Introduction

The AP-1 transcription factor is a complex of proteins composed of basic region-leucine zipper proteins that belong to the Jun, Fos, Jun dimerization partners (JDP1 and JDP2) and the closely related activating transcription factors (ATF/CREB) subfamilies. These proteins bind DNA at specific AP-1 sites and regulate the transcription of AP-1-dependent genes. AP-1 is a central component of many signal transduction pathways in a variety of cell types, and is critical for mitogenesis, apoptosis, and carcinogenesis depending on the cell type. We and others have shown that AP-1 is involved in controlling cellular proliferation, differentiation, apoptosis, and oncogene-induced transformation (Holt et al., 1986; Szabo et al., 1991; Brown et al., 1993; Brown et al., 1994; Rodgers et al., 1994; Ham et al., 1995). However, the exact molecular mechanism by which AP-1 transcription factors control cell proliferation, survival and death is still being elucidated.

In breast cells, the previous studies have suggested that growth factors and hormones, such as IGF, EGF, estrogens and retinoids, can modulate AP-1 transcriptional activity (Schule and Evans, 1991; Chen et al., 1996b; Webb et al., 1999; Lin et al., 2000). Other studies demonstrate that ER and AP-1 interact to regulate the expression of certain estrogen- and tamoxifen-regulated genes (Paech et al., 1997). Activation of AP-1 may also contribute to tumor cell invasive capacity and to tamoxifen resistance (Smith and Prochownik, 1992; Yang et al., 1997; Johnston et al., 1999; Schiff et al., 2000). In previous studies, we have used a specific inhibitor of AP-1, a dominant-negative cJun mutant, TAM67, to block AP-1 activity in breast cancer cells. Results from these studies demonstrated that TAM67 blocks AP-1 activation in normal, immortal, and malignant breast cells (Ludes-Meyers et al., 2001). We have also demonstrated that TAM67 inhibited breast cancer growth both in vivo and in vitro (Liu et al., 2002). These studies suggest that AP-1 transcription factor is an important regulator of breast cancer cell growth, invasion, and resistance to antiestrogens.

In the present study, we studied the effect of TAM67 on the expression and activity of cell cycle regulators and on markers of apoptosis. We found that TAM67 inhibited MCF7 cell growth and decreased the expression of cyclin D proteins (cyclin D1, D2, D3) and cyclin E, the main cyclins in G1 phase of cell cycle, leading to reduced CDK2 and CDK4 activities, which in turn caused hypophosphorylation of retinoblastoma (Rb) that blocked the release of E2F from Rb/E2F complex. The downregulation of cyclin D's was seen both at the RNA and protein levels, while cyclin E downregulation was seen only at the protein level. Thus, AP-1 blockade causes growth inhibition by suppressing the expression of G1 cyclins, inducing p27, and ultimately inhibiting E2F activity. These studies provide a strong rationale for the foundation for future attempts to develop specific signal transduction inhibitors to treat or prevent breast cancer effectively.

Results

AP-1 blockade induced by TAM67 inhibits MCF7 cell growth in the presence of serum

We previously showed a cJun dominant-negative mutant, TAM67, suppresses AP-1 activity, inhibits cell growth induced by several different growth factors, (EGF, IGF-1, heregulin-, bFGF, and estrogen), and MCF7 xenograft tumor growth (Ludes-Meyers et al., 2001; Liu et al., 2002). In this study, we investigated the molecular mechanism by which AP-1 blockade suppresses breast cancer cell growth using MCF7 cells that express TAM67 under the control of an inducible promoter. As shown in Figure 1, in complete medium without Dox, the expression of TAM67 is induced causing inhibition of MCF7 cell growth. These cells proliferated normally in the presence of Dox. Vector clone cells grew well both in the presence and absence of Dox.

Figure 1.

TAM67 inhibition of MCF7 cell growth. MCF7 Tet-Off TAM67 cells were cultured in the presence or absence of DOX for 5 days, starved of fetal bovine serum for two more days. The cell proliferation was determined by the MTS assay

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TAM67 blocks the cell cycle

We next investigated the effect of AP-1 blockade on DNA synthesis and the cell cycle using a ³H-thymidine incorporation assay and flow cytometry. The results of the ³H-thymidine uptake assay showed that TAM67 dramatically inhibited ³H-thymidine uptake in MCF7 cells (Figure 2). Flow cytometry also showed that expression of cells of TAM67 reduced the proportion of cells in S and G2/M phases, while increased the proportion in the G₀/G₁ phase (Figure 3). Thus, the expression of TAM67 blocks the cell cycle by causing a G₁ arrest. We also examined the effect of TAM67 on apoptosis and found that TAM67 did not induce apoptosis when cells were grown in normal grade media (data not shown).

