MOLECULAR AND CELLULAR BIOLOGY, Sept. 1996, p. 4632–4638 0270-7306/96/$04.0010 Copyright q 1996, American Society for Microbiology
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Anchorage-Dependent Transcription of the Cyclin A Gene ` S,2 SANDRINE MIDDENDORP,2 ALMUT SCHULZE,1 KARIN ZERFASS-THOME,1 JOSETTE BERGE 1 ¨ PIDDER JANSEN-DURR, AND BERTHOLD HENGLEIN2* Abteilung 620, Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany,1 and Institut Curie, F-75005 Paris, France2 Received 17 January 1996/Returned for modification 9 March 1996/Accepted 3 June 1996
NIH 3T3 cells cultured in suspension fail to express cyclin A and hence cannot enter S phase and divide. We show that loss of cell adhesion to substratum abrogates cyclin A gene expression by blocking its promoter activity through the E2F site that mediates its cell cycle regulation in adherent cells. In suspended cells, G0-specific E2F complexes remain bound to the cyclin A promoter. Overexpression of cyclin D1 restores cyclin A transcription in suspended cells and rescues them from cell cycle arrest. In suspended cells, cyclin D1 and cyclin E accumulate normally upon serum stimulation, but their associated kinases remain inactive; their substrates, pRb and p107, are not hyperphosphorylated. Concomitantly, the cyclin-dependent kinase inhibitor, p27KIP1, is stabilized. Ectopic expression of p27KIP1 blocks cyclin A promoter activity through its E2F binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27KIP1 and subsequent inactivation of the specific E2F moiety required for its induction. and normal rat kidney cells exit from G0 upon serum stimulation. They express G1 cyclins but fail to express cyclin A, which is required for S-phase entry (14, 18). Guadagno and coworkers (18) suggested that anchorage dependence of cyclin A expression may underlie anchorage dependence of S-phase entry. In adherent NIH 3T3 cells, cell cycle regulation of cyclin A gene expression is controlled by the transcription factor E2F (10, 53), and overexpression of cyclin D1 can relieve the G1 block to cyclin A transcription through an E2F-dependent mechanism (53). We show here that the block to cyclin A gene expression in response to loss of adhesion is transcriptional, is mediated by the DNA element that confers cell cycle regulation, and can be overridden by enforced expression of cyclin D1. In suspended cells, the kinase activities associated with cyclins D1 and E are low, and proteolysis of the cdi p27KIP1 is inefficient. Together with the fact that p27KIP1 overexpression blocks cyclin A promoter activity through the E2F binding site, these findings suggest that loss of anchorage abrogates the particular E2F activity required for cyclin A transcription, through p27KIP1 stabilization.
In vitro growth of most nontransformed cells requires mitogen stimulation and adhesion to substratum. Established human and rodent fibroblasts undergo cell cycle arrest in the G1 phase of the cell cycle when cultured in suspension (19, 36, 41). Beyond a defined time point in G1, cells are no longer dependent on adhesion to complete the cell cycle (6, 21). Cell adhesion and spreading involves interaction of integrins with the extracellular matrix and the actin-based cytoskeleton. In response to this interaction, integrins activate a number of growth control signal pathways (reviewed in reference 8). Loss of anchorage dependence and disorganization of the cytoskeleton are hallmarks of cellular transformation. Anchorage-independent growth in vitro is closely correlated with tumorigenicity and tumor metastasis in vivo (57). Progression through the cell cycle in mammalian cells is controlled by cyclins and cyclin-dependent kinases (cdks) (45). Cyclin gene expression is tightly regulated at the transcriptional level in a phase-specific manner. Expression of cyclin D1 is induced by serum growth factors (63). It is required for progression through early G1 (2). In late G1, cyclin E is synthesized (32); cyclin E is required and rate limiting for entry into S phase in mammalian fibroblasts (40, 51). Cyclin A gene expression starts at the G1/S transition, and the protein is required