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Involvement of p27^Kip1 in the G₁- and S/G₂-phase Lengthening Mediated by Glucocorticoids in Normal Human Lymphocytes¹

Laboratoire de Cytologie Analytique, Faculté de Médecine, 69373 Lyon CEDEX 08 (JE 1879) [N. B., A. P., E. D., P. A. B., M. F.]; and Laboratoire d’Hématologie et de Cytogénétique, Hôpital Edouard-Herriot, 69003 Lyon [P. A. B., M. F.], France

	Abstract

TOP Abstract Introduction Results Discussion Materials and Methods References

 
Glucocorticoids inhibit cell proliferation by inducing cell cycle lengthening. In this report, we have analyzed, in normal peripheral blood lymphocytes, the involvement of p27^Kip1in this slowing of proliferation. Following dexamethasone (DXM) treatment, p27^Kip1 expression and regulation varied differently with the level of lymphocyte stimulation. In quiescent cells, DXM inhibited p27^Kip1 protein expression by decreasing its rate of synthesis, whereas its half-life and mRNA steady state remained constant. In contrast, in stimulated lymphocytes, DXM increased p27^Kip1 expression by enhancing its mRNA steady state. This increase is not only a consequence of the DXM-induced interleukin 2 inhibition: we also found an increase in p27^Kip1 mRNA stability that was not observed in quiescent lymphocytes. Cyclin/cyclin-dependent kinase (CDK) complexes immunoprecipitated with p27^Kip1 are differentially modified by DXM addition: (a) G₁ kinasic complexes (cyclin D/CDK4 or CDK6) associated with p27^Kip1 are strongly decreased by DXM, (b) S-phase complexes (CDK2/cyclin E and A) remained stable or increased, and (c) the association of p27^Kip1 with the phosphorylated forms of CDK1 is increased by DXM. In addition, CDK2 kinase activity was decreased in DXM-treated cells: we suggest that p27^Kip1 might participate in inhibiting its catalytic activity. These results indicated that, in normal lymphoid cells, p27^Kip1 may be involved in DXM antiproliferative effects. The increase of p27^Kip1 expression and a decrease in G₁ mitogenic factors, together with the redistribution of p27^Kip1 to S/G₂-M regulatory complexes, may explain the lengthening of G₁ and S/G₂ after DXM treatment in lymphocytes.

	Introduction

TOP Abstract Introduction Results Discussion Materials and Methods References

 
Glucocorticoids are frequently used for their antiproliferative activity in autoimmune diseases and in lymphoid malignancies, including leukemias. This action is based upon two kinds of phenomena: apoptosis induction (1) and cell cycle slowing (2) . Although the molecular mechanisms are poorly understood, several studies have reported a G₁ accumulation of treated cells (2, 3, 4) . Moreover, Fanger et al. (5) also observed, in DXM³ -treated HeLa S₃ cells, an increase in the G₂-M phase duration, and we recently described, in normal human lymphoid cells, a lengthening of S phase. These results indicate that glucocorticoid activity is closely associated with factors involved in G₁ regulation and suggest delayed control points during S phase and G₂-M.

Cellular proliferation is regulated by positive and negative signals. The primordial role played by the CDK1–CDK8 and their regulatory subunits, cyclins A–I, in cellular growth control is now well known. These kinases are negatively regulated by CKIs, which physically associate with cyclins, CDKs, or cyclin-CDK complexes. On the basis of the specificity of the CDK targets as well as on sequence homology, two distinct families of CKIs have been identified. The p16^Ink4 family competes with cyclin D for binding to CDK4 and CDK6, thereby negatively regulating kinase activity. p27^Kip1, along with p21^Waf1 and p57^Kip2, belongs to the second family of CKIs, which universally inhibit CDKs (6) . p27^Kip1 was first identified in cell-cell contact and TGF-ß G₁-arrested cells, which were incapable of assembling active complexes containing cyclin D/CDK4–CDK6 or cyclin E (or A)/CDK2 (7) . This CKI is induced by TGF-ß (7) , cAMP (8) , or vitamin D3 (9) , whereas IL-2 inhibits its expression in T lymphocytes (10 , 11) . Several levels of regulation have been reported: (a) a translational regulation by the ubiquitin pathway was described first (12) ; (b) a transcriptional control of p27^Kip1 mRNA was also reported (9 , 13) ; and (c) more recently, Millard et al. (14) described a third mechanism of p27^Kip1 regulation involving the association of p27^Kip1 mRNA with polyribosomes. Sequestration of p27^Kip1 by cyclin D also modulates its inhibitory activity on cyclin/CDK2 complexes on proliferating cells (7) . The importance of p27^Kip1 in regulating cell growth has been largely demonstrated and emphasized by the fact that p27^Kip1 -/- mice increased their body size associated with hyperplasia of multiple organs (15, 16, 17) . Furthermore, a low expression of p27^Kip1 is correlated with a poor prognosis in several human malignancies (18 , 19) . These results indicate that p27^Kip1 is an important target in growth regulation of normal cells and, perhaps, of their transformed counterparts.

