Title: BCR/ABL Regulates Expression of the Cyclin-dependent Kinase Inhibitor p27Kip1 through the Phosphatidylinositol 3-Kinase/AKT Pathway
Abstract: Deregulation of cell cycle checkpoints is an almost universal abnormality in human cancers and is most often due to loss-of-function mutations of tumor suppressor genes such as Rb, p53, or p16INK4a. In this study, we demonstrate that BCR/ABL inhibits the expression of a key cell cycle inhibitor, p27Kip1, by signaling through a pathway involving phosphatidylinositol 3-kinase (PI3K). p27Kip1 is a widely expressed inhibitor of cdk2, an essential cell cycle kinase regulating entry into S phase. We demonstrate that the decrease of p27Kip1 is directly due to BCR/ABL in hematopoietic cells by two different approaches. First, induction of BCR/ABL by a tetracycline-regulated promoter is associated with a reversible down-regulation of p27Kip1. Second, inhibition of BCR/ABL kinase activity with the Abl tyrosine kinase inhibitor STI571 rapidly increases p27Kip1 levels. The PI3K inhibitor LY-294002 blocks the ability of BCR/ABL to induce p27Kip1down-regulation and inhibits BCR/ABL-induced entry into S phase. The serine/threonine kinase AKT/protein kinase B is a known downstream target of PI3K. Transient expression of an activated mutant of AKT was found to decrease expression of p27Kip1, even when PI3K was inhibited by LY-294002. The mechanism of p27Kip1 regulation is primarily related to protein stability, since inhibition of proteasome activity increased p27Kip1 levels in BCR/ABL-transformed cells, whereas very little change in p27 transcription was found. Overall, these data are consistent with a model in which BCR/ABL suppresses p27Kip1protein levels through PI3K/AKT, leading to accelerated entry into S phase. This activity is likely to explain in part previous studies showing that activation of PI3K was required for optimum transformation of hematopoietic cells by BCR/ABL in vitro and in vivo. Deregulation of cell cycle checkpoints is an almost universal abnormality in human cancers and is most often due to loss-of-function mutations of tumor suppressor genes such as Rb, p53, or p16INK4a. In this study, we demonstrate that BCR/ABL inhibits the expression of a key cell cycle inhibitor, p27Kip1, by signaling through a pathway involving phosphatidylinositol 3-kinase (PI3K). p27Kip1 is a widely expressed inhibitor of cdk2, an essential cell cycle kinase regulating entry into S phase. We demonstrate that the decrease of p27Kip1 is directly due to BCR/ABL in hematopoietic cells by two different approaches. First, induction of BCR/ABL by a tetracycline-regulated promoter is associated with a reversible down-regulation of p27Kip1. Second, inhibition of BCR/ABL kinase activity with the Abl tyrosine kinase inhibitor STI571 rapidly increases p27Kip1 levels. The PI3K inhibitor LY-294002 blocks the ability of BCR/ABL to induce p27Kip1down-regulation and inhibits BCR/ABL-induced entry into S phase. The serine/threonine kinase AKT/protein kinase B is a known downstream target of PI3K. Transient expression of an activated mutant of AKT was found to decrease expression of p27Kip1, even when PI3K was inhibited by LY-294002. The mechanism of p27Kip1 regulation is primarily related to protein stability, since inhibition of proteasome activity increased p27Kip1 levels in BCR/ABL-transformed cells, whereas very little change in p27 transcription was found. Overall, these data are consistent with a model in which BCR/ABL suppresses p27Kip1protein levels through PI3K/AKT, leading to accelerated entry into S phase. This activity is likely to explain in part previous studies showing that activation of PI3K was required for optimum transformation of hematopoietic cells by BCR/ABL in vitro and in vivo. chronic myelogenous leukemia acute lymphoblastic leukemia interleukin cyclin-dependent kinase cyclin-dependent kinase inhibitor phosphatidylinositol 3-kinase hemagglutinin phosphate-buffered saline polyvinylidene difluoride Tris-buffered saline TBS with 0.5% Tween polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase polyacrylamide gel electrophoresis green fluorescent protein wild type Chronic myelogenous leukemia (CML)1 is a myeloproliferative disorder associated with expression of the Philadelphia chromosome (1Nowell P.C. Hungerford D.A. J. Natl. Cancer Inst. 1960; 25: 85-109PubMed Google Scholar), a translocation between chromosomes 9 and 22 that fuses the Bcr and Abl genes (2Rowley J.D. Nature. 