BCR-ABL and Interleukin 3 Promote Haematopoietic Cell Proliferation and Survival through Modulation of Cyclin D2 and p27Kip1 Expression*

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27Kip1 to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27Kip1 expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27Kip1 on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27Kip1 have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27Kip1 can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27Kip1, cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.


Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27
to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27 Kip1 expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27 Kip1 on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27 Kip1 have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27 Kip1 can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27 Kip1 , cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.
The BCR-ABL oncogenes originate from a reciprocal translocation between the long arms of chromosomes 9 and 22, culminating in the formation of the Philadelphia (Ph) chromosome and the fusion of a truncated bcr gene to 5Ј-upstream sequences of the second exon of c-abl (1). The resultant BCR-ABL genes encode the chimeric BCR-ABL proteins of 230, 210, and 185 kDa (p230, p210, and p185, respectively) which are constitutively active tyrosine kinase activity (2), that have been linked to the pathogenesis of chronic myelogenous leukemia (CML) 1 and Philadelphia 1 (Ph1) acute lymphoblastic leukemia (3,4). The p185 BCR-ABL is predominantly found in Ph-positive acute lymphoblastic leukemia (5), while the p210 protein is linked to most cases of CML and some Ph-positive acute lymphoblastic leukemias (6,7). The less common p230 BCR-ABL protein is found primarily in a mild form of CML, defined as Philadelphia chromosome-positive chronic neutrophilic leukemia (Ph-positive CNL) (8,9).
Hemopoietic cells rely on the presence of cytokine(s) for their continued growth and survival (10,11). Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are pleiotropic cytokines which regulate various cellular functions, including proliferation, survival, and differentiation, in a variety of hemopoietic cells. Interleukin-3 (IL-3) is generated by activated T cells, monocytes/macrophages, and stromal cells, and acts as a growth and survival factor for a number of hemopoietic cells, including mast cells, eosinophils, and basophils. IL-3 mediates its effects through binding to the IL-3 receptor consisting of a IL-3 specific ␣-subunit and a ␤-chain also shared by other cytokine receptors, including those of GM-CSF and IL-5 (12,13). Mutation studies have demonstrated that the cytoplasmic tail of the common ␤-chain is critical for transducing proliferation and survival signals (10).
Although the exact mechanism of action of BCR-ABL has not been established, the biological effects of BCR-ABL have been shown to overlap with IL-3. For example, chronic administration of IL-3 or overexpression of IL-3 in mice results in hyperplasia similar to that observed in the chronic phase of CML. Moreover, it has also been shown that progenitor CD34 ϩ cells * 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  from some CML patients can survive and proliferate in vitro in the absence of exogenous growth factors (14 -16). Similarly, transformation by BCR-ABL has also been demonstrated to convert IL-3-dependent cell lines to become growth factor independent (17)(18)(19)(20)(21).
It is believed that BCR-ABL and IL-3 activate similar intracellular signaling pathways to promote proliferation and survival in cytokine-dependent hematopoietic cells. Expression of BCR-ABL activates multiple signaling cascades, including the Ras, phosphatidylinositol 3-kinase, and JAK/STAT pathways, which have also been implicated in the IL-3-dependent signaling (22). Among these, the phosphatidylinositol 3-kinase-dependent signaling pathway has been linked to the anti-apoptotic functions of both BCR-ABL and IL-3. Previous studies have suggested that survival signals triggered by IL-3 or BCR-ABL inactivate the pro-apoptotic Bcl-2 family protein Bad through direct phosphorylation by Akt, a downstream target of phosphatidylinositol 3-kinase. Nevertheless, the role of the phosphatidylinositol 3-kinase/Akt/Bad pathway in mediating the anti-apoptotic functions of IL-3 and BCR-ABL is far from clear, as BCR-ABL could also rescue IL-3-dependent cells from apoptosis in the absence of Bad phosphorylation (23).
