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 μM), 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 μM), 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.

The transition from interphase to mitosis is characterized by the condensation of chromosomes, the breakdown of the nuclear envelope, the disassembly of the cytoplasmic microtubule complex, and the formation of the mitotic spindle. These events appear to be initiated by the specific phosphorylation of protein components associated with each of these structures. These mitosisspecific phosphorylations correlate with the activation of a histone H1 serine/threonine protein kinase activity that has been identified as both the product of the fission yeast cell cycle control gene cdc2 (p34cdc2) and a component of maturation or mitotic promoting factor (MPF) (Arion et al. 1988; Dunphy et al. 1988; Gautier et al. 1988; Labbé et al. 1988; Lohka et al. 1988). Mitotic protein substrates of the activated p34cdc2 kinase must subsequently be dephosphorylated upon exit from mitosis. These dephosphorylation events are temporally associated with the onset of anaphase (Dorée et al. 1983; Vandré and Borisy, 1989), and correlate with the inactivation of p34cdc2 kinase. Therefore, a series of phosphorylation and déphosphorylation reactions are intimately involved with the regulation of mitotic processes such as nuclear envelope breakdown and re-formation (Newport, 1987; Dessev et al. 1991), and ultimately with the entry and exit of cells from mitosis.

Okadaic acid (OA), a polyether derivative of a fatty acid, has been shown to be a potent tumorpromoting substance on mouse skin; however, OA does not bind to receptors of the phorbol ester class of tumor-promoting compounds or activate protein kinase C (Suganuma et al. 1988). OA binds to and inhibits protein phosphatases present in the cell, specifically protein phosphatase 1(PP1) and protein phosphatase 2A (PP2A) (reviewed by Cohen et al. 1990). Reported ID50 values for OA range from 0.04 to 1 nM against the PP2A catalytic subunit, and 12 to 500 nM against the catalytic subunit of PPI (Hescheler et al. 1988; Cohen et al. 1989). The resulting inhibition of phosphatase activity leads to an increase in overall protein phosphorylation in treated cells (Haystead et al. 1989).

When OA was microinjected into Xenopus or starfish oocytes, at a final intracellular concentration of 0.25 μM and 1.2 μM, respectively, MPF activation and meiotic maturation were induced (Goris et al. 1989; Picard et al. 1989). This suggests that OA-sensitive phosphatases maintain MPF in its inactive precursor form, pre-MPF. Activation of MPF in these oocyte systems leads to a burst of protein phosphorylation and germinal vesicle breakdown (GVBD). Similarly, mouse oocytes treated with OA (25 nM-2.5 μM) also exhibit GVBD and chromosome condensation (Rimé and Ozon, 1990; Gavin et al. 1991). In the first of these reports, spindles were not detected in treated oocytes (Rimé and Ozon, 1990); however, in approximately 50% of treated oocytes in the second study spindles were present but they showed an abnormal morphology (Gavin et al. 1991). Spindle formation was also not observed in microinjected starfish oocytes (Picard et al. 1989). Therefore, either the cytoplasm of OA-treated oocytes was not capable of organizing metaphase microtubules in a normal fashion, or the phosphorylation state of some oocyte proteins must prevent the formation of or cause the depolymerization of spindle microtubules.

In Xenopus egg extracts, concentrations of OA that inhibit PP2A but not PPI were shown to transiently activate the p34cdc2 kinase (Felix et al. 1990a), and the activity of p34cdc2 kinase has been shown to influence the dynamics of microtubule assembly in vitro using Xenopus extracts (Verde et al. 1990). This correlates with the finding that a negative regulator of MPF activity, INH, is an OA-sensitive PP2A (Lee et al. 1991), and alteration in the activity of INH reduces the threshold level of cyclin proteins, which are required for MPF activation (Solomon et al. 1990).

Treatment of tissue culture cells with OA has demonstrated morphological changes similar to those of mitotic cells (Kipreos and Wang, 1990; Yamashita et al. 1990). In each of these studies relatively high extracellular concentrations of OA were added to the culture media, and cell rounding was observed within 60 min. Yamashita and coworkers (1990) showed in their study that the rounded BHK cells detached from the growing surface and entered a transient mitotic state characterized by premature chromosome condensation. Spindles were not observed in these cells while in the mitotic state, and upon further incubation all microtubules depolymerized. On the other hand, the rounding observed in NIH 3T3 cells by Kipreos and Wang (1990) was not associated with nuclear envelope breakdown or chromosome condensation. From these studies it was not clear whether the rounding of cells and alterations in microtubule architecture were due solely to the induction of a mitotic state. We, therefore, examined the effect of OA on cell growth, mitotic progression, and microtubule and spindle morphology, in LLC-PK cells. These results demonstrate that the effects of OA on mitotic progression and spindle morphology can be distinguished from its effects on cell shape and interphase microtubule arrays. Some of these results have appeared in preliminary form (Vandré, 1990).

Cell culture

LLC-PK cells, derived from porcine kidney, were maintained in monolayer culture at 37°C in a 5% CO2 atmosphere. Cells were grown in DMEM media supplemented with 10% fetal bovine serum, penicillin (100 units ml-1), streptomycin (0.1 mg ml-1), and 20 mM Hepes buffer (Sigma Chemical Company, St Louis, MO). Cells were subcultured 16–24 h prior to the addition of okadaic acid (OA) from a 100 μg ml-1 stock solution in dimethyl formamide (Moana Bioproducts, Honolulu, HI). Addition of equivalent amounts of dimethyl formamide to the cultures had no effect on cell growth or morphology. Toxicity of OA was determined by counting cells that remained attached to the monolayer growing surface of the flask after appropriate incubation time in OA. Attached cells were collected by treatment with trypsin-EDTA, and counted using a hemacytometer chamber. The effects of varying OA concentration and incubation time on the mitotic index, mitotic stage and number of spindle poles present in treated cells were determined. Between 100 and 300 individual cells per coverslip were examined at each experimental point. Cultures were grown on glass coverslips, incubated with OA, and then processed for immunofluorescence microscopy (see below). In some cases, cultures were synchronized at the G/S boundary following exposure to 2 mM hydroxyurea for 16 h prior to the addition of OA.

Immunofluorescence staining and microscopy

Cells remaining attached to coverslips were lysed in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgSO-O, pH 6.9, containing 0.5% Triton X-100 for 90 s. Following lysis, cells were rinsed in PHEM buffer and fixed in PHEM buffer containing 0.7% glutaraldehyde for 15 min. Fixative was aspirated and cells were rinsed in three changes of phosphate-buffered saline (PBS), pH 7.4. Unreacted aldehyde groups were reduced by two changes of NaBH (1 mg ml-1 in Tris-buffered saline, pH 7.4) over 30 min. Coverslips were rinsed three times in PBS and incubated in 4% normal goat serum for 30 min at 37°C, rinsed in PBS, and processed for tubulin immunofluorescence using the YL 1/2 rat monoclonal anti-tubulin antibody (Accurate Chemical and Scientific Corp., Westbury, NY). After three rinses in PBS, coverslips were incubated with fluorescein-conjugated anti-rat immunoglobulin for 30 min at 37°C, and subsequently rinsed three times in PBS. The next to last PBS rinse contained 5 μg ml-1 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI, Polysciences, Inc., Warrington, PA). After a final rinse in distilled water coverslips were mounted in Mowiwol (Osborn and Weber, 1982) containing 1 mg ml-1 paraphenylene diamine. For double-label immunofluorescence, cells were incubated with a mixture of the MPM-2 mouse monoclonal antibody (Davis et al. 1983) and rabbit anti-Tyr-tubulin antibody (Gunderson et al. 1984). This was followed by an incubation in a mixture of rhodamine-conjugated goat antimouse and fluorescein-conjugated goat anti-rabbit immunoglobulins.