The antiproliferative action of glucocorticoids, via their nuclear receptor, modulates the expression of genes involved in cellular activation such as c-myc in malignant lymphoid cell lines (20) or IL-2 in stimulated PBLs (21) . Their action on cell cycle regulatory proteins has been also described. Glucocorticoids down-regulate CDK4 at the transcriptional level (22) and inhibit cyclin D3 expression, probably by inducing a product that decreases the stability of its mRNA (23) . In contrast, these hormones up-regulate p21^Waf1 by inducing its mRNA in fibroblastic cells (24) or by increasing its mRNA stability in epithelial cells (25) . Our previous studies on normal human PBLs showed that glucocorticoids modified the expression of numerous cell cycle regulatory proteins. G₁ proteins (cyclin D3, CDK4, and CDK6) and G₁-S transition proteins (cyclins E and A and CDK2) were decreased under DXM treatment. After DXM exposure, whereas p21^Waf1 does not present significant variations in lymphocytes, p27^Kip1 expression varies in different ways according to stimulation: it decreases in quiescent lymphocytes and increases in stimulated lymphocytes.

The aim of this study was to define the level of action of glucocorticoids on p27^Kip1 expression according to PBL proliferation and to determine the role of this CKI in the DXM-induced cell cycle lengthening. We showed that glucocorticoids modified p27^Kip1 expression by acting on: (a) both mRNA steady state and stability in stimulated lymphocytes and (b) the rate of synthesis of the protein in quiescent lymphocytes. We also found that the G₁ and S/G₂ p27^Kip1-related protein complexes are differentially modified by the steroid. We, therefore, suggest that, in lymphocytes, p27^Kip1 is involved at different levels in the glucocorticoid induced cell cycle lengthening.

	Results

TOP Abstract Introduction Results Discussion Materials and Methods References

 
p27^Kip1 Regulation Varies with Cell Proliferation.
Before studying DXM effects on p27^Kip1, we first characterized, in normal PBLs, p27^Kip1 variations after PHA stimulation. As described in several studies (10) , p27^Kip1 presents a high level of expression in nonstimulated lymphocytes, so the protein decreases rapidly with the PHA-induced stimulation (Fig. 1A) . We measured the rate of [³⁵S]methionine incorporation into p27^Kip1 and the half-life of the labeled protein in unstimulated and stimulated lymphocytes. As shown in Fig. 1B , the rate of synthesis was identical in quiescent (4.8 ± 2.6 AU) and stimulated (4.7 ± 3.2 AU) lymphocytes. This result was also confirmed using a shorter time of incorporation, i.e., 30 min. As expected, p27^Kip1 half-life was shorter in PHA-stimulated than in unstimulated lymphocytes (Fig. 1C) . As for the mRNA, in opposition to other cellular model, Kip1 mRNA presented variations according to the level of cell proliferation (Fig. 2A) . p27^Kip1 mRNA was highly expressed in unstimulated lymphocytes (9.9 ± 6.1 AU) and then significantly decreased under stimulation by 40% (5.8 ± 3.9 AU) after 48 h of stimulation (n = 6, P = 0.031; Wilcoxon signed rank test). This increase appeared within the first 15 h of treatment, before any modification of G₁. Although the mRNA steady state was higher in quiescent than in stimulated lymphocytes, its stability appeared unchanged according to stimulation level and was estimated at 30 min (Fig. 2B) .

