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Cell Growth & Differentiation Vol. 13, 421-430, September 2002
© 2002 American Association for Cancer Research

Early Cycling-independent Changes to p27, Cyclin D2, and Cyclin D3 in Differentiating Mouse Embryonal Carcinoma Cells1

Helena Preclíková, Vítezslav Bryja, Jirí Pacherník, Pavel Krejcí, Petr Dvorák and Ales Hampl2

Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague [V. B., P. D., A. H.]; Centre for Cell Therapy and Tissue Repair, Charles University, 150 18 Prague [V. B., J. P., P. D., A. H.]; and Mendel University Brno, 613 00 Brno [H. P., J. P., P. K., P. D., A. H.], Czech Republic


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Changes to cell cycle-regulating machinery that occur during differentiation of cells are thought to be responsible mostly for withdrawal from cycling. Here, embryonal carcinoma (EC) cell lines were found that differ in their basal levels of p27 inhibitor of cyclin-dependent kinases but not in their growth rates, distribution of cells in phases of cell cycle, and their ability to differentiate. High basal levels of p27 did not substitute for up-regulation of p27 that in EC cells normally occurs early after entering a differentiation pathway. Under both standard and differentiation-supporting culture conditions, variances in the levels of p27 were strictly followed by variances in the levels of cyclins D2 and D3. In EC cells genetically manipulated to overexpress p27 protein, cyclin D3 became up-regulated and vice versa. Supposedly, titration of p27 by D-type cyclins, which prevents its inhibitory action toward cyclin-dependent kinase 2, allows for the maintenance of elevated p27 in proliferating EC cells. Increased levels of p27 in early embryonal cells thus may, at least in certain phases of embryo development, serve a differentiation-associated, rather than proliferation-associated, function.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In all multicellular organisms, appropriate progression of ontogenesis depends largely on precise coordination of cell proliferation and differentiation. To drive their proliferation, eukaryotic cells use members of the family of CDKs.3 Various mechanisms have evolved to regulate the activity of CDKs. These include their heterodimerization with a cyclin subunit to form holoenzyme, changes in phosphorylation, and interactions with regulatory molecules commonly called CKIs. In general, cells that acquire a terminally differentiated phenotype simultaneously interrupt their cycling. According to cell type, such differentiation-associated withdrawal from the cell cycle can be mediated by any of several mechanism(s), including down-regulation of G1 cyclins, up-regulation of CKIs, and down-regulation of CDKs themselves.

Although in some cell types differentiation and growth are mutually exclusive events (1) , in others differentiation occurs concomitantly with progression through the cell cycle (2) . During embryogenesis, specifically in its earliest phases, significant phenotypic changes occur in cells that are intensively proliferating. Early pluripotent embryonic cell lineages have their in vitro propagatable counterparts in ES and EC cells that are both derived from preimplantation embryos (3, 4, 5) . When they are properly manipulated, both ES and EC cells are able to follow multiple differentiation pathways in vitro (6 , 7) . RA is potent inducer of differentiation in many cell types (8 , 9) . In both ES and EC cells, RA-induced differentiation is accompanied by profound alterations to organization of the cell cycle-regulating molecular machinery. Simultaneously, the cell cycle characteristics significantly change, which manifests primarily by slowing down proliferation of these cells. On the basis of their work on mouse ES cells, Savatier et al. (10) suggested that such early changes that take place in pluripotent cells correspond with adopting a thoroughly new level in controlling the progression through the cell cycle. This sophistication of regulatory mechanisms is supposed to underlie the typical lengthening of the G1 phase that results in: (a) normalization of the cell cycle; (b) occurrence of responsiveness to mitogenic and/or antimitogenic signals; and (c) loss of cancerous nature of EC and ES cells.

Corresponding to the scenario described above, studies addressing the behavior of various cell cycle regulators in differentiation-committed EC cells confirmed that the most prominent change is the occurrence of D-type cyclins and p27 CKIs (11, 12, 13, 14) . Still, some exceptions from this rule, specifically synthesis of cyclins D and p27 in nondifferentiated EC cells, were also observed (13, 14, 15) . It emerged recently that changes in the amount of CKIs during the course of differentiation might serve some other function(s) besides simply preventing further cell proliferation via the inhibition of CDKs. Specifically, p27 was suggested to operate by such cell cycle-unrelated way, e.g., in differentiating/differentiated oligodendrocytes, cardiac myocytes, keratinocytes, and luteal cells (16, 17, 18, 19) . Notably, when the accumulation of p27 was inhibited by p27 antisense oligonucleotides in P19 EC cells, the expression of differentiation-related markers was prevented (11) . Thus, an increased amount of p27 protein observed in differentiating EC cells might also, at least in its early phase, mediate the occurrence of phenomena that are not directly linked to modification of cell cycle progression. To allow for such behavior, cells necessarily need to possess the molecular mechanism(s) protecting their CDKs from being inhibited by high amounts of p27.