Figure 2.

TAM67 inhibition of ³H-thymidine uptake in MCF7 cells. MCF7 Tet-Off TAM67 cells were cultured in the presence or absence of DOX for 5 days, starved of fetal bovine serum for two more days. The cells were then stimulated with serum and ³H-thymidine incorporation assay was performed at time points

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Figure 3.

TAM67 inhibits normal cell cycle by causing G1 arrest. MCF7 Tet-Off TAM67 cells were cultured in the presence or absence of DOX for 7 days; 48 h before harvest the medium was changed to serum-free to synchronize cells. Then the flow cytometry assay was performed. (a) TAM67 increased cell numbers in G0/G1 phase. (b) TAM67 caused reduced cell numbers in S phase. (c) TAM67 reduced the cell distribution in G2/M phase

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Effects of TAM67 on Rb phosphorylation and E2F activity

The above results show that TAM67 blocks the cell cycle by inducing a G1 cell cycle arrest. Molecules that regulate G1 to S transition include Rb, cyclins (D's, and E), CDKs (2, 4, and 6), and CDK inhibitors. We first investigated the effects of TAM67 on the phosphorylation of Rb. As seen in Figure 4, Rb is highly phosphorylated in the presence of Dox. When the MCF7 Tet-Off TAM67 cells were cultured in the absence of Dox, Rb phosphorylation was reduced (Figure 4). Given this hypophosphorylation of Rb, we predicted that E2F activity would be reduced. We therefore measured E2F transactivation activity by transfecting an E2F-responsive luciferase plasmid into the MCF7 cells in the presence and absence of Dox. These results showed that in the absence of Dox, E2F transcription factor activity was inhibited (Figure 5).

Figure 4.

TAM67 causes Rb hypophosphorylation. MCF7 Tet-Off TAM67 cells were cultured in the presence or absence of DOX for 7 days, and synchronized using nocodazole, then Rb phosphorylation status was determined by Western-blotting assay. Rb hypophosphorylation was observed in the absence of DOX condition

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Figure 5.

TAM67 decreases E2F1 activity. MCF7 Tet-Off TAM67 cells and MCF7 Tet-Off vector cells were cultured in the presence or absence of DOX for 7 days, then the cells were cotransfected with the E2F1-Luc reporter gene and pRL-TK, luciferase activity was measured and normalized with the Renilla activity. E2F1 activity was decreased in the DOX absence condition in TAM67 cells, while there is no difference in vector cells between DOX present and absent conditions

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Effects of TAM67 on expression of cell cycle regulatory proteins

Rb is phosphorylated by cyclin E–CDK2 or cyclin D–CDK4/6 complexes, and this phosphorylation is suppressed by CDK inhibitors. Therefore, we next determined the effects of TAM67 on protein expression levels of these cell cycle regulators. The cells were first synchronized in M phase by the addition of nocodazole, and then released from cell cycle block and cells harvested at different time points for Western blot analysis. As shown in Figure 6, we found that TAM67 decreased the expression of cyclin Ds (including cyclin D1, D2, and D3), cyclin E, CDK4 and CDK6, and increased p27 expression. TAM67 also decreased p21 expression.

Figure 6.

Effect of TAM67 on the expression of cell cycle regulatory proteins. MCF7 Tet-Off TAM67 cells were cultured in the presence or absence of DOX for 7 days, synchronized using nocodazole; then cells in M phase were replated in full medium and harvested at several time points. The cell cycle regulatory proteins expression was determined by Western blotting. (a) TAM67 decreased cyclin E, cyclin D1, cyclin D2, cyclin D3 expression. (b) TAM67 decreased the expression of CDK4, CDK6. (c) TAM67 decreased P21 expression, while increased p27 expression

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Effects of TAM67 on the expression of cyclin Ds and cyclin E mRNA

Our data showed TAM67 decreased the protein expression level of cyclin D1 and cyclin E. Next, we performed an RNase Protection Assay (RPA) to determine the mRNA expression of cyclin Ds (including D1, D2, D3), and cyclin E. The cells were first synchronized in M phase by the addition of nocodazole, and then released from cell cycle block and cells harvested at different time points for the RPA analysis. As shown in Figure 7, we found that TAM67 decreased the expression of cyclin Ds mRNA (Figure 7a), but did not affect the expression of cyclin E mRNA (Figure 7b).