View larger version (40K): [in this window] [in a new window]   Fig. 1. p27Kip1 expression varies with stimulation. A, p27Kip1 protein expression was measured at the indicated times after the beginning of PHA stimulation. The percentage of cells in S phase was evaluated by flow cytometry. Cells were lysed, electrophoretically separated, and transferred to a nitrocellulose membrane immunoblotted with anti-p27Kip1 antibody. B, rate of synthesis of p27Kip1 in quiescent (Lanes -) and in stimulated cells (Lanes +) was measured after incubation for 30 and 120 min with [35S]methionine. Cells were lysed, and total proteins were immunoprecipitated with p27Kip1 antibody and with preimmune serum (Lane C) used as control. The labeled proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and exposed for autoradiography. C, half-life was measured after the [35S]methionine incorporation. After labeling, cells were cultured with medium containing nonradioactive methionine. At the indicated times, cells were harvested, lysed, and immunoprecipitated with p27Kip1 antibody. The labeled proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by autoradiography. D, the amount of labeled p27Kip1 was quantified and plotted on a scale.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Kip1 mRNA expression following PHA stimulation. A, cells were harvested at indicated times after the beginning of PHA stimulation. Twenty µg of total mRNA were separated and transferred to nylon membrane. Membranes were hybridized with p27Kip1 probe as well as with GAPDH probe to verify the loading. Comparison of p27Kip1 mRNA between unstimulated and 48-h PHA-stimulated lymphocytes was repeated six times by independent experiments and showed a statistically significant difference (P < 0.031). B, the study of p27Kip1 mRNA stability was performed after actinomycin D addition (10 µg/ml). Total RNA was extracted at the indicated times after addition of actinomycin D and processed for Northern blot analysis. The filters were then hybridized to a p27Kip1 probe. Representative ethidium bromide staining of the membrane is shown as a control for RNA loading.

DXM Acts on p27^Kip1 Translational Regulation in Quiescent Lymphocytes.
Proliferation was analyzed by flow cytometry, and the mean percentage of S-phase cells was <2% in both DXM-treated (1.9 ± 0.7%) and untreated (1.2 ± 0.5%) lymphocytes.

As shown in Fig. 3A , the addition of DXM in quiescent lymphocytes had no effect upon p27^Kip1 mRNA level. Analysis of mRNA stability after actinomycin D addition demonstrated that DXM treatment does not alter the p27^Kip1 mRNA half-life (Fig. 3B) . The inhibition of p27^Kip1 expression after DXM treatment of quiescent lymphocytes does not seem to be linked either to a transcriptional mechanism or to mRNA stability alteration. We then measured the rate of synthesis and the half-life of the protein after [³⁵S]methionine cell labeling and immunoprecipitation with anti-p27^Kip1 antibody (Fig. 4) . The half-life of p27^Kip1 protein did not seem to be influenced by DXM treatment (Fig. 4, C and D) . In contrast, whatever the duration of [³⁵S]methionine labeling (30 and 120 min), the rate of synthesis of p27^Kip1 protein in DXM-added cells (3.1 ± 2.2 AU) clearly decreased compared to untreated cells (8 ± 6 AU; Fig. 4B ). These results may indicate that, in quiescent lymphocytes, DXM exerts on p27^Kip1 a translational regulation that is not linked to the stability of the protein.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Kip1 mRNA in quiescent lymphocytes according to DXM treatment. Total RNA was extracted from quiescent lymphocytes untreated (0 µM) and treated for 15 h with 5 µM of DXM. A, expression of p27Kip1 mRNA was analyzed by Northern blotting using a specific cDNA probe. Hybridization with GAPDH was also carried out as a control of the loading. All assays were repeated at least three times. B, the study of p27Kip1 mRNA stability was performed after actinomycin D addition (10 µg/ml). Total RNA was extracted at the indicated times after addition of actinomycin D and processed for Northern blot analysis. The filters were then hybridized with the p27Kip1 probe. Representative ethidium bromide staining of the membrane is shown as a control for RNA loading.

View larger version (56K): [in this window] [in a new window]   Fig. 4. p27Kip1 protein expression in quiescent PBLs according to DXM treatment. PBLs were cultured in the presence (5 µM) or absence of DXM. A, after 15 h of DXM treatment, cells were harvested and lysed. Seventy µg of total proteins were separated on a 12% gel and transferred on a nitrocellulose membrane. p27Kip1 was detected by blotting membrane with p27Kip1 antibody. B, the rate of synthesis of p27Kip1 was measured after incubation for 30 or 120 min with [35S]methionine. Cells were lysed, and total proteins were immunoprecipitated with p27Kip1 antibody and with preimmune serum (Lane C) used as control. The labeled proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and exposed for autoradiography. C, the half-life was measured after metabolic labeling with 35S. Cells were cultured with medium containing nonradioactive methionine and DXM (5 µM for treated cells). At the indicated times, cells were harvested, lysed, and immunoprecipitated with p27Kip1 antibody. The labeled proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by autoradiography. D, the amount of labeled p27Kip1 was quantified and plotted on a scale.

