Stem Cell Factor Inhibits Erythroid Differentiation by ... - CiteSeerX

Vol. 11, 269 –277, May 2000

Cell Growth & Differentiation

Stem Cell Factor Inhibits Erythroid Differentiation by Modulating the Activity of G1-Cyclin-dependent Kinase Complexes: A Role for p27 in Erythroid Differentiation Coupled G1 Arrest1 Ami Tamir,2,3 Teresa Petrocelli,2 Kendra Stetler, Wendy Chu, Jeff Howard,4 Brad St. Croix, Joyce Slingerland, and Yaacov Ben-David5 Department of Medical Biophysics, University of Toronto, Cancer Biology Research, Sunnybrook, and Women’s College Health Science Centre, Toronto, Ontario, M4N 3M5 Canada

cell cycle arrest that involves p27 is part of a broader molecular program during HB60-5 erythroid differentiation. Moreover, we suggest that SCF stimulation of erythroblasts, in addition to inhibiting erythroid differentiation, activates parallel or sequential signals responsible for maintaining cyclin/cdk activity.

Abstract

Introduction

Terminal erythroid differentiation is accompanied by decreased expression of c-Kit and decreased proliferation of erythroid progenitor cells. Using a newly established erythroleukemia cell line HB60-5, which proliferates in response to erythropoietin (Epo) and stem cell factor (SCF) and differentiates when stimulated with Epo alone, we characterized several events associated with the cell cycle during erythroid differentiation. Forty-eight h after SCF withdrawal and Epo stimulation, there was strong inhibition of cyclindependent kinase (cdk) 4 and cdk6 activities, associated with an increase in the binding of p27 and p15 to cdk6. A significant increase in the binding of p27 to cyclin E- and cyclin A-associated cdk2 correlated with the inhibition of these kinases. In addition, the expression of c-Myc and its downstream transcriptional target Cdc25A were found to be downregulated during Epo-induced terminal differentiation of HB60-5 cells. The loss of Cdc25A was associated with an increase in the phosphotyrosylation of cyclin E-associated cdk2, which may contribute to cell cycle arrest during differentiation. Although overexpression of p27 in HB60-5 cells caused G1 arrest, it did not promote terminal erythroid differentiation. Thus, the

Erythropoiesis is the process by which erythroid progenitor cells undergo terminal differentiation, resulting in the generation of RBCs. Regulation of this process is governed by a complex set of factors, notably hematopoietic growth factors and transcription factors (1). In the past two decades, the molecular mechanisms underlying erythroid differentiation have been studied extensively. However, many of these studies have used a chemical induced differentiation model, whereby Friend virus-transformed murine erythroleukemic (MEL) cell lines are induced to differentiate in response to DMSO or related hybrid polar compounds (2). In the present study, we used HB60-5, an erythroblastic cell line that proliferates in the presence of SCF6 and Epo but undergoes differentiation when grown in the presence of Epo alone (3). Epo-induced differentiation of HB60-5 cells is associated with a number of morphological changes that are typically detected during normal erythropoiesis (3). This includes the appearance of distinctive basophilic and orthochromatic normoblasts and more mature normoblasts, all of which display condensed nuclei and reduced cytoplasmic volume. After three days of Epo treatment in the absence of SCF, there is a dramatic increase in the number of mature enucleated erythrocytes. In contrast, HB60-5 cells grown in the presence of both SCF and Epo display an undifferentiated, blastic phenotype. The ability of HB60-5 cells to undergo differentiation in response to specific growth factors in the culture medium provides an excellent in vitro model for erythroid differentiation. Previous studies suggest that SCF, IL-3, and GM-CSF play important roles in the generation and self renewal of erythroid progenitors. However, analysis of mice with null mutations in either GM-CSF or IL-3 receptor genes indicate that GM-CSF and IL-3 are not crucial for erythropoiesis, or that other factors can compensate for their function (4 – 6). In contrast, mice deficient in either SCF or its receptor (c-Kit)