for DNA replication (7, 9, 15, 68) and passage through G2 (43). In addition, cyclin A may have a role in transcriptional control during S phase (33, 35). The enzymatic activity of cyclin-cdk complexes is controlled by association with cdk inhibitor (cdi) proteins (see references 28 and 56 for reviews). p21CIP1 and p27KIP1 inhibit cyclin D- and cyclin Edependent kinase activity. Growth arrest in response to DNA damage is due to transcriptional activation of p21CIP1 by p53 (12); p27KIP1 mediates G1 arrest by transforming growth factor b, cyclic AMP, and possibly by contact inhibition (31, 48). The p27KIP1 protein accumulates in serum-starved cells (24, 39) and is degraded upon serum stimulation by ubiquitin-mediated proteolysis (44). When cultured in suspension, murine and human fibroblasts
MATERIALS AND METHODS Cells and extracts. NIH 3T3 cells were grown in Dulbecco’s modified medium supplemented with 10% calf serum (CS), either in monolayer or in suspension on plates coated with agarose as described previously (18). Synchronous cell cycle progression of NIH 3T3 cells was achieved by serum starvation (0.5% CS) for 72 h followed by serum readdition (10% CS). Cellular DNA content was analyzed by flow cytometry. Cellular extracts were prepared as described previously (29). Western blotting (immunoblotting). Whole cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with polyclonal antibodies to cyclin A (a gift from M. Pagano, Cambridge, Mass.). Antibodies to cyclin D1 (HD11), cyclin E (M20), p107 (C18), pRb (C15), cdk2 (M2), cdk4 (M22), and p27KIP1 (C19) were purchased from Santa Cruz Inc., Santa Cruz, Calif. The polyclonal antibody to p21CIP1 was purchased from PharMingen, San Diego, Calif. Signals obtained in Western blot experiments were quantitated by densitometric evaluation. Northern (RNA) blotting. Total cellular RNA was extracted by the guanidinium thiocyanate-acid phenol method. Ten micrograms of total RNA was separated on 1% agarose-formaldehyde gels and transferred onto nylon membranes. Expression of cyclin D1, cyclin E, and p27KIP1 was detected by using murine cDNA probes (gifts from G. Peters, London, England, and J. Massague´, New York, N.Y.); cyclin A was detected with a 1.6-kb human cDNA probe (61). Quantitation of Northern blots was performed with a PhosphorImager (Molecular Dynamics).
* Corresponding author. Mailing address: Institut Curie, INSERM U255, 26 rue d’Ulm, F-75005 Paris, France. Phone: 33-1-44 32 42 25. Fax: 33-1-40 51 04 20. Electronic mail address:
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Transfection, reporter plasmids, and expression vectors. Cells were transfected by the calcium phosphate method as described previously (29). The reporter gene PA/111LUC was constructed by inserting the cyclin A gene promoter region from positions 27300 to 111 relative to the major (and 39most) transcription start site into the luciferase reporter plasmid pL (23). In construct PA/111LUCmut, the E2F site at 237 to 233 was mutated as described previously (53). Luciferase activity was normalized to transfection efficiency as determined by b-galactosidase activity, expressed from a cotransfected cytomegalovirus-driven b-galactosidase expression plasmid. A cytomegalovirus-based expression vector for the mouse p27KIP1 protein (48) was obtained from J. Massague´. Tetracycline-controlled expression of cyclin D1 was achieved by cotransfection of plasmids cycD1/T and CMV/T (53); in the latter, the sequence coding for the transactivator tTa (16) had been inserted. Cells were transfected and serum starved in the presence of 2 mg tetracycline per ml in the medium. Induction was obtained by incubation in tetracycline-free medium. To specifically assess the DNA content of the transfected cells, we cotransfected an expression plasmid for a truncated mouse low-affinity type II Fcg receptor (CD32) as a surface marker (1). Cells were reacted with an anti-CD32 monoclonal antibody (a gift from C. Bonnerot, Paris, France) and with fluorescein isothiocyanate-conjugated second antibodies, then DNA stained with propidium iodide, and analyzed on a FacScan (Becton Dickinson). Doublets were