We examined, in PHA-stimulated lymphocytes, the mechanisms underlying the previously observed induction of p27^Kip1 protein by DXM (26) . After 48 h of DXM and PHA treatment, lymphocytes were harvested, and the steady-state level of p27^Kip1 mRNA was assessed. DXM treatment led to an increase in the level of p27^Kip1 mRNA (Fig. 5A) . Its mRNA was significantly increased by 30% within the first 15 h of treatment and remained high as long as cells were exposed to the steroid: 6.7 ± 4.2 AU in untreated cells versus 9.8 ± 4.5 AU in treated ones (n = 10, P = 0.0020; Wilcoxon signed rank test). This increase appeared before any modification of the percentage of cells in G₁. Actinomycin D was added in the presence or absence of DXM in 48-h PHA-stimulated lymphocytes (Fig. 5B) . The p27^Kip1 mRNA half-life was increased in DXM-treated cells by 30 min (half-life was evaluated to be 1 h). To determine another possible translational level of action of glucocorticoids on p27^Kip1, we measured the turnover and the half-life of the protein after [³⁵S]methionine incorporation (Fig. 6) . The rate of synthesis of p27^Kip1 in DXM-treated cells (8.7 ± 4.6 AU) was unchanged compared to untreated stimulated lymphocytes (8 ± 4 AU), whatever the duration of methionine incorporation (15, 30, or 120 min). No significant differences of p27^Kip1 half-life between untreated and DXM-treated cells were observed (Fig. 6, B and C) .Therefore, p27^Kip1 accumulation, in response to glucocorticoid addition, appears to be modified at the mRNA level, with an increase of mRNA steady state and stability.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Kip1 mRNA in stimulated PBLs according to DXM treatment. Total RNA were extracted from PHA-stimulated lymphocytes untreated (0 µM) and treated with 5 µM of DXM. A, expression of p27Kip1 mRNA was analyzed by Northern blotting using a specific cDNA probe. Hybridization with GAPDH was also carried out as a control of the loading. Comparison of p27Kip1 mRNA between DXM-treated and untreated cells was repeated 10 times and showed a statistically significant difference (P < 0.002). B, p27Kip1 mRNA stability was studied after actinomycin D addition (10 µg/ml). Total RNA was extracted at the indicated times after addition of actinomycin D and processed for Northern blot analysis. The filters were then hybridized to a p27Kip1 probe. Representative ethidium bromide staining of the membrane is shown as a control for RNA loading.

View larger version (50K): [in this window] [in a new window]   Fig. 6. p27Kip1 protein expression in stimulated PBLs according to DXM treatment. DXM was added at the time of PHA stimulation. A, the p27Kip1 rate of synthesis of 48 h PHA-stimulated cells was measured after incubation for 15, 30, or 120 min with [35S]methionine. Cells were lysed, and an equal amount of proteins were immunoprecipitated with p27Kip1 antibody and with preimmune serum (Lane C) used as control. The labeled proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and exposed for autoradiography. B, the half-life was measured after the 35S metabolic labeling. Cells were cultured with medium containing nonradioactive methionine and DXM (5 µM for treated cells). At the indicated times, cells were harvested and lysed, and equal amounts of proteins were immunoprecipitated with p27Kip1 antibody. The labeled proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by autoradiography. C, the amount of labeled p27Kip1 was quantified and plotted on a scale.

In quiescent lymphocytes, in which p27^Kip1, cyclin D3, CDK4, and CDK6 expression was decreased after DXM addition (26) , p27^Kip1 was only found to be associated (albeit weakly) with CDK4. No cyclin D3 or CDK6 complexes were detected, even when lysis was effected with low-stringency buffer. This observation could be attributed to the weak level of expression of these proteins in quiescent cells. The results shown in Fig. 7A established that, after DXM addition, the level of CDK4 associated with p27^Kip1 was relatively unchanged.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Effect of DXM on p27Kip1 associated complexes in quiescent and in PHA-stimulated lymphocytes and on CDK2 kinase activity. A, quiescent cells were cultured in the absence (0 µM) or presence (5 µM) of DXM during 15 h and then they were harvested and lysed. Seven hundred µg of total proteins from DXM-treated or untreated cells were immunoprecipitated with anti-p27Kip1 antibody. The eluted immune complexes were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-CDK4 and anti-p27Kip1 antibody. B, Cells were stimulated with PHA and treated (5 µM) or untreated (0 µM) with DXM for 48 h. Then, they were harvested and lysed. Seven hundred µg of total proteins from DXM-treated or untreated cells were immunoprecipitated with anti-p27Kip1 antibody. The eluted immune complexes were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. Each experiment was repeated three times. Arrows represent the three CDK isoforms; the two upper bands correspond to phosphorylated forms. C, specific bands obtained in B after anti-p27Kip1 antibody immunoprecipitation and immunoblotting were measured by densitometry. Numerical values were given in AU. Columns, means; bars, SD. D, effect of DXM on CDK2-associated H1 kinase activity. PHA-stimulated cells for 48 h in the presence or absence of DXM, lysed, and then immunoprecipitated with anti-CDK2 antibody. The immunoprecipitates were tested for their kinase activity using histone H1 as substrate.

