Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation.
A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.
Qi
Y
Y
Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Wang
J K
JK
McMillian
M
M
Chikaraishi
D M
DM
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D002395:Catecholamines / Q000378:metabolism; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D016895:Culture Media, Serum-Free; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D009857:Oncogenes; D014407:Tumor Cells, Cultured
D000952:Antigens, Polyomavirus Transforming; D002395:Catecholamines; D016895:Culture Media, Serum-Free; D009419:Nerve Tissue Proteins