1973; 243: 290-293Crossref PubMed Scopus (3396) Google Scholar, 3Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1124) Google Scholar, 4McWhirter J.R. Wang J.Y. Mol. Cell. Biol. 1991; 11: 1553-1565Crossref PubMed Google Scholar). Unlike many other leukemia oncogenes, BCR/ABL does not appear to alter differentiation of granulocyte lineage cells. In contrast, recent studies have suggested that the major cellular effects of BCR/ABL are related to increased mitogenic activity (5Puil L. Liu J. Gish G. Mbamalu G. Bowtell D. Pelicci T.G. Arlinghaus R. Pawson T. EMBO J. 1994; 13: 764-773Crossref PubMed Scopus (401) Google Scholar), reduced sensitivity to apoptosis (6Bedi A. Zehnbauer B.A. Barber J.P. Sharkis S.J. Jones R.J. Blood. 1994; 83: 2038-2044Crossref PubMed Google Scholar), and altered adhesion and homing of CML progenitor cells (7Gordon M.Y. Dowding C.R. Riley G.P. Goldman J.M. Greaves M.F. Nature. 1987; 328: 342-344Crossref PubMed Scopus (396) Google Scholar). The BCR/ABL oncogene is associated with both myeloproliferative disease and acute leukemias in human and in murine models. There are three known breakpoints in the gene, resulting in three different protein products, p190, p210, and p230, which vary in the length of Bcr present in the fusion protein (8Quackenbush R.C. Reuther G.W. Miller J.P. Courtney K.D. Pear W.S. Pendergast A.M. Blood. 2000; 95: 2913-2921Crossref PubMed Google Scholar). Interestingly, the three proteins tend to be associated with different leukemias: ALL, CML, and chronic neutrophilic leukemia, respectively, for p190, p210, and p230BCR/ABL. Each of the BCR/ABL proteins have elevated Abl tyrosine kinase activity (9Konopka J.B. Witte O.N. Mol. Cell. Biol. 1985; 5: 3116-3123Crossref PubMed Scopus (193) Google Scholar), and this increased kinase activity is necessary for transformation (3Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1124) Google Scholar). Although a number of substrates of the BCR/ABL tyrosine kinase have been identified, including CBL (10Sattler M. Salgia R. Okuda K. Uemura N. Durstin M.A. Pisick E. Xu G. Li J.L. Prasad K.V. Griffin J.D. Oncogene. 1996; 12: 839-846PubMed Google Scholar), CrkL (11Oda T. Heaney C. Hagopian J.R. Okuda K. Griffin J.D. Druker B.J. J. Biol. Chem. 1994; 269: 22925-22928Abstract Full Text PDF PubMed Google Scholar), Dok (12Carpino N. Wisniewski D. Strife A. Marshak D. Kobayashi R. Stillman B. Clarkson B. Cell. 1997; 88: 197-204Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar), STAT5 (13Shuai K. Halpern J. ten Hoeve J. Rao X. Sawyers C.L. Oncogene. 1996; 13: 247-254PubMed Google Scholar, 14Carlesso N. Frank D.A. Griffin J.D. J. Exp. Med. 1996; 183: 811-820Crossref PubMed Scopus (434) Google Scholar), SHP-2 (15Tauchi T. Feng G.S. Marshall M.S. Shen R. Mantel C. Pawson T. Broxmeyer H.E. J. Biol. Chem. 1994; 269: 25206-25211Abstract Full Text PDF PubMed Google Scholar), Shc (16Pelicci G. Lanfrancone L. Salcini A.E. Romano A. Mele S. Grazia Borrello M. Segatto O. Di Fiore P.P. Pelicci P.G. Oncogene. 1995; 11: 899-907PubMed Google Scholar), and Fak (17Salgia R. Sattler M. Pisick E. Li J.L. Griffin J.D. Exp. Hematol. 