Timar J, McIntosh D P, Henry R, Cumber A J, Parnell G D, Davies A J
Institute of Pathology and Experimental Cancer Research, Semmelweis Medical University, Budapest, Hungary.
Br J Cancer. 1991 Oct;64(4):655-62. doi: 10.1038/bjc.1991.377.
Covalent linkage of the A chain of ricin to the LICR-LOND-Fib75 monoclonal antibody produced an immunotoxin, Fib75-SS-ricin A, which demonstrated immunospecific toxicity to human bladder carcinoma cells in tissue culture (Forrester et al., 1984). The present studies have shown that ricin B chain potentiates the toxicity of the immunotoxin by two orders of magnitude and also significantly increases the rate of protein synthesis inhibition. Using immunoelectron microscopy, the receptor-mediated endocytosis and intracellular routing of the immunotoxin was studied with and without ricin B chain treatment after immunolocalisation of the conjugate. Fib75-SS-ricin A was internalised by the EJ cells predominantly in uncoated pits and vesicles and directed to the endosomes. Some degradation of the complex appeared to take place in multivesicular endosomes at early timepoints and 24 h after internalisation, most of the immunotoxin was found in lysosomes. Some ricin A chain epitopes were detected in Golgi vesicles. Cells treated with immunotoxin and ricin B chain endocytosed the complex predominantly in coated pits and coated vesicles. Using pre-embedding immunoperoxidase techniques, ricin chains were found in the whole Golgi complex and most of the conjugate escaped lysosomal degradation. Internalised immunotoxin was recycled back to the plasma membrane in an active form associated with vesicles which appeared to be derived predominantly from multivesicular endosomes. A similar mode of recycling has recently been reported (McIntosh et al., 1990) for ricin holotoxin in the same cell line. These observations may explain the potentiating effect of toxin B chains in the antibody-directed targeting of toxin A chains.
蓖麻毒素A链与LICR-LOND-Fib75单克隆抗体共价连接产生了一种免疫毒素，即Fib75-SS-蓖麻毒素A，它在组织培养中对人膀胱癌细胞表现出免疫特异性毒性（Forrester等人，1984年）。目前的研究表明，蓖麻毒素B链可使免疫毒素的毒性增强两个数量级，并且还显著提高了蛋白质合成抑制率。利用免疫电子显微镜，在对缀合物进行免疫定位后，研究了有无蓖麻毒素B链处理情况下免疫毒素的受体介导内吞作用和细胞内转运途径。Fib75-SS-蓖麻毒素A主要通过未包被的凹陷和小泡被EJ细胞内化，并被导向内体。在早期时间点，复合物在多泡内体中似乎发生了一些降解，内化24小时后，大部分免疫毒素存在于溶酶体中。在高尔基体小泡中检测到了一些蓖麻毒素A链表位。用免疫毒素和蓖麻毒素B链处理的细胞主要通过包被凹陷和包被小泡内吞复合物。使用包埋前免疫过氧化物酶技术，在整个高尔基体复合物中发现了蓖麻毒素链，并且大部分缀合物逃脱了溶酶体降解。内化的免疫毒素以与小泡相关的活性形式循环回到质膜，这些小泡似乎主要来源于多泡内体。最近有报道（McIntosh等人，1990年）称，同一细胞系中的蓖麻毒素全毒素也有类似的循环模式。这些观察结果可能解释了毒素B链在抗体导向的毒素A链靶向中的增强作用。
