Pessina Augusto, Croera Cristina, Savalli Nicoletta, Bonomi Arianna, Cavicchini Loredana, Turlizzi Elisa, Guizzardi Fabiana, Guido Lucia, Daprai Laura, Neri Maria Grazia
Institute of Microbiology, University of Milan, Via Pascal 36, 20133 Milan, Italy.
Cell Res. 2006 Mar;16(3):306-12. doi: 10.1038/sj.cr.7310038.
The very different effects of Cholera Toxin (CT) on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the "in vitro" clonogenicity, the Population Doubling Time (PDT), apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone (WEHI-3B/CTRES). In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells (WEHI-3B/CTRES), Bcl-2 expression was down regulated by both CT or drug treatment (eg., ciprofloxacin, CPX) although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase (PKA) in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models (of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX) provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.
霍乱毒素（CT）对细胞生长和增殖的截然不同的影响可能取决于细胞膜上神经节苷脂受体的类型以及所触发的不同信号转导机制，但与耐药机制相关的其他功能也不能排除。在WEHI-3B细胞系及其CT抗性亚克隆（WEHI-3B/CTRES）中评估了CT处理对“体外”克隆形成能力、群体倍增时间（PDT）、细胞凋亡、PKA激活以及Bax和Bcl-2表达的影响。在WEHI-3B亲代细胞中，CT诱导的cAMP显著积累与PKA激活、PDT值增加、克隆形成能力抑制和细胞凋亡密切相关。H-89处理抑制了CT诱导的PKA激活，但并未保护细胞免受细胞凋亡和生长抑制。在WEHI-3B/CTRES中，未观察到CT依赖性的cAMP显著积累，PKA活性和PDT也未增加。在CT抗性细胞（WEHI-3B/CTRES）中，CT或药物处理（如环丙沙星，CPX）均可下调Bcl-2表达，尽管这些细胞可免受CT依赖性细胞凋亡，但不能免受药物诱导的细胞凋亡。与其他描述的细胞模型不同，Bcl-2的下调被证明与cAMP积累和PKA激活无关。我们的观察结果支持cAMP依赖性激酶（PKA）参与抑制WEHI-3B细胞生长，并表明在WEHI-3B/CTRES中，CT可在无cAMP积累的情况下调节Bcl-2表达。考虑到文献中报道的许多相互矛盾的数据，我们的细胞模型（一种敏感的亲代细胞株和两个对CT和CPX具有不同非交叉特异性抗性的克隆）为更好地研究CT信号转导机制与Bcl-2表达和功能之间的关系提供了一个新的有趣工具。
