Efficient Down-Regulation of Cyclin A-Associated Activity and Expression in Suspended Primary Keratinocytes Requires p21

When suspended in methylcellulose, primary mouse keratinocytes cease proliferation and differentiate. Suspension also reduces the activity of the cyclindependent kinase cdk2, an important cell cycle regulatory enzyme. To determine how suspension modulates these events, we examined its effects on wild-type keratinocytes and keratinocytes nullizygous for the cdk2 inhibitor p21. After suspension of cycling cells, amounts of cyclin A (a cdk2 partner), cyclin A mRNA, and cyclin A-associated activity decreased much more rapidly in the presence than in the absence of p21. Neither suspension nor p21 status affected the stability of cyclin A mRNA. Loss of p21 reduced the capacity of suspended cells to growth arrest, differentiate, and accumulate p27 (a second cdk2 inhibitor) and affected the composition of E2F DNA binding complexes. Cyclin A-cdk2 complexes in suspended p21 cells contained p21 or p27, whereas most of the cyclin A-cdk2 complexes in p21 / cells lacked p27. Ectopic expression of p21 allowed p21 / keratinocytes to efficiently down-regulate cyclin A and differentiate when placed in suspension. These findings show that p21 mediates the effects of suspension on numerous processes in primary keratinocytes including cdk2 activity, cyclin A expression, cell cycle progression, and differentiation. Introduction Most mammalian cell types grow in an adhesion-dependent manner both in vivo and in vitro . Oncogenic transformation overrides the need for attachment for cell proliferation, and anchorage-independent growth correlates strongly with tumorigenicity. When deprived of a substratum, nontransformed cells typically arrest in G1, and studies addressing this phenomenon have focused on the cyclin-dependent kinases (CDKs) and their regulatory subunits, the cyclins (1). Complexes containing the D cyclins and cdk4, cyclin E and cdk2, and cyclin A and cdk2 become active in mid-G1, in late G1, and at G1-S, respectively (2). All complexes phosphorylate the retinoblastoma (Rb) protein, an event required for S phase entry (3). When hyperphosphorylated by cdk4 and cdk2, Rb no longer functions as a transcriptional repressor, and numerous gene products (including many involved in DNA replication) are expressed (4, 5). Events that regulate the activity of cdk4 and cdk2 include the periodic synthesis and destruction of the cyclins. In addition, proteins such as p21 and p27 interact with and inhibit the activity of complexes containing cdk4 or cdk2 and their cyclin partners (6, 7). The overall abundance of p21 and p27 varies during the cell cycle, and sequestration of p21 and p27 by D cyclin-cdk4 complexes facilitates cdk2 activation. Most notably, nontransformed cells do not express cyclin A, and consequently do not form cyclin A-cdk2 complexes, when cultured in suspension (8–18). Conversely, cyclin A is readily detectable in cells that grow in suspension as a result of enforced expression of E7, c-Myc, activated Ras, cyclin D1, or integrin-linked kinase (11, 13, 15–17, 19, 20). When ectopically expressed, cyclin A allows or contributes to the anchorage-independent proliferation of NRK, Rat 1a, and ER-1-2 fibroblasts (8, 11, 13). Collectively, these studies identify cyclin A as a key target of suspension-induced growth inhibitory pathways. Transcriptional repression of the cyclin A promoter accounts (at least in part) for the absence of cyclin A from suspended cells (10, 14, 21, 22). Repression occurs when Rb sequesters a CCAAT-binding protein required for transactivation and/or when Rb or the Rb-related protein p107 interacts with a variant E2F site in the cyclin A promoter. In both instances, Rb and p107 lose their capacity to repress cyclin A gene transcription when hyperphosphorylated by cdk4 and cdk2. Suspension inhibits cyclin E-cdk2 activity in most cell types and cdk4 activity in some cell types; as a result, Rb and p107 are typically hypophosphorylated in suspended cells (9–12, 15–17, 19, 23, 24). Suspension does not alter the abundance of cyclin E or cdk2 or the assembly of cyclin E-cdk2 complexes; however, more cyclin E-cdk2 complexes contain p21 or p27 in suspended cells than in attached cycling cells (9, 19, 23, 24). p21 and p27 often accumulate in suspended cells, and in some instances, the absence of the D cyclins precludes the sequestration of p21 and p27 by D cyclin-cdk4 complexes (9, 10, 12, 16, 19, 20, 23, 24). Thus, suspension increases the availability of p21 and p27, which results Received 9/15/03; revised 12/30/03; accepted 1/8/04. Grant support: This work was supported by the Cortner-Couch Endowed Chair for Cancer Research (W. J. P.) and NIH Grants CA73780 (W. J. P.) and CA90489 (W. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: W.J. Pledger, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa FL 33612. Phone: (813) 979-3887; Fax: (813) 979-3393. E-mail: [email protected] Copyright D 2004 American Association for Cancer Research. Vol. 2, 96–104, February 2004 Molecular Cancer Research 96 on April 9, 2017. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from in cyclin E-cdk2 inactivation, Rb hypophosphorylation, and ultimately, the repression of cyclin A gene transcription. Suspension may also inhibit cyclin A expression by processes other than those described above (9, 11). Most previous studies examined the effects of suspension on cells stimulated from quiescence (8–16, 22, 23). In contrast, our studies (18) examined the effects of suspension on growing cells, which express active cyclin A-cdk2 complexes at the time of suspension. Our studies were performed on primary mouse keratinocytes, which cease proliferation and differentiate when placed in a semisolid medium (25). This system allows us to determine how suspension affects cyclin-CDK activity and how changes in cyclin-CDK activity affect both cell cycle progression and differentiation. We found that p27 accumulated in suspended keratinocytes and interacted with cyclin A-cdk2 complexes and that amounts of cyclin A and cyclin A-associated activity declined (18). Suspension of cells in the presence of p27 antisense oligonucleotides, and thus in the absence of p27 accumulation, did not prevent the loss of cyclin A or cyclin A-associated activity or cell cycle arrest; however, the differentiation-specific marker keratin 1 (K1) was not induced. On the basis of the results of our previous report (18), we suggest that p27 promotes keratinocyte differentiation by actions unrelated to the inhibition of cdk2 activity. We also suggest that p27 (or p27 accumulation) contributes to but is not required for cdk2 inactivation and growth arrest in suspended cells. To identify additional mediators of these events, we examined the effects of suspension on keratinocytes lacking p21. We show that efficient down-regulation of cyclin A and cyclin A-associated activity in suspended keratinocytes requires p21 as do growth arrest, differentiation, and maximal accumulation of p27. Determination of how suspension abrogates cell cycle controls in normal keratinocytes may be relevant to studies of cancer cells, which typically grow in suspension. Results Requirement for p21 for Efficient Down-Regulation of Cyclin A, Cyclin A Messenger RNA, and Cyclin AAssociated Activity in Suspended Keratinocytes To assess the need for p21 for suspension-induced decreases in cyclin A expression and cyclin A-associated activity, primary keratinocytes derived from wild-type and p21-null mice were suspended in methylcellulose and harvested at various times thereafter. At the time of suspension, keratinocytes in both populations were actively cycling (data not shown). Amounts of cyclin A were determined by Western blotting of cell extracts, and kinase activity was assayed in cyclin A immune complexes using histone H1 as substrate. Although cyclin A associates with both cdk2 and the mitotic CDK cdc2 (26), p21 selectively inhibits the activity of cdk2-containing complexes (27). It is noted that p21 preferentially interacts with cyclin A-cdk2 complexes rather than cyclin A or cdk2 alone (27). Cyclin A was expressed and cyclin A-associated kinases were active in attached p21 keratinocytes and, to a greater extent, in attached p21 keratinocytes (Fig. 1, A and B, lanes 1 and 6). Cyclin A-associated activity decreased within 3 h of suspension of p21 keratinocytes and was barely detectable at 6 h and times thereafter (Fig. 1B, lanes 1–5), even when the blots were overexposed (lanes 11–15). Amounts of cyclin A declined more slowly; cyclin A abundance was modestly reduced 6 h after suspension and substantially reduced 12 h after suspension of p21 cells (Fig. 1A, lanes 1–5). Importantly, cyclin A was detectable in cells suspended for 6–12 h, whereas cyclin A-associated activity was not. This indicates that factors other than (or in addition to) loss of protein account for loss of activity during this time period. In