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Carcinogenesis, Vol. 22, No. 8, 1163-1172, August 2001
© 2001 Oxford University Press

CANCER BIOLOGY

Abbreviated cell cycle progression induced by the serine/threonine protein phosphatase inhibitor okadaic acid at concentrations that promote neoplastic transformation

D.J. Messner1,, P. Ao, A.B. Jagdale and A.L. Boynton

Department of Molecular Medicine, Northwest Hospital, Bothell, WA 98021, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
We examined cell cycle-related effects of the phosphatase inhibitor okadaic acid (OA) in T51B rat liver epithelial cells under conditions chosen to mimic early stages of tumor promotion by this compound. Optimal transformation (colony formation in soft agar) was seen after prolonged culture of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated T51B cells in 7 nM OA. Paradoxically, T51B cells treated with 2–10 nM OA showed decreased, rather than increased, proliferation in response to epidermal growth factor (EGF), as measured by [3H]thymidine incorporation. Complete inhibition was observed within 24 h at 10 nM OA. This response paralleled a loss of EGF-stimulated cdk2 kinase activity and an increase in association of the inhibitors p21 (cip-1) and p27 (kip-1) with cdk2. An increase in p53 phosphorylated on serine 15 accompanied the rise in p21 (cip-1). Both phosphorylation of the retinoblastoma protein and induction of cyclin A by EGF were blocked in cells treated with OA, but there was an increase in cyclin E. Resting cells treated with OA alone also showed elevated cyclin E levels, together with reduced levels of the E2F regulator pRb2/p130. Taken together, these observations indicate transforming levels of okadaic acid elicit a G1-trapping effect by facilitating cell cycle progression to the G1/S checkpoint, where cells are trapped by mechanisms that include p21 (cip-1)-mediated inhibition of cdk2. They support the premise that disruption of cellular processes regulating the transitions from G0 to G1 to S-phase is an important early step in tumor promotion by low levels of okadaic acid.

Abbreviations: cdk, cyclin dependent kinase; cki, cyclin kinase inhibitor; EGF, epidermal growth factor; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; OA, okadaic acid; pRb, retinoblastoma gene product.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Okadaic acid (OA), a natural toxin originally isolated as the principle cause of diarrhetic shellfish poisoning, is a highly selective inhibitor of serine/threonine protein phosphatase activity (1–3). This cell permeable compound has proven very useful for characterizing protein phosphatases (4,5), as well as for identifying cellular processes regulated by serine/threonine phosphorylation (6,7). Among its novel effects is a potent tumor promoting activity in mouse skin and other in vivo systems (8,9), suggesting an inhibitory role for OA-sensitive phosphatases in neoplastic transformation. Although the mechanistic basis for this is not completely understood, the role of serine/threonine phosphatases in cell cycle regulation must be relevant, as transformation most likely involves deregulation of cell cycle control (10,11). Consistent with this prediction, cell cycle progression from G1 through S, G2 and M phases is controlled to a large extent by reversible phosphorylation of regulatory enzymes on serine/threonine residues (for reviews, see refs 11–13).

Cell cycle progression in mammalian cells is dependent on mitogen-stimulated synthesis of G1 cyclins, assembly with cyclin dependent serine/threonine protein kinases (cdks) and activation by cyclin activating kinase (CAK), a specific member of the cyclin/cdk family (11,12). The D cyclins assemble with cdk4 and/or cdk6 early in G1, while cyclin E assembles with cdk2 in late G1. These events contribute to serine/threonine phosphorylation of the retinoblastoma protein (pRb), release of E2F transcription factors, synthesis of cyclin A and S-phase entry. Further regulation of cdk activity is achieved by adjusting the levels of cyclin kinase inhibitors (cki proteins). There are two major classes of cki proteins (14). The first selectively inhibits cdk4 and cdk6, and includes the proteins p15 (INK4B), p16 (INK4A), p18 (INK4C) and p19 (INK4D). The second class includes p21 (cip-1), p27 (kip-1) and p57 (kip-2). Members of this group appear to have a dual function: they inhibit activation of cdk2-containing complexes but facilitate the assembly and activation of cdk4/6 complexes (14). Examples of growth regulatory pathways with a cki component include (i) synthesis of p27 (kip-1) induced when contact-inhibited cells reach confluence (15); (ii) synthesis of p21 (cip-1) resulting from serine/threonine phosphorylation and activation of p53 in response to DNA damage (16,17) and (iii) p21 (cip-1) induction via p19 (ARF)-mediated inhibition of p53 degradation in response to oncogenic proteins and DNA tumor viruses (17,18). Thus, transforming stimuli can also increase cki levels and elicit cell cycle arrest.

