Metabolic changes during cell growth inhibition by p27 overexpression
Metabolic changes during cell growth inhibition by p27 overexpression
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DOI：
10.1007/s00253-003-1385-5
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发表时间：
2003-12-01
影响因子：
5
通讯作者：
Carrondo, MJT
中科院分区：
文献类型：
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作者：
Carvalhal, AV;Marcelino, I;Carrondo, MJT
p27（一种细胞周期蛋白依赖性激酶（CDK）抑制剂）的过度表达已被证明可以有效抑制不同细胞系 G1 期的细胞生长，从而增强了细胞增殖控制的有效遗传策略。为了表征表达 SEAP（人类碱性磷酸酶的分泌形式）的 CHO 细胞中 p27 过表达后的能量需求，对关键代谢参数进行了评估。细胞生长抑制导致细胞大小显着增加，同时细胞蛋白质含量增加 2 倍。细胞内蛋白水解活性与蛋白质含量同时增加表明蛋白质合成较高。观察到氧气、谷氨酰胺和葡萄糖消耗率普遍增加 2 倍，同时乳酸和氨产量增加。 p27 过表达导致细胞内 AMP（8.5 倍）、ADP（6 倍）以及更不常见的 ATP（4.5 倍）池显着增加。然而，细胞能够维持三个腺嘌呤核苷酸之间的平衡，因为 ATP/ADP 比率和能量电荷值仍然与非生长抑制细胞中观察到的相似。这项工作表明，在 p27 抑制细胞生长后，观察到 SEAP 比生产率增加了 4 倍，同时细胞能量消耗增加。细胞代谢的这种特征对于证明生长抑制系统的适用性变得很重要。
The overexpression of p27, a cyclin-dependent kinase (CDK) inhibitor, has been shown to effectively inhibit cell growth at the G1-phase of different cell lines, potentiating a valid genetic strategy for cell proliferation control. In order to characterize the energy requirements after p27 overexpression in CHO cells expressing SEAP (secreted form of the human alkaline phosphatase enzyme), key metabolic parameters were evaluated. Cell growth inhibition led to a significant increase in cell size concomitant with a 2-fold increase in cell protein content. The simultaneous increase of the intracellular proteolytic activity with protein content suggests higher protein synthesis. A general 2-fold increase in oxygen, glutamine and glucose consumption rates, coupled with an increase in lactate and ammonia production was observed. p27 overexpression led to a significant increase in the intracellular pool of AMP (8.5-fold), ADP (6-fold) and, more uncommonly, ATP (4.5-fold). Nevertheless, cells were able to maintain the equilibrium among the three adenine nucleotides since both the ATP/ADP ratio and the energy charge values remained similar to those observed with non-growth inhibited cells. This work shows that the observed 4-fold increase in SEAP specific productivity after cell growth inhibition by p27, occurred concomitantly with a higher expenditure of cell energy. This characterization of cell metabolism becomes important in demonstrating the applicability of growth inhibition systems.