Media containing OA-treated cells that had rounded and detached from the coverslip were collected, and cells were deposited onto coverslips by cytocentrifugation using a Cytospin 3 (Shandon Inc., Pittsburgh, PA). Cytocentrifuged cells were fixed in PHEM buffer containing 0.7% glutaraldehyde prior to their subsequent lysis in PHEM buffer containing 0.5% Triton X-100. Lysed cells were processed for immunofluorescence as described above.

Mounted coverslips were examined with a Zeiss IM-35 microscope equipped with epifluorescence optics using a Nikon ×60 phase, 1.4 NA, planapochromat objective. Immunofluorescence micrographs were recorded on Kodak Tech-Pan 2415 film.

Okadaic acid blocks LLC-PK cells in mitosis

LLC-PK cells were treated with various concentrations of okadaic acid (OA) to determine its effect on cell growth. OA was added to the culture media 24 h after cells had been subcultured, and monolayers were maintained for an additional 24–48 h, at which time the number of cells remaining in the monolayer was determined (Fig. 1A). Cell growth was inhibited at concentrations greater than 4.0 nM, and significant cytotoxicity was noted at concentrations of 62 nM or higher. The LD50 for OA determined after 48 h of exposure was approximately 10 nM (Fig. IB). In comparison to control cultures, in drug-treated cultures a significantly greater number of cells remaining on the monolayer appeared to be in mitosis.

Fig. 1.

Effect of OA on the growth of LLC-PK cells grown in monolayer culture. (A) Cultures were incubated with various concentrations of OA and the cell numbers determined after 24 and 48 h: control (○–○); 4 Nm (▾ ▾); 8 nM (▫ ▫); 15 nM (▴–▴); 31 nM (◊–◊); 62 nM (•–•); 124 nM (▿–▿) OA. (B) Percentage of cells surviving treatment with OA for 48 h in comparison to control populations; LD5o = 10 nM.

Fig. 1.

Effect of OA on the growth of LLC-PK cells grown in monolayer culture. (A) Cultures were incubated with various concentrations of OA and the cell numbers determined after 24 and 48 h: control (○–○); 4 Nm (▾ ▾); 8 nM (▫ ▫); 15 nM (▴–▴); 31 nM (◊–◊); 62 nM (•–•); 124 nM (▿–▿) OA. (B) Percentage of cells surviving treatment with OA for 48 h in comparison to control populations; LD5o = 10 nM.

The mitotic index of treated cultures was determined by examining cells fixed and then stained with antitubulin antibodies to detect microtubule patterns and DAPI to detect DNA. A dramatic increase in mitotic index was observed after incubation in OA. This increase was dependent upon both the concentration of OA applied (Fig. 2A) and the length of exposure to drug (Fig. 2B). At these low concentrations of OA, there was a significant lag period prior to the accumulation of mitotic cells that was highly concentration dependent. At 22 nM OA, the mitotic index did not increase significantly until nearly 8 h after treatment. This lag period was longer at lower concentrations. In comparing Fig. 2A and B it is apparent that at 15 nM OA mitotic cells began to accumulate only after 14 h of exposure, while the mitotic block was readily apparent after 12 h with 22 nM. Further incubation at 15 nM OA showed that cells continued to accumulate in mitosis (data not presented). The lag time prior to the mitotic block was significantly reduced at higher concentrations of OA (see below).

Fig. 2.

Effect of OA on the mitotic index of LLC-PK cells. (A) Cultures were incubated for 14 h with various concentrations of OA, and the mitotic index of the population was determined. Only a slight effect on mitotic index was observed at 15 nM OA but the mitotic index was nearly 6 times control levels at 31 nM OA. (B) The mitotic index of treated cultures was determined following in cubation with 22 nM OA for various periods of time. An increase in mitotic index was not observed until after nearly 8 h of treatment, and the mitotic index continued to increase with longer exposures to the OA.

Fig. 2.

Effect of OA on the mitotic index of LLC-PK cells. (A) Cultures were incubated for 14 h with various concentrations of OA, and the mitotic index of the population was determined. Only a slight effect on mitotic index was observed at 15 nM OA but the mitotic index was nearly 6 times control levels at 31 nM OA. (B) The mitotic index of treated cultures was determined following in cubation with 22 nM OA for various periods of time. An increase in mitotic index was not observed until after nearly 8 h of treatment, and the mitotic index continued to increase with longer exposures to the OA.

Treated cells were examined to determine the mitotic stage blocked by OA. At concentrations of OA that had an effect on mitotic index (15–31 nM; Fig. 2A), cells accumulated in a prometaphase-like state, as defined by the absence of an intact nuclear envelope, the presence of spindle microtubules and the lack of metaphase alignment of condensed chromosomes. Almost no metaphase, anaphase or telophase cells were present in the cultures treated overnight with 31 nM OA. However, little change in the percentage of mitotic cells in prophase was detected (data not presented), suggesting that cells were continuing to enter mitosis, but were blocked in a mitotic state prior to anaphase onset. When cells were first treated overnight with hydroxyurea followed by low concentrations of OA in the continued presence of hydroxyurea, no mitotic cells were detected even after extended periods of incubation. Mitotic cells did accumulate if the hydroxyurea block was released at the time of OA addition (data not presented). Together, these results indicate that during the first few hours of exposure to OA other stages of cell cycle progression were not inhibited.

The okadaic acid-induced mitotic block is characterized by the presence of an abnormal mitotic spindle

Various spindle morphologies were represented in the blocked mitotic cells following overnight treatment with 22 nM OA acid; the most common types are shown in Fig. 3. Some cells contained a normal bipolar spindle with chromosomes aligned on the metaphase plate (Fig. 3A and B). However, some of the bipolar spindles with a typical microtubule pattern showed a displacement of some chromosomes off the metaphase plate. These displaced chromosomes were usually oriented near one of the spindle poles (Fig. 3C and D). Many cells contained bipolar spindles that had a much greater pole-to-pole separation compared to typical bipolar spindles (compare Fig. 3A with Fig. 4E and G). Chromosomes were no longer aligned at the metaphase plate but were distributed in a fairly random fashion between the spindle poles (Fig. 3F). In a few examples, the spindle fibers appeared to collapse, giving the impression of an extended narrow spindle (Fig. 3G). The chromosomes in this particular cell appear to be in three groups, one group associated with each spindle pole and one group in a metaphase position (Fig. 3H). Multipolar cells were also fairly common, with the chromosomes often randomly distributed between the spindle poles (Fig. 31 and J). The last major category of spindle morphology present in blocked cells was represented by what appeared to be a disruption in the association between the two half-spindles (Fig. 3K and L). Interzonal microtubules connecting the halfspindles were absent, and chromosomes were independently associated with each half-spindle. In each example of aberrant spindle morphology the sister chromatids remained paired.

Fig. 3.

Indirect immunofluorescence staining of tubulin in OA-treated LLC-PK cells. Cells grown on coverslips were treated with 22 nM OA for 18 h, at which time the cells fixed and processed for anti-tubulin indirect immunofluorescence staining (A, C, E, G, I and K). DNA was stained with DAP1 (B, D, F, H, J and L). Various spindle morphologies are present in OA-blocked cells, and representative examples are presented. These morphologies range from normal bipolar spindles with chromosomes aligned on the metaphase plate (A and B), to spindles with a few chromosomes displaced from the metaphase plate (C and D), bipolar spindles with a dispersed set of chromosomes (E and F), compacted narrow bipolar spindles (G and H), multipolar spindles (I and J), and disrupted spindles in which the half-spindles are no longer associated (K and L). Bar, 10 μm.

