Kaneuchi M, Yamashita T, Shindoh M, Segawa K, Takahashi S, Furuta I, Fujimoto S, Fujinaga K
Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University School of Medicine, Japan.
Mol Carcinog. 1999 Sep;26(1):44-52.
Codon 273 is one of the hot spots of missense mutation of the p53 tumor suppressor gene found in human cancers. We have previously reported that a mutation at codon 273, p53-273L (Arg --> Leu), suppresses cell growth despite its having no p53-specific transactivation activity. To further elucidate the mechanism of growth suppression caused by p53-273L, we used squamous cell carcinoma cell line HSC3 to isolate subclones containing Zn2+-inducible wild-type (wt) p53, p53-175H, and p53-273L. Northern blot hybridization of the HSC3 cells possessing an inducible function of p53 as well as a luciferase assay for the p21Waf1/Cip1/Sdi1 promoter showed that only wt p53 could induce p21Waf1/Cip1/Sdi1 transcription. Meanwhile, the expression of bax remained unchanged between, before, and after the induction of any analyzed p53s. When wt p53 was induced in HSC3 cells cultured in medium containing 5% fetal bovine serum, cell growth was suppressed through G1 arrest. On the other hand, in medium with 0.1% fetal bovine serum, the growth of HSC3 cells expressing p53-273L was suppressed to a greater degree than that of cells expressing wt p53. Flow cytometric analysis and DNA ladder formation revealed that, unlike wt p53-SN3- and p53-175H-expressing HSC3 cells, p53-273L-expressing cells contained a larger sub-G1 fraction under this culture condition. These findings suggest that p53-273L can induce apoptosis in HSC3 cells without transactivation of p21Waf1/Cip1/Sdi1 and bax.
密码子273是在人类癌症中发现的p53肿瘤抑制基因错义突变的热点之一。我们之前报道过，密码子273处的突变，即p53-273L（精氨酸→亮氨酸），尽管没有p53特异性的反式激活活性，但仍能抑制细胞生长。为了进一步阐明由p53-273L引起的生长抑制机制，我们使用鳞状细胞癌细胞系HSC3来分离包含锌离子诱导型野生型（wt）p53、p53-175H和p53-273L的亚克隆。对具有p53诱导功能的HSC3细胞进行Northern印迹杂交以及对p21Waf1/Cip1/Sdi1启动子进行荧光素酶测定，结果表明只有wt p53能够诱导p21Waf1/Cip1/Sdi1转录。同时，在诱导任何分析的p53之前、期间和之后，bax的表达均保持不变。当在含有5%胎牛血清的培养基中培养的HSC3细胞中诱导wt p53时，细胞生长通过G1期阻滞受到抑制。另一方面，在含有0.1%胎牛血清的培养基中，表达p53-273L的HSC3细胞的生长受到的抑制程度比表达wt p53的细胞更大。流式细胞术分析和DNA梯带形成显示，与表达wt p53-SN3和p53-175H的HSC3细胞不同，在这种培养条件下，表达p53-273L的细胞含有更大的亚G1期部分。这些发现表明，p53-273L可以在不反式激活p21Waf1/Cip1/Sdi1和bax的情况下诱导HSC3细胞凋亡。