Many studies have documented the varied effects of serine/threonine phosphatase inhibition by OA on cell transformation, cell proliferation, cell cycle gene expression or cell cycle protein activity (for reviews, see refs 6, 7 and 9). However, few consistent inter-relationships have emerged, largely due to the diversity of responses to OA seen among different systems. Although OA can elicit premature induction of mitosis (19,20), induce proto-oncogene expression (21) or stimulate cell cycle progression (22), most studies report inhibitory proliferative effects over the concentration range 5–100 nM (23–28). Okadaic acid induces apoptosis in some (but not all) cells (25,29–31). Even the transformation data in cultured cells vary widely, with OA acting either as a tumor promoter (32), an anti-promoter (33) or a transformation revertant (34). With respect to cell cycle proteins, OA treatment can result in increased (22,35) or inhibited (26,28) phosphorylation of pRb, increased phosphorylation of p53 (35–37), elevation of cki levels (30,37,38) and inhibition (26) or stimulation (19,24,39) of cyclin/cdk levels or activity. Experimental differences in OA concentration and timing (resulting in different phosphatase inhibition profiles) do not completely account for these different effects. There must also be differences in phosphatase function or kinase status among the different model systems examined. Because of this, the relevance of these various effects to tumor promotion is unknown. We know of no studies that simultaneously examine proliferative effects, cell cycle protein effects and transformation in a single experimental system.

We now address this issue by identifying the proliferative and biochemical effects of OA under conditions known to result in tumor promotion activity. T51B cells, a non-transformed epithelial cell line obtained from rat liver (40), are well characterized for use in tumor promotion experiments (41). Using T51B cells, we demonstrate that concentrations of OA that facilitate transformation also inhibit EGF-stimulated cell cycle progression, in parallel with activation of p53 and induction of p21 (cip-1). Further analyses reveal that transforming concentrations of OA also cause unstimulated cells to accumulate in late G1, apparently by facilitating a net entry of resting cells into the cycle.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Okadaic acid was obtained from Alexis Biochemicals (San Diego, CA). N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) was from Sigma (St Louis, MO). Epidermal growth factor (EGF) was from Collaborative Biomedical (now Becton Dickinson, Bedford, MA) or Upstate Biotechnology (Lake Placid, NY). Antibodies were purchased from commercial suppliers as noted. Other reagents were from standard suppliers or as noted.

Transformation assays
T51B cells (40) were maintained in Eagle's basal medium supplemented with 10% calf serum (Colorado Serum, Denver, CO). For the transformation assay, cells were seeded in duplicate dishes for each experimental point. One day after plating, freshly prepared MNNG, or dimethyl formamide vehicle control, was added at a concentration of 0.25 µg/ml. This initiating agent was removed after 24 h and replaced with fresh media. Okadaic acid or dimethyl formamide control was added at the indicated concentration. The cells were maintained in culture for the times indicated in the legend to Figure 1Go, with weekly media changes and replating every 2 weeks. Okadaic acid was normally added immediately upon media change and within 3 days of replating. A 10 nM experimental point was discontinued due to poor growth of the cells in this concentration of OA, and the cells treated with 7 nM were allowed recovery periods that slightly reduced their time of exposure to OA but not their time in culture.



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  		Fig. 1. Okadaic acid treatment potentiates the transforming effects of MNNG. T51B cells treated once with 0.25 mg/ml MNNG were then cultured in normal media or media containing the indicated concentrations of OA. Cells were re-plated every 2 weeks and were exposed to OA both while they were growing and after reaching confluence. At 8 weeks (open bars), 10 weeks (shaded bars) and 12 weeks (solid bars) of culture, cells exposed to each treatment condition were plated in soft agar and allowed to form colonies in the absence of OA. The number of colonies with a cross sectional area >0.1 µm2 was determined under the microscope and is presented as the mean ± SEM from two to eight replicates.

 
At 6, 8 or 10 weeks after initial plating, the cells were seeded in soft agar as follows. Cells suspended in normal media were combined with an equal volume of 0.6% agar (FMC BioProduct, now BMA, Rockland, ME) in Iscoves medium (Gibco, BRL, Life Technologies, Gaithersburg, MD) containing 10% serum and 10 ng/ml EGF. A 5 ml aliquot containing 25 000 cells was plated onto a 3 ml bottom layer of 0.5% agar in Iscoves media with 10% serum (in duplicate). The plates were kept in culture for 3 weeks and then stained and scored. Colonies larger than 0.015 mm2 were defined as positive and counted under the microscope. The data shown in Figure 1Go represent the mean ± standard error of between two and eight replicate soft agar dishes.

[3H]Thymidine incorporation
Details of this assay have been described (42). Briefly, cells were seeded on plastic coverslips and grown to confluence (see below). Following the indicated treatment in serum-free Eagle's medium with OA, 2 µCi/ml [3H]thymidine (New England Nuclear) was added with or without 25 ng/ml EGF for 20 h. The cells were rinsed with PBS and fixed in 16% formalin, 9% acetic acid for 1 h. The coverslips were rinsed with water, allowed to air dry, mounted on slides, coated with Ilford K5 emulsion (Polysciences, Warrington, PA) and allowed to develop for 3–6 days. They were developed (Kodak D-19 developer), fixed and stained with Giemsa stain. Labeled and unlabeled nuclei in duplicate representative fields were scored under the microscope to determine the percentage labeled nuclei for each data point.

Cell culture and biochemical analyses
T51B cells were seeded at 7000 cells/cm2 in Eagle's basal medium (buffered with 10 mM HEPES pH 7.4) containing 10% serum and used 1–4 days after reaching confluence, or ~1 week after plating. Okadaic acid treatments were made after replacing the media containing 10% serum with fresh Eagle's basal medium only, and the indicated concentration of OA (or vehicle control) was added. Cells were rinsed on ice with PBS containing phosphatase and protease inhibitors (0.1 mg/ml PMSF, 1 mM sodium vanadate, 10 µg/ml benzamidine and 1 µg/ml each pepstatin A, chymostatin and leupeptin) and harvested by scraping for western blotting, immunoprecipitation or FACS analyses.