Taking into account that p27^Kip1 was increased after DXM treatment, we showed that the ratio of G₁ regulatory proteins to p27^Kip1, including CDK4, CDK6, and cyclin D3, presented an important decrease. These modifications may favor the binding of p27^Kip1 to S/G₂-M regulation proteins (cyclin A, cyclin E, CDK2, and CDK1).

Because CDK2 is required for S-phase entry and is an important target for p27^Kip1, we have determined the effect of DXM on this kinase by analyzing the activity of complexes implicating CDK2. We immunoprecipitated CDK2 from lysates of stimulated cells with or without DXM treatment and tested the associated kinase activity using histone H1 as a substrate (Fig. 7D) . CDK2 activity was decreased in DXM-treated cells.

	Discussion

TOP Abstract Introduction Results Discussion Materials and Methods References

 
Our results showed that, in normal human PBLs, DXM acts on p27^Kip1 following pathways, depending on the level of proliferation. In quiescent lymphocytes, DXM exerts a negative translational control of p27^Kip1 by reducing the rate of protein synthesis. In stimulated lymphocytes, DXM induces an increase in p27^Kip1 mRNA steady state and stability. Several modifications of protein complexes involving this CKI accompany growth inhibition induced by DXM. These observations provide evidence that p27^Kip1 is involved in the DXM antiproliferative effects by multiple mechanisms that may be better understood by the analysis of p27^Kip1 regulation, according to cellular proliferation.

Progression of quiescent cells into cell cycle required mitogenic factors as well as a second signal, such as IL-2 in lymphocytes, which inactivates the inhibitory action of p27^Kip1, allowing the restoration of the kinase activity of CDK2/cyclin complexes and entry of the cells into S phase (10 , 11) . p27^Kip1 regulation during proliferation induction appears to vary with the cellular types and organisms considered. Studies conducted with different cell types (skeletal muscle and HeLa cells) showed that the level of p27^Kip1 mRNA remains relatively constant (28 , 29) . However, in CD3-stimulated murine T lymphocytes, Kwon et al. (30) found that Kip1 mRNA decreased and suggested that IL-2 influences its promoter function. p27^Kip1 protein is highly expressed in quiescent cells and decreased following stimulation. It has been reported that, during cellular stimulation, the rate of protein synthesis was constant, whereas p27^Kip1 degradation, caused by ubiquitination, increased (12) . Our results on human lymphocytes also showed a decrease of p27^Kip1 protein half-life after stimulation, indicating that p27^Kip1 was subjected to a translational control, probably through the ubiquitin-proteasome pathway. Furthermore, we showed that p27^Kip1 mRNA decreased when cells were induced with PHA to enter the cell cycle, whereas its mRNA stability seemed unchanged. This decrease of the mRNA steady state could be due to an action of IL-2 on p27^Kip1 promoter, as suggested by Kwon et al. (30) .

Glucocorticoids act partly on T-lymphocyte proliferation through an inhibition of IL-2 transcription, a decrease in its mRNA stability, and a consequent decrease of IL-2 expression (21) . The IL-2 variations according to cellular stimulation may then be taken into account to analyze the different levels of action of DXM on p27^Kip1 regulation. In quiescent lymphocytes, which are devoid of IL-2, we observed an IL-2-independent DXM effect on p27^Kip1. Our data showed that, in these quiescent cells, glucocorticoids decreased the rate of p27^Kip1 protein synthesis without changing the amount and the stability of its mRNA, suggesting a posttranscriptional mechanism of regulation. In HL60 cell-lines, TPA increased the rate of p27^Kip1 protein synthesis by increasing the amount of p27^Kip1 mRNA in polyribosomes (14) . The mechanism regulating the association ribosome/mRNA is unclear: unknown factors might either enhance or decrease the ribosome association with p27^Kip1 mRNA. We suggest that, in quiescent cells, DXM down-regulates p27^Kip1 protein by this pathway. Furthermore, we show that glucocorticoids did not modify p27^Kip1 expression by the ubiquitin pathway because no modification of its half-life was observed.

