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°C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine-synthesizing enzymes and secretory vesicleassociated 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°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°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°C, and also had the capacity to terminally differentiate into neuronlike cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non-permissive 39°C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation. Keywords: adrenal chromaffin cells, immortalization, neuronal differentiation, neurotrophic factor, temperature-sensitive SV40 T-antigen, tsAM5D cells. Address correspondence and reprint requests to Norio Kaneda, Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan. E-mail: nkaneda@ccmfs.meijo-u.ac.jp Abbreviations used 1: AADC, aromatic L-amino acid decarboxylase; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CgA, chromogranin A; CgB, chromogranin B; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; DBH, dopamine b-hydroxylase; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; GAP-43, growth-associated protein-43; GDNF, glia cell line-derived neurotrophic factor; MAP2, microtubule-associated protein 2; MTS tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NF160K, 160-kDa neurofilament; NF200K, 200-kDa neurofilament; NF68K, 68-kDa neurofilament; NGF, nerve growth factor; NSE, neuron-specific enolase; NT-3, neurotrophin-3; PNMT, phenylethanolamine N-methyltransferase; p75NTR, p75 neurotrophin receptor; SgII, secretogranin II; SV40T, simian virus 40 large T-antigen; TH, tyrosine hydroxylase; TrkA, tyrosine kinase A; tsSV40T, temperature-sens...

Effects of defective herpes simplex vectors expressing neurotrophic factors on the proliferation and differentiation of nervous cells in vivo

“…It should be noted that these data relate either to peripheral preparations 26 or to CNS in vitro cell systems. 8,18,[21][22][23][24][25]27 Thus, the present one is the first evidence of a synergy between FGF-2 and CNTF occurring in the CNS in vivo.…”

“…These data support the notion that these two NTFs, when acting together, promote proliferation and glial differentiation. 8,18,21 However, depending on the experimental conditions, the developmental stage and the nervous system region, oligodendroglial 24,26 and neuronal 22,23,25,27 differentiation have also been reported. It should be noted that these data relate either to peripheral preparations 26 or to CNS in vitro cell systems.…”

“…8,18,21 Under specific experimental conditions, however, the combination of FGF-2 and CNTF has also been reported to promote oligodendroglial or neuronal differentiation. [22][23][24][25][26][27] Thus, it was expected that vectors expressing FGF-2 and CNTF would mimic this effect both in vitro and in the adult brain. Indeed, this proved to be the case.…”

Protective Effects of Neurotrophic Factors on Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Apoptosis of Murine Adrenal Chromaffin Cell Line tsAM5D

“…Cell Culture-tsAM5D cells were seeded into type IV collagen-coated dishes and cultured in DMEM supplemented with G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, bFGF, and epidermal growth factor), 10% heat-inactivated FBS, 50 units/ml penicillin, and 50 g/ml streptomycin under a humidified atmosphere of 5% CO 2 and 95% air at 33°C (15).…”

“…On the basis of this experiment, we generated transgenic mice expressing the tsSV40T gene under the control of the tyrosine hydroxylase promoter to provide an ideal source for the establishment of catecholaminergic cell lines. We succeeded in establishing a conditionally immortalized adrenal chromaffin cell line, tsAM5D, from an adrenal tumor of an adult tyrosine hydroxylase-tsSV40T transgenic mouse (15). tsAM5D cells conditionally grow at the permissive temperature of 33°C and exhibit the dopaminergic phenotype.…”