For western blots of cell lysates, cells were scraped in 2% SDS, TBS (25 mM Tris–HCl, 120 mM NaCl, pH 7.5) with phosphatase and protease inhibitors and immediately boiled. Protein concentration was determined using a modified Lowry assay and aliquots containing the same amounts of protein were prepared for SDS–PAGE as has been described (43). Following transfer to Immobilon PVDF membranes (Bio-Rad, Richmond, CA), the membranes were blocked and labeled with primary antibodies according to the manufacturer's recommendations. Antibodies used for western blotting in this study were anti-human EGF receptor, anti-p21 (cip-1) (Upstate Biotechnology, Lake Placid, NY); anti-pRb (Pharmingen, San Diego, CA); anti-cyclin E, anti-cdk2, anti-rat p53 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p27(kip-1), anti-p130 (Transduction Laboratories, Lexington, KY); anti-cyclin A (Oncogene Research Products, Cambridge, MA); anti-phospho (ser15)-p53 (New England Biolabs, Beverly, MA). The detection system consisted of secondary antibodies conjugated with horseradish peroxidase (Jackson Immunoresearch, West Grove, PA), chemiluminescence (reagents from either Amersham, Arlington Heights, IL or Kirkegaard and Perry, Gaithersburg, MD) and exposure to Fuji RX film. In each figure, the western blot data for a given protein was taken from a single ECL film exposure, so that the effect of the various treatments on the relative band intensities can be accurately evaluated (for two part figures, however, A and B were done separately).

Cell extracts for immunoprecipitation (IP) or western blotting were prepared on ice after rinsing the cells in PBS containing phosphatase and protease inhibitors. Triton extracts were obtained by scraping in either 1% Triton X-100, 10% glycerol, 20 mM HEPES, pH 7.4, 100 mM NaCl with phosphatase and protease inhibitors (Figures 4 and 8GoGo), or in 0.1% Triton X-100, 50 mM Tris–HCl pH 7.4, 250 mM NaCl, 50 mM NaF, 5 mM EDTA, also with phosphatase and protease inhibitors including 100 nM OA (for Figure 10Go). In either case, triton-insoluble material was removed by centrifugation and the resulting extracts were used directly for western blots or IP. For the western blots (Figures 8 and 10GoGo), samples were normalized by using a constant percentage of total cell extract in each lane rather than a fixed amount of protein. For IP, antibody was added at 4°C for 2–4 h, followed by protein A–Sepharose (Sigma) for an additional 1 h and the IP material was washed and processed for SDS–PAGE and western blotting as described above.



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  		Fig. 4. Cells treated with okadaic acid retain functional EGF receptors. (A) Western blots of whole cell lysates. Cells were treated in serum-free media with 0, 7 or 10 nM OA for 24 or 48 h and then analyzed by western blotting for the EGF receptor. (B) Western blots of phosphotyrosine immunoprecipitates after stimulation with EGF. T51B cells were incubated in serum-free media with or without 10 nM OA for 42 h and EGF during the final 0–2 h. EGF was either omitted (0), added for 15 min to cells kept on ice (ice) or added for the indicated number of minutes (2–120) at 37°C. Triton-solubilized extracts were immunoprecipitated with anti-phosphotyrosine antibodies and processed for western blotting with antibodies against the EGF receptor.

 


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  		Fig. 8. Increased cellular levels and cdk2 association of cki proteins in okadaic acid treated cells stimulated with EGF. (A) Western blotting of cell extracts. Cells were pre-treated for 26 h in serum-free media containing the indicated concentration of OA. EGF was then added for an additional 17 h to allow for maximal progression to S-phase prior to harvesting. Cells were extracted with Triton X-100 and aliquots were analyzed by sequential western blotting with antibodies against cyclin E, cdk2, cyclin A, p21 (cip-1) or p27 (kip-1). (B) Western blotting of cdk2 immunoprecipitates. Aliquots of the samples shown in (A) were immunoprecipitated with antibodies to cdk2 and then processed for western blots using antibodies against p21 (cip-1) or p27 (kip-1).

 


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  		Fig. 10. The amount of hypophosphorylated p130 is decreased by EGF or okadaic acid. Cells pretreated for >24 h in serum-free media containing 0, 7 or 10 nM OA were subsequently stimulated or not with EGF. After 0, 12 or 18 h EGF (all samples exposed to OA for 42 h total), they were harvested, extracted with Triton X-100 and the soluble fraction was processed for western blots using antibodies specific for p130, the EGF receptor, pRb, cyclin A or p21 (cip-1).

 
FACS analysis
Washed cells were scraped in PBS containing phosphatase and protease inhibitors, pelleted and resuspended in PBS. The samples were adjusted to 50% ethanol, placed at –20°C overnight and centrifuged. The pellets were rinsed in PBS, adjusted to 0.1 mg/ml propidium iodide and 20 U/ml RNAse (Sigma Type I) and incubated at room temperature for 30 min. DNA profiles were measured with a Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and the cell cycle position determined using Mod Fit software (Becton Dickinson).