This study was undertaken to further investigate how the amount of p27 CKI relates to the transition from nondifferentiated to differentiating status in pluripotent cells of embryonal origin. For this purpose, we took advantage of the existence of several P19-derived cell lines (20) that we found to differ from each other in basal levels of this regulator. We demonstrate that the amounts of p27 and D-type cyclins themselves cannot determine the presence or absence of the pluripotent phenotype of EC cells, and that coregulation of their quantities may serve some specific differentiation-associated function, at least in such embryonal cell types.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
EC Cell Lines May Differ in Basal Levels of p27.
As shown previously by others, p27 protein is not detectable in nondifferentiated EC cells, and it starts to accumulate only upon the induction of differentiation in culture. To first confirm a general significance of this phenomenon, several P19-derived cell lines that differ from each other by the expression of membrane oligosaccharides (for a detailed characterization, see "Materials and Methods") were assayed in initial experiments. The original P19 EC cell line, here referred to as X.1, was also included. The basal level of p27 was determined by Western blot in cells of five EC lines that were kept for 48 h from seeding in culture under standard conditions that do not support differentiation. Surprisingly, the cell lines differed among each other in their p27 contents (Fig. 1)Citation . The amount of p27 protein varied in the range of about 10–15-fold, with the lowest amount being close to a limit of detectability. It is well established that elevated p27 may represent the reaction of cells on increased culture density. Thus, we excluded this possibility as an intrinsic property of the cell, and the observed differences between EC cell lineages stem from their different reactivity to culture density; the amount of p27 was determined at 6, 24, and 48 h after plating. Two EC cell lines from our panel, here referred to as A.1 and 1.3, containing the lowest and the highest amount of p27, respectively, were analyzed. As shown in Fig. 2Citation , the ratio between p27 amounts in EC lines remained unchanged (Fig. 2A)Citation , irrespective of the actual density of cells (Fig. 2B)Citation . Also, besides being refractory to the actual cell density, the extent of differences in p27 amount did not change with long-term passaging (not shown), which further documents the stability of this phenomenon.



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Fig. 1. Expression of p27 protein in nondifferentiated cells of several EC cell lines. Cells grown under standard conditions were harvested, and the proteins were extracted. Equal amounts of total protein of each cell line were separated on 10% SDS-PAGE and Western analyzed using anti-p27 antibody. To control the loading, the blot was reprobed using anti-mitogen-activated protein kinase antibody. Data are representative of at least three independent replicates.

 


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Fig. 2. The effect of cell density on the expression of p27 in nondifferentiated EC cells. Cells of A.1 and 1.3 lines were grown under standard conditions for 6, 24, and 48 h, respectively. A, cells were harvested, and the proteins were extracted. Equal amounts of total protein of each cell line were separated on 10% SDS-PAGE and Western analyzed using anti-p27 antibody. B, cells were pictured at the time of harvesting to document their density on dish. Data are representative of at least three independent replicates.

 
The Basal Level of p27 Influences Neither Proliferation Characteristics Nor Differentiation Status of EC Cells.
On the basis of our current understanding, we hypothesized that the differences in p27 levels translate into differences in cell cycle characteristics, the capacity of cells to differentiate, or both. A.1 and 1.3 EC cells were chosen to test this hypothesis. The karyotype and sequence of p27 cDNA were determined and found to be normal in both these EC cell lines. Normal growth rates were determined using samples of cells taken at 24, 48, 72, and 96 h after plating. EC cells are sensitive to high density, and they tend to die rather than to reach steady state. Therefore, for this analysis, cells were seeded at a density as low as 2.5 x 103 cells/cm2. A representative result is shown in Fig. 3ACitation , documenting that both cell lines follow typical exponential growth curves without being markedly different from each other. The distribution of cells in particular phases of the cell cycle was determined by flow cytometry in samples taken 48 h after seeding (Fig. 3B)Citation . In both lines, ~60% of cells were in S-phase, which is typical for nondifferentiated cells of embryonal origin. Altogether, manyfold differences in the level of p27, to our surprise, are not mirrored by major differences in the basal regulation of the proliferation of A.1 and 1.3 EC cells under normal conditions. It is well proven that, irrespective of other culture specifics, treatment with RA causes virtually all EC cells in culture to enter a differentiation program. Differentiation is typically accompanied by suppression of brachyury and up-regulation of Pax-6 transcription factors (21 , 22) . To evaluate differentiation potentials of A.1 and 1.3 EC cells, expression of both brachyury and Pax-6 was determined by reverse transcription-PCR in cells that were continuously exposed to 10-6 M RA for 48 h. As expected, although no Pax-6 message was found in nondifferentiated controls, cells treated with RA generated a strong Pax-6 signal (Fig. 4)Citation . The inverse dynamics was, as appropriate, found for brachyury(Fig. 4)Citation . Most importantly, there was no major difference between A.1 and 1.3 lines in their ability to properly regulate brachyury and Pax-6 messages.