discriminated from G2 cells by signal area/ signal width analysis using the CellQuest data acquisition program. Band shift experiments. A double-stranded oligonucleotide encompassing the E2F binding site of the cyclin A promoter (cycA wt; 59-GATCTTCAATAGTC GCGGGATACTT-39) was 39-end labeled and incubated with cell extracts as described previously (42). Oligonucleotides containing the wild-type (E2 wt; 59-GATCCACTAGTTTCGCGCGCTTTCTA-39) and mutant (E2 mut; 59-GA TCCACTAGTTTACTCAGATAACTA-39) E2F binding sites of the adenovirus E2 promoter were used as unlabeled competitors. E2F-associated proteins were analyzed by incubation of the band shift reaction mixture with specific antibodies on ice for 50 min prior to electrophoresis. p107 was detected by monoclonal antibody SD15 (a gift from N. Dyson, Charlestown, Mass.), cyclin E was detected by a polyclonal antiserum (M20; Santa Cruz), and DP-1 was detected by a polyclonal antibody (a gift from N. La Thangue, Glasgow, Scotland). In vitro kinase assays. Cyclin D1-associated kinase activity was determined as described previously (37), using a monoclonal antibody to cyclin D1 (DCS11) kindly provided by J. Bartek, Copenhagen, Denmark. Recombinant glutathione S-transferase (GST)–pRb fusion protein [GST-Rb(379-972)] was used as an in vitro substrate. Antibody DCS6, of the same immunoglobulin isotype, was used as a negative control for nonspecific kinase activity present in the immunoprecipitates. Cyclin E-associated histone H1 kinase activity was determined as described previously (65), using a polyclonal antibody to cyclin E (M20; Santa Cruz). p27KIP1 degradation assay. Recombinant human p27KIP1-His6 was expressed in Escherichia coli and purified by Ni21-agarose chromatography as described previously (48). Cellular extracts were prepared by lysis in distilled H2O and incubated with equal amounts of p27KIP1-His6 in the presence of 50 mM TrisHCl (pH 8.3)–5 mM MgCl2–2 mM dithiothreitol–1 mM ATP–10 mM phosphocreatine–40 mg of creatine phosphokinase per ml at 378C. At different time points, the reaction was stopped, samples were separated by SDS-PAGE, and p27KIP1 levels were determined by immunoblotting.
RESULTS Nonadherent cells are arrested in G1 and fail to express cyclin A. NIH 3T3 cells were serum starved for 72 h and replated in 10% serum either in monolayer or in suspension. At regular intervals, cellular DNA content was monitored by flow cytometry. While adherent cells entered S phase 14 h after serum readdition, nonadherent cells maintained 2N DNA content throughout the experiment (data not shown). As previously published by others (14, 18), we found that cells grown in suspension failed to enter S phase but did exit from G0, since they accumulated cyclin D1 and cyclin E to levels similar to those seen in adherent cells (Fig. 1). While cyclin E mRNA levels were somewhat lower in nonadherent cells, the levels and kinetics of accumulation of the protein were unaffected by loss of adhesion. Adherent cells start to synthesize cyclin A mRNA and protein 14 h after release from serum starvation; in nonadherent cells, cyclin A gene expression was not detectable. We thus examined how cell adhesion might control cyclin A expression. Anchorage-dependent activity of the cyclin A promoter is mediated by E2F. During the normal cell cycle, cyclin A gene expression is tightly regulated at the level of transcription. To
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FIG. 1. Cyclin gene expression in nonadherent cells. NIH 3T3 cells were starved by serum withdrawal for 72 h, trypsinized, and plated on normal (adherent) or agarose-coated (nonadherent) dishes in the presence of 10% serum. At the indicated time points, protein extracts and whole cell RNA were prepared. (A) Protein extracts were analyzed by Western blotting using antibodies to cyclin A, cyclin D1, and cyclin E. (B) RNA was analyzed by Northern blotting using probes for cyclin A, cyclin D1, cyclin E, and glyceraldehyde phosphate dehydrogenase (GAPDH), as indicated. Cyclin mRNA levels relative to GAPDH mRNA levels, as obtained by densitometric quantitation, are indicated by numbers above the lanes.
