Neurofilament proteins are constitutively expressed in F9 teratocarcinoma cells

Abstract

We examined neuronal differentiation of F9 teratocarcinoma cells using retinoic acid (RA) and cyclic AMP (cAMP) as inducing agents. Neuronal differentiation was monitored using (1) cDNA probes for the rat 68-kDa neurofilament gene, (2) RT-PCR for neurofilament genes and (3) antibodies against several neuronal differentiation markers. We found by Northern blotting that the uninduced F9 cells, grown in 10% serum, expressed mRNA for the 68-kDa neurofilament protein whereas the control cells, grown in 3% serum, failed to express detectable levels of the 68-kDa neurofilament transcripts. However, RT-PCR allowed detection of both the 68- and 200-kDa neurofilament gene transcripts in F9 cells with or without the inducing agents. Under serum deprivation, a prolonged (>10–15days) cultivation of the F9 cells in the presence of RA and cAMP was required for the expression of detectable levels of the 68-kDa neurofilament transcripts and immunocytochemically detectable neurofilament proteins. Treatment of the F9 cells with RA and cAMP was also required for induction of their neuronal phenotype.Immunocytochemically, the uninduced F9 cells expressed several neuronal antigens including the 68-kDa neurofilament protein, the 200-kDa neurofilament protein, neural cell adhesion molecule (N-CAM) and a neuronal specific tubulin isoform (TUJI). The control cells expressed N-CAM and TUJI, but failed to express the neurofilament proteins. A subclone, D9L2, derived from a single F9 parent cell, expressed both TUJI and neurofilament proteins, but no N-CAM molecule.The present results indicate that both the 68- and the 200-kDa neurofilament genes are constitutively active in uninduced F9 teratocarcinoma cells. Under serum deprivation both RA and cAMP are required for expression of detectable levels of neurofilament mRNA and protein. Thus, serum deprivation of the F9 cells either down-regulates the NF gene expression, stability of mRNA or degradation of the NF-proteins. Importantly, expression of a neuronal phenotype by a subpopulation of F9 cells appears to require administration of RA and cAMP, although expression of neuronal marker proteins is not dependent on these agents. Lastly, we demonstrate cloning of a novel cell line (D9L2), derived from a single F9 parent cells, capable of extending neurites and expressing several neuronal antigens under serum deprivation without the requirement of RA and cAMP. We propose that the D9L2 cell line may offer a simplified F9 cell model system to investigate the mechanisms of neuronal differentiation.