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Fig. 3. The parameters of proliferation of A.1 and 1.3 EC cells. EC cells of both lines were seeded in the same densities (2.5 x 103 cells/cm2) and cultured under nondifferentiating conditions for 24, 48, 72, and 96 h, respectively, and then harvested for analyses. A, growth rates. Cells were lysed in SDS-containing buffer, and the total amounts of protein were used as a measure of cell quantities. Micrograms of total protein are shown on the Y axis. A representative result is shown. B, the distribution of cells in cell cycle phases. Cells were fixed in Vindelov’s solution, stained with propidium iodide, and analyzed using FACSCalibur equipped with ModFit 2.0 software. The percentages of cells in G1, S, and G2 phases are expressed as the means of three independent experiments; bars, SE.

 


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Fig. 4. The changes in expression of brachyury and Pax-6 in differentiating EC cells. Both A.1 and 1.3 EC cells were cultured for 48 h with and without 10-6 M RA and then lysed for RNA isolation. Equal amounts of total RNA were reverse transcribed, and the resulting cDNAs were PCR amplified using brachyury- and Pax-6-specific primer pairs. The resulting PCR products were separated on agarose gel and visualized by staining with ethidium bromide. Data are representative of at least three independent replicates.

 
Cyclins D2 and D3 Are Elevated in EC Cells with a Higher Level of p27.
The data described above suggested the existence of molecular mechanism(s) that make EC cells behave independently of variances in the amount of p27. To approach their identification, attention was initially focused on the key partners of p27, including CDK2, CDK4, CDK6, cyclins A2, E, D1, D2, and D3, as regulators that might potentially compensate for the quantitative differences in p27. The levels of these molecules in normally proliferating A.1 and 1.3 EC cells were determined by Western blot analysis. It was found that both EC lines were indistinguishable in the amounts of all three CDKs, cyclin A2, and cyclin D1 (Fig. 5)Citation . On the other hand, profound differences in the quantities of cyclins D2, D3, and E were invariably observed under the same conditions (Fig. 5)Citation . Specifically, although the level of cyclin E was ~2-fold lower in 1.3 compared with A.1 EC cells, the levels of cyclins D2 and D3 followed the pattern similar to that of p27, both being about 10–13-fold higher in 1.3 than in A.1 EC cells.



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Fig. 5. Expression of G1-S regulators in A.1 and 1.3 EC cells. Exponentially growing cells of both lines were harvested 48 h after seeding, and the proteins were extracted. Equal amounts of total protein of each cell line were separated on 10% SDS-PAGE and Western analyzed using the appropriate antibody. Data are representative of at least three independent replicates.

 
High Levels of p27 Further Increase in EC Cells during Differentiation.
Both p27 and D-type cyclins are known to become synthesized by ES and EC cells, along with their entering a differentiation pathway. However, here it appeared that high abundance of these regulators does not have to be necessarily in major conflict with the maintenance of a nondifferentiated status in mouse EC cells. On the basis of these two facts, further attention was focused on the phenomenon of coexpression of p27 and D-type cyclins and their relation to the process of differentiation. We first asked whether expected differentiation-associated changes to quantities of these regulators are influenced by their basal levels found in nondifferentiated cells. The amounts of p27, cyclins, and also CDKs, as above, were determined in both A.1 and 1.3 cells that were cultured with and without 10-6 M RA for 48 h. As shown in Fig. 6ACitation , although the amount of cyclin D1 remained unchanged, the levels of p27, cyclin D3, and cyclin D2 in RA-treated cells were markedly increased compared with nontreated controls. Densitometric analysis revealed that the RA-induced increase of p27 and cyclin D3 was about five times higher in A.1 cells than in 1.3 cells. When these changes, however, are expressed as an absolute rather than relative increase, it becomes obvious that A.1 and 1.3 cells are in fact much more similar in the amounts of accumulated p27 and cyclin D3 (Fig. 6B)Citation . Because the same results were obtained when lysing cells directly in SDS-containing buffer (not shown), all of these findings obviously reflect the real differences in total amounts and not the different solubilities of these regulators. Besides down-regulation of cyclin E, no changes to other regulators were observed under differentiation-inducing conditions.



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Fig. 6. The changes in expression of G1-S regulators in differentiating EC cells. Both A.1 and 1.3 EC cells were cultured for 48 h with and without 10-6 M RA and then extracted for protein analysis. Equal amounts of total protein of each cell line were separated on 10% SDS-PAGE and Western analyzed using the appropriate antibody. A, regulators visualized by chemiluminescence. B, densitometric analysis of p27 and cyclin D3 data shown in A. Arbitrary units are shown on the Y axis. Data are representative of at least three independent replicates.

