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
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.