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Characterization of two pituitary GH3 cell sublines partially resistant to apoptosis induction by okadaic acid
1Institute of Pharmacology & Toxicology, University of Göttingen, Germany.
Abstract:
Pituitary GH3 cells die by apoptosis when treated with okadaic acid, a specific inhibitor of ser/thr phosphatases. Incubations starting at concentrations of 5 and 12.5 nM followed by stepwise rises resulted in two populations (the S1 and S2 sublines) that proliferated at initially lethal 30 nM. Cells were partially resistant to higher concentrations of okadaic acid and its derivative methyl okadaate. Toxicity of the structurally distinct inhibitors cantharidic acid and calyculin A was differently affected in the two resistant lines. The enhanced expression of the P-glycoprotein was one mechanism of resistance in S1 and S2. Resistance was reversed completely (S1) or partially (S2) by the addition of verapamil. In addition, phosphatase activity, presumably PP2A, was increased in S2. Therefore, pharmacokinetic and pharmacodynamic mechanisms can protect pituitary GH3 cells from apoptotic cell death by okadaic acid.
Insights
Pituitary cells developed resistance to okadaic acid, a phosphatase inhibitor, through enhanced P-glycoprotein expression and increased phosphatase activity. This resistance protects cells from apoptosis.
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- Okadaic acid, a specific inhibitor of serine/threonine phosphatases, induces apoptosis in pituitary GH3 cells.
- Understanding cellular resistance mechanisms is crucial for drug development and toxicity studies.
Purpose of the Study:
- To investigate the mechanisms of resistance in pituitary GH3 cells against okadaic acid-induced apoptosis.
- To characterize the pharmacokinetic and pharmacodynamic factors contributing to cellular resistance.
Main Methods:
- Exposure of pituitary GH3 cells to increasing concentrations of okadaic acid to select for resistant sublines (S1 and S2).
- Assessment of cellular resistance to okadaic acid, methyl okadaate, cantharidic acid, and calyculin A.
- Analysis of P-glycoprotein expression and reversal of resistance by verapamil.
- Measurement of intracellular phosphatase activity, specifically PP2A.
Main Results:
- Two resistant sublines (S1 and S2) were established, capable of proliferation at concentrations initially lethal to wild-type cells.
- Enhanced P-glycoprotein expression was identified as a key mechanism of resistance in both S1 and S2 sublines.
- Resistance was partially or completely reversed by verapamil, a P-glycoprotein inhibitor.
- Increased phosphatase activity, likely PP2A, was observed in the S2 subline.
Conclusions:
- Pituitary GH3 cells can develop resistance to okadaic acid-induced apoptosis through multiple mechanisms.
- Enhanced P-glycoprotein expression confers resistance by potentially reducing intracellular drug accumulation (pharmacokinetic).
- Increased phosphatase activity contributes to resistance, possibly by counteracting the effects of okadaic acid (pharmacodynamic).
- Combined pharmacokinetic and pharmacodynamic adaptations protect cells from apoptotic cell death.