Lebon Alexis, Seyer Damien, Cosette Pascal, Coquet Laurent, Jouenne Thierry, Chan Philippe, Leprince Jerome, Fournier Alain, Vaudry Hubert, Gonzalez Bruno J, Vaudry David
INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France.
Ann N Y Acad Sci. 2006 Jul;1070:380-7. doi: 10.1196/annals.1317.049.
The rat pheochromocytoma PC12 cell line has been widely used as a model to study neuronal differentiation. In particular, after serum depletion, PC12 cells stop to proliferate and undergo apoptosis. Under such conditions, treatment with pituitary adenylate cyclase-activating polypeptide (PACAP) promotes cell survival and induces neurite outgrowth. The identification of the proteins regulated by PACAP in PC12 cells under apoptotic conditions should provide valuable information concerning the mechanisms controlling neuronal cell survival and differentiation. To this aim, PC12 cells cultured in serum-free medium were treated with PACAP (10(-7) M), proteins were extracted, separated by two-dimensional gel electrophoresis (2-DE), and identified by MALDI-ToF mass spectrometry. The comparison between 16 2-DE maps led to the characterization of 110 proteins regulated by PACAP among which 22 have been identified by automatic query of the Mascot, Aldente, and Profound servers with the ProGeR-CDD database. Seventy-six percent of these proteins, including the p17 subunit of caspase-3, the heat shock protein hsp60, and the GTPase ran were found to be repressed whereas the others notably hsp27, tubulin beta-5, and calmodulin were overexpressed. Investigation of the putative functions indicated that some of the proteins regulated by PACAP and identified in the present article could control cell survival or differentiation.
大鼠嗜铬细胞瘤PC12细胞系已被广泛用作研究神经元分化的模型。特别是在血清剥夺后，PC12细胞停止增殖并发生凋亡。在这种条件下，用垂体腺苷酸环化酶激活多肽（PACAP）处理可促进细胞存活并诱导神经突生长。鉴定凋亡条件下PACAP在PC12细胞中调节的蛋白质，应能提供有关控制神经元细胞存活和分化机制的有价值信息。为此，将在无血清培养基中培养的PC12细胞用PACAP（10^(-7) M）处理，提取蛋白质，通过二维凝胶电泳（2-DE）分离，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-ToF）鉴定。对16张2-DE图谱的比较，确定了110种受PACAP调节的蛋白质，其中22种已通过使用ProGeR-CDD数据库自动查询Mascot、Aldente和Profound服务器得以鉴定。发现这些蛋白质中有76%被抑制，包括半胱天冬酶-3的p17亚基、热休克蛋白hsp60和GTP酶ran，而其他蛋白质，特别是hsp27、微管蛋白β-5和钙调蛋白则过表达。对推定功能的研究表明，本文中鉴定的一些受PACAP调节的蛋白质可能控制细胞存活或分化。