Kinase assays
Cells were rinsed on ice in PBS containing phosphatase and protease inhibitors and solubilized in 0.5% NP-40, 150 mM NaCl, 50 mM Tris–HCl pH 8. Insoluble material was removed by centrifugation, the supernatant was adjusted to 5 mg/ml BSA and anti-cdk2 (Santa Cruz Biotechnology) and protein A–Sepharose were added to immunoprecipitate the kinase activity as indicated above. The kinase assay was adapted from Takuwa et al. (44). The IP sample was washed in kinase buffer (5 mM Tris–HCl pH 7.4, 1 mM MgCl2, 0.1 mM DTT), added to an equal volume of 2x reaction mix containing 0.8 mg/ml H1 histone (Sigma), 120 µM ATP, 2 µCi [{gamma}-32P]ATP (NEN, Boston, MA) and the reaction was allowed to proceed for 30 min at 30°C. It was terminated by addition of SDS–PAGE sample buffer and boiling. Samples were run on SDS gels, transferred to Immobilon and analyzed by PhosphorImaging (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Okadaic acid acts as a tumor promoter in T51B cells
The T51B cell line displays anchorage-dependent and density-inhibited cell proliferation that is lost upon transformation by carcinogens. Formation of colonies in soft agar is a widely accepted indicator of the transformed phenotype that is used to estimate the potency of a carcinogenic agent (41,45). This assay was used to assess the tumor promoting properties of OA in initiated T51B cells. As shown in Figure 1Go, exposure of the cells to the initiator MNNG alone had very little effect on colony formation in soft agar. However, when this treatment was followed by prolonged exposure to 7 nM OA, the number of colonies increased dramatically. Significant differences in soft agar growth were not seen in cells exposed to OA but not MNNG (data not shown), or in MNNG-treated cells exposed to lower concentrations of OA (<=3 nM). At higher concentrations (>=10 nM), the proliferation and viability of the cells was significantly impaired in prolonged culture, preventing an assessment of tumor promotion properties using this protocol (see Materials and methods). The dependence on prior exposure to initiator, as well as the time and dose dependence of the effects, is as predicted according to the multistage model of carcinogenesis and establish conditions under which okadaic acid acts as a tumor promoter in the T51B system.

Okadaic acid acts as a G1 cell cycle blocker in T51B cells
The apparent inhibition of proliferation by OA in the transformation assays seemed paradoxical for a tumor promoter. It was at odds with previous observations in C3H/10/T1/2 cells, where similar concentrations of OA inhibited PDGF-induced proliferation (27) and inhibited (rather than promoted) transformation (33). To confirm the inhibitory effects of OA on T51B cell proliferation, we chose conditions analogous to those used in the C3H/10/T1/2 study. Figure 2Go shows EGF-stimulated incorporation of [3H]thymidine (a measure of DNA synthesis) in confluent T51B cells was completely blocked by pre-treatment with 10 nM OA for 48 h. Similar effects were observed using fresh 20% serum as the mitogen (data not shown). Many variations of cell status, mitogen supplements and OA concentration and timing failed to identify conditions under which OA either stimulated progression to S-phase on its own, or stimulated the mitogenic response to EGF. Thus, although prolonged exposure to OA resulted in transformation of T51B cells, the short-term effect of similar concentrations was clearly inhibition of cell proliferation.



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  		Fig. 2. Okadaic acid blocks EGF-stimulated progression to S-phase in T51B cells. Confluent T51B cells were pre-treated in serum-free media with or without 10 nM OA for 48 h, followed by addition of [3H]thymidine with or without 25 ng/ml EGF. The incubation was continued for 20 h to allow for S-phase entry and the cells were processed for autoradiography as described in Materials and methods. Nuclei from individual cells were scored under the microscope as having incorporated [3H]thymidine or not. The data are presented as the percent nuclei labeled under a given condition and represent the mean ± SEM of 12 experiments.

 
The close OA concentration dependence for transformation and inhibition of proliferation suggested these two responses might be related. One possible explanation is a selection model for the tumor promotion assays: inhibition of proliferation of normal cells allows for preferential growth of (and selection for) cells that are transformed. If true, then OA-transformed cells should be resistant to the anti-proliferative effects of OA, a prediction that was not supported by our preliminary analyses of cell lines derived from the transformation protocol described by Figure 1Go (A.B.Jagdale and D.J.Messner, unpublished data). It may be that pre-neoplastic cells adopt a transient resistance to the anti-proliferative effects of OA that contributes to tumor promotion but is not necessarily retained permanently. This variation of the selection model is difficult to evaluate. A different idea is that tumor promotion and inhibition of proliferation represent separate responses to a single OA-induced event, in which case there would be no requirement for OA resistance in the transformed cells. This seemed likely, especially since inhibition of proliferation is a common cellular compensatory response to toxic or transforming insults (e.g. following DNA damage induced by ionizing radiation). Therefore, as a first step towards identifying factors contributing to OA-induced tumor promotion in T51B cells, we investigated the mechanisms involved in inhibition of EGF-stimulated DNA synthesis by OA.