investigate whether the cyclin A gene promoter is responsive to cell adhesion, we transfected NIH 3T3 cells with cyclin A promoter-luciferase constructs (23, 53). Cells were starved for 72 h and then replated in medium containing 10% serum, either in monolayer or in suspension. At regular intervals, cells were harvested, and luciferase activity was determined. In adherent cells, transcription from the full promoter-reporter gene PA/111LUC was activated 14 h after serum readdition. The activity of the reporter gene remained low throughout the entire observation period in nonadherent cells (Fig. 2A). In adherent cells, the block to cyclin A transcription during G1 is mediated by the transcription factor E2F, which binds to a nonconsensus site at positions 237 to 233 in the human cyclin A promoter (53). The promoter construct harboring a mutated E2F site, PA/111LUCmut, is derepressed in G1 and not activated at the G1/S transition in adherent cells (Fig. 2B). The finding that it is also derepressed in nonadherent cells shows that the adhesion-dependent block to cyclin A transcription requires the integrity of the E2F site. Taken together, these data indicate that cell adhesion regulates cyclin A expression at the level of transcription through the transcription factor E2F. In adherent cells, E2F binding to the cyclin A promoter changes according to the activation state of the promoter (53). To determine whether anchorage affected protein binding to
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FIG. 2. Adhesion-dependent regulation of the cyclin A promoter. NIH 3T3 cells were transfected with the cyclin A promoter construct PA/111LUC (A) or PA/111LUCmut (B). Transfected cells were synchronized by serum starvation and plated on normal (squares) or agarose-coated (diamonds) dishes in the presence of 10% serum. At the indicated time points, luciferase activity was determined and normalized to b-galactosidase activity. Shown are the values relative to the value obtained with PA/111LUC at 0 h. Values represent the means of three independent experiments. The derepression of cyclin A promoter activity resulting from mutation of the E2F site (B) was previously observed in adherent NIH 3T3 cells (53).
the E2F site, we performed band shift experiments using extracts from synchronized NIH 3T3 cells grown in monolayer or in suspension (Fig. 3). E2F complex II*, containing p107, is characteristic for quiescent cells and disappears after 14 h of serum stimulation of adherent cells. In nonadherent cells, complex II* remains the predominant complex bound to the promoter throughout the experiment (Fig. 3A). In adherent cells, activation of the cyclin A promoter is paralleled by an increase of complex III (free E2F) and the appearance of complex II, which contains p107 and cyclin E (Fig. 3B and reference 53). This complex is not detectable in extracts from nonadherent cells after serum stimulation, and the level of free E2F does not increase. These data suggest that loss of anchorage prevents replacement of complex II* by complex II and limits the availability of free E2F. Overexpression of cyclin D1 can abolish the block to cyclin A transcription in nonadherent cells. We recently found that ectopic expression of cyclin D1 can relieve the G1 repression of the cyclin A promoter through its E2F binding site (53). We were thus prompted to test whether ectopic expression of cyclin D1 could also override the adhesion-dependent promoter repression. Cyclin D1 overexpression may lower the growth factor requirements of NIH 3T3 cells (49) and may thus prevent proper cell synchronization by serum starvation. To circumvent this problem, we used an inducible expression system for cyclin D1 (see Materials and Methods). Cells were cotransfected with the inducible expression plasmid for cyclin D1 together with promoter-reporter construct PA/111LUC and an expression plasmid for CD32 as a surface label of transfected cells. Cells were then serum starved for 72 h, serum refed under induced and uninduced conditions, and cultured in suspension during 20 h. At the 6-h time point, normalized luciferase activity and DNA content of the transfected cells were determined. Induced cells had luciferase activity 5.5 times higher than noninduced cells; 96% of the transfected cells had G1 DNA content in both cases. At the 20-h time point, induced cells had activated the reporter gene 34-fold, and 22% of them were in S phase (Table 1). Although S-phase entry of suspended cells upon cyclin D1 overexpression is later than in untransfected, adherent NIH 3T3 cells (23), these data show that overexpression of cyclin D1 can relieve the adhesiondependent block to cyclin A transcription and allows DNA replication in suspension culture.
