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 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.
Christman
Shelly A
SA
Department of Animal Science, University of Minnesota, 495 AnSci/VetMed, 1988 Fitch Ave., St. Paul, MN 55108, USA.
Kong
Byung-Whi
BW
Landry
Megan M
MM
Kim
Hyunggee
H
Foster
Douglas N
DN
D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002642:Chick Embryo / Q000166:cytology* / Q000502:physiology*; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000032:analysis; D003513:Cycloheximide / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005434:Flow Cytometry; D016193:G1 Phase; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D050762:Cyclin-Dependent Kinase Inhibitor p15; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D003513:Cycloheximide; D008156:Luciferases