Wu Y N, Gadina M, Tao-Cheng J H, Youle R J
Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
J Cell Biol. 1994 May;125(4):743-53. doi: 10.1083/jcb.125.4.743.
All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.
全反式维甲酸可使受体介导的蓖麻毒素A链免疫毒素中毒特异性增加10000倍以上，而单独的蓖麻毒素A链或蓖麻毒素A链免疫毒素的液相内吞作用不受维甲酸影响。维甲酸对免疫毒素的激活不需要RNA或蛋白质合成，也不是免疫毒素受体结合增加的结果。激活维甲酸受体（RAR）同源物并与类视黄醇X受体（RXR）形成异二聚体的维生素D3和甲状腺激素T3不影响免疫毒素的效力。在测试的其他类视黄醇中，既不与RAR也不与RXR结合的13-顺式维甲酸也能提高蓖麻毒素A链免疫毒素的效力。因此，激活维甲酸受体似乎不是免疫毒素活性所必需的。布雷菲德菌素A（BFA）可阻止维甲酸对免疫毒素的增强作用，这表明在存在维甲酸的情况下，免疫毒素有效地通过高尔基体转运至细胞质。用针对高尔基体标记酶甘露糖苷酶II的单克隆抗体（Mab）直接检测细胞表明，用维甲酸处理后，高尔基体从核周网络变为弥散聚集体。去除维甲酸后60分钟内，细胞重新组装出与正常对照细胞无法区分的核周高尔基体网络。C6-NBD-神经酰胺是一种用于高尔基体的活体染色剂，它表明维甲酸可阻止高尔基体的荧光染色并消除预先用C6-NBD-神经酰胺染色的高尔基体的荧光。对经维甲酸处理的细胞进行电子显微镜检查显示，特异性缺乏任何外观正常的高尔基体，以及一个与莫能菌素处理的细胞中所见非常相似的核周泡状结构。去除维甲酸后这种空泡化消失，核周高尔基体堆叠重新出现。这些结果表明，维甲酸可能通过高尔基体改变细胞内转运途径，在不依赖新基因表达的情况下增强免疫毒素活性。维甲酸似乎是一种用于操纵高尔基体和细胞内运输的新试剂。由于维甲酸和免疫毒素都在癌症治疗的临床试验中，它们在体内的联合活性值得研究。
