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Volume 17, Number 4, Issue of February 15, 1997 pp. 1217-1225
Copyright ©1997 Society for NeuroscienceCharacterization of a CNS Cell Line, CAD, in which Morphological Differentiation Is Initiated by Serum Deprivation
Received Sept. 25, 1996; revised Nov. 20, 1996; accepted Nov. 25, 1996.
Yanping Qi2, James K. T. Wang2, Michael McMillian1, and Dona M. Chikaraishi1 1 Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, and 2 Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111
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
-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.
Key words: CNS cell line; catecholamine; tyrosine hydroxylase; neuronal differentiation; T antigen; microtubule; intermediate filament; synaptic vesicle
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