Isolation of human-mouse somatic cell hybrids producing human prolactin: dominant expression of hormone secretion and regulation.
Human PRL (hPRL)-secreting adenoma cells obtained at hypophysectomy were fused with a mutant mouse fibroblast line (LMTK-) which is aminopterin sensitive due to a deficiency in the enzyme thymidine kinase. After fusion with polyethylene glycol, cells containing nuclear material from the two parental lines (heterokaryons) were selected in medium containing hypoxanthine, aminopterin, and thymidine, and resultant clones were screened for hPRL secretion. Functional human X mouse somatic cell hybrid clones secreting hPRL were isolated in order to study hPRL gene expression and regulation. Positive hybrid clones were subcultured and have sustained hPRL secretion. The hybrid nature of the cells was confirmed by fibroblastic morphology resembling the mouse parental cell, mixed karyotype of mouse and human chromosomes, and mixed isozyme banding pattern for human and mouse glucose-6-phosphate dehydrogenase and malic enzyme. Specific expression of the hPRL gene was demonstrated by the presence of electron microscopic secretory granules (650-800 nm), positive immunoperoxidase staining using anti-hPRL serum, and sustained secretion of immunoreactive hPRL, which comigrated with [125I] hPRL standard on Sephadex chromatography. Hormonal modulation of hPRL gene expression by TRH was dominantly expressed in the hybrid cell. Human chromosome 6 was identified in hybrid cells secreting hPRL, and the cells expressed human malic enzyme, a marker for this chromosome, thus confirming the chromosome assignment of the hPRL gene. The results show that functional replicating hybrids secreting hPRL can be isolated. The technique provides a useful in vitro model for the study of hPRL gene expression and modulation.
D000818:Animals; D002469:Cell Separation; D002850:Chromatography, Gel; D002906:Chromosomes, Human, 6-12 and X / Q000648:ultrastructure; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D005786:Gene Expression Regulation; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism* / Q000648:ultrastructure; D007124:Immunoenzyme Techniques; D007621:Karyotyping; D008291:Malate Dehydrogenase / Q000032:analysis; D051379:Mice; D008854:Microscopy, Electron; D011388:Prolactin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D008291:Malate Dehydrogenase