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Mol Cell Biol, May 1998, p. 3089-3100, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Gene Amplification in a p53-Deficient Cell Line Requires Cell Cycle Progression under Conditions That Generate DNA Breakage

Thomas G. Paulson,1,2,dagger Alexandru Almasan,3 Linnea L. Brody,4 and Geoffrey M. Wahl2,*

Department of Biology, University of California, San Diego, La Jolla, California 920931; Gene Expression Lab, The Salk Institute for Biological Studies, La Jolla, California 920372; Departments of Cancer Biology and Radiation Oncology, Cleveland Clinic Foundation, Cleveland, Ohio 441953; and Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 981094

Received 26 September 1997/Returned for modification 18 November 1997/Accepted 6 February 1998

Amplification of genes involved in signal transduction and cell cycle control occurs in a significant fraction of human cancers. Loss of p53 function has been proposed to enable cells with gene amplification to arise spontaneously during growth in vitro. However, this conclusion derives from studies employing the UMP synthesis inhibitor N-phosphonacetyl-L-aspartate (PALA), which, in addition to selecting for cells containing extra copies of the CAD locus, enables p53-deficient cells to enter S phase and acquire the DNA breaks that initiate the amplification process. Thus, it has not been possible to determine if gene amplification occurs spontaneously or results from the inductive effects of the selective agent. The studies reported here assess whether p53 deficiency leads to spontaneous genetic instability by comparing cell cycle responses and amplification frequencies of the human fibrosarcoma cell line HT1080 when treated with PALA or with methotrexate, an antifolate that, under the conditions used, should not generate DNA breaks. p53-deficient HT1080 cells generated PALA-resistant variants containing amplified CAD genes at a frequency of >10-5. By contrast, methotrexate selection did not result in resistant cells at a detectable frequency (<10-9). However, growth of HT1080 cells under conditions that induced DNA breakage prior to selection generated methotrexate-resistant clones containing amplified dihydrofolate reductase sequences at a high frequency. These data demonstrate that, under standard growth conditions, p53 loss is not sufficient to enable cells to produce the DNA breaks that initiate amplification. We propose that p53-deficient cells must proceed through S phase under conditions that induce DNA breakage for genetic instability to occur.

* Corresponding author. Mailing address: Gene Expression Lab, The Salk Institute for Biological Studies, La Jolla, CA 92037. Phone: (619) 453-4100. Fax: (619) 457-2762. E-mail: wahl{at}salk.edu.

dagger Present address. Fred Hutchinson Cancer Research Center, Seattle WA 98104.

Mol Cell Biol, May 1998, p. 3089-3100, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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