Cyclin D1- and cyclin E-associated kinases are inactivated in nonadherent cells. E2F activity is controlled by complex formation with the pocket proteins of the pRb family (for a review, see reference 62). The capacity of these proteins to bind to E2F components is regulated through phosphorylation, most likely by G1 cdks (4, 27, 31, 54, 59). We compared the phosphorylation kinetics of pRb and p107 in adherent and nonadherent cells. While pRb and p107 were phosphorylated as the cell cycle progressed in adherent cells, both proteins remained underphosphorylated in suspended cells (Fig. 4a). Immunoprecipitates with an antibody specific for cyclin D1 or cyclin E were then assayed for the capacity to phosphorylate their in vitro substrates pRb and histone H1, respectively. Loss of anchorage reduced cyclin D1-dependent kinase (Fig. 4b) to the same extent as did serum starvation in adherent cells (Fig. 4c). Similarly, cyclin E-dependent kinase activity was high in adherent cells and barely detectable in nonadherent cells (Fig. 4d). The p21CIP1/p27KIP1 family of cdis binds to and inhibits the cyclin D- and cyclin E-dependent kinases (13, 22, 48, 60). We thus compared the expression kinetics of these cdis in adherent and nonadherent cells. The expression pattern of p21CIP1 did not significantly differ between adherent and nonadherent cells (data not shown). In adherent cells, p27KIP1 levels decreased dramatically at the G1/S transition (Fig. 5A, upper panel); in nonadherent cells, p27KIP1 protein levels did not decline throughout the time course. p27KIP1 is stable in quiescent cells and is degraded during cell cycle progression by the ubiquitin pathway (44). Since p27KIP1 mRNA accumulation appeared to be unaffected by loss of adhesion (Fig. 5A, lower panel), we examined whether the ability of nonadherent cells to degrade p27KIP1 upon serum stimulation might be impaired. We incubated recombinant p27KIP1 protein with extracts from adherent and nonadherent cells prepared after 16 h of serum stimulation. Degradation reactions were performed as described previously (44), and degradation of p27KIP1 was monitored by immunoblotting. In extracts from monolayer cells, most of the input p27KIP1 protein was degraded after 3 h of incubation. In contrast, p27KIP1 degradation was 30-fold less efficient in extracts prepared from nonadherent cells (Fig. 5B and C). The degradation rate observed with the extracts from nonadherent cells was only slightly higher than the rate obtained with extracts from serum-starved cells.
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FIG. 3. Analysis of E2F complexes in nonadherent cells. (A) NIH 3T3 cells were synchronized by serum starvation, trypsinized, and plated on normal (adherent) or agarose-coated (nonadherent) dishes in the presence of 10% serum. At the indicated time points, protein extracts were prepared and analyzed for protein binding to the E2F site from the human cyclin A promoter (oligonucleotide cycA wt). E2F-specific protein-DNA complexes, identified by competition experiments (see panel B), are labeled II, II*, and III. (B) Complexes obtained with extracts from adherent or nonadherent cells at 24 h after serum readdition were analyzed by competition experiments, using excess unlabeled oligonucleotides cycA wt, E2 wt, and E2 mut. Specific complexes are labeled as in panel A. To identify components of the protein-DNA complexes, specific antibodies to DP-1, p107, and cyclin E were added as indicated. The oligonucleotides used are described in Materials and Methods.
