Cajot J F, Anderson M J, Lehman T A, Shapiro H, Briggs A A, Stanbridge E J
Department of Microbiology and Molecular Genetics, University of California, Irvine 92717.
Cancer Res. 1992 Dec 15;52(24):6956-60.
This study was undertaken to analyze the effect of wild-type p53 transfection on the growth potential of a human lung cancer cell line Hut292DM expressing endogenous wild-type p53. Transfection efficiencies obtained with either the wild-type or a mutant p53 complementary DNA revealed a significant decrease in the number of colonies obtained with the wild-type p53 as compared to the mutant p53 complementary DNA (27%) or control vector DNA only (20%), suggesting that wild-type p53 inhibited the growth of Hut292DM cells. A series of wild-type and mutant p53 transfection clones were then analyzed for the presence and expression of the exogenous p53 gene. Polymerase chain reaction amplification revealed that 98% of mutant p53 transfection clones analyzed contained the exogenous p53 gene as opposed to 47% for wild-type p53 clones. The majority of mutant p53 clones expressed high levels of exogenous p53 mRNA and protein as analyzed by Northern and Western blots, respectively. In contrast, all wild-type p53 clones analyzed failed to express exogenous p53 mRNA transcript or protein of a normal size. Aberrant-size p53 mRNA was detected in two wild-type p53 clones (X833.W2 and W18), and Western blot analysis revealed that these clones expressed truncated p53 proteins (M(r) 45,000 and 33,000 respectively). No difference in proliferation rates in vitro or in tumorigenic potential in nude mice were observed between mutant p53 clones or control cell lines. In contrast, a wild-type p53 clone (X833.W2) exhibited a significantly reduced tumorigenic potential in nude mice, whereas its in vitro proliferation rate was comparable to parental Hut292DM cells. The data indicate that exogenous expression of wild-type p53 is incompatible with Hut292DM lung cancer cell proliferation in vitro and suggest that p53-mediated growth control in vitro and in vivo may be dissociated and exerted by separate domains of the p53 protein.
本研究旨在分析野生型p53转染对表达内源性野生型p53的人肺癌细胞系Hut292DM生长潜能的影响。野生型或突变型p53互补DNA的转染效率显示，与突变型p53互补DNA（27%）或仅对照载体DNA（20%）相比，野生型p53获得的集落数显著减少，表明野生型p53抑制了Hut292DM细胞的生长。然后分析了一系列野生型和突变型p53转染克隆中外源p53基因的存在和表达情况。聚合酶链反应扩增显示，所分析的98%的突变型p53转染克隆含有外源p53基因，而野生型p53克隆的这一比例为47%。通过Northern印迹和Western印迹分析，大多数突变型p53克隆分别高水平表达外源p53 mRNA和蛋白质。相比之下，所有分析的野生型p53克隆均未表达正常大小的外源p53 mRNA转录本或蛋白质。在两个野生型p53克隆（X833.W2和W18）中检测到异常大小的p53 mRNA，Western印迹分析显示这些克隆表达截短的p53蛋白（分子量分别为45,000和33,000）。在突变型p53克隆或对照细胞系之间，未观察到体外增殖率或裸鼠致瘤潜能的差异。相比之下，一个野生型p53克隆（X833.W2）在裸鼠中的致瘤潜能显著降低，而其体外增殖率与亲本Hut292DM细胞相当。数据表明，野生型p53的外源表达与Hut292DM肺癌细胞的体外增殖不相容，并提示p53介导的体外和体内生长控制可能是分离的，由p53蛋白的不同结构域发挥作用。