Received 12/1/99; revised 2/15/00; accepted 3/20/00. 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 work was supported by grants from the National Cancer Institute of Canada (to Y. B-D. and J. M. S.). A. T. was supported by a fellowship from the Sunnybrook Trust for medical research. J. S. and Y. B-D. are supported by Cancer Care Ontario. 2 Equal contributions to this work were made by these authors. 3 Present address: Department of Pathology, University of Michigan School of Medicine, 1500 East Medical Center, Ann Arbor, MI 481090940. 4 Present address: HULC Cell and Molecular Laboratory, London Wound Healing Group, Lawson Research Institute, St. Joseph’s Health Centre, 268 Grosvenor Street, London, Ontario, N6A 4V2 Canada. 5 To whom requests for reprints should be addressed, at Department of Medical Biophysics, University of Toronto, 2075 Bayview Avenue, S-216, Toronto, Ontario, M4N 3M5 Canada. Phone: (416) 480-6100, extension 3359; Fax: (416) 480-5703; E-mail: [email protected].

6 The abbreviations used are: SCF, stem cell factor; Epo, erythropoietin; GM-CSF, granulocyte/macrophage-colony stimulating factor; cdk, cyclindependent kinase; HMBA, hexamethylene bisacetamide; PKC, protein kinase C; pRb, retinoblastoma protein; IL, interleukin; PVDF, polyvinylidene difluoride.

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Cell Cycle Inhibition during Erythroid Differentiation

suffer from severe anemia (7). A more severe phenotype is displayed by mice deficient for Epo or its receptor (Epo-R). These genotypes are embryonic lethal because of massive apoptosis of fetal liver colony forming units-erythroid (8 –10). Thus, SCF/c-Kit and Epo/Epo-R represent key signaling pathways required for the proliferation, differentiation, and survival of committed erythroid progenitor cells. The molecular mechanisms whereby erythroleukemic cells differentiate in response to hybrid polar compounds, like DMSO or HMBA, are poorly defined. One proposed mechanism involves PKC activation, because depletion of PKC prevents HMBA-induced erythroleukemia differentiation (11). Other studies implicate changes in the phosphorylation status of pRb and decreased cdk4 protein levels as being crucial to HMBA-induced differentiation of erythroleukemic cells (12). In the case of HB60-5 cells, very little is known about the differentiation signals evoked by Epo stimulation and SCF withdrawal. It has long been recognized that erythroid progenitor cells lose their self-renewal potential as they differentiate in association with exit from the cell cycle (2). Therefore, signaling pathways that regulate cell cycle progression through the cdks likely play an important role in this process (reviewed in Ref. 13). The cdks are themselves regulated at multiple levels. They may be regulated by activating and inhibitory phosphorylation events, by binding of specific cyclin molecules and by the binding of inhibitory subunits (14). Two families of cdk inhibitors are known to negatively regulate cdk activity (6, 15). The first, the KIP family, consists of p21Cip/WAF1 (p21), p27Kip1 (p27), and p57kip2 (p57). All of these cdk inhibitors interact with a wide range of cyclin-cdk complexes. The second family, termed the INK4 family, includes pl5INK4B (p15), pl6INK4A (p16), p18INK4C (p18), and p19INK4D (p19). INK4 family members act specifically on cyclin D-dependent kinases, cdk4 and cdk6. The cdk complexes, once activated, have been shown to phosphorylate various substrates (reviewed in Ref. 16), including the retinoblastoma tumor suppressor gene product (pRb; Refs. 17–20). pRb negatively regulates the cell cycle (21) when hypophosphorylated during G0 and early Gl, and pRb is progressively phosphorylated during mid- to late Gl (22–25). In this hypophosphorylated state, pRb functions as a transcriptional repressor. The most well-studied molecular target of pRb is the E2F family of transcription factors. Binding of pRb to E2Fs suppresses the activation of E2F target genes, which are essential for DNA replication. Phosphorylation of pRb during mid- to late Gl is accompanied by the release of E2F from the E2F-pRb complex and activation of the E2F-regulated genes (17, 26, 27). In the present study, we characterized the activities and composition of cdk2, cdk4, and cdk6 complexes during SCF withdrawal and Epo-induced differentiation of HB60-5 cells. Our findings suggest that SCF and Epo alter the composition and activities of the different cyclin-cdk complexes. Although p27 is implicated in G1 arrest of differentiated HB60-5 cells, ectopic overexpression of p27 was not sufficient to induce terminal erythroid differentiation in the presence of SCF.

