Thottassery Jaideep V, Westbrook Louise, Someya Hitoshi, Parker William B
Department of Biochemistry and Molecular Biology, Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA.
Mol Cancer Ther. 2006 Feb;5(2):400-10. doi: 10.1158/1535-7163.MCT-05-0409.
Nucleoside anticancer drugs like gemcitabine (2'-deoxy-2',2'-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4'-thio-beta-d-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53-/- HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53-/- and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis.
核苷类抗癌药物如吉西他滨（2'-脱氧-2',2'-二氟胞苷）是p53的强效诱导剂，野生型p53的异位表达使细胞对这些药物敏感。然而，也已知核苷是不表达功能性p53的肿瘤细胞中凋亡的有效激活剂。为了阐明这个问题，我们研究了吉西他滨和4'-硫代-β-D-阿拉伯呋喃糖基胞嘧啶（T-ara-C）对p73的影响，p73是p53的结构和功能同源物，其激活也可能解释核苷诱导的凋亡，因为在癌症中尚未报道p73有功能上显著的突变。用吉西他滨或T-ara-C急性处理HCT 116结肠癌细胞可诱导明显的细胞毒性以及半胱天冬酶-3和聚（ADP-核糖）聚合酶的裂解。T-ara-C和吉西他滨显著诱导p53积累以及磷酸化p53（Ser15/Ser20/Ser46）水平升高，并诱导其与共有p53反应元件结合。尽管T-ara-C和吉西他滨能强力激活p53，但我们发现野生型和p53基因敲除的HCT 116细胞对这些核苷表现出几乎相同的敏感性。对p73的检测显示，T-ara-C和吉西他滨在p53基因敲除和野生型细胞中均显著增加p73蛋白水平和p73 DNA结合活性。此外，T-ara-C和吉西他滨诱导的p73水平升高是由于p73蛋白周转减少所致。RNA干扰研究表明，核苷诱导的p73增加独立于c-Abl，c-Abl是一种最近与p73稳定化有关的核苷激活激酶。在HCT 116细胞系中，下游p53/p73靶点Bax和PUMA（p53上调凋亡调节因子）被缺失，这些细胞对T-ara-C和吉西他滨的敏感性较低。总之，这些研究表明，不依赖c-Abl的p73稳定化途径可能解释核苷诱导凋亡中不依赖p53的机制。