Fig. 3.

Indirect immunofluorescence staining of tubulin in OA-treated LLC-PK cells. Cells grown on coverslips were treated with 22 nM OA for 18 h, at which time the cells fixed and processed for anti-tubulin indirect immunofluorescence staining (A, C, E, G, I and K). DNA was stained with DAP1 (B, D, F, H, J and L). Various spindle morphologies are present in OA-blocked cells, and representative examples are presented. These morphologies range from normal bipolar spindles with chromosomes aligned on the metaphase plate (A and B), to spindles with a few chromosomes displaced from the metaphase plate (C and D), bipolar spindles with a dispersed set of chromosomes (E and F), compacted narrow bipolar spindles (G and H), multipolar spindles (I and J), and disrupted spindles in which the half-spindles are no longer associated (K and L). Bar, 10 μm.

Fig. 4.

Effect of OA on the spindle pole number in cells blocked in mitosis. The percentages of mitotic cells that were bipolar (▪), tripolar (▪) and multipolar (more than three poles) (S), were determined in cells labeled for indirect immunofluorescence with antibodies to tubulin. (A) The change in spindle pole number in relation to the concentration of OA after treatment of 16 h. Higher concentrations of OA produce a greater percentage of multipolar spindles in treated cells. (B) The length of exposure to OA effects spindle pole number in treated cells. An increase in the percentage of multipolar cells corresponds to the length of exposure to OA. Tripolar cells appear prior to multipolar cells in response to both the concentration and the time of exposure to OA.

Fig. 4.

Effect of OA on the spindle pole number in cells blocked in mitosis. The percentages of mitotic cells that were bipolar (▪), tripolar (▪) and multipolar (more than three poles) (S), were determined in cells labeled for indirect immunofluorescence with antibodies to tubulin. (A) The change in spindle pole number in relation to the concentration of OA after treatment of 16 h. Higher concentrations of OA produce a greater percentage of multipolar spindles in treated cells. (B) The length of exposure to OA effects spindle pole number in treated cells. An increase in the percentage of multipolar cells corresponds to the length of exposure to OA. Tripolar cells appear prior to multipolar cells in response to both the concentration and the time of exposure to OA.

Like the increase in mitotic index (Fig. 2), the frequency with which multipolar spindles were observed increased with the concentration of OA (Fig. 4A), and length of exposure to OA (Fig. 4B). The increase in multipolar cells appeared to proceed in a temporal sequence, with the development of tripolar cells preceding an increase in cells that contained four or more spindle poles.

LLC-PK cells arrested in mitosis with many drugs, such as colcemid or nocodazole, lack spindle microtubules; however, cells blocked in mitosis with taxol, a microtubule stabilizing agent, retain polymerized microtubules. Spindle microtubules were present in OA-blocked cells after 36 h of treatment, which suggested that the OA may, in part, block mitosis by stabilizing microtubules. To test this possibility OA-blocked cells were treated with nocodazole at concentrations that resulted in net depolymerization of microtubules in untreated mitotic cells. OA-blocked cells showed the same sensitivity to nocodazole as did control cells (data not presented). Thus, OA treatment did not appear to stabilize spindle microtubules; therefore, stabilization cannot account for the persistence of spindle microtubules in blocked cells.

Mitosis-specific phosphorylations are maintained in blocked cells

The MPM-2 antibodies (Davis et al. 1983) recognize a set of mitotic phosphoproteins some of which are localized to the centrosome (Vandré et al. 1984). These phosphoproteins are a marker for the mitotic state and are lost or dephosphorylated only after anaphase onset (Vandré and Borisy, 1989). Yamashita and coworkers (1990) have reported that MPM-specific staining was initially detected, but subsequently lost from BHK cells after a premature mitotic state was induced by relatively high concentrations of OA. Therefore, LLC-PK cells blocked in mitosis with OA were examined with the MPM-2 antibody to determine if MPM-reactive phosphoproteins characteristic of mitosis were maintained throughout the length of the blockage.

There was no increase in the MPM staining levels in any interphase cells, but MPM-reactive material was associated with spindle poles and kinetochores in all cells blocked in mitosis regardless of the length of OA treatment (Fig. 5). Typically, two of the spindle poles or microtubule nucleating centers in multipolar cells reacted more intensely with the MPM-2 antibody (Fig. 5B and D), which suggested that these were the centriole-containing structures. The staining intensity of other poles varied greatly, some reacted fairly strongly while others could not be detected. The generation of secondary poles may result from the fragmentation of pericentriolar material from the original centrosomes, which may account for the wide range of staining intensity observed with the MPM antibody. The generation of new microtubule organizing centers in these treated cells was probably not due to the separation of existing centrioles or to the formation of new centrioles, since sufficient time to complete an additional centriole cycle is not required prior to the appearance of new nucleating sites. These possibilities will be resolved only by following ultrastructural analysis, however.

Fig. 5.

MPM-reactive mitotic phosphoproteins are present in OA-blocked mitotic cells. LLC-PK cells were treated with 22 nM OA (A and B) or 31 nM OA (C and D) for 16 h and examined by double-label immunofluorescence microscopy for tubulin staining (A and C) and MPM-2 staining (B and D). Multipolar spindles were shown to have MPM-reactive material concentrated at the foci of microtubule-organizing centers. Two of these MPM-reactive centers stained more intensely (large arrowheads in D), and probably correlate with the position of the centriole-containing centrosomes. Other foci showed variable, but less intense, levels of MPM staining (small arrowheads in D). The number of microtubules nucleated from an individual organizing center varied, and the most prominent nucleating sites did not necessarily correspond to the position of the two major MPM-reactive centers. Bar, 10 μM.

Fig. 5.

MPM-reactive mitotic phosphoproteins are present in OA-blocked mitotic cells. LLC-PK cells were treated with 22 nM OA (A and B) or 31 nM OA (C and D) for 16 h and examined by double-label immunofluorescence microscopy for tubulin staining (A and C) and MPM-2 staining (B and D). Multipolar spindles were shown to have MPM-reactive material concentrated at the foci of microtubule-organizing centers. Two of these MPM-reactive centers stained more intensely (large arrowheads in D), and probably correlate with the position of the centriole-containing centrosomes. Other foci showed variable, but less intense, levels of MPM staining (small arrowheads in D). The number of microtubules nucleated from an individual organizing center varied, and the most prominent nucleating sites did not necessarily correspond to the position of the two major MPM-reactive centers. Bar, 10 μM.

Okadaic acid-treated cells can recover from the mitotic block

To determine whether the mitotic block induced by OA (22 nM) was reversible, treated cultures were washed free of the drug and incubated in fresh media. Samples were examined at hourly intervals following drug release, for the appearance of anaphase or telophase cells. As in the initial mitotic block, a significant lag period, in this case 4–5 h, was required before there was an increase in these later mitotic stages. Spindles in recovering cells were generally bipolar, but occasional multipolar anaphase cells were observed (data not presented). The number of multipolar spindles was significantly lower than the number detected prior to release, which suggested that a bipolar spindle was reestablished during recovery. Occasionally one or both of the centrosomes appeared to be detached from the spindle in recovering cells, but normal anaphase separation of chromosomes appeared to take place (data not presented). After 14 h of release nearly all cells in the treated population recovered from the OA-induced mitotic block.

