MYC abrogates p53-mediated cell cycle arrest in N-(phosphonacetyl)-L-aspartate-treated cells, permitting CAD gene amplification

Abstract

Genomic instability, including the ability to undergo gene amplification, is a hallmark of neoplastic cells. Similar to normal cells, "nonpermissive" REF52 cells do not develop resistance to N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of the synthesis of pyrimidine nucleotides, through amplification of cad, the target gene, but instead undergo protective, long-term, p53-dependent cell cycle arrest. Expression of exogenous MYC prevents this arrest and allows REF52 cells to proceed to mitosis when pyrimidine nucleotides are limiting. This results in DNA breaks, leading to cell death and, rarely, to cad gene amplification and PALA resistance. Pretreatment of REF52 cells with a low concentration of PALA, which slows DNA replication but does not trigger cell cycle arrest, followed by exposure to a high, selective concentration of PALA, promotes the formation of PALA-resistant cells in which the physically linked cad and endogenous N-myc genes are coamplified. The activated expression of endogenous N-myc in these pretreated PALA-resistant cells allows them to bypass the p53-mediated arrest that is characteristic of untreated REF52 cells. Our data demonstrate that two distinct events are required to form PALA-resistant REF52 cells: amplification of cad, whose product overcomes the action of the drug, and increased expression of N-myc, whose product overcomes the PALA-induced cell cycle block. These paired events occur at a detectable frequency only when the genes are physically linked, as cad and N-myc are. In untreated REF52 cells overexpressing N-MYC, the level of p53 is significantly elevated but there is no induction of p21waf1 expression or growth arrest. However, after DNA is damaged, the activated p53 executes rapid apoptosis in these REF52/N-myc cells instead of the long-term protective arrest seen in REF52 cells. The predominantly cytoplasmic localization of stabilized p53 in REF52/N-myc cells suggests that cytoplasmic retention may help to inactivate the growth-suppressing function of p53.

FIG. 1

Levels of exogenous N-myc mRNA and resistance to PALA or MTX in REF52/N-myc clones. (A) Bars represent the IC₅₀s of PALA or MTX. The levels of N-myc mRNA, obtained by using a PhosphorImager, were normalized to the level in clone 12. (B) Northern transfers (15 μg of total RNA) were hybridized with a human N-myc probe.

FIG. 2

Levels of cad and dhfr mRNAs in untreated REF52 and REF52/N-myc cells. (A) Northern transfers (15 μg of total RNA) were hybridized with cad or dhfr probes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was analyzed as a loading control. (B) Quantitative data from a PhosphorImager were normalized to the levels in REF52 cells.

FIG. 3

FISH of metaphase chromosomes from PALA-resistant cells. (A) Partial metaphase spreads representing cad amplification in PALA-resistant cells selected from REF52/N-myc clones 12, 8, 16, and 11 (panels 1 to 4, respectively). An unrearranged chromosome shows two green signals for cad. (B) cad amplification in PALA-resistant REF52 cells selected after preexposure to PALA. Panels 1 to 6 represent independent clones 2, 4, 7, 11, 12, and 24, respectively. (C) Coamplification of cad and N-myc in PALA-resistant REF52 cells selected after preexposure to PALA. Panels 1 and 2 represent clones 7 and 21, respectively. Chromosomes were hybridized simultaneously with a biotin-labeled cad probe (green) and a digoxigenin-labeled N-myc probe (red). Chromosomes were counterstained with DAPI (blue). An unrearranged chromosome 6, carrying single copies of cad and N-myc, is indicated by an arrow.

FIG. 4

Expression of N-myc mRNA in PALA-resistant REF52 clones. Total RNA from PALA-resistant clones selected after preexposure to PALA (lanes 1 to 4) and parental REF52 cells (lane 5) was analyzed by RNAse protection, using as a probe a 585-bp fragment derived from the second exon of the rat N-myc gene. The amount of RNA was normalized with a β-actin probe.

FIG. 5

Flow-cytometric analyses of REF52 and REF52/N-myc cells treated with PALA or MTX. (A) In panel 1, REF52 cells were treated with 30 μM PALA for 24, 48, or 72 h; fixed; stained with propidium iodide; and analyzed for cell cycle distribution with a FACScan instrument. In panel 2, the percentage of cells in each phase of the cell cycle was calculated as a function of the number of days in PALA. (B) Analysis of REF52/N-myc clones 8 and 11 treated with PALA. Cells were exposed to 50 μM (clone 8) or 100 μM (clone 11) PALA for 2, 3, or 5 days and analyzed as in panel A. REF52/p53C141Y cells were treated with 30 μM PALA for 5 days. (C) Analysis of REF52 cells and REF52/N-myc clone 16 treated with MTX. Cells were exposed to 40 nM (REF52) or 30 nM (clone 16) MTX for 2 or 5 days and analyzed as for panel A. Apoptotic cells have less DNA than G₁-phase cells.

FIG. 6

Analysis of DNA synthesis in REF52 cells treated with PALA. The cells were exposed to 10, 15, or 30 μM PALA for 24, 48, or 72 h; labeled with BrdU for 2 h; fixed; and stained with a cell proliferation kit (Amersham). The percentage of nuclei labeled with BrdU is shown at each time point.

FIG. 7

Induction of p53 and p21^waf1 in cells treated with PALA, MTX, or UV. (A) Immunoblot analysis of p53 expression in treated or untreated control (lanes C) REF52/puro and REF52/N-myc cells. The cells were exposed to PALA for 48 or 72 h (30 μM for REF52/puro, 55 μM for REF52/N-myc clone 8, 85 μM for clone 16, and 110 μM for clone 11). The film for clone 11 was exposed three times less than were the films for the other clones shown. The untransferred part of the gel, stained with Coomassie blue, is presented as a loading control. (B) REF52/puro and REF/N-myc clone 11 cells were irradiated with UV (24 J/m²), and the proteins were extracted 8 h later. p53 was detected with antibody PAb421, and p21^waf1 was detected with a mixture of antibodies C-19 and L-17. (C) REF52/puro and REF52/N-myc clone 16 cells were treated with 50 nM MTX for 48 or 72 h and analyzed as in panel A. (D) Western analysis of the relative amounts of p53 in nuclear and cytoplasmic extracts from REF52 and REF52/N-myc clone 16 cells. The amount of nuclear extract assayed corresponded to three times as many cells as the amount of cytoplasmic extract.