 
Only Negligible Inhibition of Kinase Activities of CDK2 and CDK4 Accompanies the Early Accumulation of p27 in Differentiating EC Cells.
Inhibiting the activity of CDK2, a major driving force of S-phase, is the key role of up-regulated p27 CKI. Therefore, it was obvious for us to ask whether p27 may act similarly during early phases of RA-induced differentiation of EC cells. On the basis of our data on p27 accumulation, the cells of both EC lines were exposed to RA for 48 h as described above. Then the cells were harvested and subjected to a CDK2 kinase assay. Although the activity of CDK2 in nontreated cells seemed to reflect the basal levels of p27 in A.1 and 1.3 EC cells, no obvious down-regulation of CDK2 activity was found in differentiating EC cells when compared with normally proliferating controls in either cell line (Fig. 7)Citation . In other words, differentiation-associated up-regulation of p27 CKI did not result in lowered activity of CDK2. Realizing that CDK4 instead of CDK2 might be a target for p27 in EC cells, the activity of CDK4 was also determined. Although the activities of CDK4 were different between EC cell lines (A.1 and 1.3), there was almost no down-regulation of CDK4 associated with p27 accumulation in either cells when they were induced to differentiate by RA (Fig. 7)Citation . Corresponding to such failure of p27 to inhibit CDK2 and CDK4, only minor changes in both proliferation rate and the distribution of cells in cell cycle phases were noticeable in EC cells that were treated with RA for 48 h (Fig. 8)Citation .



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Fig. 7. Comparison of the kinase activities associated with CDK2 and CDK4 in control and differentiating A.1 and 1.3 EC cells. EC cells were cultured for 48 h with and without 10-6 M RA, and then proteins were extracted. Equal amounts of total protein were entered into each CDK2- and/or CDK4-immunoprecipitation reaction. The activities of isolated CDK2 and CDK4 were assayed using the appropriate substrate (histone H1 and GST-pRb) and visualized by autoradiography. Data are representative of at least three independent replicates.

 


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Fig. 8. The parameters of proliferation in control and differentiating A.1 and 1.3 EC cells. EC cells of both lines seeded in the same densities (2.5 x 103 cells/cm2) were cultured with and without 10-6 M RA for 24, 48, 72, and 96 h, respectively. A, growth rates. Cells were lysed in SDS-containing buffer, and the total amounts of protein were used as a measure of cell quantities. Micrograms of total protein are shown on the Y axis. Data are presented as the means of three independent experiments; bars, SE. B, the distribution of cells in cell cycle phases. Cells were fixed in Vindelov’s solution, stained with propidium iodide, and analyzed using FACSCalibur equipped with ModFit 2.0 software. The percentages of cells in G1, S, and G2 phases are expressed as the means of three independent experiments; bars, SE.

 
Significant Amounts of p27 Associate with Cyclins D2 and D3 in EC Cells, and These Amounts Increase upon Their Entering Differentiation.
As mentioned above, the action of p27 and cyclins is realized via their physical associations with CDKs. To address whether accumulating p27 and/or cyclins D2 and D3 participate in formation of molecular complexes with each other and CDKs, a series of immunoprecipitation analyses was carried out using lysates from both control and RA-treated cells. Each molecule, including CDK2, CDK4, and p27, was precipitated and probed for associating partners, cyclins D2 and D3, and p27. In general, the results of these analyses, as summarized in Fig. 9Citation , have shown that: (a) the quantities of p27 and both cyclins complexed with relevant kinases are proportional to their absolute amounts present in cells; (b) physical associations exist between p27 and each of the D-type cyclins; and (c) RA-induced accumulation of p27 and both cyclins translates into increased amounts of the given complexes. In other words, both A.1 and 1.3 EC cells have a certain amount of their p27 and cyclins D2 and D3 complexed with CDK2 and/or CDK4, and this amount further increases upon entering a differentiation program.



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Fig. 9. Interactions of cell cycle regulators in control and differentiating A.1 and 1.3 EC cells. EC cells were cultured for 48 h with and without 10-6 M RA, and then proteins were extracted. Immunoprecipitation reactions were carried out using the equal amounts of total protein and the appropriate antibodies. Immunoprecipitates (IP) were electrophoresed on 10% SDS-PAGE and Western blotted using primary antibody against the appropriate associating partner. Data are representative of at least three independent replicates. A, p27 and CDK4. B, CDK2.

