Distelhorst C W, Janiga K E, Howard K J, Strandjord S E, Campbell E J
Blood. 1987 Sep;70(3):860-8.
Characterization of glucocorticoid receptors in leukemia cells is important to understand mechanisms of glucocorticoid resistance but has been impeded by receptor fragmentation in cytosol extracts. We recently found that formation of 52- and 30-kilodalton (kD) glucocorticoid receptor fragments in cytosol of leukemia cells is due to proteolysis and is blocked by diisopropylfluorophosphate (DFP). In the present study, we identify a 28-kD serine protease in cytosol of leukemia cells that binds [3H]DFP and correlates with the formation of 52- and 30-kD receptor fragments. This protease is immunoprecipitated by antiserum to neutrophil elastase. Limited digestion of [3H]dexamethasone-21-mesylate-labeled receptors by purified neutrophil elastase produces 52- and 30-kD receptor fragments. Receptor fragmentation in the cytosol of leukemia cells in inhibited by methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethylketone, a highly specific inhibitor of neutrophil elastase. The addition of as few as 5% neutrophils to a lymphoid cell suspension provides sufficient elastase to produce receptor fragmentation. Our findings indicate that neutrophil elastase is responsible for receptor fragmentation in the cytosol of leukemia cells. The neutrophil elastase may be endogenous to the leukemia cells or may come from neutrophils that contaminate leukemia cell suspensions.
白血病细胞中糖皮质激素受体的特征对于理解糖皮质激素耐药机制很重要，但一直受到胞质提取物中受体片段化的阻碍。我们最近发现，白血病细胞胞质中52千道尔顿（kD）和30 kD糖皮质激素受体片段的形成是由于蛋白水解作用，并且可被二异丙基氟磷酸酯（DFP）阻断。在本研究中，我们在白血病细胞胞质中鉴定出一种28 kD的丝氨酸蛋白酶，它能结合[3H]DFP，并与52 kD和30 kD受体片段的形成相关。这种蛋白酶可被抗中性粒细胞弹性蛋白酶的抗血清免疫沉淀。用纯化的中性粒细胞弹性蛋白酶对[3H]甲磺酸地塞米松-21-甲酯标记的受体进行有限度消化，会产生52 kD和30 kD的受体片段。甲氧基琥珀酰-丙氨酰-丙氨酰-脯氨酰-缬氨酰-氯甲基酮（一种中性粒细胞弹性蛋白酶的高度特异性抑制剂）可抑制白血病细胞胞质中的受体片段化。向淋巴细胞悬液中加入低至5%的中性粒细胞就能提供足够的弹性蛋白酶来产生受体片段化。我们的研究结果表明，中性粒细胞弹性蛋白酶是白血病细胞胞质中受体片段化的原因。中性粒细胞弹性蛋白酶可能是白血病细胞内源性的，也可能来自污染白血病细胞悬液的中性粒细胞。
