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 studies, 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 required interaction with the galactose-binding site on both cell populations, the lag time before ricin started to inhibit protein synthesis was longer in the differentiated than the undifferentiated cells. Dose-response studies and "time-addition" experiments performed with NH4Cl, which raises the pH of acidic vesicles and organelles, showed that ricin uptake as well as the movement of the toxin to the translocation site were affected in the differentiated cells. In contrast, NH4Cl acted 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 against ricin only during the very early stage of the internalization process. In contrast, the undifferentiated cells were protected during both the early and late stages of endocytosis. Moreover, electron microscopic 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 undifferentiated and differentiated cells against ricin to about the same extent. We concluded that to enter the differentiated cells, ricin followed the classical endosome-Golgi pathway. In contrast, in the undifferentiated cells, ricin reaches the cytosol by two distinct routes: the minor one involves the endosome-Golgi pathway; the major one involves a cytochalasin D-sensitive pathway.
INSERM U 180, CNRS UAC 71, UFR Biomédicale des Saints-Pères, Université René Descartes, Paris/France.
B
B
Chazaud
M P
MP
Muriel
C
C
Bauvy
P
P
Codogno
M
M
Aubery
M
M
Decastel
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000230:Adenocarcinoma / Q000473:pathology; D000643:Ammonium Chloride / Q000494:pharmacology*; D002454:Cell Differentiation; D003106:Colon / Q000187:drug effects* / Q000473:pathology; D003110:Colonic Neoplasms / Q000473:pathology; D015638:Cytochalasin D / Q000494:pharmacology*; D004705:Endocytosis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008870:Microtubules / Q000648:ultrastructure; D012276:Ricin / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D000643:Ammonium Chloride; D015638:Cytochalasin D; D012276:Ricin