In contrast, in stimulated lymphocytes, glucocorticoids may suppress or decrease the IL-2-induced p27^Kip1 down-regulation by inhibiting IL-2 production. Although p27^Kip1 was usually described as having a posttranscriptional regulation, an increase of p27^Kip1 mRNA was reported in U937 cells in response to vitamin D3 addition (9) . In the same way, neuronal differentiation induces p27^Kip1 mRNA expression (13) . A transcriptional regulation via IL-2 was then suggested for p27^Kip1, and this was also found in murine stimulated lymphocytes by Kwon et al. (30) . Our results on stimulated lymphocytes showed that DXM increased the amount of p27^Kip1 mRNA before any variation in the percentage of cells in G₁ was observed. The action of glucocorticoid treatment on IL-2 expression may partially explain these data. However, we also found that DXM enhanced p27^Kip1 mRNA half-life in stimulated lymphocytes. We hypothesize that de novo synthesis of intermediary proteins induced by DXM is required to increase mRNA stability. This modification of mRNA half-life may participate to the increase level of p27^Kip1 under DXM treatment. The modifications of mRNA half-life seem to be a frequent mechanism reported in glucocorticoid growth arrest induction. Cyclin D3 mRNA stability in lymphoid cells decreased after DXM treatment (23) , and as described in lung alveolar epithelial cells, p21^Waf1 mRNA stability was, on the contrary, increased by glucocorticoid treatment (24) . These results are in accordance with data, reported by Rogatsky et al. (31) , showing a differential action of the glucocorticoid receptor on cell cycle regulatory proteins according to cell metabolism. In this work, DXM-induced growth arrest in pRb-negative cells was due to a transcriptional activation of p21^Waf1 and p27^Kip1. In contrast, in pRb-expressing cells, a transcriptional repression of mitogenic factors (including CDK4, CDK6, and cyclin D3) was observed, whereas CKIs were unchanged. These authors suggested that, in cells expressing low levels of pRb, a combination of repression of mitogenic factors and activation of antimitogenic factors (CKIs) by glucocorticoid receptors may bring about growth arrest. Our experimental conditions seem to illustrate this intermediary situation.

Many observations suggest that p27^Kip1 is a key regulator controlling progression throughout the cell cycle. A common feature of growth arrest cells was the increased level of p27^Kip1 protein. This increase was accompanied by modifications of cyclin/CDK complexes involving p27^Kip1 (32) . Moreover, the variations of cyclins and CDKs after DXM treatment may modify the composition of the complexes. We observed that (a) G₁ cell cycle regulatory proteins (cyclin D3, CDK4, and CDK6) associated with p27^Kip1 were strongly decreased, whereas (b) the linkage of G₁-S transition proteins to p27^Kip1 remained stable or increased (cyclin A, cyclin E, and CDK2) and the overall expression of cyclins A and E decreased significantly after DXM addition (26) . After DXM treatment, p27^Kip1 may be redistributed from cyclin D3/CDK4 or CDK6 to proteins acting later during S phase (cyclins E and A and CDK2), as was also described in TGF-ß- (33) and lovastatin-treated cells (34) . The kinase activity of CDK2 on DXM-treated cells was decreased: we hypothesize that p27^Kip1 might participate to inhibit its catalytic activity after DXM addition. Furthermore, the increase of the binding of p27^Kip1 to phosphorylated forms of CDK1 in DXM-treated cells compared to untreated ones may explain the glucocorticoid-induced lengthening of G₂-M phases (5) : i.e., p27^Kip1 contains a CDK1-inhibitory domain located in the NH₂-terminal region (35) . In stimulated lymphocytes, whatever the mechanisms involved, an increased amount of p27^Kip1 appears to be available for the inhibition of kinasic complexes of S/G₂-M phases. The inhibitory effect of p27^Kip1 on these regulatory kinases, therefore, seems to be increased by a stoichiometric effect, as described Polyak et al. (7) . All these variations of p27^Kip1-associated proteins induced by DXM may explain the observed lengthening of cell cycle phases.

In conclusion, our results showed that the mechanisms of p27^Kip1 regulation in normal lymphoid cells vary with cellular metabolism, either with the level of cell stimulation and/or with the action of biological modulators such as DXM treatment in our model. Furthermore, in stimulated lymphocytes, we found that DXM may favor p27^Kip1 growth inhibition capability, associated with both an increase in p27^Kip1 expression and a decrease of G₁ mitogenic factors. The redistribution of p27^Kip1 to S/G₂-M regulatory complexes may explain the lengthening of late phases of cell cycle after DXM treatment in lymphocytes.

	Materials and Methods

TOP Abstract Introduction Results Discussion Materials and Methods References

 
Cellular Material.
Normal human lymphocytes were isolated from heparinized peripheral blood from normal subjects on Ficoll-Hypaque gradient (d = 1.077; Seromed, Biochem KG, Roskilde, Denmark) by centrifugation at 300 x g for 20 min. These cells were grown in RPMI 1640 supplemented with 10% FCS (Life Technologies, Inc., Paisley, United Kingdom), 200 mM L-glutamine (Seromed), 100 IU/ml penicillin, and 50 µg/ml streptomycin (Seromed), and then stimulated with PHA (Murex, Diagnostics, Dartford, United Kingdom)-1 µg/ml RPMI 1640 for 48 h. The study was also performed on quiescent isolated PBLs, as described previously. Viability was tested by trypan blue staining.