 
Artificial Up-Regulation of p27 in EC Cells Brings About the Elevation of Cyclin D3 and Vice Versa.
The experiments described above pointed to a striking correlation between quantities of p27 and certain D-type cyclins. Because this coregulation took place in differentiating cells without markedly affecting their proliferation, we were interested in what might be the nature of such molecular interaction. To approach this question, two constructs carrying p27 and/or cyclin D3 cDNA under the control of cytomegalovirus promoter were generated, and each of them was introduced into both A.1 and 1.3 EC cells. Transfectants with an empty vector were also prepared to serve as the controls. Several independent clones were established for each EC cell line and vector, and all of them were analyzed by Western blot for the overexpression of p27 and/or cyclin D3. Clones with the expression appropriate to the vector used were further analyzed, and the representative results are shown in Fig. 10Citation . In summary, elevating the amount of p27 protein brought about the increase of cyclin D3 protein and vice versa. The occurrence of such interrelationship between p27 and cyclin D3 is independent of the basal levels of these proteins because this phenomenon was equally well pronounced in both A.1 and 1.3 cells.



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Fig. 10. The effect of overexpression of p27 on the level of cyclin (cyc) D3 and vice versa in EC cells. Both A.1 and 1.3 EC cells were transfected with either p27 or cyclin D3 construct. EC cell clones that were found to overexpress the appropriate molecule were further grown under standard conditions, and Western blot was analyzed for the amount of potentially coregulated p27 or cyclin D3. A, EC cells overexpressing p27. B, EC cells overexpressing cyclin D3. C, negative control; EC cells transfected with empty vector. At least three independent overexpressing clones were analyzed for each molecule.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The road from a totipotent phenotype to a differentiated cell phenotype is very complex, and the significance of cell cycle regulatory molecules for passing through this program is poorly understood. In this study, we have focused on the early molecular events implicated in the differentiation of mouse EC cells. It can be concluded that in EC cells: (a) the high levels of p27 CKI and D-type cyclins themselves do not necessarily produce changes to their growth and differentiation status; (b) differentiation-inducing conditions cause profound alterations to the amounts of p27, cyclin D2, and cyclin D3, yet did not affect the progression of the cell cycle; and (c) differentiation-associated changes in gene expression occur without a prior inhibition of cell growth. Still, the most novel finding in our study is that mouse EC cells possess a molecular mechanism that links the levels of p27 and cyclin D3, and that this linkage operates in a bidirectional manner.

Studies on ES and EC cells have established the model in which switching from a "primitive" growth-regulatory mechanism to much more sophisticated one is a key component of entering a differentiation program in embryonal cells (10 , 12) . This upgrade is mediated by a new expression of several regulators, including D-type cyclins, p27, pRb, and possibly others, which all together allow for the G1-S transition to be regulatable by mitogenic and antimitogenic signals (11 , 13, 14, 15 , 23) . Still, the presence of appreciable levels of some D-type cyclins in various nondifferentiated mouse and human EC cells may suggest the existence of a larger complexity to this general "level-based" scenario (13, 14, 15 , 23 , 24) . This notion that high levels of the partners of CDK4/6, D-type cyclins, and p27 do not have to be associated with the commitment to differentiation in embryonal cells is also further supported here. Although the amounts of all cyclin D2, cyclin D3, and p27 in 1.3 cells exceeded those in A.1 cells by severalfold, no major modifications to their growth and to the expression of differentiation markers, brachyury and Pax-6, were observable in 1.3 cells compared with A.1 cells. In other words, EC cells are able to retain their highly proliferative and multipotent phenotype despite being equipped with the key molecular components of the G1-S-regulating pathway.

As mentioned above, in EC cells growing under nondifferentiating conditions, higher amounts of p27 did not translate into a lower proliferation rate. p27 is implicated in inhibiting proliferation in many cell types under various growth-restraining conditions mainly via its effect on the activity of CDK2. Unexpectedly, A.1 and 1.3 EC cells differed not only by the total level of p27 but also by the amount of p27 associated with CDK2 and by the activity of this kinase. Rather than on its accumulation in the cell, the inhibitory function of p27 may also be dependent on its redistribution from CDK4/cyclin D to CDK2/cyclin E (25) . Here we found that although the 1.3 EC cell line is typical in that it contains a high level of p27 under standard culture conditions, this level becomes further increased upon the differentiation-inducing treatment with RA, and the absolute amount of the accumulated p27 is about the same as in the "p27 low-expressing" A.1 cell line. Thus, because RA-induced changes to EC cells include up-regulation of p27 regardless of the basal level of this CKI, obviously accumulation of p27, rather than its redistribution, is required for this function to take place. Although certain parts of newly synthesized p27 became associated with both CDK2 and CDK4, no inhibition of its kinase activities was observed in either EC line at 48 h after the beginning of RA treatment. Correspondingly, in both A.1 and 1.3 EC lines the significant RA-induced retardation of cell growth took place well after this time point. Such paradoxical behavior of given regulators was observed previously in differentiating mouse and human EC cells (13 , 15) . Thus, taking our data and the data of others together, it is likely that p27 may or may not impose its inhibitory action according to the actual proliferation and/or differentiation status of EC cells. Unfortunately, technical constrains prevented us from analyzing in more detail the amounts of p27-associated and p27-nonassociated CDKs. Thus, whether this behavior is attributable to the changes in balance between p27/CDK2 interactions mediated by LFG versus FY motifs described by Blain et al. (26) or attributable to some other unknown mechanism(s) remains to be determined.

