Stem Cells, Vol. 19, No. 3, 205-211,
May 2001
© 2001 AlphaMed Press
p55Cdc/Cdc20 Overexpression Promotes Early G1/S Transition in Myeloid Cells
Michael L. Lin,
Kathleen M. Sakamoto
Department of Pediatrics, UCLA School of Medicine, Los Angeles, California, USA
Key Words. p55Cdc • Cdc20 • Cell cycle • Myeloid cells • G1 • S
Kathleen M. Sakamoto, M.D., Department of Pediatrics-Hematology/Oncology, A2-311 MDCC, 10833 Le Conte Avenue, Los Angeles, California 90095-1752, USA. Telephone: 310-794-7007; Fax: 310-206-8089; e-mail: kms{at}ucla.edu
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ABSTRACT
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p55Cdc/Cdc20 is expressed in cycling mammalian cells and has been shown to be an activator of the mitotic spindle assembly checkpoint. We previously showed that overexpression of p55Cdc/Cdc20 in myeloid cells resulted in accelerated apoptosis and inhibition of granulocyte differentiation in the murine myeloid cell line 32Dcl3. p55Cdc/Cdc20 protein expression is detected in cells at late G1 phase of the cell cycle but is maximal during G2 phase. We report in this paper that inducible expression of p55Cdc/Cdc20 in 32Dcl3 cells results in premature transition from G1 to S phase. To characterize the mechanism of this early transition, we examined the expression of critical regulatory proteins during the cell cycle. Although expression of cyclin D, cyclin E, cdk2, and cdc2 did not change significantly between p55Cdc/Cdc20-overexpressing and control cells, p27Kip1 protein levels were lower and cdk2 activity higher during G1 to S transition in p55Cdc/Cdc20-overexpressing cells compared to control cells. Cyclin B1 levels were lower at early G1 phase in cells overexpressing p55Cdc/Cdc20. Our results suggest that p55Cdc/Cdc20 may play an important role in G1 to S transition during myelopoiesis.
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INTRODUCTION
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p55Cdc/Cdc20 is a member of a family of cell cycle proteins including Cdc4 in S. cerevisiae, that has been proposed to be a regulator of DNA synthesis [1, 2]. Other members of this family are Drosophila Fzy and S. pombe Slp1 [3-5]. These proteins contain leucine-rich WD repeats, which have highly conserved tryptophan and aspartate residues [1]. The p55Cdc/ Cdc20 protein has seven WD repeats that are proposed to act as a platform for interactions between proteins regulating transcription, signal transduction, and cell cycle [1, 6-9].
p55Cdc/Cdc20 is a cell cycle protein that is expressed in proliferating mammalian cells but not in differentiated or growth-arrested cells and appears to be required for normal cell division. Mutations in the Drosophila fzy gene exhibited a metaphase arrest phenotype that is accompanied by stabilization of cyclin A and B proteins. This suggests the possibility that fzy function is required for cell cycle-regulated ubiquitin-mediated proteolysis of cyclins [3, 4]. In Chinese hamster ovary (CHO) cells, transfection of sense p55Cdc/Cdc20 cDNA resulted in increased cellular proliferation while antisense p55Cdc/Cdc20 cDNA led to decreased cellular proliferation. Transfection of antisense p55Cdc/Cdc20 cDNA into CHO cells also resulted in isolation of only those cells which demonstrated a compensatory increase in p55Cdc/ Cdc20 transcripts in the sense orientation [1]. Although p55Cdc/Cdc20 appears to be required for normal cell division, the precise role of this protein during cell cycle regulation is unknown.
p55Cdc/Cdc20 has recently been shown to interact with Mad2 (mitotic arrest deficient). It also interacts with Cdc16 and Cdc27, that are both components of the APC (anaphase promoting complex) [10]. Furthermore, p55Cdc/Cdc20 activates the APC [11], whereas Mad2 inhibits APC activity [12]. p55Cdc/Cdc20 is also phosphorylated by the kinase BubR1 [13]. The APC/p55Cdc/Mad2 complex regulates the spindle assembly checkpoint and metaphase/anaphase transition during normal cell cycle. This ensures proper chromosomal segregation prior to the onset of anaphase.
