vasoactive-intestinal-peptide and Cell-Transformation--Viral

The molecular and functional expression of serpentine membrane receptors for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and calcitonin (CT) were characterized in human thymus and thymomas from myasthenia gravis (MG) patients and thymic epithelial cells either in primary culture (PTEC) or transformed by the simian virus 40 large T (SV40LT) oncogene (LT-TEC). Using RT-PCR combined with Southern analysis, we identified the PCR products corresponding to the receptor (-R) transcripts for VIP, CGRP, and CT in thymus from control subjects and MG patients with either hyperplasia or thymoma. Similar expressions of the VIP- and CGRP-R transcripts were observed in PTEC, whereas the CT-R message was not detected. In LT-TEC, the signals for VIP-R, CGRP-R, and CT-R transcripts were seen with a lower intensity than those in control and MG thymus. In agreement with our molecular analysis, 1) VIP was the most potent peptide among VIP-related peptides (VIP > PACAP > PHM > PHV) to stimulate cAMP production through specific type 1 VIP receptors in both PTEC and LT-TEC; 2) cAMP generation was induced by CGRP in PTEC and by CT in LT-TEC; 3) in frozen thymic sections and by flow cytometry, type 1 VIP-R, CGRP-R, and CT-R were localized in epithelial cells; and 4) in parallel, the transcription of the acetylcholine receptor alpha subunit (the main autoantigen in MG) was induced by CGRP and CT in PTEC and LT-TEC, respectively. Our data suggest that the neuroendocrine peptides VIP, CGRP, and CT may exert functional roles during MG and malignant transformation of the human thymus.

Topics: Adolescent; Adult; Antigens, Viral, Tumor; Blotting, Southern; Calcitonin; Calcitonin Gene-Related Peptide; Cell Culture Techniques; Cell Line, Transformed; Cell Transformation, Viral; Child; Child, Preschool; Epithelial Cells; Flow Cytometry; Gene Expression Regulation; Humans; Immunohistochemistry; Infant; Middle Aged; Myasthenia Gravis; Receptors, Calcitonin; Receptors, Calcitonin Gene-Related Peptide; Receptors, Cholinergic; Receptors, Neuropeptide; Receptors, Vasoactive Intestinal Peptide; Reverse Transcriptase Polymerase Chain Reaction; Simian virus 40; Thymoma; Thymus Gland; Vasoactive Intestinal Peptide

Intestinal epithelial cells were isolated from a fetus with cystic fibrosis (CF) and transfected with a plasmid vector recombined with the ori- mutant of SV40. A population of proliferative cells was then subcloned and designated as CFI-3. These cells had a doubling time of 24 h and were maintained in culture for up to 25 passages. At passage 8, CFI-3 cells did not produce any tumors in nude mice. Northern blot and immunofluorescence studies indicated that the extended lifespan of CFI-3 cells results in genomic insertion of SV40 LT. Intestinal CFI-3 cells are epithelial, according to the expression of the human cytokeratin 18 gene and poorly differentiated by phase-contrast and electron microscopy. Functional membrane receptors activated by vasoactive intestinal peptide (VIP), its natural analogue pituitary adenylate cyclase activating peptide (PACAP-38), and isoproterenol were observed in CFI-3 cells. Restriction fragment length polymorphism analysis of the PstI KM19 site revealed that the cftr locus was identical in the chorionic villi and in CFI-3 cells. The manifestation of CF in this family was not related to the common mutation delta F508, since this fetus was heterozygous for the substitutions S549N and N1303K. Chloride transport, assessed by the 125I efflux, was induced in CFI-3 cells by the calcium inophore ionomycin, but not by the adenylate cyclase activator forskolin, and was inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. These results were confirmed in patch clamp studies in which the cpt cAMP analogue failed to stimulate membrane currents, while the calcium ionophore ionomycin stimulated inward currents. We conclude that intestinal CFI-3 cells retain the CF phenotype relating to defective regulation of Cl- channels, and therefore constitute a suitable model, 1) for elucidating the function of CFTR protein, 2) developing new therapeutic agents, and 3) correcting the CF defect by gene replacement therapy in vitro.

Topics: Antigens, Polyomavirus Transforming; Base Sequence; Cell Line; Cell Transformation, Viral; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Humans; In Vitro Techniques; Intestinal Mucosa; Ionomycin; Isoproterenol; Membrane Proteins; Molecular Sequence Data; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Potassium Channels; Transfection; Vasoactive Intestinal Peptide