contrast to p21 keratinocytes, p21 / keratinocytes expressed comparable amounts of cyclin A or cyclin Aassociated activity whether attached to plates or suspended for 12 h (Fig. 1A, lanes 6–10 , and Fig. 1B, lanes 6–9). Amounts of both, however, eventually declined (Fig 1B, lane 10 , and data not shown). The data in Fig. 1, A and B, indicate that loss of p21 greatly delays the suspension-induced downregulation of cyclin A and cyclin A-associated activity in primary keratinocytes. The effects of suspension on cyclin A mRNA abundance in p21 and p21 / keratinocytes were similar to its effects on cyclin A protein abundance. After suspension, amounts of cyclin A mRNA decreased rapidly and substantially in p21 keratinocytes (Fig. 1C, lanes 1–5), but remained elevated in p21 / keratinocytes for 12 h (lanes 6–10). When p21 and p21 / keratinocytes were suspended in the presence of the mRNA synthesis inhibitor DRB, the cyclin A transcript decayed with similar kinetics in both populations (lanes 11–18). Thus, differences in cyclin A mRNA stability do not account for the differences in cyclin A mRNA abundance in suspended p21 versus p21 / keratinocytes. Amounts of cyclin A mRNA also decreased with similar kinetics in p21 keratinocytes suspended in the presence (lanes 11–14) and absence (lanes 2–5) of DRB. By default, these data suggest that loss of p21 prevents suspension-induced decreases in the activity of the cyclin A promoter. This premise is consistent with previous findings showing transcriptional repression of the cyclin A gene in nonadherent fibroblasts (10, 14, 21, 22). To ensure that p21 status accounts for the different expression patterns of cyclin A in wild-type versus p21-null keratinocytes, we ectopically expressed p21 in p21 / keratinocytes and amounts of cyclin A were determined. In these experiments, p21 / keratinocytes were infected with adenovirus without insert or adenovirus encoding human p21 (Ad-p21). Cells were suspended in methylcellulose 19 h after infection and harvested 19 h later. For comparative purposes, p21 keratinocytes were also examined (Fig. 2, lanes 1 and 2). The expression of human p21 in p21 / keratinocytes was verified by Western blotting (lanes 4 and 7). After suspension, amounts of cyclin A declined but were readily detectable in uninfected p21 / keratinocytes and p21 / keratinocytes infected with virus alone (compare lanes 3 , 5 , and 6). In contrast, cyclin A was not detected in p21 / keratinocytes infected with Ad-p21 and suspended for 19 h (lane 7). These findings show that nonadherent p21 / keratinocytes gain the capacity to efficiently down-regulate cyclin A when supplied with p21. It is unclear why Ad-p21 more completely reduces cyclin A abundance in suspended keratinocytes (lane 7) than in attached keratinocytes (lane 4). Molecular Cancer Research 97 on April 9, 2017. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Submaximal Accumulation of p27 in Suspended p21-Null Keratinocytes We have shown that amounts of p27 increase in suspended keratinocytes, as do amounts of p27-associated cyclin A-cdk2 complexes (18). As shown here, loss of p21 prevents the rapid decline in cyclin A-associated activity in suspended cells. Together, these findings indicate that p27 contributes to cdk2 inactivation in suspended cells but does not compensate for the absence of p21. As a possible explanation of this phenomenon, we examined p27 expression in p21 and p21 / keratinocytes in methylcellulose. Amounts of p27 increased substantially when p21 keratinocytes were placed in suspension (Fig. 3A); (18). Increases occurred within 6 h of suspension and amounts of p27 remained elevated for at least 24 h. p27 was less abundant in p21 / keratinocytes than in p21 keratinocytes at 0 h (i.e. , before detachment) and did not accumulate appreciably in p21 / keratinocytes suspended for 12 h; submaximal increases were observed at 24 h. Unlike protein, amounts of p27 mRNA were similar in attached and suspended p21 and p21 / keratinocytes (Fig. 3B). These findings show that p21 elicits events that increase the FIGURE 1. Amounts of cyclin A protein, mRNA, and associated activity in p21 and p21 / keratinocytes suspended in methylcellulose. A and B. Proliferating p21 and p21 / keratinocytes were detached from tissue cultures plates and suspended in methylcellulose as described in ‘‘Materials and Methods.’’ Cells were harvested at the times indicated. A. Amounts of cyclin A were determined by Western blotting. B. Cell extracts were immunoprecipitated with antibody to cyclin A, and cyclin A immune complexes were assayed for kinase activity using histone H1 as substrate. Lanes 1– 10, results of a single exposure of the autoradiogram. Lanes 11– 15, darker exposure of lanes 1 –5 , respectively. The darker exposure allows normalization of the activities at time 0 for p21 (lane 11) and p21 / keratinocytes (lane 6 ). C. Asynchronously cycling p21 and p21 / keratinocytes were suspended in methylcellulose in the presence or absence of 100 AM 5,6-dichloro-1-h-D-ribofuranosyl-benzimidazole (DRB) and harvested at the times indicated. Amounts of cyclin A mRNA and L32 mRNA (loading control) were determined by RNase protection assay. FIGURE 2. Amounts of cyclin A in suspended p21 / keratinocytes ectopically expressing p21. Proliferating p21 / keratinocytes were infected with adenovirus without insert (Ad) or Ad-p21 (p21 ) at a multiplicity of infection (MOI) of 250. Cells were suspended in methylcellulose 19 h after infection and harvested 19 h later for determination of p21 and cyclin A abundance by Western blotting. Uninfected p21 and p21 / keratinocytes were also suspended in methylcellulose for 19 h. The p21 antibody used for Western blotting recognizes ectopic (human) p21 but not endogenous (mouse) p21. A, attached cells; S, suspended cells. p21 Modulates Cyclin A Expression in Keratinocytes 98 on April 9, 2017. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from translation and/or stability of p27 in both adherent and nonadherent keratinocytes. In p21 / keratinocytes, amounts of p27 are subnormal, and as a result, the potential capacity of p27 to compensate for the absence of p21 is attenuated. Similar to our findings, loss of p21 reduced the accumulation of p27 protein (but not of p27 mRNA) in interleukin-treated astrocytoma cells (28). Increased Association of CDK Inhibitors With Cyclin A Complexes in Suspended Wild-Type Keratinocytes In contrast to p27, p21 did not accumulate in suspended p21 keratinocytes (Fig. 4A). In fact, p21 decreased in abundance 6–9 h after suspension and was not detected at 24 h. Although total amounts of p21 were constant for at least 6 h after suspension, amounts of p21 not sequestered by D cyclin-cdk4 complexes (and thus available for interaction with cdk2 complexes) may increase. Consistent with this hypothesis, we have shown that amounts of cyclin D1 decrease after suspension of wild-type keratinocytes (18). To test this hypothesis, we determined the amounts of p21-associated cyclin A and p21-free cyclin A in p21 keratinocytes at various times after suspension. Cell extracts were immunoprecipitated with antibody to p21, and p21 immune complexes were Western blotted with antibody to cyclin A (p21-associated cyclin A). p21-immunodepleted extracts were immunoprecipitated and Western blotted with antibody to cyclin A (p21-free cyclin A). Western blots using p21 antibody confirmed that immunodepleted extracts contained little if any p21 (data not shown). Suspension of p21 keratinocytes for 3 or 6 h resulted in an increase in the amounts of p21-associated cyclin A and a decrease in the amounts of p21-free cyclin A (Fig. 4B, compare lanes 2 and 3 with lanes 7 and 8). Thus, a greater percentage of cyclin A complexes contain p21 in suspended keratinocytes than in attached keratinocytes. After 6 h, amounts of p21-associated cyclin A declined (lanes 4 and 5), most likely because of reductions in p21 abundance (see Fig. 4A). Although suspension reduced amounts of p21-free cyclin A, it did not eliminate p21-free cyclin A, at least for times up to 12 h (lane 10). On the other hand, little if any cyclin A remained in 12-h cell extracts immunodepleted of both p21 and p27 (Fig. 4C, top panel , lane 8). Comparison of the relative amounts of CDK inhibitor (CKI)-associated and CKIfree cyclin A at all time points shows that the ratio of associated to free increases progressively after suspension (Fig. 4C, top panel). The data in Fig. 4 support our hypothesis that suspensioninduced decreases in cyclin D1 result in the redistribution of p21 from cyclin D1 complexes to cyclin A complexes and thus in the inhibition of cyclin A-cdk2 activity in wild-type keratinocytes. Redistribution of p27 and/or increases in p27 abundance also contribute to cyclin A-cdk2 inactivation in suspended p21 cells. Our studies also explain why cyclin A-cdk2 complexes are active in suspended p21 / cells: most of these complexes lack p27, as do most of the cyclin Acdk2 