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

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Abstract
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 p21WAF1, p27Kip1, MDM‐2, and the p16INK4a alternate reading frame (ARF) sequence showed similar patterns of differential mRNA expression. Levels of p15INK4b 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.