At the low concentrations used to generate the mitotic block, OA was not cytotoxic, as shown by the recovery of cells when the drug was removed. In addition, occasional multinucleated cells (approximately 1–2% of the mitotic cells) were observed in cultures blocked with OA longer than 24 h (Fig. 6). This suggested that some treated cells escaped the mitotic block. Closer examination of the multinucleated cells indicated that they had undergone cytokinesis, as in every example a midbody was present (Fig. 6). The distribution of nuclei appeared random, as some cells cleaved off only a small portion of cytoplasm lacking nuclei or DNA (Fig. 6A-C) while others showed varying numbers of nuclei in the two daughter cells (Fig. 6D-I). In some cases it was apparent that reforming nuclei had been trapped between the daughter cells as the cleavage furrow progressed (Fig. 6D-E). The level of MPM staining in these multinucleated cells was also at interphase levels (data not presented). Thus, at the concentrations that resulted in a mitotic blockage OA did not inhibit cytokinesis.

Fig. 6.

Multinucleated cells that had escaped the OA-induced mitotic block were observed after extended incubation. Phase-contrast (A, D and G), DNA staining by DAPI (B, E and H), and anti-tubulin staining (C, F and I) of multinucleated LLC-PK cells present in cultures treated with 22 nM OA for 30 h. The 1-2% of the cells in the culture that escaped the OA-induced mitotic block were characterized by multiple micronuclei and a midbody. These morphologies suggested that chromosome segregation did not occur, but also indicated that the cells underwent cytokinesis. This cytokinesis resulted in some cells that appeared to cleave off only a small anuclear piece of cytoplasm (A-C), while in most cleavage occurred near the center of the cell (D-I). This cleavage often resulted in a micronucleus being trapped in the cleavage furrow (D-F). Bars: 10 μm (A-F); 20 μM (G-I).

Fig. 6.

Multinucleated cells that had escaped the OA-induced mitotic block were observed after extended incubation. Phase-contrast (A, D and G), DNA staining by DAPI (B, E and H), and anti-tubulin staining (C, F and I) of multinucleated LLC-PK cells present in cultures treated with 22 nM OA for 30 h. The 1-2% of the cells in the culture that escaped the OA-induced mitotic block were characterized by multiple micronuclei and a midbody. These morphologies suggested that chromosome segregation did not occur, but also indicated that the cells underwent cytokinesis. This cytokinesis resulted in some cells that appeared to cleave off only a small anuclear piece of cytoplasm (A-C), while in most cleavage occurred near the center of the cell (D-I). This cleavage often resulted in a micronucleus being trapped in the cleavage furrow (D-F). Bars: 10 μm (A-F); 20 μM (G-I).

These results suggested that in cells escaping the OA-induced mitotic block chromosome segregation did not take place but, rather, multiple nuclei formed around the chromosomes that were randomly dispersed on the spindle (see Fig. 3). A cleavage plane was established, however, since a spindle was present in the OA-blocked cells. Cells escaping the block could complete cytokinesis in the absence of chromosome separation. The dephosphorylation events associated with anaphase occurred as the cells escaped the mitotic block. It has not been determined whether sister chromatid disjunction occurred in these cells prior to nuclear envelope breakdown.

High concentrations of okadaic acid block mitosis rapidly, but are also cytotoxic

At high concentrations of OA (0.5–1.0 μM), inhibition of mitosis is observed within 30-90 min; however, there was no evidence that cells entered a premature mitotic state. Normal metaphase spindles were present in cells exposed to 0.5 gM OA for 60 min (Fig. 7A-C), but were no longer observed after 90 min. At concentrations of 1.0 gM OA, bipolar spindles were present after a 60 min incubation, but they were structurally aberrant (Fig. 7D-I). Chromosomes alignment at the metaphase plate was lost, the spindles were generally larger than normal metaphase spindles, and half-spindles were often composed of wavy or curved microtubules (Fig. 7E). MPM staining was typical for mitotic cells, and was localized to the spindle poles and kinetochores, and along spindle fibers. Further incubation led to the detachment of mitotic cells from the monolayer, and loss of spindle microtubules (data not presented).

Fig. 7.

Mitosis was inhibited rapidly at higher concentrations of OA. Treated LLC-PK cells were examined by phasecontrast (A,D and G) and double-label immunofluorescence staining with anti-tubulin (B, E and H) and MPM-2 antibodies (C, F and I). Normal bipolar metaphase spindles were still present in cells treated with 0.5 μM OA for 60 min (A-C). At 1.0 gM OA, treatment for 60 minutes resulted in an inhibition of mitosis (D-I). Chromosomes were displaced from the metaphase plate to varying degrees, but remained associated with spindle microtubules. Spindle microtubules at this higher concentration of OA were typically curved or wavy in appearance (E). MPM-2 antibodies clearly stained the spindle poles and kinetochores in these treated mitotic cells (C, F and I). Bar, 10 μm.

Fig. 7.

Mitosis was inhibited rapidly at higher concentrations of OA. Treated LLC-PK cells were examined by phasecontrast (A,D and G) and double-label immunofluorescence staining with anti-tubulin (B, E and H) and MPM-2 antibodies (C, F and I). Normal bipolar metaphase spindles were still present in cells treated with 0.5 μM OA for 60 min (A-C). At 1.0 gM OA, treatment for 60 minutes resulted in an inhibition of mitosis (D-I). Chromosomes were displaced from the metaphase plate to varying degrees, but remained associated with spindle microtubules. Spindle microtubules at this higher concentration of OA were typically curved or wavy in appearance (E). MPM-2 antibodies clearly stained the spindle poles and kinetochores in these treated mitotic cells (C, F and I). Bar, 10 μm.

The MPM staining of interphase cells at these higher OA concentrations was not increased, even though continued incubation at these elevated concentrations resulted in the rounding and detachment of cells from the monolayer. Interphase cells having typical morphology were still apparent after 60 min at 0.5 μM OA (Fig. 8A-C), but the majority of cells were rounded and floating in the culture media after 120 min of treatment. Most interphase cells were rounded after incubation with 1.0 μM OA for 60 min (Fig. 8D-F), and almost all were detached and floating in the culture media after 120 min (Fig. 8G-I). Only a few cytoplasmic microtubules remained in rounded cells (Fig. 8E), and in the detached cells what few microtubules remained were extremely short and were only associated with the centrosomes (Fig. 8H). Although in many cell types cell rounding is characteristic of mitotic cells, LLC-PK cells do not normally round up appreciably in mitosis. Rounding of cells following OA treatment was also not associated with induction of mitosis in the LLC-PK cells as determined by both the presence of intact interphase nuclei and the low levels of MPM staining that are characteristic of interphase cells. Cells that detached from the monolayer did not survive subculturing into media lacking OA (data not presented). Thus, the morphological changes associated with high OA concentrations were not due to the induction of a mitotic state, but more closely reflected the cytotoxicity of the drug at these concentrations.

Fig. 8.