We previously demonstrated that p55Cdc/Cdc20-overexpressing myeloid cells undergo accelerated apoptosis [14]. Increased p55Cdc/Cdc20 expression also inhibited granulocyte differentiation [14]. In the murine myeloid cell line 32Dcl3, p55Cdc/Cdc20 protein expression appears approximately 6 hours following synchronization and growth factor stimulation. The expression begins at late G1 and continues through S and G2 phases. These data suggest that p55Cdc/ Cdc20 may regulate G1 to S transition in addition to mitosis.
In this paper, we studied the potential role of p55Cdc/ Cdc20 during G1/S transition by overexpressing the protein under the control of a zinc-inducible metallothionein promoter in 32Dcl3 cells. The overexpression of p55Cdc/Cdc20 in hematopoietic cells synchronized by serum- and factor-starvation followed by growth factor stimulation leads to aberrant cell cycle regulation by premature entry into S phase. Our experiments demonstrate that p55Cdc/Cdc20 overexpression in myeloid cells correlated with lower levels of cyclin B1 and an earlier decrease in p27Kip1 levels without a significant change in the expression of other cell cycle regulators. We also observed that Cdk2 activity increased earlier in p55Cdc-overexpressing cells compared to control cells. These results suggest that p55Cdc may play a role during G1/S transition.
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MATERIALS AND METHODS
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Cell Culture and Preparation of Stably Transfected Cell Lines
The murine myeloid cell line, 32Dcl3, was cultured in Iscove's modified minimal essential Dulbecco's medium + 10% calf serum (Gemini Bio-Products, Inc.; Calabasas, CA; http://www.gembio.com), 100 U/ml penicillin (Sigma Chemical Co.; St. Louis, MO; http://www.sigma-aldrich.com), 100 mg/ml streptomycin (Sigma), 2 mM L-glutamine (Sigma), and COS murine GM-CSF. The COS murine GM-CSF supernatant was used at a dilution of 1:100 since this was determined by growth assays to be sufficient for cell growth. The parental 32Dcl3 cell line was stably transfected with an expression construct called pMT-p55Cdc/Cdc20 containing the pMT vector with a metallothionein promoter and rat p55Cdc/Cdc20 cDNA. This construct was obtained from Jasminder Weinstein (Amgen, Inc.; Thousand Oaks, CA) and has previously been used in growth assays [1]. Cells (2 x 107) were electroporated using a BioRad electroporator with 25 µg of DNA at 250 V and 960 µF for about 10 seconds. Approximately 102 to 103 cells were pooled into 24-well plates. Two days after transfection, cells were grown in the presence of G418 (2.5 mg/ml) (GIBCO BRL; Rockville, MD; http://www.lifetech.com). Cells stably transfected with pMT-p55Cdc/Cdc20 were examined for p55Cdc/Cdc20 expression by Northern and Western blot analysis.
RNA Extraction and Northern Blot Analysis
Parental 32Dcl3 cells at a density of 1 x 106 cells/ml were treated with water (control) or zinc sulfate (0.1 mM) for 3 hours in Iscove's modified minimal essential Dulbecco's medium + 10% calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and L-glutamine (2 mM). Approximately 108 cells were harvested and RNA was extracted using the Fast Track kit (Stratagene; La Jolla, CA; http://www.stratagene.com). For the Northern blot analysis, 3 µg of RNA were loaded on a 2% formaldehyde, 1% agarose gel and transferred onto a nylon membrane. The rat p55Cdc/ Cdc20 cDNA and choB control DNA were used as probes. The membrane was washed as previously described [15]. The blot was exposed to film overnight at -80°C.
