Protein phosphorylation of tumor suppressor gene p53 in leukemia cells and regulation of apoptosis-rated gene expression

(1)这两个辐射阿糖胞苷处理的BV 173人白血病细胞，其中含有野生型p53基因，导致细胞凋亡和诱导p53。p53下游基因Bax和Fas/APO-1在辐射中被激活，而在阿糖胞苷处理中仅Bax被激活。双向电泳结果显示，辐射和阿糖胞苷处理的BV 173细胞中p53蛋白具有不同的等电点，磷酸酶处理后p53蛋白的等电点趋于一致。P53-丝氨酸15、33和392在辐射、阿糖胞苷处理和阿霉素处理的BV 173细胞中磷酸化，没有差异。(2)K562红白血病细胞中Bax的瞬时过表达引起的凋亡细胞明显多于对照组。在转染Tet-On Bax诱导系统的野生型K562细胞中，Bax的过表达无任何细胞毒性信号，导致凋亡caspase-3激活，线粒体释放细胞色素c，线粒体膜电位改变。pancaspase抑制剂zVAD-favorite抑制线粒体上Bax过表达时的凋亡事件。这些研究结果表明，Bax蛋白，当存在高于阈值水平，是足以触发凋亡级联反应，其启动需要同时激活的半胱天冬酶不介导的线粒体细胞色素c释放在K562细胞。(3)在DLD-1结肠癌细胞中，Tet-On-Bu诱导的caspase活化和凋亡被认为是由线粒体细胞色素c释放介导的。Bax过表达也通过增加细胞色素c的释放而使KATOIII胃癌细胞对化疗药物诱导的凋亡敏感。综上所述，p53蛋白在不同的细胞毒性刺激下被不同的磷酸化，Bax诱导的caspase激活有两条途径：一条是由线粒体细胞色素c释放介导的，另一条不是由细胞色素c介导的。

(1) Both irradiated ara-C-treated BV173 human leukemia cells, which harbor the wild-type p53 gene, resulted in apoptosis and induction of p53. The downstream genes of p53, Bax and Fas/APO-1 were activated in irradiation, but only Bax in ara-C treatment. Two-dimensional gel electrophoresis revealed that the p53 proteins in irradiated and ara-C-treated BV173 cells had different isoelectric points which converged to a single isoelectric point after phosphatase treatment. P53-serine 15, 33, and 392 in irradiated, ara-C-treated and doxorubicin-treated BV173 cells were phosphorylated with no difference.(2) Transient Bax overexpression in K562 erythroleukemia cells caused significantly more apoptotic cells than the control. In wild type Bax-bearing K562 cells transfected with Tet-On Bax-inducible system, overexpression of Bax without any cytotoxic signal resulted in apoptosis caspase-3 activation, mitochondrial release of cytochrome c, and mitochondrial membrane potential change. A pancaspase inhibitor, zVAD-fmk, inhibited the apoptotic events in the presence of overexpression Bax on mitochondria. These findings suggest that Bax protein, when present above a threshold level, is sufficient to trigger apoptosis cascade, and its initiation requires simultaneous activation of caspases not mediated by mitochondrial cytochrome c release in K562 cells.(3) In DLD-1 colon cancer cells, Tet-On-Bax-induced caspase activation and apoptosis were considered to be mediated by mitochondrial cytochrome c release. Bax overexpression also sensitized KATOIII gastric cancer cells to chemotherapeutic agent-induced apoptosis through enhancing the cytochrome c release.Taken together, p53 proteins are differently phosphorylated by different cytotoxic stimuli, and Bax-induced caspase activation has two pathways ; one is mediated by mitochondrial cytochrome c release, and one not mediated by cytochrome c.