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mental in development and tissue homeostasis, and loss of the appropriate cell cycle controls is a hallmark of cellular transformation. Since progression through the mammalian cell cycle is controlled by cdks (45, 55, 56), cell cycle arrest involves inactivation of at least one cdk. Absence of mitogen stimulation, or terminal differentiation, inactivates cdk4 and cdk6 by abrogating expression of their regulatory subunit cyclin D1, and ectopic expression of cyclin D1 rescues the cells from cell cycle arrest (49, 50). In other cases, cell cycle arrest results from cdk inactivation through protein-protein interactions. In senescent cells, cyclin D1 is highly expressed, but it is sequestered in inactive complexes with cdk2 (11). Ionizing or UV irradiation leads to G1 arrest as a result of inactivation of cyclin D- and cyclin E-dependent kinases by the cdi p21CIP1 (12). Growth arrest by cell-cell contact of untransformed cells in culture is thought to be due to the inhibition of G1 cdks by p27KIP1 (47). A common denominator of all natural and experimental forms of G1 arrest observed in mammalian cells is the absence of cyclin A gene expression, resulting in the inability to replicate DNA (15, 43, 68). In this report, we have analyzed why cyclin A is not expressed in untransformed fibroblasts arrested in response to loss of adhesion, as found by Guadagno et al. (18, 19). We show that loss of anchorage abrogates cyclin A gene expression in NIH 3T3 cells at the level of transcription. In suspended cells, activity of the cyclin A gene promoter is blocked through an E2F-dependent mechanism (Fig. 2). E2F also mediates cyclin A promoter repression during G1 in adherent cells (53). In response to serum stimulation of adherent NIH 3T3 cells, increased binding of free E2F is observed, and at the G1/S transition, an activation-specific complex which contains p107 and cyclin E is formed. In contrast, in suspended NIH 3T3 cells, serum addition does not induce binding of free E2F, and the cyclin E-p107 complex is not formed (Fig. 3), although both proteins are present (Fig. 1 and 4). E2F activity is controlled by association with the pocket proteins of the pRb family (34). The hypophosphorylated forms of pRb and p107 bind and inactivate E2F-1, -2, and -3 and E2F-4 and -5, respectively. Phosphorylation of pRb (27, 30) and p107 (4) by cyclin D- and cyclin E-dependent kinases appears to be crucial to the regulation of cellular E2F activity. We thus analyzed the activity of these kinases and the phosphorylation state of pRb and p107 in suspended NIH 3T3 cells. We found that loss of adhesion did not affect the expression of
TABLE 1. Cyclin D1-mediated activation of cyclin A transcription in suspended cellsa
Expression of p27KIP1 in proliferating cells represses cyclin A promoter activity via the E2F binding site. To test whether elevated levels of p27KIP1 can limit the particular E2F activity required for activation of the cyclin A gene promoter, we transfected asynchronous NIH 3T3 cells with the wild-type and mutant promoter-reporter constructs PA/111LUC and PA/ 111LUCmut, respectively, together with different amounts of an expression vector for p27KIP1 (Fig. 6). Ectopic expression of p27KIP1 strongly repressed cyclin A promoter activity, dependent on the integrity of the E2F site. These data show that high-level expression of p27KIP1 can inhibit E2F-driven transcription of the cyclin A gene. DISCUSSION The ability of mammalian cells to arrest cell cycle progression in response to intra- and extracellular signals is funda-
Time (h) of incubation with serum
6h 1Tet 2Tet 20 h 1Tet 2Tet
%b of cells in:
Promoter induction (fold)
G1
S
1 5.5
96 96
2 2
94 71
4 22
1 34
a NIH 3T3 cells were transfected with plasmids cycD1/T and CMV/T and an expression plasmid for the Fcg receptor CD32. Transfected cells were serum starved in the presence of 2 mg of tetracycline per ml (1Tet), and induction was obtained by incubation in tetracycline-free medium (2Tet). Activity of the cotransfected reporter gene PA/111LUC was determined by measuring the luciferase activity in cellular extracts. For cell cycle analysis of transfected cells, cells were doubly stained with propidium iodide (to measure DNA content) and an anti-CD32 monoclonal antibody (for selective identification of transfected cells). Cell cycle analysis was performed by flow cytometry. b Mean of two experiments.
