Metallothionein expression prevents apoptosis: a study with antisense phosphorothioate oligodeoxynucleotides in a human T cell line.
Metallothionein expression prevents apoptosis: a study with antisense phosphorothioate oligodeoxynucleotides in a human T cell line.
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发表时间：
1998-07
影响因子：
2
通讯作者：
G. Tsangaris;F. Tzortzatou-Stathopoulou
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作者：
G. Tsangaris;F. Tzortzatou-Stathopoulou
人金属硫蛋白（hMTs）是一种低分子量的富含半胱氨酸的蛋白质，构成了细胞内蛋白质硫醇的大部分。它们的转录受金属、糖皮质激素和细胞因子的调控，在某些组织中，这是一种高度专业化的现象。尽管其生理功能尚不完全清楚，但已观察到hmt诱导与保护细胞免受重金属毒性和细胞对细胞毒性抗癌药物的抗性有关。然而，研究hMTs生理功能的主要问题是缺乏任何已知的特异性抑制剂，以及许多基因构成hMTs家族的事实。由于鉴定防止细胞凋亡的基因具有很大的兴趣，我们试图通过反义序列特异性磷酸化寡脱氧核苷酸（ODNs）抑制未成熟T细胞系CCRF-CEM中hmt的表达来研究其在细胞凋亡过程中的作用。在实验过程中，细胞被激活并在含有20% FBS而不是10% FBS的培养基中培养。我们发现，与ODNs孵育24小时后，细胞的hmt合成受到抑制，刺激了细胞凋亡过程，这一点被形态学改变和DNA断裂所证实。细胞凋亡的定量分析表明，抑制hMTs表达导致剂量依赖性和ODNs序列特异性的细胞凋亡诱导。免疫细胞化学检测hMTs和Tunel实验显示，所有Tunel阳性细胞均为hMTs阴性，表明hMTs的表达可阻止细胞凋亡。由于hmt在应激和/或环境刺激下的诱导是快速和短暂的，这些结果表明hmt构成了细胞保护机制，中和了外部凋亡信号。
Human metallothioneins (hMTs), are low molecular weight cysteine-rich proteins that constitute the majority of intracellular protein thiols. Their transcription is regulated by metals, glucocorticoids and cytokines, and in certain tissues it is a highly specialized phenomenon. Although their physiological function is not entirely understood, hMTs induction has been observed to be associated with protection from heavy metal toxicity and cellular resistance to cytotoxic anticancer drugs. However, the main problem in the investigation of the physiological function of hMTs is the absence of any known specific inhibitor, as well as the fact that many genes constitute the hMTs family. As the identification of genes preventing apoptosis is of great interest, we attempted to examine the role of hMTs in the apoptotic process by inhibiting their expression in the immature T cell line CCRF-CEM with antisense sequence-specific phosphorothioate oligodeoxynucleotides (ODNs). In the experimental procedure the cells were activated and cultured in medium containing 20% FBS instead of 10%, during maintenance. We found that the inhibition of hMTs synthesis, induced by the incubation of the cells for 24 hours with ODNs, stimulated the apoptotic process, as confirmed by the characteristic morphological alterations and DNA fragmentation. Quantitative analysis of apoptosis has shown that inhibition of hMTs expression results in a dose-dependent and ODNs sequence-specific induction of apoptosis. Immunocyto-chemical detection of hMTs followed by Tunel assay showed that all the Tunel positive cells were hMTs negative, suggesting that hMTs expression prevents apoptosis. As hMTs induction is rapid and transient in response to stress and/or environmental stimuli, these results indicate that hMTs constitute a cellular protective mechanism, neutralizing external apoptotic signals.