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Establishment of a human small-cell lung-cancer subline resistant to okadaic acid
1Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
Abstract:
Okadaic acid (OA), a specific protein phosphatase inhibitor, has various biological functions. To elucidate the mechanism of OA resistance, we have established a small-cell lung-cancer subline (H69/OA100) resistant to the growth-inhibitory effect of OA; this was done by using the parental cell line (H69) and increasing the concentration of OA. H69/OA100 was about 8 times more resistant to OA than H69. Intracellular retention of the fluorescent OA derivative in H69/OA100 was the same as that in H69. The catalytic activity of protein phosphatase from H69/OA100 was significantly reduced compared with that from H69. The protein phosphatase from H69/OA100 was 3.6 times more resistant to OA than that from H69. We examined the effect of OA on the activity of the immunoprecipitated protein phosphatase type I (PPI) and type 2A (PP2A) from the 2 cell lines. The PPI and PP2A from H69/OA100 showed more resistance to OA than those from H69. We next examined the effect of OA on the cell cycle of H69 and H69/OA100. In H69, G2/M block was observed at an OA concentration of 30 ng/ml whereas in H69/OA100, no G2/M block was observed at concentrations up to 100 ng/ml OA. We finally evaluated the amount of p34cdc2 kinase expression and the phosphorylation status of p34cdc2. There was no difference in p34cdc2 expression between H69 and H69/OA100 at several concentrations of OA. However, dephosphorylation of p34cdc2 was observed at 30 ng/ml OA in H69, but not in H69/OA100 up to 100 ng/ml OA. These data suggest that the resistance to OA and the resistance of the cell-cycle block to OA in H69/OA100 might be due to alteration of protein phosphatase activity.
Insights
This study developed okadaic acid (OA)-resistant small-cell lung cancer cells (H69/OA100). The resistance mechanism involves altered protein phosphatase activity, affecting cell cycle progression and p34cdc2 dephosphorylation.
Area of Science:
- Cell Biology
- Molecular Pharmacology
Background:
- Okadaic acid (OA) is a potent protein phosphatase inhibitor with diverse biological effects.
- Understanding resistance mechanisms to OA is crucial for its therapeutic applications and biological studies.
Purpose of the Study:
- To establish and characterize a small-cell lung cancer cell line resistant to okadaic acid (OA).
- To elucidate the molecular mechanisms underlying OA resistance in cancer cells.
Main Methods:
- Developed an OA-resistant subline (H69/OA100) from parental H69 cells by incremental OA exposure.
- Assessed OA resistance, intracellular OA retention, and protein phosphatase catalytic activity.
- Investigated the effect of OA on cell cycle progression and p34cdc2 kinase phosphorylation status.
Main Results:
- H69/OA100 cells exhibited approximately 8-fold higher resistance to OA compared to H69 cells.
- Protein phosphatase activity in H69/OA100 was significantly reduced and more resistant to OA inhibition.
- H69/OA100 cells showed resistance to OA-induced G2/M cell cycle arrest and p34cdc2 dephosphorylation.
Conclusions:
- OA resistance in H69/OA100 cells is associated with altered protein phosphatase activity.
- These alterations confer resistance to OA's effects on cell cycle regulation and kinase activity.