title: Temperature-dependent, neurotrophic factor-elicited, neuronal differentiation in adrenal chromaffin cell line immortalized with temperature-sensitive SV40 T-antigen.
abstract: We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature-sensitive simian virus 40 large T-antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33 degrees C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine-synthesizing enzymes and secretory vesicle-associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non-permissive temperature of 39 degrees C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron-like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39 degrees C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes including neuron-specific enolase, growth-associated protein-43, microtubule-associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33 degrees C, and also had the capacity to terminally differentiate into neuron-like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non-permissive 39 degrees C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.
authors.affiliation: Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya, Japan.
authors.forename: Tomiyasu
authors.initials: T
authors.lastname: Murata
authors.forename: Kiyomi
authors.initials: K
authors.lastname: Hikita
authors.forename: Masaru
authors.initials: M
authors.lastname: Tsuboi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Niwa
authors.forename: Misao
authors.initials: M
authors.lastname: Suzuki
authors.forename: Norio
authors.initials: N
authors.lastname: Kaneda
mesh_terms: D000310:Adrenal Gland Neoplasms / Q000235:genetics* / Q000473:pathology; D000311:Adrenal Glands / Q000166:cytology*; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology; D002999:Clone Cells; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009414:Nerve Growth Factors / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000502:physiology; D013696:Temperature; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
chemical_list: D000952:Antigens, Polyomavirus Transforming; D015415:Biomarkers; D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D016222:Fibroblast Growth Factor 2; D014446:Tyrosine 3-Monooxygenase