Besides the enormous variability in p27 CKIs discussed above, EC cell lines studied here also exerted variability in two D-type cyclins, cyclins D2 and D3. Moreover, there was a noticeable parallelism in the amounts of all three regulators, with a higher level of p27 always being correlated with a higher level of both D-type cyclins. This was observed both in normally proliferating A.1 and 1.3 EC cells and in the same cells induced to differentiate by RA when all three regulators were simultaneously up-regulated. Specifically, the last finding pointed to a certain kind of cross-talk between p27 and D-type cyclins, the existence of which was further evaluated here by separately overexpressing p27 and/or cyclin D3 in both A.1 and 1.3 EC lines. In this system, artificial elevation of p27 has led to up-regulation of cyclin D3 and vice versa, irrespective of the original amount of the particular molecule in nontransfected EC cells. Although our study is the first one documenting co-inducibility of p27 and cyclin D in cells of embryonal origin, several studies of others pointed to a similar phenomenon in various cancer cell lines (27 , 28) . At the time of completion of this study, Bagui et al. (29) described a new model of relationship between p27 and/or p21 and cyclin D3/CDK4. They conclude that only a nearly imperceptible portion of total cellular cyclin D3/CDK4 dimers, which are free of any CKI, exert enzymatic activity, whereas the majority of cyclin D3/CDK4 complexes present in the cell is held inactive via their association with p27 and/or p21. On the basis of these findings, we suggest that the majority of cyclin D3/CDK4 complexes is in fact devoted to sequestration of p27 and p21 CKIs from CDK2. Importantly, such a "p27-disabling" strategy seems to be used not only by various types of human cancers (30, 31, 32, 33) , but this strategy is also involved in some developmental processes (16) . Of major significance for interpreting the results of this study is that both p27 and D-type cyclins were found previously to possess activities that are unrelated to their function as regulators of CDKs (18 , 19 , 34, 35, 36, 37) . Assuming that in proliferating EC cells high levels of p27 are necessary for some differentiation-related processes to take place, as has been already suggested by Sasaki et al. (11) , it is tempting to hypothesize that titrating of p27 by D-type cyclins/CDK4 complexes fulfills this requirement. In other words, we propose that coordinated molecular changes to p27 and cyclins D2 and D3 described here are primarily to allow for the maintenance of high levels of p27 and possibly also of given cyclins, under proliferation-supporting conditions during the early phases of embryonal cell differentiation. To gain further insight into these phenomena, experiments have already started to unravel the mechanism that underlies the coregulation of p27 and cyclin D3. Although we realize that the amount of p27 and D-type cyclin proteins in a cell can be controlled at various levels, including level of gene transcription (38 , 39) , mRNA stability (40 , 41) , translation (42, 43, 44) , and proteolysis (45, 46, 47) , we currently hypothesize that it is primarily a higher stability of complexed p27 and cyclin D3 that is responsible for such behavior.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines, Cell Culture, and Treatment with RA.
The P19S1801A1, P19X1, P19XT.1.1, P19ST.1.3, and P19ST.1.5 cell lines, here referred to as A.1, X.1, 1.1, 1.3, and 1.5, respectively, were kindly provided by Dr. Petr Draber (Institute of Molecular genetics, Academy of Sciences of the Czech Republic). Both A.1 and X.1 represent sublineages of the original P19 EC line that were obtained by either selecting for the resistance to ouabain and 6-thioguanine (A.1) or passaging in living mouse (X.1). The remaining three lines were derived from the A.1 lines (1.3 and 1.5) and X.1 line (1.1) by selection for the absence of TEC-1 carbohydrate epitope (20) . The expression of TEC-1, TEC-2, and TEC-3 carbohydrate epitopes is as follows: 1.1 (TEC-1-, 2-, 3-); 1.3 (TEC-1-, 2+, 3+); and 1.5 (TEC-1-, 2+/-, 3+). Importantly, all these cell lines grow in the same way as parental cells, are able to differentiate, and are able to form tumors in nude mice that are composed almost exclusively of cells with the morphology of EC cells (20) . All EC cells were routinely cultured on gelatinized Petri dishes in DMEM supplemented with 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µM 2-mercaptoethanol under standard conditions (5% CO2, 37°C, 95% humidity). RA was prepared as 5 mM stock solution in ethanol and then diluted in culture medium to a final concentration of 1 µM. Cells were supplied with fresh RA-containing medium in 24-h intervals.

