Characterization of a CNS Cell Line, CAD, in which Morphological Differentiation Is Initiated by Serum Deprivation

Abstract

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.

Selective destruction of glioblastoma cells by interference with the activity or expression of ATF5

“…As noted above, a majority of the latter cells express ATF5 (Figure 2). In addition, two cell lines that express ATF5, HEK293 cells (Aiello et al, 1979) and CAD cells (Qi et al, 1997), showed no excess cell death when transfected with d/n ATF5 (data not shown). PC12 pheochromocytoma cells and embryonic neural progenitor cells also express high levels of ATF5 and their capacity to differentiate is accelerated by ATF5 d/n and siRNA constructs (Angelastro et al, 2003).…”

Differentiation of an adult neuron cell line increases susceptibility to rabies infection.

“…This greater percentage of infection in differentiated cell cultures is possibly because differentiated CAD-R1 cells acquire morphological and biochemical characteristics which they do not possess in their undifferentiated state (16). Moreover the cell differentiation could change enzymatic machinery involved in viral replication.…”

“…Both cell lines express biochemical neuron markers (synaptophisin 38 and neurofilament) and show neuronal physiological characteristics (voltage-dependent sodium and calcium currents) (18,19). Additionally, CAD cells express other biochemical markers such as β-tubulin III, GAP-43 and sinaptotagmin 65 with stronger intensity (16,20). The main characteristic of CAD is that it acquires neuronal morphological features when it is cultured in serum-free medium.…”

“…CVS-11 adapted to mouse neonatal brain (donated to the Colombian NIH by PAHO/WHO) was used to produce two different virus strains: BHK-21 adapted CVS (CVS-BHK) and adult ICR mouse brain-adapted CVS (CVS-MB) at 10 5.3 and 10 7.2 LD 50 titres, respectively. The virus produced was titrated by calculating the LD 50 (lethal dose 50) following intracerebral injections of increasing dilutions in young ICR mice (14)(15)(16). Viral dilutions were prepared in appropriated medium (MM or MD) from stocks of CVS-BHK and CVS-MB strain.…”

“…A CNS neuron cell line (called CAD) has recently been isolated (16), this being a variant of the CATHα catecolaminergic cell line (obtained by direct tumorogenesis in adult mouse locus coeruleus) (17). Both cell lines express biochemical neuron markers (synaptophisin 38 and neurofilament) and show neuronal physiological characteristics (voltage-dependent sodium and calcium currents) (18,19).…”