Cell Cycle Experiments
32Dcl3 cells (106 cells/ml) were serum- and growth factor-starved for 15 hours in Iscove's modified minimal essential Dulbecco's medium + 0.5% bovine serum albumin (BSA), 100 U/ml penicillin, 100 mg/ml streptomycin, and L-glutamine (2 mM) prior to stimulation with recombinant murine GM-CSF (1 nM). Cells with pMT or pMT-p55Cdc/Cdc20 were treated with zinc sulfate (0.08 mM or 0.1 mM) (Sigma) for 3 hours prior to stimulation with recombinant murine GM-CSF. At each time point, cells (106 total) were harvested and stained with hypotonic propidium iodide solution containing 0.1% sodium citrate (Sigma), 0.3% Triton X-100 (Sigma), 0.01% propidium iodide (Calbiochem; La Jolla, CA; http://www.calbiochem.com), and 0.002% RNase A (Sigma). Alternatively, cells (2 x 106) were harvested, pelleted at 1,000 rpm x 5 minutes, and resuspended in 200 µl phosphate-buffered saline (PBS) (Sigma). Cells were fixed in 2 ml 70% EtOH, 30% PBS for 1 hour at 4°C. After centrifugation (2,000 rpm x 10 minutes) and resuspension in PBS, RNase A and propidium iodide were added to final concentrations of 0.1 mg/ml and 40 µg/ml, respectively. Solutions were incubated at 37°C for 30 minutes and then stored at 4°C. Percentages of cells in each cell cycle phase were determined by flow cytometry.
Western Blot Analysis and Time Course
32Dcl3 cells overexpressing p55Cdc/Cdc20 were starved as described above. Cells (106 cells/ml) were then treated with recombinant murine GM-CSF (1 nM final concentration). They were treated with or without zinc sulfate (approximately 0.1 mM final concentration) for 3 hours prior to treatment with recombinant murine GM-CSF. Cells (106 total) were harvested at the indicated time points. Cells were lysed with boiled SDS-Laemmli buffer and boiled further for 5 minutes as previously described [14]. Samples from whole cell lysates were separated on a 10% SDS-PAGE gel and checked for protein loading using Ponceau S (Sigma) staining. Western blot was probed with antibodies (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA; http://www.scbt.com) diluted 1:100 in 1% milk in Tris-buffered saline with 0.1% Tween 20 (Sigma). p55Cdc/Cdc20 antibody was provided by Jasminder Weinstein and used at 1:200 dilution in 2% BSA, 2% milk, and 0.02% Tween 20 in PBS. p27Kip1 antibody was provided by Steve Coats (Amgen, Inc.) and used at 1:2,000 dilution in 150 mM NaCl, 25 mM Tris-HCl (pH 7.5), and 0.5% Tween 20. Biotinylated donkey anti-rabbit IgG and streptavidin horseradish peroxidase (Amersham Pharmacia Biotech; Piscataway, NJ) were used as secondary and tertiary antibodies for most primary antibodies. Protein A-peroxidase (Boehringer Mannheim Corporation; Indianapolis, IN; http://biochem.boehringer-mannheim.com) was used as secondary antibody for p27Kip1. Enhanced chemiluminescence detection kit (Amersham, Inc.) was used for all antibodies.
In Vitro Kinase Assay
32Dcl3 cells were serum- and growth factor-starved for 15 hours for cell cycle experiments as previously described. Three hours prior to release from block, cells were treated with zinc sulfate to a final concentration of 0.08 mM to induce p55Cdc/Cdc20 overexpression. Cells were released from block by addition of recombinant murine GM-CSF (1 nM). At 0, 6, 12, and 24 hours following release from block, 107 cells were collected and pelleted for analysis by kinase assay performed by Jasminder Weinstein. Pellets were lysed in TG buffer consisting of 1% Triton-X, 10% glycerol, 100 mM NaCl, 50 mM NaF, 20 mM HEPES, 10 mM Na3VO4, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 10 µg/ml soybean trypsin inhibitor. Lysate was centrifuged for 30 minutes at 20,000 x g. Protein levels in the supernatant were determined using bicinchoninic acid protein assay (Pierce Chemical Company; Rockford, IL; http://www.piercenet.com). Immunoprecipitations were completed using 100 µg lysate in 1 ml volume of TG buffer with 10 µl of cdk2 antibody (Santa Cruz, Inc.) or 4 µg pre-immune rabbit IgG for control. Incubations occurred on ice overnight, and precipitation was done with 30 µl of 50% protein G-sepharose slurry. Beads were washed three times with TG buffer and then once in kinase buffer (50 mM Tris pH 7.5, 10 mM MgCl2) without ATP. The kinase assay was performed as described previously [16]. Fifty-five µM 32P-labeled ATP (8,000 cpm/pmol) were used, and histone H1 was the substrate. Reactions were incubated at 30°C for 30 minutes. Kinase activity was determined by quantitating radioactivity from excised gel bands.