Fig. 1. Up-regulation and down-regulation of differentiation genes in HB60-5 cells in response to Epo⫹/SCF⫺ stimulation. HB60-5 cells (5 ⫻ 106) were grown in the presence of Epo or Epo ⫹ SCF for the indicated times. Total RNA (20 ␮g) prepared from these cells was subjected to Northern blot analysis, transferred to nitrocellulose, and sequentially hybridized with cDNA probes for c-Myc, ␣-globin, and finally glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for equalization of loading.

Results SCF Withdrawal Leads to Cyclin-cdk Inhibition and Blocks G1-to-S Phase Progression in HB60-5 cells. The newly established erythroleukemia cell line, HB60-5, possesses the unique characteristic of undergoing Epo-induced terminal differentiation when SCF is removed from the culture medium. Upon SCF withdrawal, Epo induces alterations in the expression of a number of erythroid-specific genes (3), most notably the induction of ␣-globin (Fig. 1). Epo stimulation and withdrawal of SCF also resulted in growth arrest and down-regulation of c-Myc expression (Fig. 1). Differentiation of erythroid precursors is usually accompanied by withdrawal from the cell cycle. Therefore, we investigated the influence of Epo after SCF withdrawal on the cell cycle in the HB60-5 cell line (referred to hereafter as Epo⫹/ SCF⫺). Asynchronously growing cultures of HB60-5 cells containing Epo and SCF were washed and transferred to fresh media without SCF. Cells were collected at intervals thereafter, and the cell cycle profile was analyzed by flow cytometry. As shown in Fig. 2A, SCF withdrawal from HB60-5 cells led to an increase in the percentage of cells in G1 and a decrease in the percentage of S phase. This G1to-S phase block was maximal at 48 h. Because SCF is a growth factor known to protect cells from apoptosis (28, 29), we examined cells for internucleosomal DNA cleavage, a characteristic of apoptosis. As shown in Fig. 2B, there was an increase in the proportion of cells undergoing apoptosis between 24 and 72 h after transfer to Epo⫹/SCF⫺. Flow cytometry also revealed a sub-G1 population between 24 and 72 h after addition of Epo⫹/ SCF⫺ (Fig. 2A). At 72 h, a large fraction of cells had com-


Fig. 2. The effect of Epo⫹/SCF⫺ stimulation on HB60-5 cell cycle distribution and apoptosis. A, asynchronously growing HB60-5 cells were transferred to medium containing Epo alone. Cells were recovered at the indicated time points, fixed in 70% ethanol, and stained with propidium iodide before fluorescence-activated cell sorter analysis. The vertical axis indicates the percentage of cells in G1 (f), S (Œ), or G2-M () from the total cells that did not suffer DNA degradation or loss of their nucleus. Bars, SE. B, HB60-5 cells were recovered at the indicated time points after Epo stimulation, and apoptosis was detected by DNA purification and resolution on 2% agarose electrophoresis followed by ethidium bromide staining and visualization with UV. Arrow, position of the 500-bp marker.

Fig. 3. Epo stimulation inhibits cdk activities in HB60-5 cells. Cell extracts were prepared from asynchronous HB60-5 cells at the indicated times after Epo stimulation as described in “Materials and Methods.” Total cellular extracts (200 ␮g) were immunoprecipitated with antibodies against cdk4, cdk6, cyclin A, and cyclin E. The associated kinase activities from these immunoprecipitates were assayed using GST-pRb (for cdk4 and cdk6) or histone H1 (for cdk2 associated with cyclin A or cyclin E) as substrate. Uptake of radioactivity in substrate at the different times was expressed as a percentage of activity found in asynchronous HB60-5 cells growing with SCF ⫹ Epo (indicated as 0 h after Epo stimulation).

pleted differentiation (3). The mechanism whereby certain cells undergo differentiation, whereas other clonally related cells undergo apoptosis in response to Epo stimulation, is not known and merits further investigation. To better understand the molecular mechanisms whereby Epo⫹/SCF⫺ medium inhibits G1-to-S phase progression, we assayed cdk4, cdk6, and cdk2 activities. As shown in Fig. 3, there was inhibition of immunoprecipitable cdk4 and cdk6 activities, consistent with G1 arrest. A reduction in immunoprecipitable kinase activity associated with cyclin E and A also occurred within 48 h of Epo stimulation (Fig. 3). Our

previous work has shown that this cell cycle arrest is associated with a loss of pRb phosphorylation (3). Changes in Cell Cycle Regulation in HB60-5 Cells during Epo-induced Differentiation. To further investigate the mechanisms leading to cyclin-cdk inactivation during G1 arrest of HB60-5 cells, we examined the levels of cell cycle regulatory proteins. Cell lysates were prepared at different times after Epo⫹/SCF⫺ stimulation, and cell cycle regulators were analyzed by immunoblotting. The levels of the cdks did not change during the course of HB60-5 differentiation (Fig. 4). Cyclin D2 and Cyclin D3 protein levels increased