DXM Treatment.
To study the action of DXM (Sigma Chemical Co., St. Louis, MO) on lymphocyte proliferation, we cultured cells in medium supplemented with 5 µM DXM. This dose was shown to be active and has been used by several authors (36 , 37) . DXM treatment was added just at the time of PHA stimulation and cells were harvested, when necessary, 15, 24, 36, and 48 h after the beginning of treatment. To analyze a potential direct action of DXM on p27^Kip1 (independent of the inhibition of proliferation), we studied the action of DXM on unstimulated PBLs, 15 h after the beginning of treatment. For each time, a PBL sample without DXM treatment (0 µM) was used as an internal reference.

Flow Cytometry DNA Analysis.
For all samples, flow cytometry analysis of cellular DNA content was performed on cell suspensions. Ethanol-fixed cells were stained with propidium iodide after RNase A III (Sigma) treatment. Stained cells were then analyzed on a Coulter flow cytometer. Percentages of cells in G₀/G₁, S, and G₂-M phases of cell cycle were then calculated on the basis of DNA distribution histograms with the software provided by the manufacturer.

RNA Extraction and Northern Blot Analysis.
Total RNA of DXM-treated and untreated lymphocytes was extracted by an acid guanidium thiocyanate-phenol-chloroform-derived method (38) using RNA Plus (Bioprobe Systems). RNA (20 µg) was then resolved by electrophoresis in a formaldehyde-1.2% agarose gel and blotted onto nylon membrane filter by capillary action. The human p27^Kip1 probe, kindly provided by Dr. K. Polyak (Memorial Sloan-Kettering Cancer Center, New York, NY), was labeled with [³²P]dCTP using the Rediprime DNA Labeling System (Amersham, Aylesbury, United Kingdom). Hybridization was carried out overnight at 42°C, after which membranes were washed to a stringency of 2x SSC at 20°C and 2x SSC-0.5% SDS and 0.2x SSC-0.5% SDS at 65°C for 30 min each. Hybridization was followed by autoradiography on Biomax MS films with specific intensifying screen (Eastman Kodak Company, Rochester, NY) at -70°C. The membranes were also sequentially hybridized with GAPDH probe (Clontech, Palo Alto, CA) as a control of equivalence of the loading. p27^Kip1 mRNA of DXM-treated and untreated lymphocytes was quantified using EDAS software from Kodak. Numerical values of sample signals were given in AU.

Kip1 mRNA Stability.
PBLs after DXM treatment (0 and 5 µM) were incubated with 10 µg/ml actinomycin D (Sigma). Cells were harvested at various times after actinomycin D addition (30 min and 1, 2, and 3 h), and Kip1 mRNA was assessed by Northern blot analysis with a Kip1-specific probe, as described previously.

Immunoprecipitation Assays.
To study the level of action of DXM on p27^Kip1 expression in stimulated or unstimulated PBL, we analyzed the turnover and stability of p27^Kip1 by metabolic labeling with [³⁵S]methionine.

For measurement of the rate of synthesis, cells were incubated for 1 h in methionine-free MEM supplemented with 10% dialyzed fetal bovine serum and 300 µCi/ml [³⁵S]methionine were added for various times (15 min to 2 h). After washing in PBS, cells were lysed in lysis buffer (buffer I) consisting of 0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 1% NP40, and protease inhibitors (10 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 µg/ml tosyl phenylalanyl chloromethyl ketone, 0.3 µg/ml benzamidine, and 75 µg/ml phenylmethylsulfonyl fluoride, all from Sigma). Lysates (equal protein amount) were precleared by the addition of preimmune rabbit serum for 30 min, followed by two incubations with protein A beads. Lysates were then incubated overnight with 1 µg of antibody anti-p27^Kip1 (Santa Cruz Biotechnology, Santa Cruz, CA). Immune complexes were collected on protein A beads and washed three times in lysis buffer. The beads were resuspended in 50 µl of 2x Laemmli sample buffer and boiled for 4 min. The [³⁵S]methionine-labeled precipitates were fractionated by electrophoresis in a 12% SDS-polyacrylamide gel. Gels were then transferred to nitrocellulose membrane in a semidry system, and membranes were exposed to Amersham ß Max films at -70°C. Controls were prepared with (a) preimmune serum instead of antibody and (b) antibody anti-p27^Kip1 preblocked by an excess of control peptide as competitor.

