Related Experiment Videos
Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing
1Department of Neurology, University of Massachusetts Medical Center, Worcester 01655, USA.
Abstract:
Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfRneg) and two lines transfected with either wild-type TfR (Tfrwt) or an internalization-deficient (TfR(delta 7-58del)) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfRwt cells (ID50 < 1 nM) while both TfRneg cells and TfR(delta 7-58del) were only minimally affected (ID50 > 100 nM). Butyrate up-regulation substantially increased receptor expression on the TfRwt and TfR(delta 7-58del) cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfRwt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR(delta 7-58del) cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number.
Insights
Immunotoxin efficacy depends more on how fast cells internalize the toxin and where it goes inside the cell, rather than just the number of transferrin receptors (TfR) present.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Immunotoxin activity relies on target cell binding, internalization, and cytosolic passage.
- Understanding the control of these steps is crucial for optimizing immunotoxin effectiveness.
Purpose of the Study:
- To evaluate the impact of internalization rate and transferrin receptor (TfR) number on immunotoxin potency.
- To investigate the relationship between TfR expression, internalization, and cytotoxicity.
Main Methods:
- Utilized an anti-TfR/ricin A chain immunotoxin (7D3-A).
- Assessed effects on Chinese hamster ovary cell lines with varying TfR expression: none (TfRneg), wild-type (TfRwt), and internalization-deficient mutant (TfR(delta 7-58del)).
- Investigated the influence of butyrate and monensin treatments on immunotoxin sensitivity.
Main Results:
- 7D3-A demonstrated potent cytotoxicity on TfRwt cells (ID50 < 1 nM) but minimal effect on TfRneg and TfR(delta 7-58del) cells (ID50 > 100 nM).
- Butyrate increased TfR expression but did not enhance sensitivity to 7D3-A.
- Monensin treatment significantly increased immunotoxin potency, especially in TfR(delta 7-58del) cells.
Conclusions:
- Internalization rate and intracellular trafficking are more critical determinants of immunotoxin efficacy than receptor number.
- Target cell internalization mechanisms play a pivotal role in immunotoxin-mediated cell killing.