p53-dependent growth arrest of REF52 cells containing newly amplified DNA.
p53-dependent growth arrest of REF52 cells containing newly amplified DNA.
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含有新扩增 DNA 的 REF52 细胞的 p53 依赖性生长停滞。
DOI：
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发表时间：
1995
影响因子：
11.1
通讯作者：
G. Stark
中科院分区：
文献类型：
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作者：
Y. Ishizaka;M. V. Chernov;C. Burns;G. Stark
大鼠细胞系REF 52不允许基因扩增。猴病毒40肿瘤（T）抗原将这些细胞转化为允许状态，p53的显性阴性突变体也是如此，这表明T抗原的作用主要是由于其与p53结合的能力。为了操纵perturbine，我们将T抗原的温度敏感突变体（tsA 58）引入REF 52细胞中，并选择对N-（膦酰基乙酰基）-L-天冬氨酸（PALA）的抗性。大多数新鲜分离的PALA抗性菌落，每个约200个细胞，在允许的温度下选择，当转移到非允许的温度时被捕集。只要T抗原不存在，生长停滞就稳定，没有细胞凋亡的证据，但当T抗原恢复时，生长停滞就会逆转。相比之下，在允许温度下生长至约10（7）个细胞的PAA抗性克隆在转移至非允许温度时不停滞。所有的PALA耐药克隆检测扩增的氨甲酰磷酸合成酶-天冬氨酸转氨甲酰酶-二氢乳清酸酶（CAD）基因，目前的结构与涉及桥断裂融合（BBF）周期的机制一致。我们认为p53介导的生长停滞仅在基因扩增的复杂过程的早期起作用，此时新形成的PALA抗性细胞含有在BBF循环中产生的断裂DNA。在允许的条件下繁殖期间，断裂的DNA末端被愈合，并且，即使p53介导的途径在非允许的温度下仍然是完整的，并且细胞含有扩增的DNA，它们在没有断裂的DNA的情况下也不会被阻止。这些数据支持这样的假设，即BBF循环是扩增的重要机制，并且每个循环中产生的断裂DNA是调节基因扩增的关键信号。
The rat cell line REF52 is not permissive for gene amplification. Simian virus 40 tumor (T) antigen converts these cells to a permissive state, as do dominant negative mutants of p53, suggesting that the effect of T antigen is due mainly to its ability to bind to p53. To manipulate permissivity, we introduced a temperature-sensitive mutant of T antigen (tsA58) into REF52 cells and selected for resistance to N-(phosphonacetyl)-L-aspartate (PALA). Most freshly isolated PALA-resistant colonies, each of approximately 200 cells, selected at a permissive temperature, arrested when shifted to a nonpermissive temperature. Growth arrest was stable, with no evidence of apoptosis, as long as T antigen was absent but was reversed when T antigen was restored. In contrast, PALA-resistant clones grown to approximately 10(7) cells at a permissive temperature did not arrest when shifted to a nonpermissive temperature. All PALA-resistant clones examined had amplified carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) genes, present in structures consistent with a mechanism involving bridge-breakage-fusion (BBF) cycles. We propose that p53-mediated growth arrest operates only early during the complex process of gene amplification, when newly formed PALA-resistant cells contain broken DNA, generated in BBF cycles. During propagation under permissive conditions, the broken DNA ends are healed, and, even though the p53-mediated pathway is still intact at a nonpermissive temperature and the cells contain amplified DNA, they are not arrested in the absence of broken DNA. The data support the hypothesis that BBF cycles are an important mechanism of amplification and that the broken DNA generated in each cycle is a key signal that regulates permissivity for gene amplification.