MYC Abrogates p53-Mediated Cell Cycle Arrest in N-(Phosphonacetyl)-l-Aspartate-Treated Cells, Permitting CAD Gene Amplification

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 p21(waf1) 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.

基因组不稳定性（包括发生基因扩增的能力）是肿瘤细胞的标志性特征。与正常细胞类似，非容许性REF52细胞（REF52 cells）无法通过扩增靶基因cad来获得对嘧啶核苷酸合成抑制剂N-(膦酰基乙酰基)-L-天冬氨酸（PALA）的耐药性，反而会经历保护性的长期p53依赖型细胞周期阻滞。外源性MYC的表达可阻断这种细胞周期阻滞，并使REF52细胞在嘧啶核苷酸匮乏时仍能进入有丝分裂阶段。这一过程会引发DNA断裂，进而导致细胞死亡，极少数情况下会引发cad基因扩增与PALA耐药性的产生。先用低浓度PALA预处理REF52细胞——该浓度可减慢DNA复制但不会引发细胞周期阻滞——随后将细胞暴露于高浓度的选择性PALA中，可促进PALA耐药细胞的形成；这类细胞中，物理连锁的cad基因与内源性N-myc基因会发生共扩增。在这些经预处理的PALA耐药细胞中，内源性N-myc的激活表达可使它们绕过未处理REF52细胞所特有的p53介导的细胞周期阻滞。本研究数据表明，生成PALA耐药的REF52细胞需要两个独立事件：一是靶基因cad的扩增（其编码产物可抵消药物的作用），二是N-myc的表达上调（其编码产物可阻断PALA诱导的细胞周期阻滞）。这两个协同事件仅在基因物理连锁的情况下（如cad与N-myc）才能以可检测到的频率发生。在过表达N-MYC的未处理REF52细胞中，p53的表达水平显著升高，但不会诱导p21(waf1)的表达或引发生长阻滞。然而，当DNA发生损伤后，激活的p53会在这些REF52/N-myc细胞中快速诱导细胞凋亡，而非像普通REF52细胞那样引发长期的保护性细胞周期阻滞。在REF52/N-myc细胞中，稳定化的p53主要定位于细胞质，这提示细胞质滞留可能有助于灭活p53的生长抑制功能。