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RESULTS
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Preparation and Verification of p55Cdc/Cdc20- Overexpressing 32Dcl3 Cells
To examine the effects of p55Cdc/Cdc20 overexpression, the murine factor-dependent myeloid cell line 32Dcl3 was stably transfected with the pMT-p55Cdc/Cdc20 construct. p55Cdc/Cdc20 clone 7S was analyzed by Northern and Western blot analysis. We observed a significant increase in p55Cdc/Cdc20 expression by Northern blot analysis (Fig. 1A
) in cells stimulated with zinc compared to control cells (stimulated with water). We observed that one clone of cells overexpressing p55Cdc/Cdc20 had a higher level of choB (Fig. 1B, right panel). Several other 32Dcl3 cell clones stably transfected with pMT-p55Cdc/ Cdc20 were also examined for p55Cdc/Cdc20 expression following zinc stimulation by Western blot analysis (Fig. 1B). The level of p55Cdc/Cdc20 protein was minimal in uninduced cells.
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Figure 1. Inducible expression of p55Cdc/Cdc20 in 32Dcl3 cells. (A) Two Northern blots showing p55Cdc/Cdc20 induction. Left panel shows parental and zinc-inducible 32Dcl3 cells. Right panel shows zinc-inducible without and with zinc stimulation. Zinc-inducible 32Dcl3 cells were treated for 3 hours with zinc sulfate (0.075 mM). RNA was isolated from 108 cells overexpressing p55Cdc/Cdc20 or parental 32Dcl3 cells. Three micrograms of RNA were loaded on a 1% formaldehyde gel and transferred to nylon membrane as previously described [15]. The rat p55Cdc/Cdc20 cDNA and choB control were used as probes. Blot was placed on film overnight at -80°C. (B) To detect protein expression of p55Cdc/Cdc20 in 32Dcl3 cells, 32Dcl3 cells were stably transfected with pMT-p55Cdc/Cdc20 and treated with 0.1 mM zinc sulfate for 24 hours to induce p55Cdc/Cdc20 overexpression. Cells were lysed in SDS-Laemmli buffer at a concentration of 1 x 106 cells/100 µl buffer. Samples were run on 10% SDS-PAGE and probed with p55Cdc/Cdc20 antibody. 4S, 5S, 6S, and 7S are different clones isolated after pMT-p55Cdc/Cdc20 transfection.
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p55Cdc/Cdc20-Overexpressing Cells Enter S Phase Earlier Than Control Cells
To determine the effect of p55Cdc/Cdc20 overexpression on cell cycle regulation, we serum- and growth factor-starved 32Dcl3 cells for 15 hours to synchronize cells in G0 phase. To induce p55Cdc/Cdc20 expression, we added 0.1 mM zinc sulfate to media 3 hours prior to release from block using 1 nM recombinant murine GM-CSF. We previously demonstrated that greater than 80% of cells arrested in G0 phase following serum- and growth factor-starvation [14]. Figure 2A illustrates a single representative cell cycle experiment of three independently performed experiments. Within 3 hours following GM-CSF stimulation, 32% of p55Cdc/Cdc20-overexpressing cells entered S phase, compared to 25% in control cells. Between 3 to 15 hours, a 7% difference in S phase cells was observed between the p55Cdc/Cdc20-overexpressing cells and uninduced cells. Figure 2B shows results from the same cell cycle experiment for percent cells in G0/G1 and G2/M in addition to S phase represented as a graph. Our data indicate that the increase of cells in S phase correlated with the decrease of cells in G0/G1 phase. Percentages of cells in G2/M phase in p55Cdc/Cdc20-overexpressing cells remain relatively unchanged from control cells until the 24-hour time point. This could be due to increased numbers of cells in S phase entering G2/M.
