Vandré D D, Wills V L
Department of Cell Biology, Ohio State University, Columbus 43210.
J Cell Sci. 1992 Jan;101 ( Pt 1):79-91. doi: 10.1242/jcs.101.1.79.
The effects of the protein phosphatase inhibitor okadaic acid were examined using the pig kidney cell line LLC-PK. At relatively low concentrations of the inhibitor (8-40 nM), cells became blocked in a metaphase-like mitotic state beginning 6-8 h after initial treatment. Spindle microtubules were present throughout the period of the mitotic block, but were not stabilized since they remained sensitive to nocodazole depolymerization. With increasing length of the mitotic block chromosome alignment at the metaphase plate was disrupted and multipolar spindles developed. Cells continued to accumulate in mitosis for at least 24 h, indicating that at these low concentrations okadaic acid was not cytotoxic, but rather acted as a cytostatic agent. Upon release of the okadaic acid block, mitotic LLC-PK cells recovered and completed anaphase. After extended periods of treatment some cells were able to escape the okadaic acid-induced mitotic block. These cells were multinucleate and had undergone cytokinesis in the absence of chromosome segregation. At higher concentrations of okadaic acid (0.5-1.0 microM), mitosis was blocked within 30-60 min of treatment. However, within 90-120 min treated cells rounded up and detached from the monolayer, regardless of whether they were in interphase or mitosis. Cytoplasmic microtubules were depolymerized in the detached cells, and these cells could not recover from the cytotoxic effects of such high concentrations of okadaic acid. Thus, differential effects of the phosphatase inhibitor could be demonstrated, depending upon the concentration of okadaic acid applied to the cultures. The okadaic acid-induced mitotic blockage was probably due to the inhibition of a type 2A protein phosphatase that is involved in the transition from metaphase to anaphase.
利用猪肾细胞系LLC - PK研究了蛋白磷酸酶抑制剂冈田酸的作用。在相对较低浓度的抑制剂（8 - 40 nM）作用下，细胞在初始处理后6 - 8小时开始被阻滞在类似中期的有丝分裂状态。在有丝分裂阻滞期间纺锤体微管一直存在，但并未稳定，因为它们对诺考达唑解聚仍敏感。随着有丝分裂阻滞时间延长，中期板处的染色体排列被破坏，多极纺锤体形成。细胞在有丝分裂中持续积累至少24小时，表明在这些低浓度下冈田酸无细胞毒性，而是起到细胞生长抑制剂的作用。解除冈田酸阻滞作用后，有丝分裂的LLC - PK细胞恢复并完成后期。长时间处理后，一些细胞能够逃脱冈田酸诱导的有丝分裂阻滞。这些细胞是多核的，并且在没有染色体分离的情况下经历了胞质分裂。在较高浓度的冈田酸（0.5 - 1.0 microM）作用下，处理30 - 60分钟内有丝分裂被阻滞。然而，90 - 120分钟内，处理的细胞变圆并从单层细胞上脱离，无论它们处于间期还是有丝分裂期。脱离的细胞中细胞质微管解聚，这些细胞无法从如此高浓度冈田酸的细胞毒性作用中恢复。因此，根据应用于培养物中冈田酸的浓度，可以证明磷酸酶抑制剂的不同作用。冈田酸诱导的有丝分裂阻滞可能是由于抑制了参与从中期到后期转变的2A类蛋白磷酸酶。
