P53 ALTERATION IS A COMMON EVENT IN THE SPONTANEOUS IMMORTALIZATION OF PRIMARY BALB/C MURINE EMBRYO FIBROBLASTS
P53 ALTERATION IS A COMMON EVENT IN THE SPONTANEOUS IMMORTALIZATION OF PRIMARY BALB/C MURINE EMBRYO FIBROBLASTS
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DOI：
10.1101/gad.5.12b.2375
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发表时间：
1991-12-01
影响因子：
10.5
通讯作者：
LEVINE, AJ
中科院分区：
文献类型：
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作者：
HARVEY, DM;LEVINE, AJ
先前已经表明，当突变体 p53 与选择性标记一起共转染到原代大鼠胚胎成纤维细胞中时，可以充当永生化基因。为了确定 p53 位点的突变是否是导致自发细胞永生化途径中的常见事件，通过按 3T3 计划传代建立了 11 个克隆衍生的 BALB/c 小鼠胚胎成纤维细胞系，并检查了 p53 的改变。根据以下标准，所有 11 个独立建立的品系均含有至少一个 p53 突变等位基因。其中 7 个品系具有 PAb240 反应性 p53 物种，并通过脉冲追踪分析测定显示出延长的 p53 半衰期。这些品系的一部分中的 p53 蛋白种类也能够与组成型热休克蛋白 hsc70 形成复合物。通过细胞质 RNA 逆转录和 PCR 扩增，克隆了其中几个品系的 p53 细胞质 DNA (cDNA)，并通过 DNA 序列分析绘制了突变图谱。 p53 保守结构域中的点突变似乎是这些品系中的常见改变，尽管一个已建立的品系携带 p53 的 24 bp 框内缺失。其余四种细胞系不表达可检测到的 p53 蛋白。对于每个品系，p53 表达缺乏背后都有一个不同的分子事件：(1) 两个 p53 等位基因的至少前 6 个外显子缺失； (2)编码第173位氨基酸终止密码子的单个p53 mRNA的表达； (3) 未检测到p53 mRNA； (4)p53 mRNA的表达大大减少。这些发现表明，p53 改变通常发生在按 3T3 计划传代的自发永生化 BALB/c 小鼠胚胎成纤维细胞中，因此可能是永生化过程的重要事件。
It has been shown previously that mutant p53 can act as an immortalizing gene when cotransfected into primary rat embryo fibroblasts along with a selectable marker. To determine whether a mutation at the p53 locus is a common event in the pathways leading to spontaneous cellular immortalization, 11 clonally derived BALB/c murine embryo fibroblast lines were established by passage on a 3T3 schedule and examined for p53 alterations. By the following criteria, all 11 independently established lines contain at least one mutant allele of p53. Seven of these lines have a PAb240-reactive p53 species and exhibit an extended p53 half-life as determined by pulse-chase analysis. The p53 protein species in a subset of these lines is also capable of complex formation with the constitutive heat shock protein hsc70. p53 cytoplasmic DNAs (cDNAs) from several of these lines have been cloned by reverse transcription of cytoplasmic RNA followed by PCR amplification, and the mutations have been mapped by DNA sequence analysis. Point mutation in conserved domains of p53 appears to be a common alteration in these lines, although one established line carries a 24-bp in-frame deletion of p53. The remaining four cell lines do not express detectable p53 protein. For each line there is a different molecular event underlying the lack of p53 expression: (1) deletion of at least the first 6 exons of both p53 alleles; (2) expression of a single p53 mRNA encoding a stop codon at amino acid position 173; (3) no detectable p53 mRNA; and (4) greatly diminished expression of p53 mRNA. These findings indicate that p53 alteration commonly occurs in spontaneously immortalized BALB/c mouse embryo fibroblasts passaged on a 3T3 schedule and, therefore, may be an important event for the immortalization process.