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Figure 2. Increased percentage of cells in S phase in p55Cdc/Cdc20-overexpressing cells. 32Dcl3 cells were serum- and growth factor-starved for 15 hours in Iscove's modified Dulbecco's medium + 0.5% BSA and treated with zinc sulfate (0.1 mM) for 3 hours. Cells were stimulated with recombinant murine GM-CSF (1 nM), and 106 cells were harvested for each time point. (A) Percentage of cells in S phase at each time point was determined by staining cells with propidium iodide and analyzing by flow cytometry. This is a representative graph of one of three independent experiments. (B) Percentages of cells in G0/G1, S, and G2/M phases for the same cell cycle experiment as shown in (A).
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We previously demonstrated that 50% of parental 32Dcl3 cells undergo G1 to S transition between 12 to 24 hours [14]. In cells inducibly overexpressing p55Cdc/Cdc20, the majority of cells entered S phase as early as 9 hours following GM-CSF stimulation compared to 15 hours in uninduced cells (Fig. 2A
). Our results indicate that cells overexpressing p55Cdc/Cdc20 enter S phase prematurely by up to 6 hours. Furthermore, a greater percentage of p55Cdc/Cdc20-overexpressing cells are in S phase within the first 5 to 15 hours following growth factor stimulation compared to control cells. Although the difference is less than 10%, the pattern was highly reproducible in multiple experiments.
Cyclin D1, Cyclin E, cdk2, and cdc2 Levels Are Similar in p55Cdc/Cdc20-Overexpressing and Control Cells
Among the most critical cell cycle regulatory proteins in G1/S transition are cyclin D1, cyclin E, and cdk2. To determine the mechanism of G1 to S regulation by p55Cdc/Cdc20, we analyzed the expression of these proteins by Western blot analysis. Whole cell lysates were analyzed from zinc-induced, vector control, or parental 32Dcl3 cells treated with zinc. Our results demonstrated that the levels of these cell cycle proteins were not altered in the presence of p55Cdc/ Cdc20 overexpression compared to control cells (Fig. 3).
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Figure 3. Western blot analyses of proteins that regulate G1 to S transition in 32Dcl3 cells overexpressing p55Cdc/Cdc20. p55Cdc/Cdc20-overexpressing ("p55Cdc"), vector control ("vector"), or parental ("control") 32Dcl3 cells were treated with zinc sulfate (0.1 mM) for 3 hours and starved for 15 hours in serum-free, growth factor-free media. Cells were treated with GM-CSF in the presence of zinc sulfate (0.1 mM) and harvested at 0, 6, 12, and 24 hours. Cells were lysed with boiling SDS-Laemmli buffer and boiled for 5 minutes. Proteins from approximately 2 x 105 cells were separated on a 10% SDS-PAGE gel. Western blot was probed with respective antibodies (Santa Cruz, Inc.). Arrow indicates the protein of interest.
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Cyclin B1 Levels are Lower in p55Cdc/Cdc20-Overexpressing Cells
During metaphase-anaphase transition, p55Cdc activates the APC, which promotes cyclin B1 degradation [11]. This degradation is essential for mitotic exit. Therefore, we also examined expression of cyclin B1 in p55Cdc/Cdc20-overexpressing cells. Western blot analysis using antibody against cyclin B1 (Santa Cruz, Inc.) was performed with cell lysates from zinc-induced and parental 32Dcl3 cells. Cyclin B1 levels were lower at 0, 6, and 12-hour time points in cells overexpressing p55Cdc/Cdc20 compared to control cells (Fig. 3).