The morphology of interphase cells was altered following treatment with high concentrations of OA. Treated LLC-PK cells were examined by phase-contrast (A,D and G) and double-label immunofluorescence staining with anti-tubulin (B, E and H) and MPM-2 antibodies (C, F and I). Typical interphase morphology, microtubule arrays and MPM-staining were still present in cells treated with 0.5 μM OA for 60 min (A-C). Interphase cells in cultures treated with 1.0 μM OA for 60 min had rounded up (D), and this was associated with a loss of many of the cytoplasmic microtubules (E). The MPM staining intensity remained at low levels typical of interphase cells. After 2 h in the presence of either 0.5;<M or 1.0 μM OA almost all cells in treated cultures had detached from the monolayer. The nuclear envelope remained intact (G), and the levels of MPM staining remained low in the majority of the detached cells (I). The cytoplasmic microtubules present were short, few in number, and radiated from the centrosome, giving the appearance of an aster in some cells (H). In other detached cells only a few very short microtubules were present (arrowhead, H). The rounding of cells and asterlike appearance was not associated with a premature mitosis in the LLC-PK cells.

Fig. 8.

The morphology of interphase cells was altered following treatment with high concentrations of OA. Treated LLC-PK cells were examined by phase-contrast (A,D and G) and double-label immunofluorescence staining with anti-tubulin (B, E and H) and MPM-2 antibodies (C, F and I). Typical interphase morphology, microtubule arrays and MPM-staining were still present in cells treated with 0.5 μM OA for 60 min (A-C). Interphase cells in cultures treated with 1.0 μM OA for 60 min had rounded up (D), and this was associated with a loss of many of the cytoplasmic microtubules (E). The MPM staining intensity remained at low levels typical of interphase cells. After 2 h in the presence of either 0.5;<M or 1.0 μM OA almost all cells in treated cultures had detached from the monolayer. The nuclear envelope remained intact (G), and the levels of MPM staining remained low in the majority of the detached cells (I). The cytoplasmic microtubules present were short, few in number, and radiated from the centrosome, giving the appearance of an aster in some cells (H). In other detached cells only a few very short microtubules were present (arrowhead, H). The rounding of cells and asterlike appearance was not associated with a premature mitosis in the LLC-PK cells.

When cells were treated with high OA concentrations after first being synchronized at Gj/S phase with hydroxyurea, rounding and detachment of the hydroxyurea-blocked cells was observed. There was no evidence in these rounded cells for chromosome condensation or nuclear envelope breakdown (data not presented), indicating that the rounding of cells was more likely to be the result of additional drug effects that were not related to the mitotic blockage observed at lower concentrations of OA.

Treatment of LLC-PK cells with low concentrations of OA (8–40 nM) results in a unique metaphase-like mitotic block that is characterized by the presence of an intact mitotic spindle. Deleterious effects of OA at these concentrations were not apparent, since mitotic cells continued to accumulate for at least 24 h and the mitotic block was reversible. This is in contrast to the effects of OA at higher concentrations (0.5–x1.0 μM), which resulted in cell rounding and detachment from the monolayer followed by loss of cell viability. Spindle morphology in OA-blocked cells demonstrated progressive alterations, which were dependent upon both the length of time an individual cell remained blocked in mitosis and the concentration of drug. The various spindle morphologies represented in treated cell populations suggested a temporal sequence for the development of the abnormal spindle structures. Treated cells entered prophase at a fairly constant rate even after OA treatment. As prophase was completed a bipolar spindle was formed with chromosomes aligned on the metaphase plate in a fairly normal fashion. At this point the transition from metaphase to anaphase was blocked and there was no further mitotic progression. Chromosome displacement from the metaphase plate followed. Initially only a few chomosomes were affected, but continued loss of chromosomes from their metaphase alignment accompanied the further disruption of spindle structure. Multipolar spindles or dissociated half-spindles developed after an extended period of blockage in mitosis.

Chromosomes in blocked cells appear to remain attached to spindle microtubules via an interaction at the kinetochore even after extended periods in the mitotic block; however, they appear to lose the ability to position themselves properly. This may reflect a disruption in the normal function or regulation of motor molecules required for proper movement of chromosomes, a change in the dynamics of spindle microtubules, or a combination of these events as a result of the elevated state of phosphorylation that is maintained in the blocked cells. Although in some blocked cells groups of chromosomes would appear to be clustered around the spindle poles, there was no evidence of sister chromatid separation. Similar morphologies have been observed following microinjection of OA into prometaphase PtK1 cells (Vandré and Borisy, unpublished observations). Cells that escaped the mitotic block exhibited a unique morphology; they underwent cytokinesis but in the absence of chromosome segregation resulting in daughter cells with a variable number of micronuclei.

Cells blocked in mitosis also showed the presence of MPM-reactive mitosis-specific phosphoproteins (Davis et al. 1983; Vandré et al. 1984). These phosphoproteins were only detected in treated cells that entered mitosis, and the level of MPM-reactive material associated with centrosomes and kinetochores was maintained throughout the mitotic block. Generally, only two major MPM-reactive sites were typically detected in multipolar cells. This major MPM staining indirectly reflects the number of centriole-containing MTOCs as indicated by our preliminary ultrastructural analysis of OA-treated cells (Vandré, unpublished observations). Therefore, the increase in MTOCs either reflects the fragmentation of pericentriolar material from existing centrosomes or the aggregation of additional microtubule-nucleating material from other extracentrosomal locations within the cell rather than the splitting of existing centriole pairs and/or the generation of new centrioles and centrosomes.

There is a significant lag period between addition of OA to the culture medium and the initial appearance of cells blocked in mitosis. This lag period is concentration dependent, with an effect on mitosis being detected within 30 min at 1 μM OA, whereas several hours were required to observe an initial mitotic block at 22 nM OA. In general, higher concentrations of OA (0.25–15 μM) have been used to elicit rapid responses in intact cells. For example, OA has been shown to activate a kinase activity that phosphorylates microtubule-associated protein in quiescent fibroblasts with a maximal effect at 10–20 μM following a 15 min incubation (Gotoh et al. 1990) and a 10-fold stimulation of myelin basic protein kinase activity has been reported in adipocytes following treatment with 10 μM OA for 20 min (Haystead et al. 1990). At these high OA concentrations no apparent toxic effects are observed in shortterm incubations (Cohen et al. 1990). While higher concentrations of OA are required to generate a response in intact cells measured in terms of minutes rather than hours, these higher concentrations are also cytotoxic. Kim et al. (1990) reported the stimulation of c-fos expression in the A-549 human lung adenocarcinoma cell line by OA after exposure for 24-48 h at concentrations around 50 nM, but these authors noted OA cytotoxicity at 125 nM against the A-549 cells and 60 nM against human synoviocytes in similar assays. OA toxicity was also shown to be high against 3T3 cells, ranging from 10 to 20 nM (Herschman et al. 1989). In contrast to intact cells, in cell extracts lower concentrations of OA may be used to observe rapid inhibition of phosphatase activity (Cohen et al. 1989).

To observe a more rapid response to OA, LLC-PK cells were treated with high concentrations of OA (1 μM) for 30-60 min. At this concentration of drug, however, nearly all cells in the population also rounded up and subsequently detached from the surface of the culture flask. Rounded cells, either before or after detachment from the monolayer, exhibited no apparent induction of a mitotic state, since both the nuclear envelope remained intact and the level of MPM antibody staining remained at interphase levels. Detachment was followed by a generalized depolymerization of microtubules, which ultimately resulted in a total loss of microtubules. Microtubule dynamics can be influenced by phosphorylation events regulated by p34cdc2 kinase as has been demonstrated in Xenopus oocyte extracts (Verde et al. 1990), which may be mediated through a MAP kinase (Gotoh et al. 1991). Phosphorylation of microtubule-associated proteins (MAPs) has also been shown to influence the stability of microtubules (Jameson et al. 1980), and the phosphorylation state of several MAPs has been shown to be modulated through the cell cycle (Gard and Kirschner, 1987; Vandré et al. 1991). It is likely that the depolymerization of interphase microtubules results from the inhibition of phosphatase activities in addition to these phosphatase activities responsible for the mitotic block in LLC-PK cells. Thus, cell rounding that resulted from OA treatment is not an indicator of entry into a mitotic-like state, but may reflect a generalized increase in the phosphorylation state of MAPs and other proteins involved in maintaining cell shape. A similar disruption of the interphase microtubule array was not observed at concentrations of OA that blocked mitosis but did not cause cell rounding and detachment.