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associated with cyclin D1 was not affected by loss of adhesion (14); furthermore, Bohmer et al. recently reported that cyclin D1 gene expression is blocked in nonadherent NIH 3T3 cells (5). These data indicate that the response to loss of anchorage may vary between different cell lines and even between different subclones of one cell line. We previously reported that overexpression of cyclin D1 can relieve the E2F-mediated block to cyclin A transcription in the G1 phase of attached cells, through activation of the cdk4 kinase (53). We now find that enforced expression of cyclin D1 can also overcome the adhesion-dependent block of cyclin A transcription. Ectopic expression of cyclin D1 leads to phosphorylation of pRb (27) and p107 and induces dissociation of E2F-p107 complexes (4). Cyclin D1 overexpression may
FIG. 4. Modulation of cdk activities by loss of adhesion. (a) Synchronized NIH 3T3 cells were plated on normal (lane N) or agarose-coated (lanes A) dishes in medium containing 10% serum. After 24 h, protein extracts were prepared, subjected to high-resolution SDS-PAGE, and analyzed by Western blotting using antibodies to p107 and pRb as indicated. Bands representing the hypophosphorylated (pRb and p107) and hyperphosphorylated (ppRb and pp107) forms of these proteins are marked. (b) Samples were taken at 16 h after serum stimulation, and cyclin D1 was immunoprecipitated with monoclonal antibody DCS11. Associated kinase activity was assayed by using GST-Rb (379-792) as a substrate (upper panel). As a control, 50% of the immunoprecipitate (IP) was analyzed by Western blotting using anti-cyclin D1 monoclonal antibody HD11. (c) As a control, cyclin D1 kinase activity from proliferating (10% serum) and serumstarved (0.5% serum) NIH 3T3 cells was determined, using the same experimental conditions as for panel b. Extracts were also incubated with nonspecific antibody DCS6 (control) and subjected to the immunoprecipitation procedure. The low degree of GST-Rb phosphorylation, obtained with the DCS6 immunoprecipitate, represents unspecific background activity. (d) Samples were taken at 16 h after serum stimulation and cyclin E, was precipitated with polyclonal antibody M20. Associated kinase activity was assayed by using histone H1 as a substrate. As a control, 50% of the immunoprecipitate was analyzed by Western blotting using anti-cdk2 polyclonal antibody M2 to determine the amount of coprecipitated cdk2.
cyclins D1 and E. Cyclin D1- and cyclin E-dependent kinases, however, were inactivated. Loss of anchorage stabilized the cdi p27KIP1 (Fig. 5A), which can inactivate both cyclin D- and cyclin E-dependent kinases (48). We show that overexpression of p27KIP1 efficiently represses cyclin A transcription through an E2F-dependent mechanism. p27KIP1 may do so by inhibiting the phosphorylation of the pocket proteins by cdks and thus blocking the release of E2F. Alternatively, p27KIP1 may act directly by interfering with the formation of E2F complexes on the cyclin A promoter. It was shown that both p27KIP1 and p21CIP1 can compete with p107 for binding of cyclin-cdk complexes, by virtue of a conserved cyclin-binding domain (58, 67), thereby preventing the association of cyclin-cdk with E2F complexes. Our preliminary results indicate that addition of recombinant p27KIP1 to band shift reactions dissociates the cyclin E-containing p107-E2F complex (65a). Although the precise molecular mechanism mediating the E2F-dependent block to cyclin A transcription in suspended cells remains to be established, our results suggest that loss of cell adhesion blocks cell cycle progression by inhibiting G1 cdks and by preventing E2F-dependent transcription of cyclin A through stabilization of p27KIP1. How integrinmediated signals (for a recent review, see reference 8) control the stability of p27KIP1 remains to be elucidated. Stabilization of p27KIP1 was also observed in suspended human fibroblasts (14). However, in this particular cell type, the kinase activity