Mammalian cell proliferation is susceptible to regulation by extracellular signals during the early/mid G 1 phase of the proliferative cell cycle. After passing the restriction point (R) at mid/late G 1 , cells become refractory to growth inhibitory signals or do not require growth factors to progress into S phase (24). In mammalian cells, the two families of G 1 cyclins: D-type cyclins (cyclin D1, D2, and D3) and cyclin E (cyclin E1 and E2) (25,26), and their dependent kinases (CDKs) control the transition through R. The D and E types of cyclins have specificity for different CDK subunits. The D-type cyclins bind and activate CDK4 and CDK6 exclusively, while cyclin E forms kinase complexes predominantly with CDK2 (26,27). Cyclin E/CDK2 kinase is believed to function downstream of cyclin D (28,29). The principal cellular targets of the G 1 cyclin-dependent CDKs are the retinoblastoma protein (pRB) family of "pocket proteins," consisting of pRB, p107, and p130 (30 -32). In their hypophosphorylated forms, these pRB-related pocket proteins associate with members of the E2F family of transcription factors, thereby negatively regulating transcription activity of E2F-regulated genes, which are important for entry into the S phase of the cell cycle (30,(33)(34)(35). The activities of CDKs are negatively regulated by the WAF/CIP family of CDK inhibitors (CKIs) (p21 Cip1 , p27 Kip1 , and p57 Kip2 ), which target a broad spectrum of CDKs, and the INK4 family of CKIs (p15 INK4b , p16 INK4a , p18 INK4c , and p19 INK4d ), which specifically inhibit cyclin D-CDK4/6 interaction (26,27). In summary, the pRB pathway (i.e. cyclin D, CDKs, CKIs, pRB, and E2F), whose components are important for restriction point control, links the positive and negative proliferative signals to the cell cycle machinery, and inactivation of this pathway, which occurs frequently in human cancer, may alter the growth factor dependence of a cell (26,27).
The exact mechanism by which the proliferative/apoptotic signals transduce from BCR-ABL and IL-3 to the pRB pathway is not defined. Here, we use the IL-3-dependent pro-B cell line BaF3 (36) to investigate the role and regulation of the pRB pathway in mediating signals from IL-3 and BCR/ABL signals.

EXPERIMENTAL PROCEDURES
BaF3 Cell Lines and Tissue Culture-The BaF3 cells were cultured at 10 6 cells/ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin/streptomycin and if necessary, 10% (v/w) WEHI-3B (37) conditioned medium as a source of IL-3. BaF3-p210 cells were generated by retroviral infection of BaF3 cells with retroviruses encoding the bcr-abl gene (38) followed by selection with 1 mg/ml G418 (Life Technologies, Inc.) for 2 weeks. Expression of BCR-ABL was confirmed with anti-ABL antibodies (Pharmingen). The pBabe-puro cyclin D2 was constructed by inserting full-length mouse cyclin D2 cDNA (39) into the BamHI cloning site of the pBabepuro expression vector. BaF3-cyclin D2 and BaF3-p210-cyclin D2 cell lines were established (39) after transfecting BaF3 and BaF3-p210 cells by electroporation (0.25 V; 960 microfarads) with pBabe-puro cyclin D2 and selection with 0.7 mg/ml puromycin. The TonB210.1 BaF3 cell line contains tetracycline-inducible BCR-ABL p210 and was a kind gift from Dr. G. Daley (40). TonB210.1 cells were routinely cultured with IL-3, except during IL-3 starvation, when the cells were incubated without IL-3 in the presence of 1% fetal calf serum and RPMI medium. BCR-ABL expression was induced by adding 1 g/ml doxycyclin (Sigma). The anti-BCR-ABL drug STI571 was provided by Elisabeth Buchdunger, Novartis, and administered to BaF3 cells at a concentration of 10 M.
Preparation and Transduction of TAT Fusion Proteins-The expression plasmids for TAT-p27 Kip1 WT, TAT-p27 Kip1 KK, TAT-eGFP, and TAT-␤-galactosidase were obtained from Dr. S. Dowdy, Washington University School of Medicine, St. Louis, MO (41,42). Each TAT fusion protein was expressed in Escherichia coli and the proteins were purified as described previously (41). Cell lines were transduced by adding the appropriate TAT-protein to the medium at a final concentration of 100 -500 nM as stated in the text. Experiments with TAT-␤-galactosidase showed that almost all BAF3 and BaF3-p210 cells in the culture were transduced. The transduced cells were detected by staining with 5-bromo-4-chloro-3pindoyl ␤-D-galactoside (X-gal) (Sigma) (data not shown).