complexes in attached p21 / cells. This finding is consistent with our data showing subnormal amounts of p27 in p21 / keratinocytes (Fig. 3A). FIGURE 3. Amounts of p27 in suspended wild-type keratinocytes. A and B. Exponentially growing p21 and p21 / keratinocytes were suspended in methylcellulose and harvested at the times indicated. A. Amounts of p27 protein were determined by Western blotting. B. Amounts of p27 mRNA and L32 mRNA (loading control) were determined by RNase protection assay. FIGURE 4. Association of cyclin A complexes with p21 and p27 in attached and suspended keratinocytes. A. Asynchronously cycling p21 keratinocytes were suspended in methylcellulose and harvested at the times indicated. Cell extracts were immunoblotted with antibody to mouse p21. B. Cycling p21 keratinocytes were suspended in methylcellulose for the times indicated. Lanes 1 –4, cell extracts were immunoprecipitated with antibody to p21, and p21 immune complexes were Western blotted with antibody to cyclin A. Lanes 5– 8, after removal of p21, cell extracts were immunoprecipitated with antibody to cyclin A, and cyclin A immune complexes were Western blotted with antibody to cyclin A. C. As in B, with the exception that cell extracts were immunoprecipitated with antibody to both p21 and p27 (p21 ) or with antibody to p27 (p21 / ). Molecular Cancer Research 99 on April 9, 2017. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Reduced Capacity of p21 / Keratinocytes to Growth Arrest and Differentiate in Suspension Although suspension typically arrests cells in G1 (1), suspended primary mouse keratinocytes arrest in G1, S, and G2-M (29). To determine whether cell cycle arrest requires p21, p21 and p21 / keratinocytes attached to tissue culture plates received bromodeoxyuridine (BrdUrd) for 2 h to label S-phase cells. Cells were then suspended in methylcellulose in the absence of BrdUrd and harvested 12 h later, and the position of the BrdUrd-labeled cells in the cell cycle was determined by flow cytometry. The percentages of cells remaining in S phase 12 h after suspension were 52% and 31% for p21 and p21 / populations, respectively (Fig. 5). Moreover, a greater percentage of p21 / cells were in G1 at this time than were p21 cells. These data suggest that p21 / cells cycle for longer periods of time when placed in suspension than do p21 cells. In this respect, p21 / keratinocytes mimic MK cells, which are immortalized mouse keratinocytes (30) and which express little if any p21 (data not shown). To assess the need for p21 for differentiation, we placed p21 and p21 / keratinocytes in suspension, and amounts of two differentiation markers, K1 (Fig. 6A) and loricrin (Fig. 6B), were determined. Both proteins increased in abundance after suspension of p21 keratinocytes. Increases in the abundance of loricrin were especially pronounced. Amounts of loricrin did not increase in suspended p21 / keratinocytes, whereas amounts of K1 increased to a lesser extent and in a delayed manner. At 48 h, K1 was still much less abundant in p21 / keratinocytes than in p21 keratinocytes (data not shown). These findings show that efficient differentiation of primary keratinocytes requires p21. To confirm that the differentiation-defective phenotype of p21 / keratinocytes reflects the absence of p21, p21 / keratinocytes were infected with Adp21 and suspended in methylcellulose. Nineteen hours after suspension, Ad-p21-infected p21 / cells (Fig. 6C, lane 4) expressed more K1 than did uninfected p21 / cells (lane 2) and p21 / cells infected with adenovirus alone (lane 3). E2F Complexes in Wild-Type and p21-Null Keratinocytes in Suspension Cyclin A is a member of a DNA binding complex that contains cdk2, the transcription factor E2F, the E2F binding partner DP1, and p107, an Rb family member (31–33). Previous studies have shown that p21 disrupts this complex both in vivo and in vitro (21, 34, 35). Thus, as another potential consequence of p21 loss, we monitored E2F DNA binding activity in p21 and p21 / keratinocytes before and after suspension. As demonstrated by electrophoretic mobility shift assays, attached keratinocytes (wild-type and p21-null) expressed an activity that interacted with an E2F binding site; this DNA/protein complex is designated complex I (Fig. 7A, lanes 1 and 5). Between 3 and 6 h after suspension of p21 keratinocytes, complex I disappeared and a more rapidly migrating complex (complex II) appeared (Fig. 7A, lanes 3 and 4). Complex II was not seen in