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Effects of low concentrations of okadaic acid in HeLa cells
S K Chaudhuri1, S Ghosh, N Paweletz
1Centre of Advanced Study in Botany, University of Calcutta, India.
Abstract:
HeLa cells treated for prolonged period with okadaic acid (OA; 5-10nM) inhibiting protein phosphatase 2A (PP2A) and also protein phosphatase 1 (PP1) partially showed prolonged effects on mitotic progression. In the presence of OA cells progressed normally in mitosis almost upto 4 hr, then a progressive accumulation of mitotic cells could be noticed. Most of the mitotic cells seemed to be arrested at the metaphase-anaphase transition point. In arrested mitotic cells the chromosomes remained arranged at the equiatorial plate, but with prolonged treatment the chromosomes got either scattered or clumped. However, a slow release into anaphase could also be observed after 15 hr treatment. Immunofluorescence studies for microtubules and electron microscope investigations indicated the dearrangement of spindle fibres, and a prolonged treatment led to the formation of multipolarity. This was also confirmed by spread preparations of chromosomes and the formation of multinucleate cells in preparations released from the mitotic block. Chromosomes became highly condensed showing mostly nondisjunction, but separation of sister chromatids could be observed in many cells. Immunoblot assays indicated a degradation of cyclin A, but the cyclin B1 level was significantly higher in the arrested mitotic cells after 12 hr treatment. After 24 hr of treatment the cyclin B1 level was slightly lower in arrested cells. Possible roles of protein phosphatase 2A inhibition and a prolonged partial inhibition of PP1 on the mitotic progression and the cyclin degradation at the metaphase-anaphase transition have been discussed.
Insights
Okadaic acid (OA) prolonged mitotic arrest in HeLa cells by inhibiting protein phosphatases 2A (PP2A) and 1 (PP1), disrupting spindle fibers and causing cell cycle arrest at metaphase-anaphase transition.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein phosphatases, particularly PP2A and PP1, play critical roles in regulating cell cycle progression.
- Disruptions in phosphatase activity can lead to aberrant mitotic events and cell cycle abnormalities.
Purpose of the Study:
- To investigate the effects of prolonged inhibition of protein phosphatase 2A (PP2A) and partially protein phosphatase 1 (PP1) using okadaic acid (OA) on mitotic progression in HeLa cells.
- To elucidate the molecular mechanisms underlying mitotic arrest and its consequences on chromosome behavior and cell division.
Main Methods:
- HeLa cells were treated with okadaic acid (OA) at concentrations of 5-10 nM for prolonged periods.
- Mitotic progression was monitored, and cell cycle arrest was analyzed.
- Immunofluorescence microscopy was used to study microtubule organization and spindle fibers.
- Electron microscopy provided ultrastructural details of cellular changes.
- Chromosome spread preparations and immunoblot assays were performed to assess chromosome behavior, cyclin levels, and protein degradation.
Main Results:
- Prolonged OA treatment led to a progressive accumulation of mitotic cells, primarily arrested at the metaphase-anaphase transition.
- Observed defects included dearrangement of spindle fibers, formation of multipolar spindles, chromosome scattering/clumping, and sister chromatid nondisjunction.
- Degradation of cyclin A was noted, while cyclin B1 levels remained high in arrested cells, with a slight decrease after 24 hours.
- A slow release into anaphase was observed after 15 hours of treatment, leading to the formation of multinucleate cells.
Conclusions:
- Inhibition of PP2A and partial inhibition of PP1 by OA significantly disrupts mitotic progression and leads to cell cycle arrest at the metaphase-anaphase transition.
- The observed defects in spindle organization and chromosome segregation highlight the critical role of phosphatases in maintaining mitotic fidelity.
- Altered cyclin dynamics, particularly the accumulation of cyclin B1, contribute to the prolonged mitotic arrest and subsequent cellular abnormalities.