Cell Cycle and Apoptosis Analysis-Cell cycle analysis was determined by fluorescence-activated cell sorting (FACS) following staining with propidium iodide as described before (43). Cells were collected by centrifugation, washed with phosphate-buffered saline, and permeabilized in 90% ethanol, 10% phosphate-buffered saline prior to DNA staining. The permeabilized cells were incubated with 50 g/ml propidium iodide, 0.1 mg/ml RNase A (Sigma), 0.1% Nonidet P-40, and 50 g/ml trisodium citrate for 30 min prior to analysis using a Becton Dickinson FACSort analyzer. The cell cycle profile was analyzed using the Cell Quest software. Assessment of apoptotic cells was carried out by annexin V staining as recommended by the manufacturer (R & D Systems Europe Ltd., Oxon, United Kingdom). Briefly, centrifuged cells were resuspended in binding buffer (100 mM HEPES, pH 7.4, 1.5 mM NaCl, 50 mM KCl, 10 mM MgCl 2 , and 18 mM CaCl 2 ) and incubated with 0.5 g/ml fluorescein-conjugated annexin V and 20 g/ml propidium iodide for 30 min at room temperature prior to FACS analysis.
Northern Blot Analysis-Total RNA was isolated using the RNeasy Kit (Qiagen, UK) and quantified by absorbance at 260 nm. Twenty g of RNA, prepared as above, was resolved on 1.5% formaldehyde-agarose gels. Following electrophoresis, RNA were transferred to Hybond-N membrane (Amersham Pharmacia Biotech) and subjected to Northern blotting as previously described (43). Cyclin D2 (39), cyclin D3 (39), and p27 Kip1 (44) mRNA was detected by hybridization with their respective full-length 32 P-labeled mouse cDNA probes kindly provided by Dr. Charles Sherr.
Western Blot Analysis and Antibodies-Western blot extracts were prepared by lysing cells with 4 times packed cell volume of lysis buffer (20 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM MgCl 2 , 5 mM EDTA, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 5 mM sodium orthovanadate) on ice for 20 min. Protein yield was quantified by Bio-Rad Dc protein assay kit (Bio-Rad). Fifty g of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and specific proteins were recognized by the following antibodies. The antibodies against pRB (C-15), p107 (C-  (17), and p27 Kip1 (C-19) were purchased from Santa Cruz Biotechnology. The anti-phospho pRB(Ser 807/811 ) antibody were purchased from New England Biolabs and the anti-phospho-pRB (Thr 821 ) antibody was acquired from BIOSOURCE, International. The anti-ABL monoclonal antibody 8E9 was purchased from PharMingen. The antibodies were detected using horseradish peroxidase-linked goat anti-mouse or anti-rabbit IgG (Dako), or mouse absorbed goat anti-rat IgG (Southern Biotechnology Associates, Inc.) and visualized by the enhanced chemiluminescent (ECL) detection system (Amersham Pharmacia Biotech).

BCR-ABL Protects BaF3
Cells from Undergoing G 1 Cell Cycle Arrest and Subsequent Apoptosis following IL-3 Withdraw-al-BCR-ABL has been shown to be able to abrogate the IL-3 requirement in the pre-B hematopoietic cell line BaF3 (45). In order to investigate the roles of BCR-ABL and IL-3 in cell proliferation and survival, the parental BaF3 cells and BaF3 cells expressing BCR-ABL (i.e. BaF3-p210) were cultured in the absence or presence of IL-3. Propidium iodide staining indicated that in the presence of IL-3 both the BaF3 and BaF3-p210 cells proliferated normally with no detectable sign of cell cycle arrest or apoptosis after 24 h. The results also showed that 24 h after deprivation of IL-3, the BaF3 cells arrested at the G 1 phase of the cell cycle (Fig. 1A). Furthermore, detailed analysis of the BaF3 cells after IL-3 withdrawal revealed that following the withdrawal of IL-3, the parental BaF3 cells accumulated at the G 1 phase of the cell cycle, before undergoing apoptosis (Fig. 1B). By 24 h, over 80% of the cells were arrested in G 1 , and nearly all of the cells displayed detectable sign of apoptosis (Ͻ2N DNA content) after 72 h of IL-3 depletion (Fig.  1B). In contrast, none of the BaF3-p210 cells underwent cell cycle arrest or apoptosis following the withdrawal of IL-3, thus confirming that expression of BCR-ABL could indeed protect BaF3 cells from the G 1 cell cycle arrest and subsequent apoptosis induced by IL-3-depletion (Fig. 1A).
