Modulation of p53 expression and its role in the conversion to a fully immortalized chicken embryo fibroblast line

We have established a spontaneously immortalized chicken embryo fibroblast (CEF) cell line (SC-1) that has been in continuous culture for more than three years. This is only the second report of a spontaneously immortalized reverse transcriptase (RT)-negative chicken cell line. The SC-1 cells emerged from crisis (at about passage 29-31) with a slower growth rate than primary cells. Passage 50 SC-1 cells expressed similar levels of p53 mRNA, but slightly lower levels of p53 protein than passage 6 CEF cells. By passage 120, p53 mRNA levels were significantly decreased in the SC-1 cells, while protein levels were slightly increased compared to passage 6 CEF cells. However, functional analysis of p53 revealed reduced activity in later passage SC-1 cells. Other p53-related genes including p21^WAF1, p27^Kip1, MDM-2, and the p16 ^INK4a alternate reading frame (ARF) sequence showed similar patterns of differential mRNA expression. Levels of p15^INK4b mRNA and protein were dramatically decreased in SC-1 cells, suggesting that the Rb pathway also has been compromised. Telomerase expression was undetectable in SC-1 cells. Fluorescence-activated cell sorting analysis showed that SC-1 and primary cells contained a similar proportion of G0/G1 phase cells, unlike the only other spontaneously immortalized chicken cell line (DF-1). The present study suggests that alterations in the p53 and Rb pathways cause fluctuations in expression levels of important cell-cycle regulatory genes during crucial transition periods as the SC-1 spontaneously immortalized chicken fibroblast cells progress toward becoming a fully committed cell line.