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Fig. 4. The effect of Epo⫹/SCF⫺ stimulation on cell cycle-regulated proteins. Total cell extracts (30 ␮g) prepared from HB60-5 cells grown in the presence of Epo and absence of SCF for the indicated times were separated using SDS-PAGE. The proteins were transferred to PVDF membranes and immunoblotted with specific antibodies. Proteins were visualized by use of a secondary horseradish peroxidase-conjugated antiimmunoglobulin antibody and enhanced chemiluminescence technology.

slightly in the first 24 h after Epo⫹/SCF⫺ stimulation but declined sharply after 48 h (Fig. 4). Cyclin D1 expression was not detected in HB60-5 cells as reported previously for other erythroleukemic cells (30). Cyclin E and A levels were not significantly altered during the first 48 h after Epo⫹/SCF⫺ stimulation. Although down-regulation of the D-cyclins during differentiation of HB60-5 cells may contribute to the inhibition of cdk4 and cdk6, this event occurred late and may result from rather than induce the observed G1 arrest. Analysis of the protein expression of cdk inhibitors was also carried out during Epo-induced differentiation of HB60-5 cells. After 12 h of Epo stimulation and SCF withdrawal, there was a strong increase in p15 expression, followed by a reduction in its expression level by 48 h. Both p21 and p16 levels fell during HB60-5 differentiation. In contrast,

p27 levels were significantly increased (Fig. 4). p27 has been shown to inhibit cdk2, cdk4, and cdk6 (29 –31). It also appears to be an important regulator of differentiation in various tissues (31–35). Changes in the composition of the G1 cyclin-cdk complexes were assayed by immunoprecipitation (Western) at various intervals during differentiation of HB60-5 cultures. The levels of cdk6-associated cyclin D2 and D3 rose substantially within 12 h of Epo⫹/SCF⫺ stimulation (Fig. 5A). Low levels of p27 were associated with cdk6 in proliferating HB60-5 cells. However, Cdk6-bound p27 increased dramatically by 12 h after Epo⫹/SCF⫺ stimulation and remained stably associated with cdk6 over the next 36 h. The level of cdk6 bound p21 rose at 12 h but fell by 48 h as cells entered quiescence. No change in the levels of p16 bound to cdk6 was detected during differentiation. However, there was a significant increase in levels of p15 bound to cdk6 within 12 h of transfer to Epo⫹/SCF⫺ medium, which persisted until 48 h. In contrast to cdk6, there were no apparent changes in binding of p27 to cdk4 complexes. p15 was not detectable in cdk4 complexes during differentiation (data not shown). Cyclin E and cyclin A were immunoprecipitated from HB60-5 cell lysates at various times after Epo⫹/SCF⫺ stimulation and the associated proteins detected by immunoblot analysis (Fig. 5B). p21 was not readily detected in either cyclin E or cyclin A immunoprecipitates prepared from HB60-5 cells. In contrast, p27 was readily detectable, and its association with both cyclin E-cdk2 and cyclin A-cdk2 increased dramatically during Epo-induced differentiation of HB60-5 cells. Down-Regulation of Cdc25A during Differentiation. The G1-S phase checkpoint can also be regulated by cdk phosphorylation. Cdk2 activation requires phosphorylation of threonine 160, which is catalyzed by cdk activating kinase (reviewed in Ref. 36). In addition, the inhibitory phosphates on threonine 14 and/or tyrosine 15 are removed by a family of Cdc25 phosphatases. Cdc25A activates cyclin E-bound cdk2 and is essential for the G1-to-S phase transition (37, 38). In addition, Cdc25A has been recently identified as a transcriptional target of c-Myc (39). Because c-Myc is downregulated during Epo-induced differentiation of HB60-5 cells (Fig. 1), we examined the expression levels of Cdc25A in these differentiating cells. During differentiation of HB60-5 cells, there was a gradual loss of Cdc25A expression (Fig. 6A), which correlated with an increase in the inhibitory phosphotyrosine content of cyclin E-associated cdk2 (Fig. 6B). Therefore, down-regulation of Cdc25A could also contribute to the inhibition of cyclin E-cdk2 during this form of differentiation in HB60-5 cells. p27-mediated Cell Cycle Arrest Was Not Sufficient to Cause Terminal Differentiation. To further investigate the role of p27 up-regulation during erythroid differentiation, HB60-5 cells were transfected with an inducible p27 vector, MTMp27. Clones from these cells were isolated by limiting dilution. The clone showing the strongest expression of p27 after induction by zinc and cadmium (ZnCd; clone HB60p27-C4; Fig. 7A) was chosen for subsequent experimental analysis. Flow cytometry of ZnCd-treated cells showed a progressive increase in the percentage of cells in G1, indi-


