EGF-stimulated polyamine accumulation in the colon carcinoma cell line, caco-2
EGF-stimulated polyamine accumulation in the colon carcinoma cell line, caco-2
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DOI：
10.1159/000007763
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发表时间：
2000-01-01
期刊：
影响因子：
3.2
通讯作者：
Stein, J
中科院分区：
文献类型：
--
作者：
Bauske, R;Milovic, V;Stein, J
背景：多胺（腐胺、亚精胺和精胺）是细胞增殖所必需的普遍存在的分子。在肠腔中，它们以大量存在。肠粘膜增殖细胞中的多胺积累是高的，并且其通过增强的合成和增加的从管腔的摄取而发生。目的：为了研究有丝分裂原诱导的多胺在肠道中的积累，我们用表皮生长因子（EGF）处理增殖的Caco-2细胞，并测量鸟氨酸脱羧酶（ODC）和腐胺摄取的活性。此外，我们调查是否EGF诱导的顶膜的变化可能是负责EGF对Caco-2细胞多胺摄取的影响。方法：在100 ng/ml EGF存在下，检测腐胺摄取、ODC活性和细胞内多胺含量。为了研究EGF刺激多胺摄取的机制，分离顶端膜囊泡，并测量囊泡中腐胺的摄取。可能的富集刷状缘膜细胞骨架蛋白（ezrin和绒毛蛋白）进行了评估，通过Western印迹。结果如下：EGF处理诱导ODC活性增加，其发生在处理的第一分钟内，并在3小时后达到峰值。相反，腐胺摄取的增加更持久，在12小时达到峰值水平。EGF处理后，细胞内腐胺和亚精胺的合成和摄取均增加了60%以上。在EGF处理的Caco-2细胞中，顶端膜细胞骨架没有可检测到的变化，这是由于缺乏ezrin和绒毛蛋白富集而得出的结论。然而，在从EGF预处理的细胞中分离的顶端膜囊泡中，腐胺摄取增强了两倍。结论：EGF刺激Caco-2细胞合成和摄取多胺。增强的合成似乎可以确保快速供应与多胺在生长的最早阶段，而摄取是负责维持高多胺细胞内水平在后期生长阶段。EGF刺激的多胺摄取显然不是顶端膜结构变化的结果，但很可能是由一个独特的EGF诱导的多胺转运蛋白本身的改变发生。版权所有（C）2000 S. Karger AG，巴塞尔。
Background: Polyamines (putrescine, spermidine and spermine) are ubiquitous molecules indispensable for cell proliferation. In the intestinal lumen they are present in high amounts. Polyamine accumulation in proliferating cells of the intestinal mucosa is high, and it occurs both by enhanced synthesis and by increased uptake from the lumen. Aims: To study mitogen-induced polyamine accumulation in the gut, we treated proliferating Caco-2 cells with epidermal growth factor (EGF) and measured the activity of ornithine decarboxylase (ODC) and putrescine uptake. Furthermore, we investigated whether EGF-induced changes in the apical membrane could be responsible for the effect of EGF on polyamine uptake in Caco-2 cells. Methods: Putrescine uptake, ODC activity and intracellular polyamine content were evaluated in the presence of 100 ng/ml EGF. To study the mechanisms of EGF-stimulated polyamine uptake, apical membrane vesicles were isolated, and putrescine uptake into the vesicles measured. Possible enrichment in brush border membrane cytoskeleton proteins (ezrin and villin) was assessed by Western blot. Results: Treatment with EGF induced an increase in ODC activity, which occurred within the first minutes of treatment and reached peak values after 3 h. In contrast, an increase in putrescine uptake was more sustained, with peak levels at 12 h. Both synthesis and uptake contributed to an over 60% increase in intracellular putrescine and spermidine after EGF treatment. There were no detectable changes in apical membrane cytoskeleton las concluded by the absence of ezrin and villin enrichment in EGF-treated Caco-2 cells). However, in apical membrane vesicles isolated from EGF-pretreated cells, putrescine uptake was enhanced twofold. Conclusions: EGF stimulates both synthesis and uptake of polyamines in Caco-2 cells. Enhanced synthesis seems to ensure rapid supply with polyamines in the earliest stages of growth, while the uptake is responsible for the maintenance of high polyamine intracellular levels during late growth phases. EGF-stimulated polyamine uptake is apparently not a consequence of structural changes in the apical membrane, but is likely to occur by a distinct EGF-induced alteration of the polyamine transporter itself. Copyright (C) 2000 S. Karger AG, Basel.