Growth Rate and Cell Cycle Analyses.
The increase in total cellular protein was used as a measure of cell growth. Cells on culture dish were washed twice with PBS (pH 7.2) and lysed in 100 mM Tris/HCl (pH 6.8), 20% glycerol, and 1% SDS. Protein concentrations in lysates were determined by a DC Protein Assay kit (Bio-Rad, Hercules, CA), and then they were used for calculating total protein/culture. For the cell cycle analysis, cells were harvested by trypsinization, washed with PBS (pH 7.2), fixed in 70% ethanol, and stained by incubation with Vindelov’s solution [10 mM Tris buffer (pH 8), 0.7 mg/ml RNase, 50 µg/ml propidium iodide, 0.1% Triton X-100, and 10 mM NaCl; Ref. 48 ] for 30 min at 37°C. For each measurement, 1.5 x 104 cells were collected and analyzed by flow cytometry (FACSCalibur;, Becton-Dickinson, San Jose, CA; 488 nm laser beam for excitation). Single cells were identified and gated by pulse-code processing of the area and the width of the signal. Cell debris was eliminated by appropriately raising the forward scatter threshold. The percentages of cells in the individual cell cycle phases were analyzed using ModFit 2.0 software (Verify Software House, Topsham, ME).

Antibodies, DNAs, and Reagents.
Rabbit polyclonal antibody to human CDK2, which cross-reacts with the mouse homologue (sc-163); rabbit polyclonal antibody to mouse CDK4 (sc-260); goat polyclonal antibody to human CDK4, which cross-reacts with the mouse homologue (sc-601); rabbit polyclonal antibody to human CDK6, which cross-reacts with the mouse homologue (sc-177); rabbit polyclonal antibody to human cyclin A2, which cross-reacts with the mouse homologue (sc-751); rabbit polyclonal antibody to rat cyclin E, which cross-reacts with the mouse homologue (sc-481); mouse monoclonal antibody to mouse cyclin D1 (sc-450); rabbit polyclonal antibody to mouse cyclin D2, which cross-reacts with the mouse homologue (sc-593); and rabbit polyclonal antibody to human p27, which cross-reacts with the mouse homologue (sc-528), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to mouse p27 (K25020) was purchased from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibody against rat mitogen-activated protein kinase, which cross-reacts with the mouse homologue (#9102), was purchased from New England Biolabs (Beverly, MA). Mouse monoclonal antibody against a COOH-terminal part of human cyclin D3, which cross-reacts with the mouse homologue (DCS-22), and a substrate for the kinase assay, GST [GST-pRb (773–928)], were generously provided by Dr. Jiri Lukas (Danish Cancer Society, Copenhagen, Denmark). cDNAs of mouse p27 and cyclin D3 were generously provided by Drs. Joan Massagué (Sloan Kettering Institute, New York, NY) and Charles Sherr (St. Jude Children’s Research Hospital, Memphis, TN), respectively. Expression plasmid pEGFP-C1 was purchased from Clontech (Palo Alto, CA). Restriction and modifying enzymes were purchased from MBI Fermentas (Vilnius, Lithuania), Roche Diagnostics (Mannheim, Germany), and Promega Corp. (Madison, WI). Taq DNA polymerase and reverse transcriptase were purchased from TopBio (Prague, Czech Republic). RNeasy mini kit was purchased from Qiagen (Valencia, CA). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or Fluka (Buchs, Switzerland). Anti-immunoglobulins and Protein A agarose beads were from Sigma; polyvinylidene difluoride membrane Hybond-P, [32P]ATP, and chemiluminescence detection reagents (ECL+Plus) were purchased from Amersham (Aylesbury, United Kingdom).

Immunochemical Analyses.
Cells were washed twice with PBS (pH 7.4), trypsinized, pelleted, and lysed for 30 min in ice-cold lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM sodium chloride, 0.5% NP40, 1 mM EDTA, 0.1 mM dithiotreitol, 50 mM sodium fluoride, 8 mM ß-glycerophosphate, 100 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml tosylphenylalanine chloromethane]. Then lysates were cleared by centrifugation at 15,000 x g for 15 min at 4°C and stored at -80°C until use. Concentrations of total protein were determined using a DC Protein Assay kit (Bio-Rad). Extracts were equalized for total protein and then used for Western blot analyses, immunoprecipitations, and kinase assays. For Western analysis, samples were mixed with double-strength Laemmli sample buffer, boiled for 5 min, and subsequently applied onto 10% SDS-PAGE. After being electrotransferred onto Hybond-P membrane, proteins were immunodetected using appropriate primary and secondary antibodies and visualized by ECL+Plus reagent according to the manufacturer’s instructions. When required, membranes were stripped in 62.5 mM Tris/HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol, washed, and reblotted with another antibody from this selection. For immunoprecipitation, extracts were first subjected to initial absorption with protein A agarose beads and then incubated with appropriate antibodies for 1 h in an ice bath. Immunoprecipitates were collected on protein A agarose beads by overnight rotation, washed five times with lysis buffer, resuspended in double-strength Laemmli sample buffer, boiled for 5 min, and applied onto 10 and 12.5% SDS-PAGE for p27 and D-type cyclin detection. Films that resulted from Western blot and immunoprecipitation analyses were scanned (ULTRA-LUM, Paramount, CA), and the signals were quantified using the Intelligent Quantifier (Bio Image, Ann Arbor, MI) when appropriate.