Fig. 5. Interaction of cell cycle regulators with cdks during differentiation of HB60-5 cells. Total cellular protein (200 ␮g) prepared from HB60-5 cells that were cultured for the indicated times with Epo were immunoprecipitated with antibodies against cdk6 (A) and cyclin A and cyclin E (B) and subjected to SDS-PAGE. The immunoprecipitates were transferred to PVDF membranes, immunoblotted with antibodies against the indicated proteins (right panel), and visualized as described in Fig. 4.

cating inhibition of the G1-to-S phase transition over the 72 h of treatment (Fig. 7B). This was also reflected by a decrease in the number of ZnCd-treated cells in comparison with untreated cells during the same 72-h interval (data not shown). Although ZnCd-mediated induction of p27 caused a loss of pRb phosphorylation, in the parental HB60-5 cells, pRb phosphorylation remained unaltered (Fig. 7C). To determine whether the block in cell cycle progression seen in HB60p27-C4 cells was accompanied by differentiation, mRNA was isolated at different time points after p27 induction by ZnCd and the levels of ␣-globin assayed by Northern blot analysis. No increase in ␣-globin mRNA expression was observed after p27 induction (Fig. 7D). Furthermore, enforced expression of p27 did not generate the morphological changes seen in HB60-5 cells induced to undergo terminal differentiation by Epo (data not shown). These data suggest that overexpression of p27 can block the cell cycle but is not sufficient to induce terminal differentiation in HB60-5 cells.

Discussion In this study, we have analyzed the cell cycle changes associated with Epo-induced erythroid differentiation in a newly established erythroblastic cell line HB60-5. This cell line was chosen for cell cycle analysis because it is capable of undergoing terminal erythroid differentiation in response to the physiological growth factor Epo when SCF is with-

drawn from the culture medium. Terminal erythroid differentiation in this line is associated with dephosphorylation of pRb, inhibition of cdk4, cdk6, and cdk2 activities, and upregulation of p27. Furthermore, we have shown that although enforced expression of p27 in HB60-5 cells resulted in cell cycle arrest, erythroid differentiation was not induced in these cells, suggesting that commitment to erythroid differentiation is a complex process that requires alteration in other pathways in addition to the cell cycle. Withdrawal of SCF from HB60-5 Growth Media Causes Cell Cycle Arrest. HB60-5 cells, cultured in media containing Epo alone, undergo withdrawal from the cell cycle as a result of a block in the transition from G1 to S. This G1 arrest involves the accumulation of hypophosphorylated pRb (40), which can bind to and inhibit the activity of the E2F transcription factor, the function of which is required for cellular proliferation (16, 26, 27). Hypophosphorylated pRb can also promote growth arrest by recruiting histone deacetylase, which inhibits transcription (41, 42). In HB60-5 cells, the amount of hyperphosphorylated pRb did not change during the first 24 h of Epo⫹/SCF⫺ stimulation but was significantly reduced between 24 and 48 h (3). In these cells, the decrease in hyperphosphorylated pRb was correlated with an inhibition of cdk2, cdk4, and cdk6 activities (17–20). Our finding that Epo⫹/SCF⫺ stimulation did not reduce cdk4 differs from the HMBA-induced erythroleukemia cell differentiation model, where decreased levels of cdk4 protein expression