Figure 7.

Effect of TAM67 on the expression of cyclin Ds (D1, D2, D3) and cyclin E mRNA. MCF7 Tet-Off TAM67 cells were cultured in the presence or absence of DOX for 7 days, synchronized using nocodazole; then cells in M phase were replated in full medium and harvested at several time points. The cyclin D1 and cyclin E mRNA level was measured by performing RPA. TAM67 decreased the expression of cyclins mRNA including cyclin D's (a), but did not affect the expression level of cyclin E mRNA (b)

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TAM67 inhibits CDK2 and CDK4 kinases activity

The observed hypophosphorylation of Rb induced by TAM67 suggested a defect in activation of essential G1 CDKs. Accordingly, the activity of the CDK2 and CDK4 kinases was measured using immunoprecipitates from MCF7 Tet-Off TAM67 cells grown in the presence or absence of Dox. The cells were then synchronized in M phase and restimulated to enter the cell cycle. A significant reduction of CDK2 activity was observed when cells were cultured in the absence of Dox at different time points (Figure 8a). CDK4 activity was also decreased in cells expressing TAM67 (Figure 8b). These immunoprecipitation–Western blot experiments also showed that CDK2-associated cyclin E and CDK4-associated cyclin D1 were also reduced in cells expressing TAM67 (cultured in the absence of Dox, Figure 8a and b).

Figure 8.

Effect of TAM67 on CDK2 and CDK4 kinase activity. MCF7 Tet-Off TAM67 cells were cultured in the presence or absence of DOX for 7 days, synchronized using nocodazole for 18 h, then cells in M phase were replated in full medium and harvested at several time points. CDK2 and CDK4 kinase assay were performed as described in Materials and methods. CDK2 and cyclin E protein expression in CDK2/cyclin E complex, CDK4 and cyclin D1 proteins expression in CDK4/cyclin D1 complex were determined by immunoprecipitation–Western blotting. (a) TAM67 suppressed cyclin E expression and CDK2 kinase activity, while did affect CDK2 protein expression. (b) TAM67 inhibited cyclin D1 and CDK4 expression, and suppress CDK4 activity in some time points

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Discussion

The above results demonstrate that blockade of the AP-1 transcription factor in MCF7 breast cancer cells leads to inhibition of cell growth. The effects of AP-1 blockade are shown schematically in Figure 9. AP-1 blockade causes decreased expression of D and E cyclins, the main cyclins in G1 phase of cell cycle, and increases the expression of the CDK inhibitor p27. These changes in the expression of cell cycle regulators leads to reduced CDK2 and CDK4 activity, which in turn causes hypophosphorylation of Rb and inhibition of E2F activity, ultimately inducing a G1 cell cycle block. In combination with our previous results showing that expression of TAM67 blocks signal transduction by multiple growth factors (Liu et al., 2002), these studies demonstrate that AP-1 blockade can effectively block signal transduction and inhibit the growth of breast cancer cells.

Figure 9.

Mechanism of AP-1 blockade in breast cancer cells causes cell cycle arrest. AP-1 blockade causes decreased expression of D and E cyclins, and increases the expression of the CDK inhibitor p27, leads to reduced CDK2 and CDK4 activity, which in turn causes hypophosphorylation of Rb and inhibition of E2F activity, ultimately inducing a G1 cell cycle block

Full figure and legend (108K)

We and others have previously shown that AP-1 transcription factors are critical for cell proliferation and transformation of several cell types (Holt et al., 1986; Brown et al., 1994; Chen et al., 1996a; Liu et al., 2002). Studies of jun and fos-null cells and animals indicate that c-Fos and c-Jun are critical growth-promoting components of AP-1 (Johnson et al., 1993; Brown et al., 1998; Schreiber et al., 1999; Wisdom et al., 1999), whereas JunB and JunD are negative regulators of cell proliferation (Weitzman et al., 2000; Potapova et al., 2001). In fibroblasts, c-Jun is required for transit beyond the G1/S interphase (Smith and Prochownik, 1992; Schreiber et al., 1999). The most severe defects are exhibited by c-Jun-/- fibroblasts, which can be passed only once or twice in culture before they exhibit a pseudosenescent phenotype and their cell cycle transit time increases dramatically (Johnson et al., 1993; Schreiber et al., 1999; Wisdom et al., 1999).

