Effects of low concentrations of okadaic acid in HeLa cells.

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.

Post-metaphase mitotic events in cells treated with dinitrophenol.

The sequence of post-metaphase mitotic events, such as anaphase movement A and B, chromosome decondensation, nuclear envelope reformation and cytokinesis, has been studied in 2,4-initrophenol (DNP)-treated HeLa cells. The effects of DNP were found to be dose dependent and at concentrations higher than 3 mM, both anaphase A and B movements were totally and nearly instantaneously arrested. It could be shown that cytokinesis did not depend on the completion of anaphase movements. This was also true for nuclear envelope reformation which could take place even around condensed chromosomes arrested in anaphase. The post-metaphase mitotic events do not follow a strict causal sequence, but they can be dissociated from each other in anaphase-arrested cells.