Indole-3-Carbinol (I3C) Inhibits Cyclin-dependent Kinase-2 Function in Human Breast Cancer Cells by Regulating the Size Distribution, Associated Cyclin E Forms, and Subcellular Localization of the CDK2 Protein Complex
2004; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês
10.1074/jbc.m407957200
ISSN1083-351X
AutoresHanh H. Garcia, Gloria A. Brar, David H.H. Nguyen, Leonard F. Bjeldanes, Gary L. Firestone,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoIndole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a significant shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E. I3C treatment caused no change in the level of CDK2 inhibitors (p21, p27) or in the inhibitory phosphorylation states of CDK2. The effects of I3C are specific for this indole and not a consequence of the cell cycle arrest because treatment of MCF-7 breast cancer cells with either the I3C dimerization product DIM or the anti-estrogen tamoxifen induced a G1 cell cycle arrest with no changes in the associated cyclin E or subcellular localization of the CDK2 protein complex. Taken together, our results have uncovered a unique effect of I3C on cell cycle control in which the inhibition of CDK2 kinase activity is accompanied by selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex. Indole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a significant shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E. I3C treatment caused no change in the level of CDK2 inhibitors (p21, p27) or in the inhibitory phosphorylation states of CDK2. The effects of I3C are specific for this indole and not a consequence of the cell cycle arrest because treatment of MCF-7 breast cancer cells with either the I3C dimerization product DIM or the anti-estrogen tamoxifen induced a G1 cell cycle arrest with no changes in the associated cyclin E or subcellular localization of the CDK2 protein complex. Taken together, our results have uncovered a unique effect of I3C on cell cycle control in which the inhibition of CDK2 kinase activity is accompanied by selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex. Considerable epidemiological evidence show that diets high in vegetable and fiber lead to low cancer risks and confer protection from various forms of cancers, including breast cancer (1Birt D.F. Pelling J.C. Nair S. Lepley D. Prog. Clin. Biol. Res. 1996; 395: 223-234PubMed Google Scholar, 2Lopez-Otin C. Diamandis E.P. Endocr. Rev. 1998; 19: 365-396Crossref PubMed Scopus (181) Google Scholar). In particular, consumption of vegetables belonging to the Brassica genus, which includes broccoli, cabbage, and cauliflower, have been reported to correlate with a decrease in mammary tumor incidence (3Bresnick E. Birt D.F. Wolterman K. Wheeler M. Markin R.S. Carcinogenesis. 1990; 11: 1159-1163Crossref PubMed Scopus (66) Google Scholar). These epidemiological studies suggest the existence of naturally occurring compounds in dietary sources that represent a largely untapped source of potential chemotherapeutic molecules. 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Chem. 1998; 273: 3838-3847Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Our previous studies have shown that I3C down-regulates CDK6 transcription by disrupting the functional interactions of the Sp1 transcription factor with an Sp1-Ets composite DNA element in the CDK6 promoter (19Cram E.J. Liu B.D. Bjeldanes L.F. Firestone G.L. J. Biol. Chem. 2001; 276: 22332-22340Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). I3C also causes a pronounced decrease in CDK2 specific enzymatic activity and inhibited phosphorylation of endogenous Rb proteins (20Cover C.M. Hsieh S.J. Cram E.J. Hong C. Riby J.E. Bjeldanes L.F. Firestone G.L. Cancer Res. 1999; 59: 1244-1251PubMed Google Scholar), although the precise mechanism underlying this process has not been characterized. The activity, accessibility and cellular utilization of CDK2/cyclin E can be regulated at multiple levels, any one of which could be potentially targeted by I3C treatment. For example, in addition to cyclin association, the activation of CDK2 also requires the dephosphorylation of Thr14 and Tyr15 by Cdc25A (47Sebastian B. Kakizuka A. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3521-3524Crossref PubMed Scopus (212) Google Scholar) and an activating phosphorylation event at Thr160 by CDK-activating kinase (CAK) (48Gu Y. Rosenblatt J. Morgan D.O. EMBO J. 1992; 11: 3995-4005Crossref PubMed Scopus (556) Google Scholar). Another mode of regulating CDK2 kinase activity is through the association with cyclin-dependent kinase inhibitors (CKI) such as p21, p27, and p57. Recent studies have also shown that the subcellular compartmentalization of either cyclin E or CDK2 greatly alters its enzymatic activity and accessibility to nuclear residing CAK, Cdc25A, and known substrates of CDK2 (49Malumbres M. Carnero A. Prog. Cell Cycle Res. 2003; 5: 5-18PubMed Google Scholar, 50Keenan S.M. Bellone C. Baldassare J.J. J. Biol. Chem. 2001; 276: 22404-22409Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 51Brown K.A. Roberts R.L. Arteaga C.L. Law B.K. Breast Cancer Res. 2004; 6: R130-R139Crossref PubMed Google Scholar). In this study we demonstrate for the first time that I3C selectively controls the size distribution of the CDK2-cyclin E protein complex with concomitant alterations in cyclin E interactions and subcellular localization that results in an inhibition of CDK2 enzymatic activity in human breast cancer cells. Materials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, calcium and magnesium-free PBS, l-glutamine, penicillin/streptomycin, and trypsin-EDTA were supplied by Cambrex/Biowhittaker (Walkersville, MD). I3C and DIM were purchased from LKT Laboratory Inc. (St. Paul, MN). [γ-32P]ATP (3,000 Ci/mmol) was purchased from PerkinElmer Life Science Products. Insulin (bovine), tamoxifen, and tryptophol were purchased from Sigma. Flow Cytometry Analysis of DNA Content—MCF-7 cells were plated at 30% confluency on 100-mm tissue culture plates and treated for the indicated time points with 100 μm I3C in complete media. An aliquot of harvested cells were hypotonically lysed in 0.5–1 ml of DNA staining solution (0.5 mg/ml propidium iodide, 0.1% sodium citrate, 0.05% Triton X-100). Cell debris was filtered and DNA content was analyzed as described previously (21Cover C.M. Hsieh S.J. Tran S.H. Hallden G. Kim G.S. Bjeldanes L.F. Firestone G.L. J. Biol. Chem. 1998; 273: 3838-3847Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Tissue Culture and Cell Lines—MCF-7 human breast cancer cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 10 μg/ml insulin, 1.25 ml of 20,000 units/ml penicillin and streptomycin. Cells were maintained at subconfluency at 37 °C in humidified air containing 5% CO2. I3C, DIM, tamoxifen, and tryptophol were dissolved in Me2SO (99.9% high performance liquid chromatography grade; Aldrich) at concentrations 1000-fold higher than the final concentrations used. Western Blot Analysis—After the indicated treatments, cells were lysed in either CDK2 lysis buffer (250 mm NaCl, 0.1% Triton X-100, 50 mm Tris/HCl, pH 7.3) or RIPA buffer (150 mm NaCl, 0.5% deoxycholate, 0.1% Nonidet P-40, 0.1% SDS, 50 mm Tris-HCl) containing protease and phosphatase inhibitors (50 μg/ml phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 5 μg/ml leupeptin, 0.1% sodium fluoride (NaF), 10 μg/ml β-glycerophosphate, and 0.1 mm sodium orthovanadate). Proteins were electronically transferred to nitrocellulose membranes (Micron Separations, Inc., Westboro, MA) and blocked as described previously (21Cover C.M. Hsieh S.J. Tran S.H. Hallden G. Kim G.S. Bjeldanes L.F. Firestone G.L. J. Biol. Chem. 1998; 273: 3838-3847Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Blots were subsequently incubated at room temperature for an hour with anti-CDK2 (sc 748), CDK4 (sc260), and cyclin E1 (sc 198) antibodies. Blots probed with anti-p-Tyr (sc 7020), anti-p21(sc 398) or anti-p27 (sc 528) antibodies were either incubated at room temperature for 2–3 h or incubated overnight. All antibodies were used at a concentration of 1 μg/ml in TBST with the exception of anti-pTyr antibody, which was made in 1% nonfat dry milk/TBST. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoprecipitation and CDK2 Kinase Assays—After the indicated treatments, cells were lysed for 15 min at 4 °C in CDK2 lysis buffer with protease and phosphatase inhibitors (50 μg/ml PMSF, 10 μg/ml aprotinin, 5 μg/ml leupeptin, 0.1% sodium fluoride, 10 μg/ml β-glycerophosphate, and 0.1 mm sodium orthovanadate). Samples (500–800 μg of protein) were precleared as described previously (21Cover C.M. Hsieh S.J. Tran S.H. Hallden G. Kim G.S. Bjeldanes L.F. Firestone G.L. J. Biol. Chem. 1998; 273: 3838-3847Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) followed by incubation with 0.5 μg of anti-CDK2 or anti-cyclin E1 antibodies (Santa Cruz Biotechnology) for 2 h. Thirty microliters of a 1:1 bead slurry was added to each sample and left on a rocking platform for 30 min. The beads were washed with CDK2 lysis buffer and twice with kinase buffer (50 mm HEPES (pH 7.3), 5 mm MnCl2, 10 mm MgCl2) with protease and phosphatase inhibitors. Half of the immunoprecipitation was checked by Western blot analysis to confirm the efficiency of the immunoprecipitation. The