p27Kip1 Levels Also Decrease Earlier in p55Cdc/Cdc20-Overexpressing Cells
Activation of the cyclin E/cdk2 complex is critical for G1 to S transition. Cdk2 activity is tightly regulated by levels of p27Kip1 [17, 18]. Therefore, we analyzed levels of p27Kip1 in p55Cdc/Cdc20-overexpressing cells. To assess p27Kip1 expression in p55Cdc/Cdc20-overexpressing cells, we performed a Western blot analysis with p27Kip1 antisera (Fig. 4) or control rabbit antisera (data not shown). p27Kip1 levels decreased earlier in zinc-induced p55Cdc/Cdc20-overexpressing cells compared to control cells (Fig. 4). Expression of p27Kip1 significantly decreased at the 13-hour time point compared to control cells. These results were confirmed in three independent experiments. We previously observed that 32Dcl3 cells constitutively overexpressing p55Cdc/Cdc20 also demonstrated premature entry into S phase, which correlated with an earlier decrease in p27Kip1 protein levels (data not shown).
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Figure 4. p27Kip1 expression in p55Cdc/Cdc20-overexpressing 32Dcl3 cells. 32Dcl3 cells (p55Cdc/Cdc20-overexpressing and vector control) were serum- and factor-starved for 14 hours and treated with zinc sulfate (0.08 mM) for 3 hours. Cells were incubated in Iscove's modified Dulbecco's medium + 0.5% BSA, zinc sulfate, recombinant murine GM-CSF (1 nM) and harvested at 0, 6, 13, and 24 hours. Cells (2 x 106) were harvested and boiled in SDS-Laemmli buffer for 5 minutes. Western blot was probed with pre-immune rabbit serum (data not shown) or p27Kip1 antiserum. Arrows indicate presence of p27Kip1 in both vector control and p55Cdc/Cdc20-overexpressing cells and overexpression of p55Cdc/Cdc20 in 7S cells induced by zinc sulfate.
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p55Cdc/Cdc20 Overexpression Leads to Earlier Increase in cdk2 Activity
Since cdk2 activity is inhibited by p27Kip1, an in vitro kinase assay was performed to examine cdk2 activity. 32Dcl3 cells were synchronized in G0 phase by serum and growth factor starvation followed by addition of recombinant murine GM-CSF to allow cells to progress through the cell cycle. Samples were collected at 0, 6, 12, and 24 hours after release from G0/G1 block. With zinc-induced overexpression of p55Cdc/Cdc20, there is an earlier increase in cdk2 activity compared to vector control cells (Fig. 5). These data represent an average of duplicate samples from this single experiment. Our results suggest that an earlier increase in cdk2 activity correlates with the premature G1/S transition and decrease in p27Kip1 levels in p55Cdc/Cdc20-overexpressing cells.
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Figure 5. cdk2 activity increases earlier in cells overexpressing p55Cdc/Cdc20 compared to control cells. 32Dcl3 cells (p55Cdc/ Cdc20-overexpressing and vector control) were deprived of serum and growth factor for 15 hours in Iscove's modified Dulbecco's medium + 0.5% BSA and treated with 0.08 mM zinc sulfate for 3 hours prior to addition of 1 nM recombinant murine GM-CSF. At indicated times, 1.5 x 107 cells were collected for in vitro kinase assay. Cdk2 activity was determined as shown on y-axis. M2 indicates cell line with pMT control vector, and 7S indicates cell line with pMT-p55Cdc/Cdc20 construct overexpressing p55Cdc/Cdc20. Error bars indicate one standard deviation value above and below average of data from two samples.
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DISCUSSION
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In mammalian cells, G1 to S transition is regulated by activation of cyclin-cdk complexes [19-22]. Interaction of cyclins with cdks is required for activation of cdks. Cdk activity leads to phosphorylation of proteins such as pRB and activation of transcription factors such as E2F [23]. Cdk inhibitors repress and regulate the activity of cyclin-cdk complexes. The strict control of cdk activity is critical since it determines the commitment of cells to enter S phase.
