Piedrafita F J, Pfahl M
Sidney Kimmel Cancer Center, San Diego, California 92121, USA.
Mol Cell Biol. 1997 Nov;17(11):6348-58. doi: 10.1128/MCB.17.11.6348.
Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis. Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of certain cancer cells. We recently identified a novel class of synthetic retinoids with strong anti-cancer cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an electrophoretic mobility shift assay, we analyzed the DNA binding activity of several transcription factors in T cells treated with apoptotic retinoids. We found that the DNA binding activity of the general transcription factor Sp1 is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1 protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells contain a protease activity which specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates cleavage of Sp1. Furthermore, inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests that caspases are directly involved in this event. In fact, caspases 2 and 3 are activated in T cells after treatment with apoptotic retinoids. The peptide inhibitors also blocked retinoid-induced apoptosis, as well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells. Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved, triggering cell death in a transcriptional activation-independent, caspase-dependent manner.
维生素A及其衍生物类视黄醇是多种重要生物学功能的关键调节因子，这些功能包括细胞生长与分化、发育、体内平衡以及致癌作用。天然类视黄醇如全反式维甲酸可诱导细胞分化并抑制某些癌细胞的生长。我们最近鉴定出一类新型合成类视黄醇，其在体外和体内均具有强大的抗癌细胞活性，可诱导多种癌细胞系发生凋亡。利用电泳迁移率变动分析，我们分析了经凋亡类视黄醇处理的T细胞中几种转录因子的DNA结合活性。我们发现，在经历凋亡的经类视黄醇处理的T细胞中，通用转录因子Sp1的DNA结合活性丧失。通过免疫印迹分析检测到一种截短的Sp1蛋白，并且从凋亡细胞制备的胞质蛋白提取物含有一种蛋白酶活性，该活性在体外可特异性切割纯化的Sp1。Sp1的这种蛋白水解可被N-乙基马来酰亚胺和碘乙酰胺抑制，表明一种半胱氨酸蛋白酶介导Sp1的切割。此外，ZVAD-fmk和ZDEVD-fmk对Sp1切割的抑制表明，半胱天冬酶直接参与了这一事件。事实上，经凋亡类视黄醇处理后，T细胞中的半胱天冬酶2和3被激活。这些肽抑制剂还阻断了类视黄醇诱导的凋亡，以及完整细胞中半胱天冬酶的加工处理、Sp1的蛋白水解和聚（ADP-核糖）聚合酶的蛋白水解。Sp1的降解在凋亡早期发生，因此可能对细胞的基础转录状态产生深远影响。有趣的是，类视黄醇诱导的凋亡不需要从头合成mRNA和蛋白质，这表明涉及一种新型的类视黄醇信号传导机制，以一种不依赖转录激活、依赖半胱天冬酶的方式触发细胞死亡。