suspended p21 / keratinocytes, although amounts of complex I decreased somewhat (lanes 6–8). Infection of p21 / keratinocytes with Ad-p21 resulted in the conversion of complex I to II (Fig. 7B). These findings show that suspension modulates the composition of E2F DNA binding complexes in a p21dependent manner. Antibody to cyclin A blocked the formation of complex I (or its entry into the gel; Fig. 7C, compare lanes 1 and 4 , and lanes 7–9 with lanes 10–12) but had no effect on complex FIGURE 5. Proliferative capacity of suspended wild-type and p21null keratinocytes. Cycling p21 and p21 / cells received 4 AM BrdUrd for 2 h. Cell pellets were processed as described in ‘‘Materials and Methods,’’ and cell cycle distribution was determined by flow cytometry. Percentages of BrdUrd-labeled cells in G1, S, and G2 are shown. FIGURE 6. K1 and loricrin induction in p21 and p21 / keratinocytes suspended in methylcellulose. A and B. p21 and p21 / keratinocytes were suspended in methylcellulose for the indicated times, and cell extracts were immunoblotted with antibody to K1 (A) or loricrin (B). C. p21 / keratinocytes were infected with control adenovirus (Ad ) or Ad-p21 (p21 ) at an MOI of 250. Cells were suspended in methylcellulose 21 h after infection and harvested 19 h later for determination of K1 abundance by Western blotting. Lane 1, uninfected cells attached to plates. p21 Modulates Cyclin A Expression in Keratinocytes 100 on April 9, 2017. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from II (compare lanes 2 and 3 with lanes 5 and 6). Both complexes were supershifted with antibodies to p107 and E2F4 but were unaffected by antibodies to Rb, the Rb-related protein p130, or other E2F family members (Fig. 7D). Thus, complex I contains E2F4, p107, and cyclin A, whereas complex II contains E2F4 and p107 but not cyclin A. Discussion Our data show that suspension reduces the abundance and associated activity of cyclin A in primary keratinocytes much more rapidly in the presence than in the absence of p21. Suspension-induced decreases in cyclin A abundance apparently result (at least in part) from the transcriptional repression of the cyclin A gene. In wild-type keratinocytes, suspension reduced the abundance of the cyclin A transcript without affecting its half-life. This transcript also decayed with similar kinetics in p21 and p21 / keratinocytes. Others have shown that suspension represses cyclin A expression in rodent fibroblasts by a process involving cdk2 inactivation and the consequent interaction of Rb with the cyclin A promoter (10, 14, 21, 22). In our studies, loss of cyclin A activity preceded loss of cyclin A protein. Thus, the primary target of p21 in suspended keratinocytes is cyclin A-associated activity rather than cyclin A expression. We show that p21 inhibits cyclin A-associated activity in suspended keratinocytes by two mechanisms: it interacts with cyclin A-cdk2 complexes, and it promotes the accumulation of p27. Suspension of p21 keratinocytes for 3 or 6 h increased the percentage of cyclin A complexes containing p21. Such increases presumably result from the redistribution of p21 from cyclin D1-cdk4 complexes to cyclin A-cdk2 complexes. First, as presented here, amounts of p21 remained constant for 6–9 h after suspension of wild-type keratinocytes and then decreased. Second, as described previously, amounts of cyclin D1 declined within 3 h of suspension of wild-type keratinocytes and were essentially undetectable at 6 h (18). p21 may interact with both cyclin A-cdk2 and cyclin E-cdk2 complexes in suspended keratinocytes; we have shown that suspension reduces cyclin E-associated activity and cyclin A-associated activity with similar kinetics (18). Others have shown that p21 associates with cyclin E-cdk2 complexes in suspended NIH 3T3 cells and that this association correlates with decreases in cyclin D1 abundance rather than increases in p21 abundance (9). Amounts of p27 increased within 6 h of suspension of wild-type keratinocytes and were substantially elevated at 9–24 h. p21 keratinocytes expressed much more p27 than did p21 / keratinocytes whether attached or suspended. FIGURE 7. Composition of E2F DNA binding complexes in suspended p21 and p21 / keratinocytes. A–D. Electrophoretic mobility shift assays were performed on cell extracts using a fragment of the dihydrofolate reductase promoter containing an E2F binding site. Antibodies to cyclin A (C), E2F1, E2F2, E2F3, E2F4, E2F5, p130, p107, or Rb (D) were added