In breast cells, AP-1 is important for regulating cell growth (Ludes-Meyers et al., 2001; Liu et al., 2002), invasion (Smith et al., 1999), chemotherapy resistance (Potapova et al., 2001), and tamoxifen resistance (Schiff et al., 2000). We have also shown that AP-1 blockade induced by TAM67 suppresses AP-1 activity induced by different peptide growth factors, including EGF, IGF-1, heregulin-, b-FGF, and estrogen. The present results showing that TAM67 inhibits MCF7 cell growth by inducing a cell cycle block are consistent with these previous results showing that AP-1 blockade causes general inhibition of growth factor signal transduction.

The results presented here show that blockade of AP-1 leads to reduction in critical G1 cell cycle regulators (cyclins D and E), and an increase in the CDK inhibitor p27. The results of Hennigan and Stambrook (2001) are consistent with our present study. These investigators used GFP-TAM67 to study the role of AP-1 in human fibrosarcoma cells. They demonstrated that GFP-TAM67 caused pRB hypophosphorylation and arrested cells in the G1 phase of the cell cycle, findings similar to those presented here. However, unlike our present findings, this group found that GFP-TAM67 did not inhibit the expression of cyclin D1, cyclin E in fibrosarcoma cells. These differences may be due to the different types of cell used for these two studies (fibrosarcoma vs breast cancer cells).

The human cyclin D1 gene regulatory sequences contain two AP-1 binding sites (Herber et al., 1994; Albanese et al., 1999). Results from previous studies have suggested that c-Jun induces while JunB inhibits cyclin D1 transcription (Bakiri et al., 2000). It has been suggested that AP-1 family members regulate cell cycle by inducing the expression of cyclin D1 via AP-1 sites in its promoter region (Brown et al., 1998). Our data showed that expression of TAM67 reduced the expression of cyclin D1, suggesting that downregulation of cyclin D is responsible, at least partially, for TAM67's inhibition effects. Since the cyclin D1 promoter contains typical AP-1-binding sites, this inhibitory effect of TAM67 could be due to direct binding of TAM67 to these sites. Cyclins D2 and D3 show considerable structural and functional homologies with cyclin D1, and in certain instances, they may complement each other functionally. Thus, the downregulation of cyclins D2 and D3 may also contribute to the inhibitory effect of TAM67.

Cyclin E is thought to act as a rate-limiting factor after cyclin D1 at the G1–S transition. In breast cancer, cyclin E also drives proliferation (Yu et al., 2001), its overexpression is a negative prognostic factor (Nielsen et al., 1996) and an independent risk factor of visceral relapse in breast cancer (Kim et al., 2001). In multivariate analysis, a high level of cyclin E is significantly correlated with poor outcome (Keyomarsi et al., 2002). Our studies demonstrated that TAM67 inhibits cyclin E protein expression and cyclin E–CDK2 kinase activity. Thus, cyclin E downregulation is also likely to contribute to the cell cycle arrest induced by TAM67. How TAM67 inhibits cyclin E expression and reduces cyclin E–CDK2 activity is currently under investigation. However, in the future, agents that inhibit the expression of both cyclin D and cyclin E (as does TAM67) may be particularly effective drugs for the treatment of breast cancer.

AP-1 transcription factors have also been implicated in the control of cell death and survival. Increased AP-1 activity may promote apoptosis in some cell types, while promoting survival in other cell types. Ectopic expression of c-Jun or c-Fos can induce apoptosis in sympathetic neurons as well as in mouse fibroblasts, Syrian hamster embryo cells and a human colorectal carcinoma cell line (Ham et al., 1995; Preston et al., 1996; Bossy-Wetzel et al., 1997). In breast cancer cells, AP-1 sensitizes cells to Vitamin E succinate-induced apoptosis (Zhao et al., 1997). On the other hand, in some circumstances inhibition of AP-1 activity may also promote apoptosis. We have found that AP-1 inhibition induced by TAM67 sensitizes MCF7 breast cancer cells to apoptosis when these cells are starved of serum (unpublished observation). Previous data results showing that reducing AP-1 activity in breast cancer cells caused increased cell death after treatment with UV light or cisplatin chemotherapy are consistent with these results (Sauter et al., 1999; Smith et al., 1999; Potapova et al., 2001).