1996; 24: 310-313PubMed Google Scholar), the signaling pathways that result in dysregulated growth, viability, and adhesion are not yet well defined. The mitogenic effects of BCR/ABL are likely to be important in the pathogenesis of CML. BCR/ABL reduces growth factor requirements of primary hematopoietic stem cells (18Sanchez-Garcia I. Grutz G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5287-5291Crossref PubMed Scopus (197) Google Scholar), converts IL-3-dependent murine hematopoietic cell lines to growth factor independence (19Mandanas R.A. Boswell H.S. Lu L. Leibowitz D. Leukemia ( Baltimore ). 1992; 6: 796-800PubMed Google Scholar), and is mitogenic in fibroblasts (20Sawyers C.L. McLaughlin J. Witte O.N. J. Exp. Med. 1995; 181: 307-313Crossref PubMed Scopus (248) Google Scholar). When compared with normal progenitor cells, CML progenitor cells are more likely to be in S phase, both in the marrow and blood, and the fraction of cells in G0 is reduced (21Eaves A.C. Cashman J.D. Gaboury L.A. Kalousek D.K. Eaves C.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5306-5310Crossref PubMed Scopus (176) Google Scholar). Thus, BCR/ABL is likely to deregulate checkpoints at one or more sites within the cell cycle. Previous studies have shown that several immediate-early genes are induced by BCR/ABL, including myc (22Sawyers C.L. Callahan W. Witte O.N. Cell. 1992; 70: 901-910Abstract Full Text PDF PubMed Scopus (356) Google Scholar), fos, andjun (23Mandanas R.A. Leibowitz D.S. Gharehbaghi K. Tauchi T. Burgess G.S. Miyazawa K. Jayaram H.N. Boswell H.S. Blood. 1993; 82: 1838-1847Crossref PubMed Google Scholar). The rapid induction of these genes correlates with an enhanced rate of transition from G0 to G1. The increased fraction of cells in S phase suggests that G1/S transition checkpoints are also suppressed. A number of molecules play a key role in regulating cell cycle progression from G1 to S, including the G1cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CKIs). CKIs can be grouped in two categories based on similarities of sequence and actions: the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the CIP/KIP family (p21WAF1/CIP1, p27Kip1, and p57Kip2), reviewed in Sherr and Roberts (24Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5155) Google Scholar). INK4 family members specifically inhibit the activity of cdk4 and 6, whereas the CIP/KIP family members have a broader action. Overexpression of each of these CKI have been shown to induce a G1 arrest. p21CIP1 has recently been directly shown to be important for regulating hematopoiesis in vivo in mice (25Cheng T. Rodrigues N. Shen H. Yang Y. Dombkowski D. Sykes M. Scadden D.T. Science. 2000; 287: 1804-1808Crossref PubMed Scopus (1085) Google Scholar, 26Mantel C. Braun S.E. Reid S. Henegariu O. Liu L. Hangoc G. Broxmeyer H.E. Blood. 1999; 93: 1390-1398Crossref PubMed Google Scholar). Although PI3K and AKT have previously been reported to play essential roles in BCR/ABL transformation (27Skorski T. Bellacosa A. Nieborowska-Skorska M. Majewski M. Martinez R. Choi J.K. Trotta R. Wlodarski P. Perrotti D. Chan T.O. Wasik M.A. Tsichlis P.N. Calabretta B. EMBO J. 1997; 16: 6151-6161Crossref PubMed Scopus (558) Google Scholar), the mechanisms and downstream signaling targets have been unclear. PI3K and AKT have been linked to enhanced cell survival through the phosphorylation and subsequent inhibition of the pro-apoptotic molecule Bad (28Neshat M.S. Raitano A.B. Wang H.G. Reed J.C. Sawyers C.L. Mol. Cell. Biol. 2000; 20: 1179-1186Crossref PubMed Scopus (165) Google Scholar). However, it has been difficult to demonstrate phosphorylation of Bad in some cell types transformed by BCR/ABL, so identification of other downstream targets is of interest. In the present study we demonstrate that BCR/ABL regulates the expression of p27Kip1 in a proteasome-dependent manner and through activation of PI3K and AKT. Anti-Abl monoclonal antibody 3F12 was a gift from R. Salgia (Dana Farber Cancer Institute). Monoclonal antibodies against p27Kip1 (K25020) and Rb (14001A) were purchased from Transduction Laboratories (Pharmingen/Transduction Laboratories, San Diego, CA). Anti-p85 antiserum (06–195) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-HA monoclonal was purchased from Babco (Richmond, CA). AKT constructs, inserted in a pCDNA3.1 backbone, were described previously (29Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). RNase A, lactacystine, andN-acetyl-leucyl-leucine norleucinal (LLnL) were purchased from Sigma. E64 and calpain inhibitors were purchased from Calbiochem. The IL-3-dependent Ba/F3 cell line was maintained in RPMI 1640 (Mediatech Cellgro, Herndon, VA) supplemented with 10% fetal calf serum, 1 mg/ml l-glutamine, penicillin-streptomycin, and 10% WEHI-3B conditioned medium (WEHI-3B-CM) as a source of IL-3. Ba/F3 is commonly used as a model for BCR/ABL signaling because it is non-leukemic and factor-dependent in the absence of BCR/ABL-transformation but becomes leukemic in syngeneic mice and factor-independent after transformation by BCR/ABL. p210BCR/ABL-transformed Ba/F3 cells (Ba/F3-p210) are maintained in culture in the medium described above, except without IL-3. All cells were maintained at 37 °C in a 5% CO2 humidified incubator. Ba/F3 cells expressing the reverse tet-transactivator pUHD172–1 (Ton.B.1) and Ton.B.210 cells in which p210BCR/ABL expression is induced by the addition of doxycycline were obtained from G. Daley (Whitehead institute, Cambridge, MA) and grown as described previously (30Gesbert F. Griffin J. Blood. 2000; 96: 2269-2276Crossref PubMed Google Scholar). In experiments using transiently transfected cells, 1 × 107 cells were transfected by electroporation (Gene-Pulser Bio-Rad, 960 microfarads, 350V). 40 μg of the indicated plasmids were cotransfected with 10 μg of a pEGFP plasmid (CLONTECH, Palo Alto, CA). 24-h post-transfection, the green fluorescent protein-expressing cells were sorted on a high speed cell sorter (Coulter Electronics, Miami, FL). After sorting, cells were pelleted, resuspended in culture medium, and kept in culture for 24 h with or without treatment as indicated. For protein analysis, cells were harvested, washed in PBS and lysed at 5 × 107 cells/ml in cold lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 10 mm NaF, 1 mm EDTA, 1 mmEGTA, 1 mm phenylmethylsulfonyl fluoride, 1 mmNaVO3, 1 μg/ml each leupeptin and aprotinin) for 30 min. Lysates were clarified by centrifugation at 15,000 ×g for 20 min at 4 °C. The protein concentration was determined by Bradford assay, and equivalent amounts of proteins were separated by gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA). Filters were blocked for 2 h at room temperature with either 5% nonfat dry milk or 3% bovine serum albumin in Tris-buffered saline (TBS), 0.5% Tween (TBS-T). Filters were washed three times in TBS-T and incubated for 1 h with optimal concentrations of primary antibodies diluted in TBS, 0.1% Tween. After four additional washes in TBS-T, filters were further incubated 45 min with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). Visualization was performed using PerkinElmer Life Sciences, Renaissance system and Kodak X-Omat blue film (Eastman Kodak Co.). Ton.B.210 cells were either left untreated or treated with 1 μg/ml doxycycline for at least 24 h before cycloheximide treatment in RPMI 1640 medium supplemented with 10% fetal calf serum and 10% WEHI-CM. 8 h before treatment, the cells were harvested, washed twice in 1× PBS, and resuspended in RPMI 1640 supplemented with 1% bovine serum albumin at a cell density of 1 × 106cells/ml with or without doxycycline. After 8 h of IL-3 deprivation, 10 μm cycloheximide was added to the culture, and an aliquot of the cells was harvested at the indicated times. Cells were lysed as described above. For cell cycle analysis, cells were treated as specified. 