School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia.
Proteomics Clin Appl. 2012 Jun;6(5-6):279-90. doi: 10.1002/prca.201200003.
Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 μM) undergo apoptosis with accumulation of p53 in the nuclei as multiple phosphorylated isoforms and C-terminal truncated derivatives. Changes induced by 2-FaraA in the levels of p53, p63 and p73 in the nuclear, cytosolic and mitochondrial fractions have been determined in four human B-lymphoid cell lines that are TP53-functional (Raji and IM9) and TP53-mutated (MEC1 and U266).
The B-lymphoid cell lines were treated with 2-FaraA (3 μM, 24 h, 48 h) and viability determined. Protein extracts of subcellular fractions from 2-FaraA-treated cells were analysed by 1D and 2D electrophoresis; multiple phosphorylated isoforms and truncated derivatives were identified by Western blots for p53, p63 and p73.
p53 and p63 were present in all three fractions, while p73 was only detected in nuclei. After treatment with 2-FaraA, nuclear p53, p63 and p73 accumulated as multiple phosphorylated isoforms and truncated derivatives. The association of p63 with mitochondria in human cells is novel.
Comprehensive information on the subcellular distributions and responses of p53, p63 and p73 to 2-FaraA provides additional insight into mechanisms for induction of apoptosis in the treatment of B-lymphoproliferative disorders with fludarabine.
用氟达拉滨核苷（2-FaraA，3μM）处理人类 Raji 细胞会导致细胞凋亡，细胞核中 p53 积聚，并呈现出多种磷酸化异构体和 C 端截断衍生物。在四个具有功能性 TP53（Raji 和 IM9）和 TP53 突变（MEC1 和 U266）的人 B 淋巴细胞系中，确定了 2-FaraA 诱导的 p53、p63 和 p73 在核、胞质和线粒体部分水平的变化。
用 2-FaraA（3μM，24 小时，48 小时）处理 B 淋巴细胞系，测定细胞活力。用 1D 和 2D 电泳分析经 2-FaraA 处理的细胞亚细胞部分的蛋白质提取物；用 Western blot 鉴定 p53、p63 和 p73 的多个磷酸化异构体和截断衍生物。
p53 和 p63 存在于所有三个部分，而 p73 仅存在于核中。用 2-FaraA 处理后，核 p53、p63 和 p73 作为多种磷酸化异构体和截断衍生物积聚。p63 在人细胞中与线粒体的结合是新颖的。
对 2-FaraA 诱导的 p53、p63 和 p73 的亚细胞分布和反应的综合信息提供了对用氟达拉滨治疗 B 淋巴细胞增生性疾病诱导细胞凋亡机制的额外认识。