For measurement of p27^Kip1 half-life, after the [³⁵S]methionine labeling, cells were washed and incubated in RPMI 1640 [containing an excess of nonradioactive methionine (100 mg/liter), 1 µg/ml PHA for stimulated lymphocytes, and 5 µM DXM for treated cells] at 37°C up to 18 h. Cells were collected at several times by centrifugation and washed in PBS. Cells were then lysed and immunoprecipitated, as described previously. Following autoradiography, bands corresponding to p27^Kip1 were quantified by densitometry using the EDAS software and data were used to calculate the half-life of the p27^Kip1 protein according to the formula: T_1/2 = 0.3t/log(D1/D2).

Analysis of p27^Kip1-related Protein Complexes.
To detect modifications of cyclin-CDK complexes associated with p27^Kip1 after DXM addition, we performed immunoprecipitations followed by immunoblotting procedures. DXM-treated as well as untreated lymphocytes were harvested, washed in PBS, and lysed in buffer I, as described above, or in buffer II [low stringency; 50 mM HEPES (Sigma; pH 7.2), 150 mM NaCl, 0.1% Tween 20, 1 mM EDTA, and 1 mM EGTA].

(a) Protein complexes were immunoprecipitated, as described previously, with anti-p27^Kip1, anti-cyclin D3, anti-cyclin E, anti-CDK6, anti-CDK2, or anti-CDK1 antibodies electrophoretically separated and transferred to nitrocellulose membrane as described previously.

(b) Seventy µg of whole protein lysates were also precipitated by acetone, resolved on SDS-PAGE, and subjected to Western blotting for comparisons. To verify the equivalence of the loading, a labeling of ß-actin was performed.

Membranes were blocked with a 5% solution of nonfat milk in PBS containing 0.5% Tween 20 and probed for 1 h with the following antibodies: mouse monoclonal anti-p34^cdc2 (1:500), anti-cyclin E (1:250), rabbit polyclonal anti-cdk2 (1:500), anti-cdk4 (1:2000), anti-cdk6 (1:1000), anti-cyclin D3 (1:250), and anti-p27^Kip1 (1:1000), all from Santa Cruz Biotechnology (Santa Cruz, CA); and mouse monoclonal IgE anti-cyclin A (1:1000), from PharMingen (San Diego, CA). Membranes were then incubated with either biotinylated rabbit antibody to mouse immunoglobulins (Dako), biotinylated goat antibody to rabbit immunoglobulins or biotinylated rat anti mouse immunoglobulin E (Calbiochem, Birmingham, AL) for cyclin A. The final step was incubation with streptavidin-biotinylated horseradish peroxidase complex (Amersham) and development with an enhanced chemiluminescence detection system. The specific bands of each proteins studied were quantified using EDAS software, and the numerical values were given in AU. All assays were repeated at least three times.

Kinase Assays.
Forty-eight-h PHA-stimulated lymphocytes, treated or untreated with DXM, were washed in PBS and lysed as described above in radioimmunoprecipitation buffer supplemented with antiproteases and phosphatase inhibitors (50 mM NaF, 0.5 mM Na₃VO₄, and 8 mM ß-glycerophosphate). Samples corresponding to 700 µg of total proteins were immunoprecipitated with anti-CDK2 antibody, as explained previously. After incubation with protein A-Sepharose, beads were washed then resuspended in 50 µl of kinase buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 1 mg of histone H1, and 74 µM ATP (10 µCi of [-³²P]ATP) and left to phosphorylate for 20 min at 37°C. Samples were finally denatured in 12.5 µl of 4x Laemmli buffer and analyzed by 12% SDS-PAGE. Gels were stained, dried, and autoradiographed.

	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 The work was financially supported by Hospices Civils de Lyon and the CNRS (Center National de Recherche Scientifique). We are also grateful to the "Ligue contre le Cancer" (Rhône, Ardèche, Saône et Loire, Drôme) and the "Association pour la Recherche sur le Cancer" for their financial help.

² To whom requests for reprints should be addressed, at Laboratoire d’Hématologie et de Cytogénétique, Hôpital Edouard-Herriot, Place d’Arsonval, 69003 Lyon, France. Phone: 33 04 72 11 73 58; Fax: 33 72 11 73 05.

³ The abbreviations used are: DXM, dexamethasone; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; TGF-ß, transforming growth factor-ß; IL-2, interleukin 2; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin; AU, arbitrary unit(s).

Received for publication 11/10/98. Revision received 3/ 4/99. Accepted for publication 4/ 9/99.

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