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Fig. 6. Erythroid differentiation induces a loss of Cdc25A phosphatase. A, protein lysates isolated from HB60-5 cells cultured with Epo for the indicated times were immunoprecipitated with monoclonal anti-Cdc25A, blotted with polyclonal anti-Cdc25A, and visualized as described in Fig. 4. B, to assay changes in phosphotyrosine content of cyclin E-associated cdk2 after Epo stimulation, cyclin E was immunoprecipitated from the above cell extracts and resolved by SDS-PAGE, and the associated cdk2 was immunoblotted with an anti-phosphotyrosine-specific cdc2 antibody, as described in “Materials and Methods.”

accompanied HMBA-induced differentiation. Furthermore, enforced expression of cdk4 in these erythroleukemic cells conferred resistance to HMBA-induced differentiation (12). Together, these results suggest that Epo and HMBA induce differentiation of erythroleukemia cells through distinct mechanisms with respect to cdk4. To further understand how SCF withdrawal triggers the inhibition of cdk activity, we examined G1 cyclin cdk complexes during Epo-induced differentiation of HB60-5 cells. cdk6 complexes were significantly altered by Epo⫹/SCF⫺ stimulation, with a dramatic increase in the binding with cyclin D2 and cyclin D3 to cdk6. Paradoxically, the increase in D-type cyclin binding to cdk6 was associated with a loss of cdk6 activity. It is noteworthy that D-type cyclins are also increased in other forms of cellular differentiation in which D-type cdks are inhibited (43). The mechanisms by which Epo⫹/SCF⫺ stimulation leads to increased cyclin D2 and D3 association with cdk6 is unknown. This could result from posttranslational modifications of cyclins or cdk6. The increased cellular p27 concentration may lead to increased cyclin D2 and D3 binding to cdk6, because it has been

shown that both p21 and p27 can facilitate cyclin D/cdk assembly (44, 45). During this form of erythroid differentiation, p27 may play roles both to facilitate assembly of cyclin D2 and D3 cdk6 complexes and also lead to their inhibition. Some of the inhibition of cdk6 may also result from its increased association with p15. Although the elevation of p15 protein was not sustained, the binding of p15 to cdk6 was increased during differentiation. This may reflect posttranslational modification of p15, leading to its stable association with cdk6. Examination of cdk4 complexes revealed no changes in association with cyclin D2, cyclin D3, p21, and p27. In addition, neither p15 nor p16 could be detected in cdk4 complexes (data not shown). The mechanism of cdk4 inhibition is not clear but could reflect loss of an activating Cdc25 phosphatase or changes in cdk4 phosphoregulatory events. As observed during other forms of cellular differentiation (33–35, 46 – 49), p27 levels rose dramatically during erythroid differentiation of the HB60-5 line. We observed a striking increase in p27 binding to cyclin A and cyclin E complexes and inhibition of these kinases. Although the primary role of p27 may be cyclin E- and cyclin A-cdk2 inhibition, p27 may also contribute to the inhibition of cdk6 complexes during Epo-mediated differentiation. p27 is an inhibitor of the G1to-S transition in several cell types (reviewed in Refs. 15 and 50). Overexpression of p27 in HB60-5 cells resulted in G1 arrest, even in the presence of both Epo and SCF. Taken together, these findings suggest that the increase in p27 contributes importantly to the induction of cell cycle arrest during erythroid differentiation. In addition to cyclin binding, cdk activation requires phosphorylation by cdk activating kinase on a conserved threonine residue in the substrate binding site and dephosphorylation of threonine and tyrosine in the ATP binding domain (36). Inhibitory phosphorylation of the conserved threonine and tyrosine residues is carried out by human homologues of Wee1 and Myt-1, whereas removal of these inhibitory phosphates involves the activity of dual-specific Cdc25 phosphatases (reviewed in Refs. 14 and 36). Epo-induced differentiation of HB60-5 cells may modulate the balance of inhibitory and activating phosphorylations on cdks contributing to cdk inhibition. Cdc25A expression has been shown to be up-regulated by c-myc (39). The loss of c-myc expression during differentiation of HB60-5 cells is associated with a gradual loss of Cdc25A. Moreover, the down-regulation of Cdc25A protein levels was associated with an increase in the inhibitory phosphotyrosine content of cyclin E-bound cdk2 after Epo⫹/SCF⫺ stimulation. Thus, the increase in p27 binding to cdk2 complexes and Cdc25A down-regulation may both contribute to the inhibition of G1 cyclin/cdk activity during this form of differentiation. Cell Cycle Arrest Is Not Sufficient for Differentiation of HB60-5 Cells. SCF binding to c-Kit results in the activation of a signaling pathway that promotes proliferation and prevents apoptosis of hematopoietic progenitors. Although the exact nature of this signal is unknown, c-Kit can activate Ras, phosphatidylinositol 3⬘-kinase, and extracellular signalregulated kinase (51). In addition, it has been proposed that c-Kit activates or changes the signal emanating from the Epo