Thus, differential effects of OA on interphase and mitotic microtubules could be distinguished by using different drug concentrations. These effects could reflect the differential specificity of OA for the catalytic subunits of PPI and PP2A. The low concentrations of OA necessary to generate a mitotic block suggest that a specific, highly sensitive phosphatase activity, perhaps a form of PP2A, is involved in processes that trigger the onset of anaphase. The phosphatase activity involved in anaphase onset has yet to be identified, but this could be a specific isoform of PP2A or a unique phosphatase. Multiple isozymes of PP2A have been cloned as well as several novel protein phosphatases related to PPI and PP2A (Cohen and Cohen, 1989). Recently, eight to ten different protein serine/threonine phosphatases have been shown to be expressed in a single cell type (Wadzinski et al. 1990), suggesting a multiplicity of regulatory roles for various phosphatases. Most recently, a unique OA-sensitive phosphatase designated PP3 has been described that may be involved in mitotic progression (Honkanen et al. 1991). The initial effects of OA, such as an inhibition of mitosis, are observed more rapidly at higher concentrations of the drug. However, as the intracellular concentration of OA continues to increase, there is also the potential for the rapid inhibition of additional PP2A activities and/or PPI activities. The inhibition of these additional phosphatase activities could be involved in the rearrangement of microtubules and cell rounding that are observed at higher OA concentrations.

Recently, Yamashita and coworkers (1990) reported on the treatment of BHK cells with 0.5 μM OA. Cells synchronized in early S phase by isoleucine deprivation and hydroxyurea treatment rounded and detached from the monolayer after exposure to OA, similar to the behavior of LLC-PK cells at equivalent drug concentrations. Unlike LLC-PK cells, many of the detached BHK cells exhibited a transient premature mitotic state characterized by nuclear envelope breakdown, premature chromosome condensation, and an increase in MPM antibody staining. A spindle, however, was not formed during this induced mitotic state. Rounded cellular morphology suggesting a mitotic phenotype has also been observed in 3T3 cells following treatment with a high concentration of OA (Kipreos and Wang, 1990). However, like the LLC-PK cells, the rounding of 3T3 fibroblasts was not associated with nuclear envelope breakdown or chromosome condensation. How can the apparent differences in the cellular response to OA be reconciled? Activation of p34cdc2 kinase, which regulates entry into mitosis, requires both the accumulation of the cyclin protein A and/or B to a critical threshold level, and the subsequent post-translational modification of p34cdc2. These modification events involve both the phosphorylation and déphosphorylation of the protein. Levels of OA that specifically inhibit PP2A activities have been shown to transiently activate p34cdc2 in Xenopus oocyte extracts (Felix et al. 1990a). Subsequent studies have demonstrated that OA inhibits a negative regulator of p34cdc2 activity termed INH (Solomon et al. 1990), and the INH has been shown to be a PP2A (Lee et al. 1991). If INH activity is inhibited by OA, the threshold level of cyclin B is reduced, and once threshold levels are achieved the delay in cyclin B activation of p34cdc2 is eliminated (Solomon et al. 1990). In BHK cells, cyclin B is present in early S phase and continues to accumulate throughout S phase (Yamashita et al. 1990). In conjunction with the accumulation of cyclin B, if threshold levels of cyclin B were lowered by OA treatment, premature entry into mitosis would occur in many of the treated BHK cells. This correlation between cyclin B levels and premature mitosis has recently been established in BHK cells (Steinmann et al. 1991). The transient nature of the premature mitotic state reported by Yamashita et al. (1990) may be a result of cytotoxic effects from the high concentrations of OA used in these studies. Cyclin B does not accumulate in S phase HeLa cells, however, but accumulates later in G2 phase (Pines and Hunter, 1989). This correlates with the inability to induce chemically premature mitosis in human cells with OA (Steinmann et al. 1991). Therefore, if cyclin levels in LLC-PK and 3T3 cells more closely resemble those of HeLa cells, few cells in an unsynchronized population would enter a premature mitosis following exposure to OA. It will be of interest to determine if lower concentrations of OA would be able to induce a more stable premature mitotic state in BHK cells in the absence of cell rounding and detachment from the monolayer.

Owing to the low concentrations of OA required, the mitotic block obtained with LLC-PK cells suggests the involvement of a PP2A-like phosphatase in the regulation of anaphase onset. A role for PP2A, in the form of INH, has been clearly established in mitotic regulation (Solomon et al. 1990; Lee et al. 1991). However, the regulation of mitotic progression may also involve the activity of PPI. Mutations in genes with sequence homology to mammalian PPI like those that have been identified in Aspergillus nidulans, the bimG gene (Doonan and Morris, 1989); the fission yeast Schizosaccharomyces pombe, the dis2 gene (Ohkura et al. 1989); and Drosophila, the PPI 87B gene (Axton et al. 1990), support this concept. Each of these mutations appears to block stages of mitosis subsequent to metaphase. The defects in spindle morphology observed in Drosophila PPI mutants are strikingly similar to spindle abnormalities observed in LLC-PK cells blocked in mitosis with concentrations of OA that would not be expected to affect PPI activity. It is possible that PPI is involved in the dephosphorylation of mitotic phosphoproteins, and mutations or inhibition of PPI would mimic the effects of stabilized p34cdc2. In each case elevated levels of mitosis-specific phosphorylation would be maintained.

In LLC-PK cells blocked in mitosis with either nocodazole (Vandré and Borisy, 1989) or low concentrations of OA (this study), MPM-reactive mitosisspecific phosphorylations are maintained for extended periods, suggesting that p34cdc2 kinase activity is stabilized under these conditions. MPF kinase activity also remains elevated in nocodazole-blocked cells (Moria et al. 1989; Yamashita et al. 1990), and in cytostatic factor (CSF)-blocked oocytes (Newport and Kirschner, 1984; Sagata et al. 1989). Microinjection of OA in to starfish oocytes also induces a stable mitotic block (Picard et al. 1989). Loss of mitotic activity requires the inactivation of p34cdc2 kinase, and loss of kinase activity coincides with the proteolytic degradation of cyclin (Murray et al. 1989; Draetta et al. 1989; Felix et al. 1990b). A proteolysis-resistant mutant cyclin molecule that contains a truncated N terminus has been shown to prevent exit from mitosis and loss of MPF kinase activity (Murray et al. 1989). In a similar fashion, the activity of cytostatic factor (CSF) is responsible for maintaining MPF activity and the meiotic block typical of oocytes (Newport and Kirschner, 1984). The active component of CSF has recently been identified as the c-mos proto-oncogene product (Sagata et al. 1989), which is a serine/threonine kinase that is capable of phosphorylating cyclin B (Roy et al. 1990). These results suggest that phosphorylated cyclin is more resistant to proteolytic degradation, and that déphosphorylation of the cyclin is involved in the cyclin degradation pathway. The results obtained with the LLC-PK cells suggest that a phosphatase responsible for the dephosphorylation of cyclin was inhibited at low concentrations of OA. Thus, an OA-sensitive phosphatase could be involved in triggering the metaphase to anaphase transition by inhibiting the signal regulating cyclin degradation. The result of this inhibition would be an extended metaphase. While consistent with the present results, this hypothesis requires the measurement of p34c<Jc2 kinase activity, cyclin levels and phosphatase activities in cells blocked in mitosis with OA.

