GENE AMPLIFICATION IN A P53-DEFICIENT CELL-LINE REQUIRES CELL-CYCLE PROGRESSION UNDER CONDITIONS THAT GENERATE DNA BREAKAGE

Citation
Tg. Paulson et al., GENE AMPLIFICATION IN A P53-DEFICIENT CELL-LINE REQUIRES CELL-CYCLE PROGRESSION UNDER CONDITIONS THAT GENERATE DNA BREAKAGE, Molecular and cellular biology, 18(5), 1998, pp. 3089-3100
Citations number
84
Categorie Soggetti
Biology,"Cell Biology
ISSN journal
02707306
Volume
18
Issue
5
Year of publication
1998
Pages
3089 - 3100
Database
ISI
SICI code
0270-7306(1998)18:5<3089:GAIAPC>2.0.ZU;2-I
Abstract
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-phosphona cetyl-L-aspartate (PALA), which, in addition to selecting for cells co ntaining extra copies of the CAD locus, enables p53-deficient cells to enter S phase and acquire the DNA breaks that initiate the amplificat ion process. Thus, it has not been possible to determine if gene ampli fication occurs spontaneously or results from the inductive effects of the selective agent. The studies reported here assess whether p53 def iciency leads to spontaneous genetic instability by comparing cell cyc le responses and amplification frequencies of the human fibrosarcoma c ell line HT1080 when treated with PALA or with methotrexate, an antifo late that, under the conditions used, should not generate DNA breaks, p53-deficient HT1080 cells generated PALA-resistant variants containin g amplified CAD genes at a frequency of >10(-5). By contrast, methotre xate selection did not result in resistant cells at a detectable frequ ency (<10(-9)). However, grow th of HT1080 cells under conditions that induced DNA breakage prior to selection generated methotrexate-resist ant clones containing amplified dihydrofolate reductase sequences at a high frequency. These data demonstrate that, under standard growth co nditions, p53 loss is not sufficient to enable cells to produce the DN A breaks that initiate amplification. We propose that p53-deficient ce lls must proceed through S phase under conditions that induce DNA brea kage for genetic instability to occur.