Nuclear exclusion of wild-type p53 in immortalized human retinoblastoma cells.
Nuclear exclusion of wild-type p53 in immortalized human retinoblastoma cells.
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永生化人视网膜母细胞瘤细胞中野生型 p53 的核排除。
DOI：
10.1093/jnci/89.20.1530
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Nickells,RW
中科院分区：
文献类型：
--
作者：
Schlamp,CL;Poulsen,GL;Nork,TM;Nickells,RW
背景：视网膜母细胞瘤是儿童眼部最常见的肿瘤，起源于视网膜母细胞瘤易感基因(RB1)两个拷贝均有缺陷的细胞。大多数视网膜母细胞瘤细胞最终经历细胞程序性死亡(即细胞凋亡)；然而，一些细胞可以获得转移并成为永生的能力。将编码野生型P53蛋白的DNA序列导入永生化的视网膜母细胞瘤细胞，可诱导细胞死亡，提示RB1和P53功能的丧失可能是细胞永生化所必需的。我们通过对6个独立分离的永生化视网膜母细胞瘤细胞系中P53蛋白和信使RNA的特征分析来研究这种可能性。方法：用Western blotting方法检测每个细胞系中P53蛋白的水平，并用互补DNA的裂解酶片段长度多态性分析来筛选P53信使RNA的突变。结果：6株永生化细胞株均表达野生型P53信使RNA，表达高水平的P53蛋白。虽然P53在正常情况下是一种核蛋白，但在6个细胞系中，有4个细胞系中的P53主要位于细胞质中；在其余两个细胞系中，P53同时定位于细胞核和细胞质中。P53在视网膜母细胞瘤标本中的胞浆定位很少见，通常局限于侵袭邻近眼组织的细胞，提示肿瘤转移的早期阶段。结论：某些永生化的视网膜母细胞瘤细胞可能通过排除野生型P53蛋白而表现出P53功能障碍。
Background:Retinoblastoma is the most common childhood tumor of the eye, arising from cells that are defective in both copies of the retinoblastoma susceptibility gene (RB1). Most retinoblastoma tumor cells eventually undergo programmed cell death (i.e., apoptosis); however, some cells can acquire the ability to metastasize and become immortal. Transfection of immortal retinoblastoma cells with DNA sequences encoding wild-type p53 protein induces cell death, suggesting that the loss of both RB1 and p53 functions may be required for cell immortalization. We have examined this possibility by characterizing the p53 protein and messenger RNA in six independently isolated, immortalized retinoblastoma cell lines.Methods:Western blotting methods were used to assess p53 protein level in each cell line, and Cleavase Fragment-Length Polymorphism analysis of complementary DNAs was used to screen for mutations in p53 messenger RNA. Localization of p53 protein in cells of the immortalized lines and in specimens of retinoblastoma tumors was achieved by means of indirect immunofluorescence and immunocytochemistry, respectively.Results:All six immortalized cell lines expressed wildtype p53 messenger RNA and high levels of p53 protein. Although p53 is normally a nuclear protein, the p53 in four of the six cell lines was located predominately in the cytoplasm; in the remaining two cell lines, p53 was localized in both the nucleus and the cytoplasm. Cytoplasmic localization of p53 in retinoblastoma tumor specimens was rare and usually restricted to cells that had invaded adjacent ocular tissues, indicative of the early stages of metastasis.Conclusions:Some immortalized retinoblastoma cells may exhibit p53 dysfunction through nuclear exclusion of wild-type p53 protein.