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Chinese hamster ovary cell mutants defective in the internalization of ricin
Abstract:
Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.
Insights
Researchers isolated Chinese hamster ovary mutants resistant to ricin and Pseudomonas toxin. These mutants show defects in toxin internalization, suggesting a shared pathway for both toxins entry into cells.
Area of Science:
- Cell Biology
- Toxicology
- Molecular Biology
Background:
- Ricin and Pseudomonas toxin are potent protein toxins.
- Understanding toxin entry mechanisms is crucial for developing countermeasures.
- Previous studies suggested potential similarities in toxin processing.
Purpose of the Study:
- To isolate and characterize mutants of Chinese hamster ovary cells resistant to both ricin and Pseudomonas toxin.
- To investigate the specific cellular defect responsible for resistance.
- To determine if ricin and Pseudomonas toxin share a common internalization pathway.
Main Methods:
- Isolation of toxin-resistant mutants using selection assays.
- Assessment of toxin binding to mutant cell surfaces.
- Biochemical assays to measure toxin internalization.
- Electron microscopic autoradiography to visualize toxin uptake.
- Cytotoxicity assays with and without ionophore treatment.
Main Results:
- Two mutant cell lines (4-10 and 11-2) were identified with simultaneous resistance to ricin and Pseudomonas toxin.
- These mutants exhibited normal binding of both toxins.
- A significant defect in the internalization of ricin was observed in mutant cells.
- Ionophore treatment, which enhances wild-type cell sensitivity, did not affect mutant cell susceptibility.
Conclusions:
- The findings indicate a common step in the internalization process for ricin and Pseudomonas toxin.
- The isolated mutants provide a valuable tool for studying toxin entry mechanisms.
- This research contributes to understanding cellular pathways involved in protein toxin uptake.