to binding reactions 30 min before addition of the radiolabeled DNA probe. A. p21 and p21 / keratinocytes were suspended for the indicated times. B. p21-null keratinocytes were infected with control adenovirus (Ad ) or Ad-p21 (p21 ) at an MOI of 250. Cells were suspended 19 h after infection and harvested 9 h later. Uninfected wild-type keratinocytes were also suspended for 9 h. C. p21 and p21 / keratinocytes were suspended for the indicated times. D. p21 / keratinocytes (top panel ) and p21 keratinocytes (bottom panel ) were suspended for 6 h. The top panel examines the composition of complex I, and the bottom panel examines the composition of complex II. Molecular Cancer Research 101 on April 9, 2017. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Consistent with these results, only a small percentage of cyclin A complexes contained p27 in suspended p21 / keratinocytes. Immunodepletion of p21 from extracts of p21 cells suspended for 6 or 12 h partially removed cyclin A complexes, whereas immunodepletion of both p21 and p27 removed almost all cyclin A complexes. Thus, both CKIs contribute to the formation of inactive cyclin A-cdk2 complexes in suspended wild-type keratinocytes. In this regard, p21 may be important at early times after suspension (i.e. , at times before p27 increases in abundance), whereas p27 is important at later times (i.e. , at times when p27 is abundant and p21 is not appreciably expressed). The data of Di Cunto et al . (36) show that enforced expression of p21 blocks the terminal stages of keratinocyte differentiation. Thus, the ‘‘p21 to p27 switch’’ may represent a means of keeping cdk2 inactive while allowing the completion of differentiation. How p21 modulates p27 abundance in keratinocytes is not known. p21 and p21 / keratinocytes express comparable amounts of p27 mRNA whether attached or suspended. cdk2 phosphorylates p27, which targets p27 for destruction in the preteasome (37–39). Thus, the greater amounts of p27 in p21 than in p21 / keratinocytes may be the indirect result of reduced amounts of cdk2 activity and consequent p27 stabilization. On the other hand, Carrano and Pagano (16) found that suspension of cycling fibroblasts accelerated p27 degradation in the absence of cdk2 activity; whether cdk2-independent p27 destruction required p21 was not determined. We have shown that wild-type keratinocytes withdraw from the cell cycle regardless of whether they are in G1, S, or G2-M at the time of suspension (29). Results of experiments using BrdUrd to tag S phase cells show that more p21 / cells exit S phase when placed in suspension than do p21 cells. Thus, loss of p21 appears to increase the capacity of keratinocytes to proliferate in suspension. Using a similar method, others (40) showed that ectopic expression of p21 prevented SAOS-2 cells from exiting S phase. p21 may arrest suspensioncultured keratinocytes in S phase arrest by inhibiting the activity of cdk2, as was shown in the SAOS-2 study (40), and/or by directly interfering with DNA replication (41). p21-null mice develop normally (42), thus indicating that p21 is not required for keratinocyte differentiation during development. However, our data suggest that loss of p21 impedes differentiation to some extent in suspension-cultured keratinocytes. We show that suspended p21 / keratinocytes express much less K1, a marker of differentiation, than do suspended p21 keratinocytes or p21 / keratinocytes ectopically expressing p21. A second differentiation marker, loricrin, increased in suspended p21 keratinocytes but not in p21 / keratinocytes. The differentiation-defective phenotype of p21 / keratinocytes may reflect their impaired capacity to growth arrest in suspension and/or to accumulate p27. We have shown that keratinocytes treated with p27 antisense oligonucleotides or derived from p27-null mice do not differentiate when placed in suspension (18, 43). Similar to our results, Missero et al . (44) found that loss of p21 reduced the expression of a set of differentiation markers in calciumtreated mouse keratinocytes. E2F-p107 complexes associate with cyclin E-cdk2 and cyclin A-cdk2 complexes in G1 and S, respectively (31–33, 45). Our data show that suspension of wild-type keratinocytes results in the removal of cyclin A from E2F-p107 DNA binding complexes, whereas suspension of p21-null keratinocytes does not. Similarly, others have shown that cyclin E and cdk2 associate with E2F-p107 complexes in attached but not suspended NIH 3T3 cells (10, 19) and that p21 releases cyclin-cdk2 complexes from E2F-p107 complexes in vitro (34, 35). The physiological relevance of cyclin-cdk2 interaction with E2F-p107 complexes is not known at present (46). However, it is clear that among its other effects, p21 modulates the composition of E2F-p107 complexes in primary keratinocytes. Although the p21 gene is rarely mutated in human tumors (47), p21 may be expressed at subnormal amounts or dysfunctional in some human tumors (48, 49). Importantly, inefficient expression of p21 is indicative of short survival times for patients with acute lymphoblastic leukemia or colorectal cancer (48, 50). We have shown that irradiated p21-null mice develop significantly more metastatic tumors than do irradiated wild-type mice (51). A key step in the metastatic process is the loss of adherent-dependent growth control (52). Thus, loss of p21 may contribute to tumor progression by increasing the capacity of cells to grow in suspension. Materials and Methods Cell Culture p21-null mice (from Dr. Tyler Jacks; 53) and p21wild type mice (The Jackson Laboratory, Bar Harbor, ME), both from the same mixed C57BL/6 and 129 genetic background, were used in our study. Keratinocytes were isolated from the epidermis of newborn mice as described previously (25, 54) and cultured in keratinocyte basal medium (KBM; Clonetics Corp., San Diego, CA) supplemented with 0.05 mM calcium, 0.5 Ag/ml insulin, 0.05 Ag/ml hydrocortisone, 1.5 ng/ml epidermal growth factor, 7.5 Ag/ml bovine pituitary extract, and 2% dialyzed FCS. Confluent 3-day-old keratinocytes were refed with KBM containing the above ingredients 12–15 h before suspension. Cells were suspended in 50-ml conical tubes in KBM containing 1.45% methylcellulose (Sigma, St. Louis, MO) and all of the above ingredients except epidermal growth factor. Preparation of Cell Extracts and Western Blotting Adherent keratinocytes were trypsinized from the plates, collected by centrifugation, and washed with KBM containing 5% serum to inactivate the trypsin. Keratinocytes suspended in methylcellulose were diluted 1/10 with PBS and collected by centrifugation. Cells were rinsed with PBS, resuspended in immunoprecipitation buffer [50 mM HEPES (pH 7.2), 150 mM NaCl, 0.1% Tween 20, 1 mM EDTA, 1 mM EGTA, 0.1 mM orthovanadate, 0.5 mM NaF, 0.1 mM phenylmethylsulfonyl fluoride, 2.5 Ag/ml leupeptin, and 1 mM DTT], and sonicated for 5 s. Insoluble material was removed by centrifugation. Cell extracts (25–50 Ag protein) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were p21 Modulates Cyclin A Expression in Keratinocytes 102 on April 9, 2017. © 2004 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from blocked in PBS containing 0.1% Tween 20 and 5% instant milk and incubated with primary antibody in PBS containing 0.1% Tween 20 for 1 h at room temperature. Proteins recognized by the primary antibody were detected by enhanced chemiluminescence using a horseradish peroxidase-coupled secondary antibody as specified by the manufacturer (Amersham Biosciences, Piscataway, NJ). In Vitro Kinase Assays Cell extracts (20–40 Ag of protein) were incubated with antibody to cyclin A for 2–10 h at 4jC and subsequently with protein A-agarose beads for 30 min at 4jC. Immune complexes were collected by centrifugation and washed twice with immunoprecipitation buffer and once with kinase buffer [50 mMHEPES (pH 7.5) and 10mMMgCl2]. Washed complexes were incubated for 5 min at 30jC in kinase buffer containing 0.5 ACi [g-P]ATP, 1 AM ATP, and 100 Ag/ml histone H1. Reaction mixtures were applied to SDS-polyacrylamide gels, and phosphoproteins were visualized by autoradiography. Detection of Cells Labeled With BrdUrd Cells received 4 AM BrdUrd for 2 h. Rinsed cell pellets were fixed in 60% cold ethanol and incubated in 0.04% pepsin (5 ml in 0.1 N HCl) for 40 min at 37jC. Cells were centrifuged, resuspended in 3 ml of 2 N HCl, and incubated for 20 min at 37jC. Cells were pelleted after addition of 6 ml of 0.1 N sodium borate to the samples, washed with PBS containing 0.1% Tween 20, and incubated in the dark for 1 h at room temperature in 0.2 ml of PBS containing 0.1% Tween 20 and 0.4 Al of BrdUrd-FITC antibody (Boehringer-Mannheim, Indianapolis, IN ). Cells were washed twice with PBS containing 0.1% Tween 20 and incubated for 25 min at room temperature in PBS containing 0.1% Tween 20, 0.05% BSA, 20 Ag/ml propidium iodide, and 50 Ag/ml RNase. Cell cycle distributions were determined using a Becton Dickinson