Fig. 7. Overexpression of p27 in HB60-5 cells induces cell cycle arrest in the absence of differentiation. A, the p27-inducible HB60-p27-C4 cells were grown in the presence of ZnCd for 24 and 48 h in medium containing SCF ⫹ Epo. The cells were then lysed and immunoblotted with p27 antibody. Equal loading was determined by hybridizing the same filter with an Erk-2-specific antibody. B, HB60-p27-C4 cells were harvested at the indicated time points after the addition of ZnCd to asynchronously growing cells. Cells were fixed in 70% ethanol and stained with propidium iodide before flow cytometric analysis. The vertical axis indicates the percentage of cells in G1 (f) S (Œ), or G2-M () from the total cells. C, HB60-5 and HB60-p27-C4 cells were grown in the presence of ZnCd for 48 h, and the phosphorylation state of pRb expression was determined by immunoblotting with anti-pRb antibody. D, total RNA and protein extracted from HB60-5 and HB60-p27-C4 cells after the indicated treatments were analyzed for ␣-globin, as described in Fig. 1. RNA from HB60-5 cells growing with Epo ⫹ SCF (T0) or Epo alone for 48 h was used as control (C).

receptor (52). Our results suggest that Epo stimulation triggers cell cycle arrest at least in part through an increase in p27 levels. Although enforced expression of p27 resulted in a G1-S cell cycle arrest, it did not result in terminal differentiation of HB60-5 cells. These data suggest that induction of erythroid differentiation by Epo stimulation requires alteration in multiple pathways in addition to cell cycle arrest. Indeed, we have shown recently that Epo⫹/SCF⫺ stimulation causes rapid changes in the levels of several transcription factors in HB60-5 cell line, including Fli-1, p45 NF-E2, and GATA-1 (3). Among these, Fli-1 has been reported recently to bind to the Rb promoter and suppress its expression in HB60-5 cell in response to SCF stimulation (3). The signal(s) that regulate these transcriptional events during differentiation may be activated in parallel or sequentially with cyclin-cdk inhibition. For example, the expression of GATA-1 can activate the expression of several erythroid genes that inhibit cell cycle progression indirectly (53). The investigation of the role of these transcription factors in the HB60-5 cells may help to further dissect the mechanisms leading to terminal erythroid differentiation. In summary, the present study suggests that terminal differentiation of HB60-5 cells induced by Epo requires signaling via several pathways, one of which results in a decreased cdk activity and withdrawal from the cell cycle. Although the mechanisms associated with cdks inhibition are partly characterized in this study, up-regulation of p27 appears to play a pivotal role in the cell cycle arrest of HB60-5 cells. The

HB60-5 cell model may prove useful for identifying the biochemical events involved in erythroid differentiation.