Expression of Cyclins, CDKs, and CKIs in BaF3 and BaF3-BCR-ABL Cells in the Absence or Presence of IL-3-To investigate the molecular mechanisms underlying the responses to IL-3 and BCR-ABL, we examined the expression levels of molecules along the pRB pathway in both BaF3 and BaF3-p210 in the presence and absence of IL-3 for 24 h. Western blot analysis showed that in IL-3-deprived BaF3 cells, the two pocket proteins, pRB and p107, were present in their respective faster migrating hypophosphorylated forms (Fig. 2). In the presence of IL-3 and/or BCR-ABL, both pRB and p107 were present at comparatively higher levels and in their hyperphosphorylated forms, whereas the expression of p130 was down-regulated. We next studied the expression patterns of cyclins, CDKs, and CKIs, which are known to be responsible for controlling pocket protein phosphorylation (26,27). The expression of cyclin D2, D3, E, and A was either undetectable or present at low levels in IL-3-deprived BaF3 cells, while these cyclins were present at significantly higher levels in the presence of IL-3 and/or BCR-ABL (Fig. 2). In contrast to the cyclins, their kinase partners, CDK2, CDK4, and CDK6 were expressed at similar levels in BaF3 and BaF3-p210 cells in the presence or absence of IL-3. We next investigated if the up-regulation of cyclin D and E expression was associated with a corresponding increase in their dependent kinase activity, which is responsible for pocket

FIG. 2. Expression of cell cycle regulators in BaF3 cells in the absence and presence of IL-3 and/or BCR-ABL.
Cell lysates extracted from BaF3 and BaF3-p210 cultured with and without IL-3 for 24 h, as described in the legend to Fig. 1A, were Western blotted for BCR-ABL, cyclin D2, -D3, -E, and -A, CDK2, -4, and -6, CDC2, pRB, p107, p130, p16 INK4a , and p21 Cip1 expression using the respective antibodies and for CDK4/6 and CDK2 activity using the phospho-pRB antibodies. Total RNAs from BaF3 and BaF3-p210 cells ϩ IL-3 were also extracted for Northern blot analysis for expression of cyclin D2, cyclin D3, and p27 Kip1 mRNA (lower right panel). Equal loading of total RNAs was confirmed by staining with ethidium bromide.

FIG. 1. Effects of IL-3 and/or BCR-ABL on cell cycle progression and survival of BaF3 cells.
A, cell cycle analysis of BaF3 and BaF3-p210 cells in the absence or presence of IL-3 (upper panel). BaF3 and BaF3-p210 cells were incubated for 24 h in the absence or presence of IL-3. The resultant cells were collected, permeablized, and stained with propidium iodide. The cell cycle profile was expressed as "number of cells" against "DNA content" and the percentages of cells in each phases (ϽG 1 , G 1 , S, and G 2 /M) were indicated. Cells undergoing apoptosis was represented by those with ϽG 1 DNA content. B, cell cycle analysis of BaF3 cells after IL-3 withdrawal (lower panel). Cycling BaF3 cells previously cultured in IL-3-supplemented medium were incubated in IL-3-free growth media and sampled at 0, 6, 12, 24, 48, and 72 h for cell cycle analysis as described above.