Pre-treatment for at least 24 h prior to addition of EGF was required for maximum mitogenic block by 10 nM OA, as illustrated in Figure 3Go. Treatment of the cells for shorter times gave only partial inhibition. Essentially complete inhibition could also be attained at slightly lower OA concentrations using a longer pre-treatment time, for example, 8 nM OA for 48 h (data not shown). These results could be due either to a very slow rate of entry of OA into the cells, or because the cellular effect itself takes time to develop, or both. The half time for diffusion into cells and saturation of PP2A by 100 nM OA at 37°C has been estimated to be ~1 h (46), suggesting that intracellular levels are 98% equilibrated within 6 h. Although this rate will be slower at lower OA concentrations, the time course shown in Figure 3Go suggests there are additional time-dependent cellular factors besides diffusion that contribute to the block of EGF-induced cell cycle progression by 10 nM OA.



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  		Fig. 3. Time of pre-treatment with okadaic acid influences the extent of proliferative block. Confluent control cells were switched to serum-free media for 48 h and stimulated with EGF for an additional 20 h before being processed for [3H]thymidine incorporation. Experimental cells were tested the same way, except OA (10 nM) was added 48, 24, 16, 8, 4 or 0 h prior to EGF. The data points represent mean values compiled from three experiments.

 
Okadaic acid inhibits EGF-stimulated DNA synthesis at a post-receptor step in T51B cells
In C3H/10/T1/2 cells, OA was reported to down-regulate receptors for EGF and PDGF, and this was thought to be the basis for its anti-mitogenic effects (27). To evaluate whether a similar mechanism explains the anti-proliferative effects seen in T51B cells, we measured EGF receptor levels by western blot analysis. Figure 4AGo illustrates that compared with untreated cells at each time point (0 nM lanes), the amount of EGF receptor protein was not decreased in cells treated with 7 or 10 nM OA. To show this receptor is accessible and responsive to extracellular EGF, we treated cells with or without 10 nM OA, added EGF for various times, and then measured the amount of EGF receptor in anti-phosphotyrosine immunoprecipitates. Figure 4BGo illustrates that treatment with 10 nM OA for 44 h did not inhibit ligand-stimulated receptor autophosphorylation or significantly alter down-regulation of activated receptors. This indicated that the EGF receptor itself is not impaired in OA-treated T51B cells.

The presence of a functional receptor in OA-treated cells combined with the lack of progression to S-phase shifted our attention to evaluating the effect of OA on the G1 cyclin/cdk regulatory system. The G1/S restriction point marks the part of the cell cycle at which extracellular growth signals are no longer required (10,11). As illustrated in Figure 5Go, T51B cells crossed this point 12–16 h after EGF addition, as evidenced by phosphorylation of pRb and elevated levels of cyclin A. Treatment with OA at concentrations that blocked EGF-stimulated DNA synthesis (10 nM for 24 h) also blocked EGF-induced changes in pRb and cyclin A (Figure 5Go). Cyclin E, however, was increased by OA treatment in the absence of stimulation by EGF (Figure 5Go, compare 0 EGF lanes after 10 nM OA versus 0 OA). Many experiments indicated cyclin E levels in cells treated with OA alone were nearly the same as in EGF-treated cells that progress on to S-phase, and often higher in cells treated with both OA and EGF (e.g. Figures 5, 8 and 9GoGoGo).



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  		Fig. 5. Okadaic acid treatment modulates EGF-stimulated changes in cell cycle proteins. Cells were pre-treated with or without 10 nM OA in serum-free media for 24 h, followed by addition of EGF. They were harvested at the indicated times and whole cell lysates were processed for sequential western blotting with antibodies against pRb, cyclin E, cyclin A and cdk2.

 


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  		Fig. 9. Phosphorylation of p53 is increased following treatment with okadaic acid. T51B cells were incubated in serum-free media with 0, 7 or 10 nM OA for 48 h and EGF as indicated during the final 15 min (0.25 h) or 16 h. They were harvested and processed for sequential western blotting with antibodies against cyclin E, cyclin A, p21 (cip-1), p27 (kip-1), total p53 and phospho-ser15-p53 (P-ser15-p53).

 
A key indicator of G1/S transition is activation of the cyclin dependent kinase cdk2. To evaluate whether the OA block precedes this point of the cell cycle, cdk2 from cells treated or not with OA and EGF was immunoprecipitated and assayed in vitro for histone kinase activity. Figure 6Go illustrates that stimulation of cdk2 activity was initiated within 12 h of addition of EGF to T51B cells and was blocked in OA-treated cells. The lack of pRb phosphorylation (Figure 5Go) is consistent with this effect. As expected, the OA concentration-dependence of inhibition of cdk2 activation paralleled that of entry into S-phase (Figure 7Go). While incubation in 2 nM OA for 24 h had little or no effect, both measures of cell cycle progression were significantly inhibited by 10 nM OA. This concentration range is very similar to what we have observed in transformation assays for the tumor promotion property of OA (Figure 1Go and P.Ao, A.L.Boynton, unpublished data). Thus despite increased cyclin E levels, T51B cells treated with 10 nM OA remained in G1 following stimulation with EGF; cdk2 was not activated and they did not progress on to S-phase.