p55Cdc/Cdc20 interacts with Mad2 during mitosis at the spindle assembly checkpoint and is required for interaction between Mad2 and APC subunits Cdc27 and Cdc16 [10]. p55Cdc/Cdc20 activates the APC during G2 phase [11]. This checkpoint is an important step in the cell cycle since it ensures proper chromosomal separation during anaphase. However, that does not preclude a different function for this protein. We have previously shown that p55Cdc/Cdc20 protein expression in 32Dcl3 cells begins approximately 6 hours after release from G0 block [14]. This coincides with G1/S transition in these cells. Our findings suggest that p55Cdc/Cdc20 activity begins upon initiation of expression instead of remaining inactive until mitosis. As described in this paper, we are proposing a novel role for p55Cdc/Cdc20 during G1/S transition. Thus, p55Cdc/Cdc20 protein may have multiple functions throughout the cell cycle.
p55Cdc/Cdc20 is a phosphoprotein that becomes increasingly phosphorylated upon progression from G1 to G2 phase [24]. We did not examine the status of or changes in phosphorylation of this protein upon zinc induction. Our current studies focused on the cell cycle effects of induced expression of p55Cdc/Cdc20 in myeloid cells. We plan to study the regulation of p55Cdc/Cdc20 phosphorylation during G1/S transition in future experiments.
Although proteins homologous to p55Cdc/Cdc20 are essential for normal exit from the cell cycle in mammalian and Drosophila cells, the precise mechanism of this regulation has not been elucidated. Our results suggest that inducible expression of p55Cdc/Cdc20 may play a role in late G1 and S phase. In previous experiments, premature entry into S phase and a shortened G1 phase were observed in cells that constitutively overexpress p55Cdc/Cdc20 (data not shown). These data are consistent with published data that CHO cells overexpressing p55Cdc/Cdc20 resulted in enhanced cellular proliferation [1].
To further characterize G1 to S transition in p55Cdc-overexpressing cells, we examined the expression of cell cycle proteins that are known to regulate G1 to S transition by Western blot analysis. Cyclin D, cyclin E, and cdk2 levels did not change significantly in p55Cdc/Cdc20-overexpressing cells compared to control cells. Cyclin B1 and cdc2 participate at the G2 and M phases. We examined the protein levels for cyclin B1, which appeared lower at 0 and 6 hour time points (Fig. 3). p55Cdc/Cdc20 has been shown to activate the APC and lead to ubiquitination of cyclin B1 [11]. Thus, induced expression of p55Cdc/Cdc20 in 32Dcl3 cells could result in decreased cyclin B1 protein at early G1 as shown in our data.
Further experiments were performed to determine if the observed decrease in p27Kip1 was a cause or effect of G1/S transition. Aphidicolin was used to inhibit 32Dcl3 cells from entering S phase. In aphidicolin-treated cells, p27Kip1 protein levels decrease even though the majority of cells remain in G1 phase (data not shown). This suggests that the p27Kip1 protein decrease is more likely an effect of G1/S transition than a cause.
Given our results, we hypothesize that p55Cdc/Cdc20 not only regulates mitotic progression but may also play a role during G1 by promoting cyclin B1 degradation and cdk2 activation. Future studies will define the mechanism of p55Cdc/ Cdc20 regulation during G1/S transition.
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SUMMARY
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p55Cdc/Cdc20 is a cell cycle protein which is related to a family of proteins including Fzy in Drosophila and Slp1 in S. pombe. It is a known activator of the APC, which participates at the spindle assembly checkpoint during mitosis. Our data demonstrated that overexpression of p55Cdc/Cdc20 in 32Dcl3 cells leads to premature G1/S transition compared to control cells. Cyclin B1 and p27Kip1 protein levels decreased in 32Dcl3 cells overexpressing p55Cdc/Cdc20 compared to control cells although the decrease in p27Kip1 levels may be a result of the premature G1/S transition. Cdk2 activity appeared to increase earlier in p55Cdc/Cdc20-overexpressing cells compared to control cells. Thus, p55Cdc/Cdc20 may play a role in G1/S transition.
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ACKNOWLEDGMENTS
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The authors thank Kathy Shin and Lisa Kane for their technical assistance and Evelyn Kwon and Patricia Mora-Garcia for their helpful comments. We are also grateful to Steve Coats and Grace Chung for providing the p27Kip1 antisera and technical advice.