Phosphatase inhibitors must be used under carefully controlled conditions to establish which phosphatases are directly involved in regulating specific events such as mitosis. In general, phosphatases have a broad substrate specificity, and inhibition of some phosphatases may have pleiotropic effects. While OA demonstrates a higher specificity for PP2A than PPI, this difference is influenced by the concentrations of the phosphatases within the cell. It is not clear whether individual phosphatase isozymes within each of these classes would be more or less sensitive to OA inhibition, but this may also be influenced by intracellular concentrations of the individual isozymes. We have demonstrated, however, that the effects of OA on cell cycle progression and cell morphology can be distinguished at specific drug concentrations. The mitotic block observed in LLC-PK cells following treatment with low levels of OA appears to be a general phenomenon, as similar mitotic effects have been observed in CHO and HeLa cells (Vandré, unpublished observations), and most recently in human leukemia K562 cells (Zheng et al. 1991). These results indicate that the transition from metaphase to anaphase may involve the activity of a specific phosphatase, and inhibition of this phosphatase activity blocks cells in an extended metaphase-like state. This phosphatase activity is distinct from other phosphatase activities that may be involved in the maintenance of microtubules and cell shape. Whether the phosphatase involved in regulating the metaphase to anaphase transition is related to the PP2A phosphatase INH, which is involved in the activation of p34cdc2 kinase, remains to be determined.

The authors thank April Smart for her technical assistance in the early phases of this work and Dr John Robinson for his comments regarding the manuscript. This work was supported by a National Science Foundation grant DCB-8902338, and American Cancer Society grant IRG-16–30.

