamus is related to neurophysin. Thus, monoclonal antibody L6, which is highly reactive with a membrane antigen of human lung cancer cell line LX-1, recognizes a cytoplasmic epitope in hypothalamic neurons identified as neurophysin by immunohistochemistry and Western analysis.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Hipotálamo/análise , Neurofisinas/análise , Adsorção , Animais , Anticorpos Monoclonais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas Imunoenzimáticas , Ocitocina , Ratos , Ratos Nus , Células Tumorais Cultivadas , VasopressinasRESUMO
An immunoblotting method to detect low-molecular-weight peptides with monoclonal antibodies that normally fail to demonstrate immunoreactivity using conventional blotting techniques is described. Detection of neurophysin, insulin, calcitonin, vasopressin, and beta-endorphin electroblotted on nitrocellulose membranes was optimized after introducing four modifications into the conventional procedure. These include renaturing the gels after sodium dodecyl sulfate electrophoresis, electroblotting the renatured gels in basic transfer buffer, fixing and/or heating the blots, and using avidin/alkaline phosphatase conjugates for antigen/antibody detection. This technique likely enables the denatured peptides to regain their native conformation and, therefore, restores antigenicity and recognition by highly structural specific monoclonal antibodies. Although the most dramatic improvement with this technique is with monoclonal antibodies, a modest improvement in sensitivity can be obtained when immunoblots are probed with polyclonal antibodies. The high resolution of this system will be useful in probing blots of partial proteolytic digests of proteins with both monoclonal and polyclonal antibodies.
Assuntos
Anticorpos Monoclonais , Peptídeos/análise , Fosfatase Alcalina , Animais , Anticorpos/imunologia , Avidina , Soluções Tampão , Calcitonina/análise , Humanos , Concentração de Íons de Hidrogênio , Immunoblotting/métodos , Insulina/análise , Peso Molecular , Neurofisinas/análise , Peptídeos/imunologia , Suínos , Vasopressinas/análiseRESUMO
The physical properties of the cardiac muscarinic acetylcholine receptor (mAcChR) purified from porcine atria as recently described [Peterson, G.L., Herron, G.S., Yamaki, M., Fullerton, D.S., & Schimerlik, M.I. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4993-4997] have been examined by D2O/H2O sucrose gradient sedimentation and Sephacryl S-300 gel filtration in Triton X-405 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the sedimentation experiments the partial specific volume and sedimentation constant for the mAcChR-Triton X-405 complex were determined to be 0.813 cm3/g and 5.30 S, respectively, which lead to an estimate of the molecular weight of the complex of 143 000. Gel filtration in Triton X-405 gave an estimate of the Stokes radius (4.29 nm) and an apparent molecular weight of 116 000. Combination of sedimentation and gel filtration gave an apparent molecular weight of 137 000 and a frictional ratio (f/f0) of 1.21 for the complex. The partial specific volume of the receptor calculated from composition was 0.717 cm3/g assuming 26.5% by weight carbohydrate. The amount of bound Triton X-405 was estimated at 1.011 g/g of mAcChR, which gave an apparent molecular weight of 70 900 (sedimentation) or 68 200 (sedimentation plus gel filtration) for the uncomplexed receptor. SDS-PAGE experiments at acrylamide concentrations ranging from 6% T [monomer plus bis(acrylamide)] to 17% T gave a linear range of apparent molecular weight from 67 600 (6% T) to 98 600 (17% T), and calibration against the retardation coefficient, Kr, determined from Ferguson plots gave an apparent molecular weight of 89 100 +/- 6700. From a newly developed, novel evaluation scheme the anomalous migration of the mAcChR in SDS-PAGE was found to be due to both an excess charge density and an abnormally large shape parameter (Kr), and the true molecular weight of the protein portion of the mAcChR ligand binding polypeptide was estimated to be between 50 000 and 60 000.