Kinase Assays.
Appropriate CDK was first immunopurified from cell extracts as described for immunoprecipitation, except that the last two washes were done using kinase buffer containing 50 mM HEPES (pH 7.5), 10 mM magnesium chloride, 10 mM manganese chloride, 8 mM ß-glycerophosphate, and 1 mM DTT. Immune complexes were collected by centrifugation and used directly for the assay. For CDK2, kinase reactions were carried out for 30 min at 37°C in a total volume of 25 µl of kinase buffer supplemented with 100 µg/ml histone H1 (type III-S; Sigma) and 40 µCi/ml [32P]ATP. For CDK4, kinase reactions were carried out for 30 min at 30°C in a total volume of 25 µl of kinase buffer supplemented with 80 µg/ml GST-pRb and 40 µCi/ml [32P]ATP. Reactions were terminated by cooling in an ice bath, followed by mixing with double-strength Laemmli sample buffer. Total reaction mixes were boiled for 5 min and subjected to 10% SDS-PAGE, followed by autoradiography.

Reverse Transcription-PCR Analysis.
Cells were washed twice with PBS, trypsinized, and pelleted, and total RNA was isolated using RNeasy mini kit according to the manufacturer’s instructions. Random-primed cDNA was synthesized from 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase. The primer pairs used for specific PCR amplification were as follows: brachyury(forward, 5'-GAGAGAGAGCGAGCCTCCAAAC-3'; reverse, 5'-GCTGTGACTGCCTACCAGAATG-3', 230bp); and Pax-6(forward, 5'-TGCCCTTCCATCTTTGCTTG-3'; reverse, 5'-TCTGCCCGTTCAACATCCTTAG-3', 178bp). PCR reactions were performed using Taq DNA polymerase as follows: brachyury(94°C/30 s, 56°C/30 s, 72°C/30 s; 29 cycles); and Pax-6(94°C/30 s, 54°C/30 s, 72°C/30 s; 33 cycles). The products were subjected to 2% agarose electrophoresis and visualized by ethidium bromide staining.

Plasmid Construction and Cell Transfection.
Mouse p27 cDNA was cut of the plasmid EXlox(+)-mp27-FL using EcoRI and ApaI and cloned into pNeoI (derived from pEGFP-C1 by removing EGFP fragment with NheI and XhoI, blunt ending, and religating) that resulted in pNeop27. pNeoCycD3 was constructed by cloning mouse cyclin D3 cDNA into the EcoRI site of pNeoI. The expression of p27 and cyclin D3 cDNAs, respectively, as driven by cytomegalovirus promoter in eukaryotic cells, was confirmed by transient transfection into quail fibroblast QT6, followed by Western blotting. A.1 and 1.3 EC cells were transfected using the Ca-precipitation method (49) with 10 µg of either pNeop27 or pNeoCycD3. Transfected cells were then selected in medium containing 400 µg/ml of G418, cloned, and screened for the expression of appropriate protein by Western blotting.


    Acknowledgments
 
We are grateful to Drs. Draber, Lukas, Massagué, and Sherr for the cells and reagents, to Milan Esner for communicating brachyury and Pax-6 amplification conditions, to Dr. John J. Eppig for his comments on the manuscript, and to Iveta Nevriva for technical assistance.


    Footnotes
 
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.

1 This research was supported in part by Grant GA 204/01/0905 from the Grant Agency of the Czech Republic, Grant AV 0Z5039906 from the Academy of Sciences of the Czech Republic, and Grants MSM 432100001 and LN 00A065 from the Ministry of Education, Youth, and Sports. Back

2 To whom requests for reprints should be addressed, at Department of Molecular Embryology, Mendel University Brno, Zemedelská 1, 613 00 Brno, Czech Republic. Phone: (+420-5)45133298; E-mail: hampl{at}mendelu.cz Back

3 The abbreviations used are: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; EC, embryonal carcinoma; ES, embryonic stem; RA, all-trans retinoic acid; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase. Back

Received for publication 8/ 2/01. Revision received 6/24/02. Accepted for publication 6/24/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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