FIG. 5. Metabolic stability of p27KIP1 in nonadherent cells. (A) Serumstarved NIH 3T3 cells were refed serum under nonadherent or adherent conditions. Protein extracts (upper panel) and cellular RNA (lower panel) were analyzed for p27KIP1 expression. The relative abundance of p27KIP1 mRNA is given below each lane; the values were obtained by densitometric analysis and subsequent normalization to the corresponding glyceraldehyde phosphate dehydrogenase (GAPDH) signal. (B) NIH 3T3 cells were grown for 16 h under nonadherent or adherent conditions as described for panel A. Extracts were prepared from these cells and incubated with a fixed amount of recombinant p27KIP1-His6 for the indicated times. Degradation of the input protein was monitored by Western blotting using an antibody to p27KIP1. For comparison, extracts of quiescent cells were also assayed. (C) The relative abundance of undegraded input p27KIP1 shown in panel B was quantitated densitometrically and plotted against the incubation time. The signal intensity at 0 min represents 100% of protein input. Squares, serum-starved cells; diamonds, adherent cells; circles, nonadherent cells.
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FIG. 6. Repression of cyclin A promoter activity by p27KIP1. Increasing amounts of p27KIP1 expression plasmid were transfected into NIH 3T3 cells together with cyclin A promoter construct PA/111LUC or PA/111LUCmut. Values represent the means of three independent experiments and are normalized to the activity of a cotransfected cytomegalovirus (CMV)-driven b-galactosidase reporter gene construct.
thereby release free E2F from inhibition by pRb (25) or p107 (64) and thus activate transcription of the cyclin A gene in suspended cells. Cyclin D1-cdk4 and cyclin E-cdk2 compete for binding to p27KIP1 (20). Overexpression of cyclin D1 may thus titrate the inhibitor in suspended cells. This would restore cyclin E-dependent kinase activity (38), which can activate cyclin A transcription (52, 65b). Overexpression of cyclin D1 or inactivation of the specific inhibitor of cyclin D-dependent kinases, p16INK4, is linked to transformation (26) and tumorigenesis (3). Loss of anchorage dependence is a property of most transformed cells and may contribute to tumor outgrowth and metastasis. Overexpression of cyclin D1 was found to be correlated with anchorage independence of cells derived from natural tumors (17), and it was shown that expression of antisense cyclin D1 leads to the loss of tumorigenicity and of anchorage independence of human esophageal cancer cells (66); these findings suggest that overexpression of cyclin D1 in certain tumor cells can facilitate anchorage-independent growth. In keeping with these observations, we show here that overexpression of cyclin D1 at least partially restores the potential of nonadherent cells to transcribe the cyclin A gene and to replicate DNA (Table 1). The results reported here indicate that a critical transforming function of cyclin D1 overexpression might be the abolishment of an E2F-dependent negative growth control mechanism that prevents cyclin A transcription and cell cycle progression in the absence of adhesion. ACKNOWLEDGMENTS We thank J. Massague´ for the gift of the p27KIP1 expression plasmid, J. Bartek, J. Lukas, and N. La Thangue for antibodies, and R. Assoian for communicating results prior to publication. Work in P.J.-D.’s laboratory was supported by a research grant from the Human Frontiers Science Program Organisation. Work in B.H.’s laboratory was supported by a grant from the Association pour la Recherche sur le Cancer. REFERENCES 1. Amigorena, S., C. Bonnerot, J. R. Drake, D. Choquet, W. Hunziker, J. G. Guillet, P. Webster, C. Sautes, I. Mellman, and W. H. Fridman. 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science 256:1808–1812. 2. Baldin, V., J. Lukas, M. J. Marcote, M. Pagano, and G. Draetta. 1993. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 7:812–821.
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