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  		Fig. 6. Okadaic acid induced block of cdk2 kinase activation. Cells were pre-treated in serum-free media with 10 nM OA ({circ}) or without OA (•) for 24 h and harvested after an additional incubation with EGF as indicated (0, 8, 12 or 16 h). Cdk2 was immunoprecipitated from cell lysates and used as an enzyme source in an in vitro kinase assay with histone substrate as described in Materials and methods. The data represent mean values from three separate experiments expresssed as percent maximal activity (seen at 16 h EGF without OA).

 


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  		Fig. 7. Inhibition of EGF-stimulated cell cycle progression and cdk2 activation each occur over a narrow concentration range of okadaic acid. Cells pre-treated for 24 h with 0–10 nM OA were stimulated with EGF for an additional 18 h and processed for FACS analysis or cdk2 immunoprecipitation and kinase assay. The cells in S-phase ({circ}) and the kinase activity (•) at each OA concentration were normalized to values obtained from control cells (no OA). The mean values of two experiments are presented.

 
Okadaic acid treatment influences the levels and cdk2 association of endogenous inhibitor proteins
Neither cyclin E nor the two components of cdk activating kinase (CAK: cdk7 and cyclin H) were diminished in OA-treated cells (Figure 5Go and data not shown). Although total cdk2 protein was slightly decreased in OA-treated cells, this is unlikely to account for the decrease in cdk2 activity, since the cyclin partner (not the cdk) is generally the limiting factor (12). This prompted us to look for inhibitor proteins in the cdk2 complex that might prevent cdk2 activation in response to EGF; two well-studied examples are p21 (cip-1) and p27 (kip-1) (14). Treatment of T51B cells with increasing amounts of OA, followed by EGF, resulted in increased levels of p21 (cip-1) and p27 (kip-1) in cells, as shown in Figure 8AGo. Both proteins increased over the range 2–10 nM OA, the concentration range needed to move from partial to full block of EGF-stimulated DNA synthesis. This was reflected in these samples by the inhibition of EGF-stimulated cyclin A expression over a similar concentration range (Figure 8AGo).

Further support for the conclusion that p21 (cip-1) and/or p27 (kip-1) contribute to the cell cycle block came from demonstrating their association with cdk2. T51B cells treated with 0–10 nM OA were lysed under conditions similar to the cdk2 kinase assay and the cdk2 immunoprecipitates were analyzed for p21 (cip-1) and p27 (kip-1) by western blot (Figure 8BGo). Both ckis were elevated in cdk2 complexes immunoprecipitated from OA-treated cells. A similar effect of OA was seen on ckis associated with cyclin E immunoprecipitates (data not shown). Thus, OA treatment resulted in an increase in the association of p21 (cip-1) and p27 (kip-1) with cyclin E-cdk2, which should prevent activation by CAK and block EGF-stimulated cells at the G1/S restriction point (14).

The best-characterized mechanism for elevating p21 (cip-1) protein levels involves increased levels of p21 (cip-1) gene expression mediated by the tumor suppressor protein p53 (14). A number of pathways lead to the activation of p53 via either an increase in the amount of p53 protein, activation of existing p53, or both (17). We examined the levels of p53 by western blot using antibodies that detect total p53, as well as antibodies specific for activated p53 (phosphorylated at Ser15). By either measure, p53 was increased by OA treatment protocols (7 or 10 nM for 48 h) that also increased p21 (cip-1) (Figure 9Go). Thus, although we have not excluded the involvement of p53-independent pathways (38), increased stability of p53 triggered by phosphorylation at Ser15 likely contributes to induction of p21 (cip-1) and cell cycle arrest caused by transforming concentrations of OA in T51B cells.

Treatment of confluent T51B cells with okadaic acid alone shifts the population from a quiescent phenotype to one that is trapped in late G1
The data thus far implicated p53-dependent induction of p21 (cip-1) in the G1/S block seen in OA-treated cells stimulated by EGF. Interestingly, confluent (G0) cells treated with OA alone also showed similar changes. This suggested that additional OA-sensitive processes governed an exit from quiescence that in turn resulted in p53-dependent induction of p21 (cip-1). As this aspect of OA action seems most consistent with its tumor-promoting properties, we next sought to identify indicators that would confirm the shift from G0 to late G1. As described earlier, one effect of our standard OA treatment protocol was elevated cyclin E levels (Figures 5 and 9GoGo); the levels induced by 7 or 10 nM OA alone were comparable with the maximal levels achieved after stimulation of the cells with EGF. This was not seen with agents that inhibit EGF-stimulated cell cycle progression by other mechanisms, including the PI3-kinase inhibitor LY 294,002 or the tyrphostin AG879 (data not shown).