protein phosphorylation. Since pRB is the key physiological substrate for cyclin/CDKs, and specific sites are phosphorylated in vivo by distinct G 1 /S cyclin-CDKs (26,27), we therefore used specific phospho-pRB antibodies to probe for in vivo cyclin D-and E-associated kinase activity in these cells. The Western blot result showed that up-regulation of cyclin D and E expression in response to IL-3 and/or BCR-ABL was associated with induction of cyclin D-and E-dependent kinase activity (Fig. 2). CDK activity is also regulated by inhibitor proteins and the CKI p27 Kip1 accumulated in IL-3-starved BaF3 cells, but was substantially down-regulated in the presence of IL-3 and by BCR-ABL expression. Interestingly, p21 Cip1 was undetectable in IL-3-starved BaF3 cells, but was abundantly expressed in cells with IL-3 and/or BCR-ABL. A similar expression pattern was observed for p16 INK4a (Fig. 2). Thus, it is likely that p27 Kip1 , but not p21 Cip1 or p16 INK4a , has a role in repressing CDK activity in the BaF3 cells when deprived of growth and survival signals. These results led us to hypothesize that the IL-3 or BCR-ABL-derived signals up-regulate cyclin D2 and D3 expression and down-regulates the expression level of p27 Kip1 , culminating in induction of cyclin-dependent kinase activity, pocket protein hyperphosphorylation, activation of E2F activity, and continued cell proliferation.
To explore the mechanisms for regulating cyclin D2 and D3 and p27 Kip1 expression, we performed Northern blot analysis on these BaF3 cells in the presence or absence of IL-3 (Fig. 2). Cyclin D2 mRNA was up-regulated while the p27 Kip1 mRNA was down-regulated in the presence of IL-3 and/or BCR-ABL, suggesting that their regulation may be in part at the transcriptional level. In contrast, the level of cyclin D3 mRNA was largely unchanged with or without IL-3, but was down-regulated in the BAF3 cells expressing BCR-ABL. Kip1 -We next studied the kinetics of expression of these cell cycle proteins following IL-3 withdrawal. Down-regulation of both cyclin D2 and cyclin D3, upregulation of p27 Kip1 , hypophosphorylation of pocket proteins, and down-regulation of CDK2 and CDK4/6 activity (Fig. 3) all occurred between 12 and 24 h, concomitant with G 1 cell cycle arrest (Fig. 1B). Thus, down-regulation of D-type cyclins and up-regulation of p27 Kip1 could have a role in mediating the cell cycle arrest and apoptosis triggered by IL-3 withdrawal.

IL-3 Withdrawal Induced the Down-regulation of Cyclin D2 and the Accumulation of p27
Expression of Cyclin Ds and p27 Kip1 Is Associated with BCR-ABL Expression-To confirm that BCR-ABL is sufficient to account for the molecular changes observed in BaF3-p210, we examined the cell cycle status and expression of cell cycle regulatory molecules in a BaF3 cell line (TonB210.1) where the expression of BCR-ABL is under the regulation of a doxycycline-inducible promoter (40). The TonB210.1 cells were first starved of IL-3 for 24 h, before doxycycline was added to induce BCR-ABL expression. After IL-3 starvation, the majority of the TonB210.1 cells accumulated in the G 1 phase of the cell cycle, but these IL-3-starved cells re-entered the cell cycle 24 h after adding doxycycline (Fig. 4A), coincident with the induction of BCR-ABL protein (Fig. 4B). Cyclin D2, D3, and E were detectable at low levels in IL-3-depleted TonB210.1 cells, but were induced at 24 h following doxycycline addition, while the expression levels of their dependent-kinases, CDK4, -6, and -2, remained largely unchanged (Fig. 4B). Again, the expression of p27 Kip1 was inversely correlated with that of BCR-ABL and p21 Cip1 . In agreement with the results from the Western blotting, the Northern blot analysis revealed that the level of p27 Kip1 mRNA decreased, while the cyclin D2 mRNA level increased, in response to BCR-ABL induction. Consistent with our previous data, the level of cyclin D3 mRNA was decreased upon BCL-ABL induction (Fig. 4B).
STI571 Down-regulates the Expression of Cyclin Ds and Induces p27 Kip1 Expression-STI571, a 2-phenylaminopyrimidine derivative, selectively inhibits the tyrosine kinase activity of c-ABL and BCR-ABL (46,47), blocks the proliferation of BCR-ABL-positive cell lines and tumors (46,47), and induces these cells to undergo apoptosis (48,49). We used this drug to investigate whether the activity of BCR-ABL is necessary to rescue the cell cycle arrest and apoptosis caused by IL-3 deprivation. Whereas BaF3-p210 cells continued to proliferate with or without IL-3, IL-3-starved BaF3-p210 cells arrested in the G 1 phase of the cell cycle in the presence of STI571 (Fig.  5A). Consistent with previous findings, the Western blot re-

FIG. 4. Effects of inducible BCR-ABL expression on cell cycle profile and expression of cell cycle regulators in TonB210 BaF3 cells.