remaining half of the immunoprecipitation was used to assess for kinase activity as described above. I3C Directly Added to CDK2 IP/Kinase Assay—Immunoprecipitation of CDK2 was carried out as described above followed by a preincubation of the CDK2-immunoprecipitated protein complex with the indicated concentrations of I3C for 15 min at 30 °C. Half of the CDK2 immunoprecipitate was resolved on SDS-PAGE and Western blot analyzed for CDK2 while the remaining half was used to assess CDK2 kinase activity as described above. In a separate set of CDK2 immunoprecipitation, I3C was added concurrently with the [γ-32P]ATP and histone H1 followed by a 15-min incubation at 30 °C and assayed for activity. CDC25A Activity Assay—The constructs for GST-Cdc25A were a kind gift from Dr. Gregory Pierce's laboratory (Cancer Research Institute). Purified GST-Cdc25A was bacterially synthesized, sonicated in extraction buffer (1× PBS, 5 mm DTT, 10 mm Tris-HCl, pH 8), and the lysates were clarified by centrifugation. GST-Cdc25A fusion proteins were then absorbed onto glutathione-Sepharose beads, washed three times and eluted with 20 mm reduced glutathione. Cells were treated with either I3C or Me2SO for 48 h followed by a CDK2 immunoprecipitation as described above. Half of the CDK2 immunoprecipitation was used for Western blot analysis while the remaining half was used for the Cdc25A kinase assay; CDK2 immune complexes were either incubated with 2 μg of purified GST-Cdc25A or GST-alone for 15 min at 30 °C in phosphate buffer (50 mm Tris, pH 8, 150 mm NaCl, 2 mm DTT, 2.5 mm EDTA). Half of the CDK2 immunoprecipitate was analyzed for CDK2 protein levels, and the remaining half was used to assess CDK2 kinase activity as described above. Gel Filtration Column Chromatography—Gel filtration chromatography was carried out using a 50-cm length, 1.0-cm wide Kontes column (Fisher) packed with Superose 12 prep grade beads (Amersham Biosciences). Superose 12 beads were washed four times in column running buffer (250 mm NaCl, 0.1% Triton X-100, 50 mm Tris/HCl, pH 7.3) followed by degassing for 1 h via vacuum suction. The bead slurry was packed onto the Kontes column for 8 min at 2.3 ml/min and 55 min at 0.5 ml/min. Uniform packing was monitored using blue dextran (2000 kDa) followed by calibration of the column using the following molecular mass standards purchased from Amersham Biosciences: catalase (250 kDa), aldolase (158 kDa), albumin (67 kDa), and ovalbumin (45 kDa). Cells were either treated with 100 μm I3C, 30 μm DIM, 1 μm tamoxifen, 100 μm tryptophol, or Me2SO (control) for 48 h. Each sample was lysed in CDK2 lysis buffer with phosphatase and protease inhibitors and cleared of cellular debris with by centrifugation at 15,000 × g. Cell lysates (800 μg) were then applied immediately onto a Kontes size exclusion column packed with Superose 12 beads and a total of 80 fractions were collected. Forty microliters of column lysate was taken for immunoblot analysis of CDK2 and 400 μl were precipitated (15 μlof 1 μg/ml bovine serum albumin, 1 ml of acetone, 400 μl of column lysate, mixed, and incubated at –70 for 3 h or overnight, followed by a 10 min spin at 15,000 × g at 4 °C), resuspended in 20 μl of 2× protein loading buffer used for immunoblotting for cyclin E and p21. For the column lysate immunoprecipitation/kinase assays, 2 consecutive gel filtration fractionations were conducted for each of the treatment groups. Column lysates were pooled, followed by the CDK2 immunoprecipitation/kinase assay described above. Indirect Immunofluorescence—MCF-7 cells were plated on 8-well Lab-Tek Permanox slides (Nage Nunc International, Naperville, IL) at 20% confluency and treated for 48–72 h with either Me2SO, I3C, DIM, tamoxifen or tryptophol (1 μl/ml of 1000× stock). Cells were washed with PBS, fixed with 3.7% formaldehyde, 0.01% glutaraldehyde for 15 min, and permeabilized with cold 50/50 acetone for 1 min. Cells were blocked for 5 min with PBS containing 4% goat serum (Jackson ImmunoResearch, West Grove, PA) followed by incubation with rabbit anti-CDK2 or mouse anti-cyclin E antibodies at a 1:150 dilution for 1.5 h (CDK2) at 25 °C or overnight for cyclin E at 4 °C. After five washes with cold PBS, cells were blocked and incubated with anti-rabbit rhodamine-texas red-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.; diluted 1:300 in PBS) or anti-mouse FITC-conjugated secondary antibody (1:300) for 30 min. Cells were washed, mounted with clear nail polish and visualized on a Nikon Optiphot fluorescence microscope. Images were captured using Adobe Photoshop and a Sony DKC-5000 digital camera. Nonspecific fluorescence, as determined by incubation