Arion
,
D. L.
,
Meijer
,
L.
,
Brizuela
,
L.
and
Beach
,
D.
(
1988
).
cdc2 is a component of the M-phase specific-H1 kinase: evidence for identity with MPF
.
Cell
55
,
371
378
.
Alton
,
J. M.
,
Dombrádi
,
V.
,
Cohen
,
P. T. W.
and
Glover
,
D. M.
(
1990
).
One of the protein phosphatase 1 isozymes in Drosophila is essential for mitosis
.
Cell
63
,
33
46
.
Cohen
,
P.
and
Cohen
,
P. T. W.
(
1989
).
Protein phosphatases come of age
.
J. biol. Chem
.
264
,
21435
21438
.
Cohen
,
P.
,
Holmes
,
C. F. B.
and
Tsukitani
,
Y.
(
1990
).
Okadaic acid: a new probe for the study of cellular regulation
.
Trends Biochem. Sci
.
15
,
98
102
.
Cohen
,
P.
,
Klump
,
S.
and
Schelling
,
D. L.
(
1989
).
An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues
.
FEBS Lett
.
250
,
596
600
.
Davis
,
F. M.
,
Tsao
,
T. Y.
,
Fowler
,
S. K.
and
Rao
,
P. N.
(
1983
).
Monoclonal antibodies to mitotic cells
.
Proc. Nat. Acad. Sci. U.S.A
.
80
,
2926
2930
.
Dessev
,
G.
,
lovcheva-Dessev
,
C.
,
Bischoff
,
J. R.
,
Beach
,
D.
and
Goldman
,
R.
(
1991
).
A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamina and disassembles nuclei of clam oocytes in vitro
.
J. Cell Biol
.
112
,
523
533
.
Doonan
,
J. H.
and
Morris
,
N. R.
(
1989
).
The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1
.
Cell
57
,
987
996
.
Dorée
.
M.
,
Peaucelller
,
G.
and
Picard
,
A.
(
1983
).
Activity of the maturation-promoting factor (MPF) and the extent of protein phosphorylation oscillate simultaneously during meiotic maturation of starfish oocytes
.
Develop. Biol
.
99
,
409
501
.
Draetta
,
G.
,
Luca
,
F.
,
Westendorf
,
J.
,
Brizuela
,
L.
,
Ruderman
,
J.
and
Beach
,
D.
(
1989
).
cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF
.
Cell
56
,
829
838
.
Dunphy
,
W. G.
,
Brizuela
,
L.
,
Beach
,
D.
and
Newport
,
J.
(
1988
).
The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis
.
Cell
54
,
423
431
.
Felix
,
M.-A.
,
Cohen
,
P.
and
Karsenti
,
E.
(
1990a
).
cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid
.
EMBO J
.
9
,
675
683
.
Felix
,
M.-A.
,
Labbé
,
J.-C.
,
Dorée
,
M.
and
Karsenti
,
E.
(
1990b
).
Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase
.
Nature
346
,
379
382
.
Gard
,
D. L.
and
Kirschner
,
M. W.
(
1987
).
A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus end
.
J. Cell Biol
.
105
,
2203
2215
.
Gautier
,
J.
,
Norbury
,
C.
,
Lohka
,
M.
,
Nurse
,
P.
and
Mailer
,
J.
(
1988
).
Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+
.
Cell
54
,
433
439
.
Gavin
,
A-C.
,
Tsukitani
,
Y.
and
Schorderet-Slatkine
,
S.
(
1991
).
Induction of M-phase entry of prophase-blocked mouse oocytes through microinjection of okadaic acid, a specific phosphatase inhibitor
.
Exp. Cell Res
.
192
,
75
81
.
Goris
,
J.
,
Hermann
,
J.
,
Hendrix
,
P.
,
Ozon
,
R.
and
Merevede
,
W.
(
1989
).
Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes
.
FEBS Lett
.
245
,
91
94
.
Gotoh
,
Y.
,
Nishida
,
E.
,
Matsuda
,
S.
,
Shiina
,
N.
,
Kosako
,
H.
,
Shiokawa
,
K.
,
Akiyama
,
T.
,
Ohta
,
K.
and
Sakai
,
H.
(
1991
).
In vitro effects of microtubule dynamics of purified Xenopus M phase-activated MAP kinase
.
Nature
349
,
251
254
.
Gotoh
,
Y.
,
Nishida
,
E.
and
Sakai
,
H.
(
1990
).
Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells
.
Eur. J. Biochem
.
193
,
671
674
.
Gunderson
,
G. G.
,
Kalnoski
,
M. H.
and
Bulinski
,
J. C.
(
1984
).
Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo
.
Cell
38
,
779
789
.
Haystead
,
T. A. J.
,
Sim
,
A. T. R.
,
Carling
,
D.
,
Honner
,
R. C.
,
Tsukitani
,
Y.
,
Cohen
,
P.
and
Hardie
,
D. G.
(
1989
).
Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism
.
Nature
337
,
78
81
.
Haystead
,
T. A. J.
,
Welel
,
J. E.
,
Litchfield
,
D. W.
,
Tsukitani
,
Y.
,
Fisher
,
E. H.
and
Krebs
,
E. G.
(
1990
).
Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes
.
J. Biol. Chem
.
265
,
16571
16580
.
Herschman
,
H. R.
,
Lim
,
R. W.
,
Brankow
,
D. W.
and
Fujiki
,
H.
(
1989
).
The tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid differ in toxicity, mitogenic activity and induction of gene expression
.
Carcinogenesis
10
,
1495
1498
.
Hescheler
,
J.
,
Mieskes
,
G.
,
Rüegg
,
J. C.
,
Takai
,
A.
and
Trautwein
,
W.
(
1988
).
Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes
.
Pflilgers Arch. ges. Physiol
.
412
,
248
252
.
Honkanen
,
R. E.
,
Zwiller
,
J.
,
Dally
,
S. L.
,
Khatra
,
B. S.
,
Dukelow
,
M.
and
Boynton
,
A. L.
(
1991
).
Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain
.
J. Biol. Chem
.
266
,
6614
6619
.
Jameson
,
L. T.
,
Frey
,
T.
,
Zeeberg
,
B.
,
Dalldorf
,
F.
and
Caplow
,
M.
(
1980
).
Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins
.
Biochemistry
19
,
2472
2479
.
Kim
,
S-J
,,
Lafyatis
,
R.
,
Kim
,
K. Y.
,
Angel
,
P.
,
Fujika
,
H.
,
Karin
,
M.
,
Sporn
,
M. B.
and
Roberts
,
A. B.
(
1990
).
Regulation of collagenase gene expression by okadaic acid, an inhibitor of protein phosphatases
.
Cell Regln
1
,
269
278
.
Klpreos
,
E. T.
and
Wang
,
J. Y. J.
(
1990
).
Differential phosphorylation of c-abl in cell cycle determined by cdc2 kinase and phosphatase activity
.
Science
248
,
217
220
.
Labbé
,
J.C.
,
Picard
,
A.
,
Karsen ti
,
E.
and
Dorée
,
M.
(
1988
).
An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation
.
Develop. Biol
.
127
,
157
169
.
Lee
,
T. H.
,
Solomon
,
M. J.
,
Mumby
,
M. C.
and
Kirschner
,
M. W.
(
1991
).
INH, a negative regulator of MPF, is a form of protein phosphatase 2A
.
Cell
64
,
415
423
.
Lohka
,
M. J.
,
Hayes
,
M. K.
and
Mailer
,
J. L.
(
1988
).
Purification of maturation-promoting factor, an intracellular regulator of early mitotic events
.
Proc. Nat. Acad. Sci. U.S.A
.
85
,
3009
3013
.
Moria
,
A. O.
,
Draetta
,
G.
,
Beach
,
D.
and
Wang
,
J. Y. J.
(
1989
).
Reversible tyrosine phosphorylation of cdc2: Dephosphorylation accompanies activation during entry into mitosis
.
Cell
58
,
193
203
.
Murray
,
A. W.
,
Solomon
,
M. J.
and
Kirschner
,
M. W.
(
1989
).
The role of cyclin synthesis and degradation in the control of maturation promoting factor activity
.
Nature
339
,
280
286
.
Newport
,
J. W.
(
1987
).
Nuclear reconstitution in vitro: stages of assembly around protein-free DNA
.
Cell
48
,
219
230
.
Newport
,
J. W.
and
Kirschner
,
M. W.
(
1984
).
Regulation of the cell cycle during early Xenopus development
.
Cell
37
,
731
742
.
Ohkura
,
H.
,
Kinoshita
,
N.
,
Miyatani
,
S.
,
Toda
,
T.
and
Yanagita
,
M.
(
1989
).
The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases
.
Cell
57
,
997
1007
.
Osborn
,
M.
and
Weber
,
K.
(
1982
).
Immunofluorescence and immunocytochemical procedures with purified antibodies: tubulin-containing structures
.
Meth. Cell Biol
.
24
,
97
132
.
Picard
,
A.
,
Capony
,
J. P.
,
Brautigan
,
D. L.
and
Dorée
,
M.
(
1989
).
Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish
.
J. Cell Biol
.
109
,
33473354
.
Pines
,
J.
and
Hunter
,
T.
(
1989
).
Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2
.
Cell
58
,
833
846
.
Rimé
,
H.
and
Ozon
,
R.
(
1990
).
Protein phosphatases are involved in the in vivo activation of histone H1 kinase in mouse oocyte
.
Develop. Biol
.
141
,
115
122
.
Roy
,
L. M.
,
Singh
,
B.
,
Gautier
,
J.
,
Arlinghaus
,
R. B.
,
Nordeen
,
S. K.
and
Mailer
,
J. L.
(
1990
).
The cyclin B2 component of MPF is a substrate for the c-mos*c proto-oncogene product
.
Cell
61
,
825831
.
Sagata
,
N.
,
Watanabe
,
N.
,
VandeWoude
,
G. F.
and
Ikawa
,
Y.
(
1989
).
The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs
.
Nature
342
,
512
518
.
Solomon
,
M. J.
,
Glotzer
,
M.
,
Lee
,
T. H.
,
Philippe
,
M.
and
Kirschner
,
M. W.
(
1990
).
Cyclin activation of p34c<k2
.
Cell
63
,
1013
1024
.
Steinmann
,
K. E.
,
Belinsky
,
G. S.
,
Lee
,
D.
and
Schlegel
,
R.
(
1991
).
chemicallyinduced premature mitosis: Differential response in rodent and human cells and the relationship to cyclin B synthesis and p34”k2/cyclin B complex formation
.
Proc. Nat. Acad. Sci. U.S.A
.
88
,
6843
6847
.
Suganuma
,
M.
,
Fujiki
,
H.
,
Suguri
,
H.
,
Yoshizawa
,
S.
,
Hirota
,
M.
,
Nakayasu
,
M.
,
OJika
,
M.
,
Wakamatsu
,
K.
,
Yamada
,
K.
and
Sugimura
,
T.
(
1988
).
Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter
.
Proc. Nat. Acad. Sci. U.S.A
.
85
,
1768
1771
.
Vandré
,
D. D.
(
1990
).
Effect of okadaic acid on mitotic progression and microtubule organization in cultured cells
.
J. Cell Biol
.
111
,
280a
.
Vandré
,
D. D.
and
Borisy
,
G. G.
(
1989
).
Anaphase onset and dephosphorylation of mitotic phosphoproteins occur concomitantly
.
J. Cell Sci
.
94
,
245
258
.
Vandré
,
D. D.
,
Davis
,
F. M.
,
Rao
,
P. N.
and
Borisy
,
G. G.
(
1984
).
Phosphoproteins are components of mitotic microtubule organizing centers
.
Proc. Nat. Acad. Sci. U.S.A
.
81
,
4439
4443
.
Vandré
,
D. D.
,
Centonze
,
V. E.
,
Peloquin
,
J.
,
Tombes
,
R. M.
and
Borisy
,
G. G.
(
1991
).
Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis
.
J. Cell Sci
.
98
,
577
588
.
Verde
,
F.
,
Labbé
,
J.
,
Dorée
,
M.
and
Karsenti
,
E.
(
1990
).
Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs
.
Nature
343
,
233
238
.
Wadzinski
,
B. E.
,
Heasley
,
L. E.
and
Johnson
,
G. L.
(
1990
).
Multiplicity of protein serine-threonine phosphatases in PC12 pheochromocytoma and FTO-2B hepatoma cells
.
J. Biol. Chem
.
265
,
21504
21508
.
Yamashita
,
K.
,
Yasuda
,
H.
,
Pines
,
J.
,
Yasumoto
,
K.
,
Nishitani
,
H.
,
Ohtsubo
,
M.
,
Hunter
,
T.
,
Sugimura
,
T.
and
Nishlmoto
,
T.
(
1990
).
Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells
.
EMBO J
.
9
,
43314338
.
Zheng
,
B.
,
Woo
,
C. F.
and
Kuo
,
J. F.
(
1991
).
Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter
.
J. Biol. Chem
.
266
,
10031
10034
.