TonB210 cells were cultured in IL-3-free and 1% fetal calf serum medium for 24 h, before doxycyclin was added to induced BCR-ABL expression. A, TonB210 cells treated with doxycyclin were collected at 0, 3, 12, 24, and 48 h, permeablized, and stained with propidium iodide for cell cycle analysis as described in Fig. 1A. B, Western and Northern blot analyses (bottom panel) was performed on protein and total RNA extracted from TonB210 cells treated with doxycyclin for 0, 3, 12, 24, and 48 h, as described in the legend to Fig. 2. sults showed that STI571 down-regulated the expression of cyclin D2 and D3 and p21 Cip1 , but induced the expression of p27 Kip1 , in the absence of IL-3 (Fig. 6A). This was accompanied by the down-regulation of both cyclin D-CDK4/6 and cyclin E-CDK2 activity, as revealed by the specific anti-phospho-pRB antibodies. The expression of CDK-4 and -6 was not affected by STI571 treatment. Consistent with earlier results, the pocket proteins pRB and p107 were down-regulated and hypophosphorylated following STI571 treatment, but p130 expression was induced (Fig. 6A). The expression of these cell cycle regulators was not significantly affected by STI571 in the presence of IL-3 (Fig. 6A), which we showed above compensates for the loss of BCR-ABL. The Northern blotting results again demonstrated that BCR-ABL repressed p27 Kip1 , but induced cyclin D2, mRNA expression, as the addition of STI571 increased the mRNA level of p27 Kip1 but decreased that of cyclin D2 in the IL-3-deprived BAF3-p210 cells (Fig. 6B). It is notable that even though the cyclin D3 protein level was significantly downregulated by STI571, the cyclin D3 mRNA level was not greatly affected (Fig. 6B). This again indicates that BCR-ABL modulates the expression of cyclin D3 predominantly at the posttranscriptional level.
Expression of p27 Kip1 Induces Cell Cycle Arrest and Apoptosis in BaF3 Cells-We wished to determine whether the induction of p27 Kip1 was sufficient to cause cell cycle arrest of BaF3 cells and to induce apoptosis. Cells have been shown to take up proteins fused to the 11-amino acid transduction domain of the human immunodeficiency virus (HIV) TAT protein (50,51). To investigate the functional significance of p27 Kip1 , we transduced the BaF3 cell lines with biologically active TAT-p27 Kip1 , or with TAT-mutant p27 Kip1 (TAT-p27 Kip1 KK) and TAT-eGFP as controls (41, 42) (Fig. 7). Cells incubated with TAT-eGFP and TAT-mutant p27 Kip1 proliferated normally, but the majority of the p27 Kip1 -transduced BaF3 displayed sub-G 1 (Ͻ2 N) DNA content, indicative of apoptosis (Fig. 7A). The ability of wild-type p27 Kip1 , but not mutant p27 Kip1 , to promote apoptosis in BaF3 cells, even in the presence of IL-3, was confirmed by annexin V staining (Fig. 7B). These observations indicated that similar to IL-3 depletion, expression of p27 Kip1 alone is sufficient to induce apoptosis in BaF3 cells. The propidium iodide staining also showed that the TAT p27 Kip1 -transduced BaF3-p210 cells ceased growth and accumulated in G 1 , whereas control cells multiplied normally. Interestingly, only low levels of apoptosis were detected in these cells expressing BCR-ABL (Fig. 7B). The presence of TAT-p27 Kip1 fusion proteins in the BaF3 and BaF3-p210 cells were confirmed by Western blotting for p27 Kip1 protein (Fig. 7C).