The present results demonstrate that in the presence of serum, TAM67 inhibits breast cancer growth predominantly by inducing the expression of cyclin-dependent kinase inhibitors (such as p27), by reducing the expression of the G1 cyclins, and by reducing CDK activity, thus leading to Rb hypophosphorylation, inhibition of E2F activity, and a G1 cell cycle block. This effectively blocks breast cell proliferation. These studies lay the foundation for future attempts to inhibit the activation of the AP-1 transcription factor for the prevention or treatment of cancer.

Materials and methods

Cell culture and transfection

The generation of the MCF7 Tet-Off TAM67 Clones #62, #67 and vector clones #1, #3 has been previously described (Ludes-Meyers et al., 2001). The cells were maintained in the improved MEM (high-zinc option, Life Technologies, Grand Island, NY, USA) with 100 g/ml of geneticin and 100ug/ml hygromycin. The cells were transfected using the Fugene 6 reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's recommendations.

Cell growth assays

The CellTiter 96™ AQueous Non-Radioactive Cell Proliferation Assay (MTS assay; Promega, Madison, WI, USA) was used to measure breast cancer cell growth according to the protocol provided by the manufacturer. Approximately 12 000 cells were seeded in a 24-well plate and doxycycline was added or removed to block or induce the expression of TAM67 of MCF7 Tet-Off TAM67 cells. A solution containing a 20 : 1 ratio of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and PMS (phenazine methosulfate) was added to the cells for 2 h at 37°C and absorption at 550 nm was determined. Each data point was performed in quadruplet, and the results were reported as mean absorptionstandard error.

³H-thymidine incorporation assay

MCF7 Tet-Off TAM67 Clone #62 cells were kept in -Dox and +Dox media to induce or block the expression of TAM67 for a total of 7 days. At 48 h before harvest (thus after 5 days in the absence or presence of Dox), the medium was changed to serum-free medium. After 2 days, the cells were stimulated with serum for 0, 6, 12, 24, 36, or 48 h, and then labeled with ³H-thymidine (2 Ci/ml) for 3 h. The cells were then incubated with 5% TCA at 4°C for 30 min, and were then lysed by addition of 0.1 N NaOH. The level of protein in the lysates was determined using a BCA assay (Pierce, Rockford, IL, USA). ³H-thymidine uptake was measured by counting ³H c.p.m. in a scintillation counter. Each data point was performed in triplet, and the results were reported as mean c.p.m.standard error. All results were normalized to protein content.

Flow cytometry

MCF7 Tet-Off TAM67 Clone #62 cells were kept in -Dox or +Dox media to induce or block the expression of TAM67 for a total of 7 days. At 48 h before harvest (thus after 5 days in the absence or presence of Dox), the medium was changed to serum-free medium. After 48 h, the cells were stimulated with serum, harvested at 0, 6, 12, 24, 36, and 48 h after serum stimulation, and fixed in 95% ethanol for 30 min in room temperature. The cells were then stained with propidium iodide in PBS. Stained cells were analysed using the EPICS XL-MCL flow cytometer (Coulter Co.).

Luciferase assay

E2F1 transcriptional activity in cells was measured using the Dual-Luciferase™ Reporter Assay (Promega, Madison, WI, USA) as previously described (Ludes-Meyers et al., 2001). The cells were cotransfected with the E2F1-luc reporter gene and pRL-TK, a Renilla construct for normalizing of transfection efficiency. Transfected cells were lysed 36 h after transfection and luciferase activity was measured with equal amounts of cell extract using the microplate luminometer (Labsystems, Helsinki, Finland) and normalized with the Renilla activity.