Cyclin E gene expression is activated by E2F transcription factors, which are sequestered in unstimulated cells by binding to pRb and related pocket proteins (13,47,48). Expression is stimulated by cell cycle-dependent phosphorylation of pocket proteins and release of active E2F. Phosphorylation of pRb accompanies the G1/S transition and was not altered by 10 nM OA in T51B cells (Figure 5Go). A different result was seen for the p130 pocket protein, however, which is involved in the transition from G0 to G1 in many cells (49,50). As shown in Figure 10Go, p130 from untreated quiescent T51B cells migrated in SDS–PAGE as a doublet. Treatment with EGF for 12 h resulted in significant loss of the lower (hypophosphorylated) band that appeared complete by 18 h. The amount of the upper band appeared relatively constant or increased slightly over this time course. Cells treated with 7 or 10 nM OA alone showed pronounced loss of both p130 bands. In particular, the lower, hypophosphorylated band reached levels comparable with that seen upon 12 h EGF treatment. Further effect of EGF on the lower band was blocked in OA-treated cells (Figure 10Go), perhaps due to inhibition of cdk2–p130 association by increased p21 (cip-1) (data not shown). As was seen in earlier experiments, OA treatment also resulted in elevated EGF receptor levels, loss of EGF-stimulated pRb phosphorylation and cyclin A induction, and increased p21 (cip-1) levels (Figure 10Go). Loss of hypophosphorylated p130, together with increased cyclin E and a lack of S-phase indicators, are good evidence for accumulation of OA-treated cells in mid to late G1.


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The purpose of this work was to identify cell cycle-related effects of OA relevant to tumor promotion in rat liver T51B cells. We found that low nanomolar levels of this phosphatase inhibitor acted as a tumor promoter in a two-stage transformation assay. At earlier time points this same concentration range facilitated progression of OA-treated cells from a quiescent (G0) state to a state resembling mid-G1. Among the indicators of this progression were okadaic acid-induced changes in cyclin E and the G0/G1 regulator p130. In the simplest model, phosphatase inhibition by OA would result directly in increased phosphorylation and down-regulation of p130. This effect of 7 nM OA partially mimics the changes seen after 12 h exposure to EGF (summarized in Figure 11Go). It seems logical to propose that the p130 effect is key to both cell cycle entry (early on) and promotion of transformation (over the long-term) induced by low levels of OA.



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  		Fig. 11. Cell cycle progression induced by EGF or okadaic acid. Treatment of quiescent cells with EGF leads to changes in levels of hypophosphorylated p130 and cyclin E at 10–12 h that indicate G1 entry, and in pRb phosphorylation and cyclin A levels at >16 h that indicate S-phase entry. Treatment with 7–10 nM OA alone results in p130 and cyclin E changes, but not pRb or cyclin A. Activation of p53 and induction of p21 (cip-1) in OA-treated cells prevents progression to S-phase and contributes to G1 trapping by this serine/threonine phosphatase inhibitor.

 
To ensure the relevance of the cell cycle experiments to tumor promotion, we used very low concentrations of OA (<=10 nM). Consequently, lengthy pre-treatment times were required to observe complete inhibition of EGF-induced proliferation (Figure 3Go) and other cell cycle effects. There are several reasons to expect this. First, it is likely to take at least several hours to equilibrate intracellular compartments with OA added to the extracellular media (46). Second, effects of this phosphatase inhibitor may depend on the rate of an opposing kinase activity, which may be slow in resting cells. Third, even strongly mitogenic treatments (such as 20% serum) require 10–12 h to induce progression to mid-G1 (data not shown) and OA is unlikely to act more quickly than this. Additional work will be required to determine the extent to which each of these factors contribute to the cellular changes associated with OA-induced G1-trapping.

The ability to see significant biochemical effects at <=10 nM OA is a clear advantage of this study when trying to identify the relevant phosphatase(s), as most serine/threonine phosphatases are not inhibited at this concentration (for reviews, see refs 4 and 5). Of the major classes, type 2A (PP2A) enzymes show the greatest sensitivity to OA in vitro (IC50 < 0.1 nM), followed by the type 1 (PP1) enzymes (IC50 ~50 nM). Types 2B and 2C are insensitive (no effect at 1 µM). Of the minor classes, type 4 (PP4) has comparable sensitivity to type 2A (IC50 ~0.1 nM) (51), type 5 (PP5) is slightly less sensitive (IC50 below 10 nM) (52,53) and type 7 is insensitive to OA (54). T51B cells are known to contain PP1, PP2A and PP5 (D.J.Messner, unpublished data), while information on PP4 is not available. The low concentration of OA used indicates that inhibition of PP2A, PP4 and PP5 are sufficient to cause the effects we observed. We conclude that inhibition of PP1 is not required for promotion of transformation or for the cell cycle effects reported here.

The proposal that phosphatase inhibition affects cell cycle entry by influencing p130 levels depends on p130 being uniquely important for maintaining a quiescent state in T51B cells; at least three of its properties favor this idea. First, it is a pocket protein capable of sequestering the transcription factors E2F4 and E2F5 (50). E2F4 is thought to be an activator of cyclin E expression but not cyclin A expression (47,50). Second, it acts as a repressor for the expression of growth inducing genes such as E2F1 (55). Elevated E2F1 can also increase cyclin E levels and has been shown to result in tumors in p53-deficient mice (56). Finally, it can act as a cdk inhibitor (57,58). This combination of effects is not found in pRb or p107, the other two pocket proteins. Thus, in addition to control of transcription, p130 may maintain a quiescent state by forming a mitogen-sensitive inhibitory complex with the cyclin/cdks found at low levels in resting cells. Each of these properties must be significantly decreased by the loss of p130 in OA-treated T51B cells. That OA treatment resulted in a loss of p130 rather than a phosphorylation-induced shift in gel migration is not unexpected; down-regulation of hyperphosphorylated p130 is known to occur as cells progress through a mitogen-induced cell cycle (50).

