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p53-dependent growth arrest of REF52 cells containing newly amplified DNA

Y Ishizaka1, M V Chernov, C M Burns

  • 1Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195, USA.

Proceedings of the National Academy of Sciences of the United States of America
|April 11, 1995
PubMed

Insights

Simian virus 40 tumor antigen enables gene amplification in rat cells by binding to p53. Broken DNA from bridge-breakage-fusion cycles triggers p53-mediated growth arrest, regulating gene amplification permissivity.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Rat REF52 cells are generally resistant to gene amplification.
  • Simian virus 40 tumor (T) antigen and dominant-negative p53 mutants can induce permissivity for gene amplification.
  • T antigen's effect is primarily attributed to its interaction with p53.

Purpose of the Study:

  • To investigate the role of p53 in regulating gene amplification permissivity.
  • To explore the mechanism of gene amplification using a temperature-sensitive T antigen mutant.
  • To understand the relationship between DNA breaks and growth arrest during amplification.

Main Methods:

  • Introduction of a temperature-sensitive simian virus 40 T antigen (tsA58) into REF52 cells.
  • Selection of cells resistant to N-(phosphonacetyl)-L-aspartate (PALA) to identify gene amplification.
  • Analysis of PALA-resistant colonies at permissive and non-permissive temperatures to assess growth arrest and DNA integrity.

Main Results:

  • Freshly isolated PALA-resistant cells (amplifying CAD genes via BBF cycles) arrested at non-permissive temperatures (T antigen absent), with arrest reversed upon T antigen restoration.
  • Established PALA-resistant clones (approx. 10^7 cells) did not arrest at non-permissive temperatures, despite containing amplified DNA.
  • All examined clones showed amplified carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) genes, indicative of bridge-breakage-fusion (BBF) cycles.

Conclusions:

  • p53-mediated growth arrest is active early in gene amplification, specifically when cells contain broken DNA generated by BBF cycles.
  • Healed DNA ends in established clones prevent p53-mediated arrest, even with amplified DNA and intact p53 pathway at non-permissive temperatures.
  • Bridge-breakage-fusion (BBF) cycles are a key amplification mechanism, and the resulting DNA breaks act as critical signals for regulating gene amplification permissivity.

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