Western blot analysis

For cell cycle study, the cells were cultured in the medium for 7 days with or without Dox to block or induce the expression of TAM67, then synchronized in 50 ng/ml of nocodazole for 18 h. After synchronization, the floating cells (in M phase) were collected and washed three times in PBS. The cells were then cultured in full medium. At time points 6, 12, 18, 24, 36, and 48 h, the cells were harvested and cells lysates were prepared. An amount of 20 g of total cellular protein extract were electrophoresed on SDS–PAGE gel and transferred by electroblotting onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The following antibodies were used: 14001A for Rb (Pharmingen, 1 : 1000); 14561A for cyclin D1 (Pharmingen, 1 : 300); C17 for cyclin D2 (Santa Cruz, sc-181, 1 : 300), C16 for cyclin D3 (Santa Cruz, sc-182, 1 : 200), HE-12 for cyclin E (Santa Cruz, sc-247, 1 : 200); H-164 for p21 (Santa Cruz, sc-756, 1 : 1000); F-8 for p27 (Santa Cruz, sc-1641, 1 : 200); M2 for CDK2 (Santa Cruz, sc-163, 1 : 200); H-22 for CDK4 (Santa Cruz, sc-601, 1 : 200); B-10 for CDK6 (Santa Cruz, sc-7961, 1 : 200); MAB1501 for Actin (Chemicon, 1 : 2000). Anti-rabbit or anti-mouse antibody (1 : 4,000, Amersham, Piscataway, NJ, USA) was used as secondary antibody. Blots were developed using the enhanced chemiluminescence (ECL) system (Amersham, Piscataway, NJ, USA).

RNAase protection

RNA was isolated from MCF7 Tet-Off TAM67 cells under the conditions of DOX presence or absence using the Qiagen MidiEasy Kit. RNA yield was determined by UV absorption at 260 nm after dissolving in sterile H₂O. RNAase protection was performed following the Pharmingen protocol using the BD RiboQuant™ RPA kit.

Kinase assay

The methods for the kinase assay have been previously described (Yang et al., 2001). For the CDK2 kinase assay, cells were lysed in a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 1% Triton X-100, 10% glycerol, 10 mM -glycerophosphate, 100 mM NaF, 0.2 mM NaVO₃, 1.5 mM MgCl₂, 10 g/ml aprotinin, 10 g/ml leupeptin, and 0.1 mM PMSF. For the CDK4 kinase assay, the cells were lysed in a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8.0, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween 20, 10% glycerol, 10 mM -glycerophosphate, 1 mM NaF, 0.1 mM NaVO₃, 1.5 mM MgCl₂, 2 g/ml aprotinin, 10 g/ml leupeptin, and 0.1 mM PMSF. Protein G agarose (Life Technologies, Gaithersburg, MD, USA) was incubated with CDK2 or CDK4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for an hour followed by incubating with 500–800 g of protein extracts at 4°C overnight. The agarose mixture was pelleted and washed in lysis buffer for four times. For immunoprecipitation–Western blotting, the agarose was resuspended in 40 l 1 sample buffer (125 mM Tris pH 6.8, 4% SDS, 0.005% bromophenol blue, 20% glycerol, 0.7 M -mercaptoethanol) and 20 l was loaded on 12% SDS–PAGE. Western blotting was performed as described above. For the CDK2 kinase assay, the agarose mixture was washed in 1 cold kinase buffer (20 mM Tris pH 7.5, 5 mM MgCl₂, 2.5 mM MnCl₂, and 1 mM DTT) and resuspended in final volume of 25 l containing 5 l 5 kinase buffer, 1 l -³²P-ATP and 20 g of histone H1 (Roche, Indianapolis, IN, USA). For the CDK4 kinase assay, the agarose mixture was washed in 1 cold kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 10 mM -glycerophosphate, 1 mM NaF, and 0.1 mM NaVO₃) and resuspended in final volume of 25 l containing 5 l 5 kinase buffer, 1 l -³²P-ATP and 1.5 g GST-Rb (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The kinase reaction was performed at 30°C for 30 min and was stopped by adding 25 l of 2 sample buffer. The samples were heated at 90°C for 5 min and 25 l of reaction mixture was loaded on 10% SDS–PAGE gel. The gel was dried and exposed to X-ray film. The intensity of the bands was quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).

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Acknowledgements

We thank Dr Kendall Wu, David Denardo, and Dr Gu Kong, for their helpful discussions and critical reading of the manuscript. We would also like to thank Linda Kimbrough for her assistance in preparing this manuscript. This work was supported by the Department of Defense Grant (DAMD-17-96-1-6225 to PHB) and the Department of Defense Postdoctoral Fellowship Award (BC-000322).