Expression of Cyclin D2 Overcomes the G 1 Arrest and Apoptosis Induced by IL-3 Deprivation and STI571 in BaF3 and
BaF3-p210 Cells, Respectively-We wished to determine whether cyclin D2 is indeed an important downstream target of IL-3 and BCR-ABL-derived signals and plays an important role in mediating proliferation and survival. To this end we generated stable transfectants of BaF3 and BaF3-BCR-ABL cells constitutively overexpressing cyclin D2 (Fig. 8C and 9). Expression of cyclin D2 rendered BaF3 and BaF3-p210 cells largely insensitive to cell cycle arrest and apoptosis induced by IL-3 deprivation and STI571 treatment, respectively (Fig. 8, A and  B). As predicated, growth arrest and apoptosis induced by IL-3 withdrawal or STI571 treatment was unaffected in control transfectants harboring the empty expression vector (Fig. 8, A  and B). This provides further evidence that cyclin D2 is a crucial component of signal transduction pathways downstream of the IL-3 receptor and BCR-ABL. Furthermore, these results also imply that the induction of cyclin D2 expression is causative for cell cycle entry in BaF3 and BaF3-p210.
Cyclin D2 Functions Upstream of the pRB Pathway-To evaluate the mechanism by which cyclin D2 promotes proliferation and survival, we examined the effects of ectopic cyclin D2 expression on the expression of downstream molecules along the pRB/E2F pathway after IL-3 withdrawal. Expression of cyclin D2 maintained the phosphorylation status of pocket proteins after IL-3 withdrawal and, consistent with this, the
CDK2 and CDK4/6-dependent kinase activity remained unchanged (Fig. 9). These data further suggest that cyclin D2 expression is responsible and rate-limiting for the phosphorylation of pocket proteins and the induction of E2F activity in response to IL-3 and BCR-ABL. Our results also show that overexpression of cyclin D2 can also prevent the down-regulation of cyclin D3 and cyclin E and the accumulation of p27 Kip1 triggered by removal of growth factor (Fig. 9). Thus, the expression of cyclin E, p27 Kip1 , and cyclin D3 is also regulated by cyclin D2 and suggest that cyclin D2 functions upstream of cyclin D3, cyclin E, and p27 Kip1 .
It is noteworthy that some of the BaF3-cyclin D2 cells arrested in G 1 and underwent apoptosis after prolonged (72 h) IL-3 withdrawal (Fig. 8A). Nevertheless, this cell cycle arrest and apoptosis was concomitant with a decline in cyclin D2 expression (Fig. 9). This further supports the idea that cyclin D2 expression has a role in regulating the cell cycle and survival of hematopoietic cells, since the induction of cell cycle arrest and apoptosis was again associated with down-regulation of cyclin D2. DISCUSSION In the present study, we investigate the mechanisms by which cytokines and BCR-ABL mediate proliferation and survival in hematopoietic progenitor cells using the IL-3-dependent pre-B cell line, BaF3. Using the parental BaF3 cells and a BaF3 cell line stably expressing BCR-ABL (i.e. BaF3-p210), we obtained data that expression of BCR-ABL can rescue BaF3 cells from cell cycle arrest and apoptosis following IL-3 withdrawal. Our results also showed that withdrawal of IL-3 induces the down-regulation of cyclin D2 and D3, and up-regulation of p27 Kip1 expression, which can be blocked by forced expression of BCR-ABL.
To ensure that the properties observed in the BaF3 cells stably expressing BCR-ABL are related to BCR-ABL and not a result of mutations introduced during the establishment of the BaF3-p210 cell line, we verified and extended our findings by two other independent approaches. First, we employed a BaF3 cell line where the expression of BCR-ABL is inducible by addition of doxycycline and second, we used the drug STI571 to inhibit BCR-ABL activity in the BaF3 cells constitutively ex- pressing BCR-ABL. Results from both these systems confirmed our earlier findings that expression of BCR-ABL results in the up-regulation of cyclin D2 and the down-regulation of p27 Kip1 expression at both protein and mRNA levels. We also demonstrated that although cyclin D3 protein is induced by BCR-ABL or IL-3, its mRNA level is either unchanged or down-regulated in response to IL-3 or BCR-ABL expression, indicating that the accumulation of cyclin D3 induced by IL-3 and BCR-ABL is regulated predominantly at post-transcriptional levels. Our results further showed that the accumulation of cyclin D2 and D3 and down-regulation of p27 in response to IL-3 and/or BCR-ABL expression correlates with the activation of CDK4/6 and CDK2 activity, pocket protein phosphorylation, and the kinetics of cell cycle entry. Given that the catalytic partners of cyclins, CDK2, -4, and -6, are expressed constitutively in BaF3 in the absence or presence of IL-3 and BCR-ABL expression, these data suggest that the expression of cyclin Ds and p27 Kip1 can be rate-limiting for pocket protein phosphorylation and thus S phase entry in response to IL-3 and BCR-ABL expression. These results also implicate cyclin Ds and p27 Kip1 as important effectors of the proliferative and survival signals emanating from IL-3 and BCR-ABL.
