Chazaud B, Muriel M P, Wantyghem J, Aubery M, Decastel M
INSERM U 180, CNRS UAC 71, Centre Universitaire des Saints-Pères, Université René Descartes, Paris, France.
Exp Cell Res. 1995 Nov;221(1):214-20. doi: 10.1006/excr.1995.1369.
We previously showed that ricin, a toxin commonly used in the construction of immunotoxins, was more toxic to undifferentiated than to differentiated HT-29 tumoral cells. This results from differences in the intracellular routings of the toxin. As these studies concerned the entry through the apical pole of differentiated polarized HT-29 cells, we investigated and compared the intracellular routing of ricin from the apical and basolateral membranes of differentiated HT-29 cells and the toxicity of ricin depending on the pole of administration. For this purpose, we developed the culture of polarized HT-29 cells on porous membrane filters and demonstrated that differentiated HT-29 cells can establish a leakproof monolayer. Ricin is 2.5-fold less toxic when it is added at the basolateral than at the apical pole of the cells, which may result from different observations: (1) less ricin is bound at the basolateral membrane than at the apical one, leading to a lesser internalization of the toxin; (2) ricin sorting in the apical and basolateral endocytic compartments of HT-29 cells differs: apically internalized ricin is targeted intracellularly while basolaterally internalized ricin uses mainly the transcytotic pathway; using NH4Cl and monensin, we observed that ricin follows the same pathway from both sides of the cells, namely the endosomal system, to reach the Golgi apparatus from which toxin intoxication occurs; (3) kinetics studies showed that a delay exists before an efficient intoxication by the basolateral pole is observed. The use of monensin at low concentration in order to perturb only the Golgi functions indicated that this delay could account for a different presentation of the toxin toward the membrane of the apical and basolateral endocytic compartments. Together, our results showed that, in differentiated HT-29 cells, if the pathways carrying ricin from the apical and basolateral membranes to the Golgi apparatus appear identical, ricin exerts differentially its toxicity depending upon the surface of administration, i.e., the apical or the basolateral surface of the cells.
我们之前表明，蓖麻毒素是免疫毒素构建中常用的一种毒素，对未分化的HT - 29肿瘤细胞的毒性比对分化的HT - 29肿瘤细胞更强。这是由于毒素在细胞内的转运途径不同所致。由于这些研究涉及毒素通过分化的极化HT - 29细胞顶端进入，我们研究并比较了蓖麻毒素从分化的HT - 29细胞顶端和基底外侧膜进入细胞内的转运途径，以及蓖麻毒素根据给药部位的毒性。为此，我们在多孔膜滤器上培养极化的HT - 29细胞，并证明分化的HT - 29细胞可以形成无渗漏的单层。当蓖麻毒素添加到细胞的基底外侧时，其毒性比添加到顶端时低2.5倍，这可能源于不同的观察结果：(1) 基底外侧膜上结合的蓖麻毒素比顶端膜上少，导致毒素内化较少；(2) HT - 29细胞顶端和基底外侧内吞区室中蓖麻毒素的分选不同：顶端内化的蓖麻毒素在细胞内靶向运输，而基底外侧内化的蓖麻毒素主要利用转胞吞途径；使用氯化铵和莫能菌素，我们观察到蓖麻毒素从细胞两侧遵循相同的途径，即内体系统，到达高尔基体，毒素在高尔基体引发中毒；(3) 动力学研究表明，在观察到基底外侧给药有效中毒之前存在延迟。使用低浓度的莫能菌素仅干扰高尔基体功能表明，这种延迟可能是由于毒素对顶端和基底外侧内吞区室膜的不同呈现方式所致。总之，我们的结果表明，在分化的HT - 29细胞中，如果从顶端和基底外侧膜将蓖麻毒素运输到高尔基体的途径看起来相同，蓖麻毒素根据给药表面，即细胞的顶端或基底外侧表面，发挥不同的毒性作用。
