p53-Dependent Growth Arrest of REF52 Cells Containing Newly Amplified DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1995-04, Vol.92 (8), p.3224-3228
• Main Authors: Ishizaka, Yukihito, Chernov, Michail V., Burns, Carmel M., Stark, George R.
• Format: Article
• Language: English
• Subjects: Animals; Antigens, Viral, Tumor - genetics; Antigens, Viral, Tumor - physiology; Aspartate Carbamoyltransferase; Aspartic Acid - analogs & derivatives; Aspartic Acid - pharmacology; Carbamyl Phosphate - metabolism; Cell Cycle - genetics; Cell Cycle - physiology; Cell growth; Cell Line; Cell lines; Cells; Cellular biology; CHO cells; Chromosomes; Cyclin-Dependent Kinase Inhibitor p21; Cyclins - genetics; Daughter cells; Deoxyribonucleic acid; Dihydroorotase; DNA; DNA Damage; DNA Replication; Drug Resistance; Gene Amplification; Genes; Hepatocytes; Hot Temperature; In Situ Hybridization, Fluorescence; Ligases - genetics; Mutation; Phosphonoacetic Acid - analogs & derivatives; Phosphonoacetic Acid - pharmacology; Rats; Recombinant Proteins; RNA, Messenger - biosynthesis; Selection, Genetic; Simian virus 40 - genetics; Tumor Suppressor Protein p53 - physiology; Viral tumor antigens
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.8.3224

The rat cell line REF52 is not permissive for gene amplification. Simian virus 40 tumor (T) antigen converts these cells to a permissive state, as do dominant negative mutants of p53, suggesting that the effect of T antigen is due mainly to its ability to bind to p53. To manipulate permissivity, we introduced a temperature-sensitive mutant of T antigen (tsA58) into REF52 cells and selected for resistance to N-(phosphonacetyl)-L-aspartate (PALA). Most freshly isolated PALA-resistant colonies, each of ≈200 cells, selected at a permissive temperature, arrested when shifted to a nonpermissive temperature. Growth arrest was stable, with no evidence of apoptosis, as long as T antigen was absent but was reversed when T antigen was restored. In contrast, PALA-resistant clones grown to ≈107cells at a permissive temperature did not arrest when shifted to a nonpermissive temperature. All PALA-resistant clones examined had amplified carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) genes, present in structures consistent with a mechanism involving bridge-breakage-fusion (BBF) cycles. We propose that p53-mediated growth arrest operates only early during the complex process of gene amplification, when newly formed PALA-resistant cells contain broken DNA, generated in BBF cycles. During propagation under permissive conditions, the broken DNA ends are healed, and, even though the p53-mediated pathway is still intact at a nonpermissive temperature and the cells contain amplified DNA, they are not arrested in the absence of broken DNA. The data support the hypothesis that BBF cycles are an important mechanism of amplification and that the broken DNA generated in each cycle is a key signal that regulates permissivity for gene amplification.