To test these ideas, we overexpressed p27 Kip1 using the HIV TAT fusion protein tranduction system and found that overex- pression of p27 Kip1 can induce cell cycle arrest and apoptosis in BaF3 cells, even in the presence of cytokines. It is notable that only the wild-type, but not a mutant p27 Kip1 which cannot interact with CDK2, can promote cell cycle arrest and apoptosis in BaF3 cells, indicating that the ability of p27 Kip1 to bind to and inhibit CDK2 is important for mediating cell cycle arrest and apoptosis. Interestingly, overexpression of p27 Kip1 causes G 1 arrest but not apoptosis in BaF3 cells stably expressing BCR-ABL. The significance for this is unclear and it could reflect the predominant anti-apoptotic function of BCR-ABL or that the signals emanating from IL-3 and BCR-ABL are not exactly identical. Consistent with these results are several recent studies reporting that BCR-ABL represses p27 Kip1 expression in hematopoietic progenitor cells (52)(53)(54). However, here, we extended these findings by showing that the downregulation of p27 Kip1 expression is functionally important for the cell cycle arrest. Moreover, we also demonstrated further that p27 Kip1 functions not only as a regulator of cell cycle arrest but also of apoptosis. This idea is supported by previous data showing that induction of p27 Kip1 expression cannot only cause cell cycle arrest but also apoptosis in T-and B-lymphocytes (55). Furthermore, this result is also corroborated by our previous finding that apoptosis is inhibited in p27 Kip1 null mouse Sca 1 ϩ hematopoietic progenitor cells compared with wild-type after growth factor withdrawal (56).
We obtained data showing that forced expression of cyclin D2 is sufficient to prevent the cell cycle arrest as well as the associated apoptosis caused by IL-3 withdrawal or inhibition of BCR-ABL activity by STI571 in BaF3 cells, indicating that IL-3 and BCR-ABL signals target cyclin Ds to promote cell growth and survival. This observation is consistent with a previous report demonstrating that overexpression of cyclin D3 can abrogate leukemic T cells from undergoing apoptosis following T-cell receptor activation (57). Further analysis of the BaF3 cell line expressing cyclin D2 shows that cyclin D2 expression is responsible and rate-limiting for CDK4/6-dependent kinase activity, pocket protein hyperphosphorylation, and S-phase entry in these cells. Furthermore, our data also show that forced expression of cyclin D2 can prevent the down-regulation of cyclin E and the accumulation of p27 Kip1 expression in response to IL-3 withdrawal, indicating that cyclin D2 also acts upstream of cyclin E and p27 Kip1 . It is possible that expression of cyclin D2 induces down-regulation of p27 Kip1 through induction of cyclin E expression and thus cyclin E-CDK2 activity (34). This concept is also in agreement with our previous data obtained from primary B-lymphocytes showing that the expression of p27 Kip1 is specifically repressed by cyclin D2 expression and that the level of p27 Kip1 is down-regulated in response to proliferative signals in normal but not in cyclin D2 Ϫ/Ϫ B-cells (58). Interestingly, our data also demonstrate that constitutive expression of cyclin D2 can also override the cyclin D3 downregulation caused by IL-3 withdrawal, indicating that cyclin D2 expression also influences the expression of cyclin D3 (58).
In summary, our results evidently demonstrate that both cyclin D2 and p27 Kip1 are important for mediating the proliferative signals from IL-3 and BCR-ABL. Moreover, we also show that both cyclin D2 and p27 Kip1 also have a role in cell survival, as ectopic expression of cyclin D2 is sufficient to prevent apoptosis induced by IL-3 withdrawal or BCR-ABL inactivation (55), while overexpression of p27 Kip1 can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27 Kip1 , cyclin E, and also cyclin D3 and play an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.