Materials and Methods Cell Lines. HB60-5 cells were derived from a primary erythroleukemia induced after injection of BALB/c mice at birth with clone 57 of F-MuLV helper virus, as described previously (3). Cells were maintained in ␣-MEM supplemented with 15% FCS, 10% supernatant from SCF-producing cells BHK-MKL (54), and 0.5 unit Epo/ml as described elsewhere (3). To induce differentiation, HB60-5 cells were washed twice with PBS and cultured in ␣-MEM medium containing 15% FCS and 1 unit Epo/ml, and cells were harvested at intervals. RNA Extraction and Northern Blotting. Total cellular RNA was isolated using TRIzol reagent as described by the supplier (Life Technologies, Inc.). Total RNA (20 ␮g) was dissolved in 2.2 M formaldehyde, denatured at 65°C for 5 min, and electrophoresed in a 1% agarose gel containing 0.66 M formaldehyde. After transfer to Zetaprobe membranes (Bio-Rad Laboratories), the filters were hybridized with 2 ⫻ 106 cpm/ml of [␣-32P]dCTP-labeled probe. DNA Probes. The c-Myc probe was a 2.6-kb XbaI-HindIII fragment derived from pSP65 vector (55). The 750-bp PstI-XbaI fragment of mouse glyceraldehyde-3-phosphate dehydrogenase cDNA was used to check the amount of RNA loaded. DNA probes were freed from plasmid sequences, gel purified, and labeled by random priming. Antibodies and Immunoblotting. Antibodies to cdk2, cdk4, cdk6, Erk-2, and p21 were obtained from Santa Cruz Biotechnology. Anti-pRb monoclonal antibody was obtained from PharMingen, anti-p27 from Transduction Laboratories, and anti-cyclin D2 and cyclin D3 were purchased from Neomarkers. Monoclonal antibody JC-6 to p16 was obtained from J. Koh and E. Harlow (Massachusetts General Hospital Cancer Center, Boston, MA). For Western blot analysis, cells were harvested and lysed at the indicated times in ice-cold RIPA lysis buffer containing 0.5% NP40, 50 mM

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Tris-HCl (pH 8.0), 120 mM NaCl, 50 mM NaF, 10 ␮g/ml aprotinin, 100 ␮g/ml leupeptin, and 10 mM phenylmethylsulfonyl fluoride. Lysates were sonicated and clarified by centrifugation. Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories), and 30 ␮g of protein/ lane were resolved by SDS-PAGE. After electrophoresis, proteins were transferred onto PVDF membrane (Millipore). Blots were reacted with appropriate primary and secondary antibodies, and proteins were detected using enhanced chemiluminescence (Amersham). For analysis of cyclin/cdk complexes, immunoprecipitates were carried out in 400 ␮l of total volume containing 200 ␮g of protein and 30 ␮l of a 50% slurry of protein A-Sepharose and specific antibodies. After three washes with RIPA buffer, Laemmli buffer was added to recovered beads, and complexes were resolved by SDS-PAGE. For detection of Cdc25A, 500 ␮g of protein were immunoprecipitated and then Western blotted with the same anti-Cdc25A polyclonal antibody (Upstate Biotechnology). To assess the phosphotyrosine content of cyclin E-bound cdk2, cyclin E complexes from 400 ␮g of total extracts were immunoprecipitated, as above, and Western blotted with a tyrosine-15 phospho-specific cdc2 antibody (New England Biolabs), which crossreacts with tyrosine-15 phosphorylated cdk2. Vector and Transfection. The pMTp27 vector was constructed by cloning the HindIII-BamHI fragment containing the entire 889-bp murine p27 cDNA into the HindIII-BamHI site of the plasmid pcDNA3-MT, as described (31). For stable transfection, 5 ⫻ 106 HB60-5 cells were mixed with 30 ␮g of pMTp27 expression vector in 0.8 ml PBS and then subjected to electroporation (Bio-Rad Laboratories) in 960 mF and 280 V. After 48 h recovery in medium containing Epo and SCF, the cells were selected for neomycin resistance by G418 (0.8 mg/ml; Life Technologies, Inc.). Clones from these cells were isolated by limiting dilution, and one of them, with the greatest p27 expression (HB60-p27-C4) after induction with ZnCd (100 ␮M ZnSO4 and 2 ␮M CdCl2) was chosen for subsequent experimental analysis. cdk Assays. Cyclin E-cdk2 and cyclin A-cdk2 complexes were immunoprecipitated with specific antibodies, collected on protein A-Sepharose beads, washed, and reacted with [␥-32P]ATP and histone H1 as described (56). Reaction products were resolved by SDS-PAGE, and quantitation of radioactivity incorporated in histone substrate was performed using Molecular Dynamics PhosphorImager and Image Quant software. cdk4- or cdk6-associated kinase activities were determined as described (45), using a GST-pRb substrate. Flow Cytometric Analysis. To examine changes in cell cycle parameters, cells were harvested at different times after SCF withdrawal, fixed in 70% ethanol, and stained with propidium iodide as described (57). Cell cycle profiles were detected using a Becton Dickinson flow cytometer using Modfit software.

Acknowledgments

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We thank Dr. J. Koh for providing the JC-6 antibody and Dr. V. A. Flørenes for discussions during the course of this work.

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