This work was supported by the University of California Biotechnology STAR Project in collaboration with Amgen, Inc. and California Cancer Research Program. M.L. was a recipient of National Research Service Award #CA09056 from the National Institutes of Health. K.M.S. is a scholar of the Leukemia and Lymphoma Society of America.
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References
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|---|
-
Weinstein J, Jacobsen F, Hsu-Chen J et al. A novel mammalian protein, p55CDC, present in dividing cells is associated with protein kinase activity and has homology to the Saccharomyces cerevisiae cell division cycle proteins Cdc20 and Cdc4. Mol Cell Biol 1994;14:3350-3363.[Abstract/Free Full Text]
-
Choi WJ, Clark MW, Chen JX et al. The CDC4 gene product is associated with the yeast nuclear skeleton. Biochem Biophys Res Commun 1990;172:1324-1330.[CrossRef][Medline]
-
Dawson IA, Roth S, Akam M et al. Mutations of the fizzy locus cause metaphase arrest in Drosophila melanogaster embryos. Development 1993;117:359-376.[Abstract]
-
Dawson IA, Roth S, Artavanis-Tsakonas S. The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae. J Cell Biol 1995;129:725-737.[Abstract/Free Full Text]
-
Matsumoto T. A fission yeast homolog of CDC20/p55CDC/ Fizzy is required for recovery from DNA damage and genetically interacts with p34cdc2. Mol Cell Biol 1997;17:742-750.[Abstract]
-
Henning K, Li L, Iyer N et al. The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH. Cell 1995;82:555-564.[CrossRef][Medline]
-
Komachi K, Redd MJ, Johnson AD. The WD repeats of Tup1 interact with the homeo domain protein alpha 2. Genes Dev 1994;8:2857-2867.[Abstract/Free Full Text]
-
Chen RH, Miettinen P, Maruoka E et al. A WD-domain protein that is associated with and phosphorylated by the type II TGF-beta receptor. Nature 1995;377:548-552.[CrossRef][Medline]
-
van der Voorn L, Ploegh H. The WD-40 repeat. FEBS Lett 1992;307:131-134.[CrossRef][Medline]
-
Kallio M, Weinstein J, Daum JR et al. Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J Cell Biol 1998;141:1393-1406.[Abstract/Free Full Text]
-
Fang G, Yu H, Kirschner MW. Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. Mol Cell 1998;2:163-171.[CrossRef][Medline]
-
Fang G, Yu H, Kirschner MW. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev 1998;12:1871-1883.[Abstract/Free Full Text]
-
Wu H, Lan Z, Li W et al. p55CDC/Cdc20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase. Oncogene 2000;19:4557-4563.[CrossRef][Medline]
-
Kao CT, Lin M, O'Shea-Greenfield A et al. Over-expression of p55Cdc inhibits granulocyte differentiation and accelerates apoptosis in myeloid cells. Oncogene 1996;13:1221-1229.[Medline]
-
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159.[Medline]
-
Pines J, Hunter T. Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell 1989;58:833-846.[CrossRef][Medline]
-
Koff A, Giordano A, Desai D et al. Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 1992;257:1689-1694.[Abstract/Free Full Text]
-
Coats S, Flanagan WM, Nourse J et al. Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science 1996;272:877-880.[Abstract]
-
Sherr CJ. G1 phase progression: cycling on cue. Cell 1994;79:551-555.[CrossRef][Medline]
-
Heichman KA, Roberts JM. Rules to replicate by. Cell 1994;79:557-562.[CrossRef][Medline]
-
Hunter T, Pines J. Cyclins and cancer. II: CYCLIN D and CDK inhibitors come of age. Cell 1994;79:573-582.[CrossRef][Medline]
-
Sherr CJ. Mammalian G1 cyclins. Cell 1993;73:1059-1065.[Medline]
-
Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149-1163.[Free Full Text]
-
Weinstein J. Cell cycle-regulated expression, phosphorylation, and degradation of p55Cdc. J Biol Chem 1997;272:28501-28511.[Abstract/Free Full Text]
Received December 22, 2000;
accepted for publication February 15, 2001.
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Abstract
Introduction