Connections between p130 phosphorylation/dephosphorylation and regulation of its levels and functional properties are beginning to be made: two levels of p130 phosphorylation have been identified (59). In the first level, intermediate phosphorylation within a specific `loop' region of the protein accompanies the onset of quiescence (G1 to G0) stimulated by cell–cell contact or serum starvation (60). However, engineered deletion of this loop region results in no loss of function, as measured by entry of cells into quiescence, E2F4 binding or mitogen-stimulated hyperphosphorylation of p130 (60). Modulation of these phosphorylation sites, therefore, is unlikely to account for the effects of OA we observed in T51B cells. In contrast, addition of mitogen to quiescent cells results in a second level of phosphorylation: the hyperphosphorylation that triggers loss of E2F binding and other functional changes responsible for progression back into and through G1 (50). The simplest model predicts that this step is modulated by OA in T51B cells, but it is not yet clear whether OA inhibits a phosphatase that acts directly on p130 or more indirectly influences mitogen-sensitive kinase activity acting on p130. Precedence for a p130-selective phosphatase comes from the finding that PP1 is a pRb-specific phosphatase important in the G1/S transition and in the exit from mitosis (61,62). Additional work will be required to sort out the complex relationships between p130, p-130 kinases, OA-sensitive phosphatases and the regulation of the quiescent state in T51B cells.

Okadaic acid treatment by itself did not provide sufficient stimulus to cause progression of T51B cells through G1 to S-phase. Furthermore, EGF-stimulated progression to S-phase was blocked by OA. In both cases there are probably a number of contributing factors, one important effect we observed was induction of the cdk2 inhibitor p21 (cip-1). How might this occur? We observed increased phosphorylation of p53 at Ser15, which is associated with p53 activation and p21 (cip-1) induction in response to DNA damage (16,17,63,64). Perhaps an OA-sensitive phosphatase is particularly important in directly regulating phosphorylation at this site. Alternatively, OA-sensitive phosphatases may be involved in upstream regulation of kinases active at Ser15; candidates include the ATM kinase, the ATR kinase or DNA activated protein kinase (63,64). Phosphorylation of Ser15 is thought to be required for subsequent phosphorylation of Thr18 and/or Ser20, which in turn leads to increased levels of p53 transcriptional activity through inhibition of p53 binding to the regulatory protein Mdm2 (63–68). Although there are no reports of increased DNA damage in cells exposed to 10 nM OA alone, this treatment may sensitize cells to subsequent damage by mutagens (69), perhaps by inhibiting aspects of DNA repair. Aspects of these pathways may be activated in OA-treated T51B cells, particularly those in which transformation has been initiated by MNNG.

An alternative scenario postulates that p53 activation and p21 (cip-1) induction represents a compensatory response to aberrant cell cycle entry triggered by loss of p130. This mechanism is consistent with the ability of p53 to control the outgrowth of disregulated cells through cell cycle arrest and/or apoptosis (16,17,64,70). Support for this scheme comes from the work of Milczarek et al. (37), who observed OA-induced mitoses and no effect of OA on p21 (cip-1) levels in fibroblasts overexpressing dysfunctional p53. Fibroblasts having wild-type p53 showed increased p21 (cip-1) and G2/M block in response to OA, similar to what we saw in T51B cells. The block at G2/M (rather than G1/S) may have been due to the higher OA concentration (50 nM) used by these authors, which most likely results in inhibition of type 1 serine/threonine protein phosphatase (PP1). This would be expected to facilitate the G1/S transition by increasing pRb phosphorylation (61,62). Similar activation of p53 is caused by a variety of oncogenic stimuli and involves induction of p19-ARF, which binds to and sequesters Mdm2 directly (16–18,63,64). However, the ARF pathway does not require p53 Ser15 phosphorylation (71), and we have not been able to detect elevated ARF in OA-treated cells, suggesting other processes are involved. The main point is that OA-induced cell cycle progression was halted in a p53-dependent fashion. Assuming those findings can be extrapolated to the less toxic OA concentrations we examined, p53 activation should also inhibit tumor promotion induced by OA and overcoming or evading p53 is likely to be a necessary step in the tumor promotion process. In summary, the data presented here implicate p53 activation and p21 (cip-1) induction in the second phase of G1-trapping by OA, and may represent either a direct effect of OA or a compensatory response to cell cycle progression induced by this tumor promoter. Identification of the relevant p53 activation pathways, as well as a clear view of how these effects are mechanistically linked to tumor promotion by OA, are important goals for the future.


   Notes
 
1 To whom correspondence should be addressed Email: dmessner{at}nwhsea.org Back


   Acknowledgments
 
Portions of this work have been presented in abstract form (Messner,D., Jagdale,A., Ao,P. and Boynton,A., 1999, Mol. Biol. Cell., 10S, 45a). The authors thank Dr Sandra Rossie (Purdue University) for helpful comments on the manuscript and Michelle Bates for photographic assistance. This work was supported in part by NIH Grant CA39745.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 10, 2000; revised April 9, 2001; accepted April 27, 2001.


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