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

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-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.

High rate of CAD gene amplification in human cells deficient in MLH1 or MSH6

“…The simplest interpretation of these observations is that PALA selects for preexisting CAD amplification events that occur spontaneously in HCT116 cells, in agreement with previous conclusions concerning the origin of CAD amplification events which are obtained in fluctuation tests (29,42). However, it has been shown that PALA exposure can induce CAD amplification in some cell lines during the selection step (43). This issue will be considered in Discussion.…”

Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells

“…Several recent studies have suggested the involvement of chromosome breakage in gene amplification (22)(23)(24)(25)(26)(27). Our study presents a possible mechanism through which a DSB can initiate palindrome formation and gene amplification.…”

“…Short IRs also are found at the genomic sites that form the junctions of palindromes in other organisms, including Leishmania and the fission yeast (15,16). In mammalian cells, the importance of DSB in gene amplification has been suggested in recent studies (22)(23)(24)(25)(26)(27), although little is known about its direct role. If palindrome formation in mammalian cells also depends on a similar short IR-mediated mechanism, the susceptibility of a gene to undergo gene amplification could be influenced by its proximity to short IR sequences, which are abundant in mammalian genomes (28).…”

Transcriptional Repression of Protein Kinase Cα via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation

“…Cell Lines-The cell lines SKLMS-1 (a human leiomyosarcoma cell line harboring a p53 point mutation at codon 245) (24) and HT1080 (a fibrosarcoma cell line deficient in p53) (25) were obtained from the American Type Culture Collection and were cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum (complete culture medium; Invitrogen) in 5% CO 2 at 37°C. Also, the cell lines Saos-2 (a p53-null osteosarcoma cell line) and U2-OS (an osteogenic sarcoma cell line with WT p53) were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum.…”