with secondary antibody alone was negligible. Subcellular Fractionations—Cell pellets were resuspended at 5× volume in buffer A (0.01 m HEPES, pH 7.9, 1.5 mm MgCl2, 0.01 m KCl, 0.5 mm DTT, 0.5 mm PMSF), incubated on ice for 30 min follow by a 5-min spin at 4 °C (1000 rpm). Cell pellets were resuspended at 2× volume in buffer A and dounced 100 strokes with type B pestle. Cells were checked with trypan blue to ensure at least 80% of cells were lysed, followed by a 20-min spin at 550 rpm. Supernatants were transferred to a new tube followed by 100,000 rpm centrifugation for 30 min in a ultracentrifuge to clear cytosolic extract of cellular debris. Nuclear pellets were resuspended in 1.5× cell volume with buffer B (20 mm HEPES, pH 7.4, 25% glycerol, 1.5 mm MgCl2, 0.4 m NaCl, 0.2 mm EDTA. 0.5 mm DTT, 0.5 mm PMSF) and dounced 20 strokes with type B pestle. Extracts were nutated at 4 °C for 30 min followed by a 17,500 rpm centrifugation. The supernatants were dialyzed against 1000× volume of cold buffer C (0.02 m HEPES, pH 7.9, 5% glycerol, 1.5 mm MgCl2, 0.1 m KCl, 0.2 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF), followed by a 17,500 rpm centrifugation. Extracts were normalized and analyzed by Western blot. Quantification of Autoradiography—Autoradiographic exposures were scanned with a UMAX UC630 scanner, and band intensities were quantified using the NIH Image program. Autoradiographs from a minimum of three independent experiments were scanned per time point. Time Course of I3C Inhibition of CDK2 Kinase Activity and G1 Cell Cycle Arrest—To determine the kinetics of the inhibition of CDK2 kinase activity by I3C, estrogen responsive human MCF-7 breast cancer cells were treated with or without 100 μm I3C over a 96-hour time course. This concentration of I3C was previously shown to be the optimal dose for the inhibition of cell growth without affecting cell viability (21Cover C.M. Hsieh S.J. Tran S.H. Hallden G. Kim G.S. Bjeldanes L.F. Firestone G.L. J. Biol. Chem. 1998; 273: 3838-3847Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). CDK2 protein complexes were immunoprecipitated from total cell lysates by using a CDK2-specific antibody, and half of each sample was assayed in vitro for kinase activity and the other half analyzed for CDK2 protein by Western blot analysis. CDK2 protein kinase activity was monitored by the ability of the immunoprecipitated CDK2-cyclin protein complexes to phosphorylate the C-terminal domain of Rb fused to GST. As shown in Fig. 1A, electrophoretic analysis of the phosphorylated GST-Rb showed that the I3C suppression of CDK2 kinase activity can be observed in cells treated as early as 24-h post-treatment, with near maximal inhibition observed by 72 h in I3C. A nonspecific IgG was used in one set of immunoprecipitations (no IP lane) as a negative control to demonstrate the specificity of the CDK2 kinase assay. A similar inhibition of CDK2 kinase activity was observed in estrogen receptor-negative breast cancer cells (data not shown). During the sample time course, the level of G1 phase MCF-7 cells was examined by flow cytometry of propidium iodide-stained nuclei. As shown in Fig. 1B, I3C treatment induces a significant increase in the number of G1 phase-arrested cells compared with untreated cells, which correlates closely to the kinetics of I3C inhibition of CDK2 enzymatic activity. Given the chemical nature of I3C, it is conceivable that this indole could act as a direct enzymatic inhibitor of CDK2, and thereby prevents the CAK phosphorylation of CDK2. To examine this possibility, a range of I3C concentrations was added to individual CDK2 immunoprecipitates either prior to or simultaneous with the addition of the histone H1 substrate and [γ-32P]ATP. As shown in Fig. 1C, I3C has no significant effect on the in vitro CDK2 kinase activity. These results demonstrate that I3C does not inhibit CDK2 kinase activity through direct interactions with the CDK2 protein immunocomplex. Western blot analyses of CDK2 immunoprecipitates show that I3C treatment did not alter the total level of CDK2 protein (Fig. 1A), indicating that I3C inhibits total cellular CDK2 specific enzymatic activity. CDK2 protein migrates as a doublet, and the faster migrating CDK2 band represents the CAK-phosphorylated Thr160 form of CDK2, which is necessary for enzymatic activity (48Gu Y. Rosenblatt J. Morgan D.O. EMBO J. 1992; 11: 3995-4005Crossref PubMed Scopus (556) Google Scholar). The Western blots also revealed that in MCF-7 breast cancer cells, the CAK phosphorylated form of CDK2 is modestly reduced in samples immunoprecipitated from t
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