Zugaza J L, Casabiell X A, Bokser L, Eiras A, Beiras A, Casanueva F F
Department of Medicine, Faculty of Medicine, Santiago de Compostela, Spain.
Exp Cell Res. 1995 Jul;219(1):54-63. doi: 10.1006/excr.1995.1204.
We have previously demonstrated that pretreatment of several cell lines with cis-unsaturated fatty acids, like oleic acid, blocks epidermal growth factor (EGF)-induced early ionic signals, and in particular the [Ca2+]i rise. In the present work we show that this blockade does not alter EGF-stimulated cellular proliferation evaluated by direct cell counting, but induces a powerful enhancement in the pulsed thymidine incorporation assay. The lack of effect of oleic acid on EGF-stimulated cellular proliferation was confirmed by repeated cell counts, cumulative thymidine incorporation, and protein synthesis, but a clear synergistic effect between oleic acid and EGF was again obtained by means of time course experiments with pulsed thymidine. Combined flow cytometry analysis and cell counts at earlier times in EGF-stimulated cells showed that oleic acids accelerates the entrance of cells into the replicative cycle leading to an earlier cell division. Afterward, these oleic acid-pretreated cells became delayed by an unknown compensatory mechanism in such a way that at 48 h post-EGF, the cell count in control and oleic acid-pretreated cells was equal. In conclusion (a) oleic acid accelerates or enhances the EGF mitogenic action and (b) in the long term cells compensate the initial perturbation with respect to untreated cells. As a side observation, the widely employed pulsed thymidine incorporation method as a measure of cell division could be extremely misleading unless experimental conditions are well controlled.
我们之前已经证明，用顺式不饱和脂肪酸（如油酸）对几种细胞系进行预处理，会阻断表皮生长因子（EGF）诱导的早期离子信号，尤其是细胞内钙离子浓度（[Ca2+]i）的升高。在本研究中，我们发现这种阻断不会改变通过直接细胞计数评估的EGF刺激的细胞增殖，但在脉冲胸苷掺入试验中会诱导其显著增强。通过重复细胞计数、累积胸苷掺入和蛋白质合成，证实了油酸对EGF刺激的细胞增殖没有影响，但通过脉冲胸苷的时间进程实验，再次获得了油酸与EGF之间明显的协同效应。在EGF刺激的细胞中，早期进行的流式细胞术分析和细胞计数相结合表明，油酸加速细胞进入复制周期，导致细胞更早分裂。之后，这些经油酸预处理的细胞会因一种未知的补偿机制而延迟，以至于在EGF处理后48小时，对照细胞和经油酸预处理的细胞中的细胞计数相等。总之，（a）油酸加速或增强了EGF的促有丝分裂作用，（b）从长期来看，细胞相对于未处理的细胞会补偿最初的扰动。作为一个附带观察结果，除非实验条件得到很好的控制，否则广泛使用的脉冲胸苷掺入法作为细胞分裂的一种测量方法可能会极具误导性。
