REQUIREMENT OF EITHER THE NH4CL-SENSITIVE OR THE CYTOCHALASIN D-SENSITIVE PATHWAY FOR RICIN TOXICITY DEPENDS UPON THE ENTEROCYTIC STATE OF DIFFERENTIATION OF HT-29 CELLS

During the course of the present biochemical and ultrastructural studi es, we found that the expression of either the undifferentiated or the differentiated HT-29 cell phenotype determined the intracellular fate of ricin. Although the recognition of ricin at the cell surface requi red interaction with the galactose-binding site on both cell populatio ns, the lag time before ricin started to inhibit protein synthesis tva s longer in the differentiated than the undifferentiated cells. Dose-r esponse studies and ''time-addition'' experiments performed with NH4Cl , which raises the pH of acidic vesicles and organelles, showed that r icin uptake as well as the movement of the toxin to the translocation site were affected in the differentiated cells. In contrast, NH4Cl act ed on only post internalization events in the undifferentiated cells. When the addition of cytochalasin D, an actin-depolymerizing drug, was staggered, the differentiated cells were found to be protected agains t ricin only during the very early stage of the internalization proces s. In contrast, the undifferentiated cells were protected during both the early and late stages of endocytosis. Moreover, electron microscop ic examination showed that cytochalasin D altered the structure of the Golgi apparatus only in the undifferentiated cells. 3-Methyladenine, a specific inhibitor of the autophagic pathway, protected the undiffer entiated and differentiated cells against ricin to about the same exte nt. We concluded that to enter the differentiated cells, ricin followe d the classical endosome-Golgi pathway. In contrast, in the undifferen tiated cells, ricin reaches the cytosol by two distinct routes: the mi nor one involves the endosome-Golgi pathway; the major one involves a cytochalasin D-sensitive pathway.