1–2 × 106 cells were harvested, washed once in 4 ml of PBS, and fixed in 1 ml of 70% ethanol solution. Fixed cells were kept at −20 °C and stained just before analysis. For staining, fixed cells were pelleted, washed once in PBS, and resuspended in 1 ml of propidium iodide-staining solution (PBS, 0.1% Triton X-100, 20 μg/ml propidium iodide, and 100 units/ml RNase A added extemporaneously). Cells were left in propidium iodide staining solution for 30 min at room temperature and analyzed immediately. DNA content and hence the cell cycle distribution was determined by flow cytometry. Repartition of the cells in the various stages of cell cycle was determined with cell cycle analysis software. For cDNA synthesis, 1 μg of total RNA was reverse-transcribed in a 20 μl of reaction mixture containing 250 μm each dNTP, 20 units of RNase inhibitor, 50 units of murine leukemia virus reverse transcriptase, 2.5 μm random hexamers, and 1× buffer (1.5 mmMgCl2) (all reagents were purchased from PE Applied Biosystems, Foster City, CA). The reaction mix was incubated at 42 °C for 45 min and then denatured at 99 °C for 5 min. For each sample, a control reaction not containing the reverse transcriptase enzyme was also performed. Specific primers and probe for p27 (forward: 5′-GGTGGACCAAATGCCTGACT-3′; reverse: 5′-GCCCTTTTGTTTTGCGAAGA-3′; probe: 5′ AATCTTCTGCCGCAGGTCGCTTCC-3′) were designed from sequences in the GenBankTM data base using the Primer Express 1.0 Software (PE Applied Biosystems). The hybridization probe spanned an intron to exclude annealing to genomic DNA. The gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control to standardize the amount of RNA in each reaction (Taqman Rodent GAPDH control reagents). All primers and probes were synthesized by PE Applied Biosystems. PCR was performed on the cDNA samples using an ABI PRISM 7700 sequence detector (PE Applied Biosystems). The Taqman® PCR Core reagent kit (PE Applied Biosystems) was used according to the manufacturer's protocol with the modification that dUTP was replaced by dTTP, and incubation with AmpErase was omitted. For each sample tested, PCR reaction was carried out in a 50-μl volume containing 1 μl of cDNA reaction (equivalent to 50 ng of template RNA) and 2.5 units of AmpliTaq Gold. Oligonucleotide primers and fluorogenic probe were added to a final concentration of 100 nm each. The amplification step consisted of 60 cycles of 94 °C for 45 s, 58 °C for 45 s, and 65 °C for 1 min. In each experiment, additional reactions with 7 serial 2-fold dilutions of Ton.B.210 cDNA, prepared from cells induced or not with doxycycline, as template were performed with each set of primers and probes on the same 96-well plate to generate standard curves, which related the threshold cycle (CT) to the log input amount of template. All samples were amplified in triplicate. The relative amount of p27 transcripts in each sample was determined by using the standard curve method and by normalizing for GAPDH mRNA expression levels, as described previously (ABI PRISM sequence detection system user bulletin No. 2 (PE Applied Biosystems and Ref. 31Fink L. Seeger W. Ermert L. Hanze J. Stahl U. Grimminger F. Kummer W. Bohle R.M. Nat. Med. 1998; 4: 1329-1333Crossref PubMed Scopus (524) Google Scholar). The ability of BCR/ABL to promote survival and proliferation in the absence of growth factors has been well documented in certain hematopoietic-derived cell lines. The Ba/F3 cell line used in our studies is a pre-B cell fully dependent on the presence of IL-3 for survival and proliferation. IL-3 withdrawal for 16 h induces a partial G1 arrest in parental Ba/F3 cells but not in Ba/F3 cells transformed by p210BCR/ABL (Fig. 1, upper panels). Similar results were obtained with the previously described Ton.B.210 cell line (Fig. 1, lower panels). This cell line, derived from Ba/F3 cells, expresses p210BCR/ABL in response to the addition of doxycycline in the culture medium. After withdrawal of IL-3 for 16 h, non-induced Ton.B.210 cells arrest at G0/G1, whereas BCR/ABL-expressing Ton.B.210 cells progress through G1 to S phase (Fig. 1, lower panels). These results demonstrate that BCR/ABL expression regulates cell cycle progression in hematopoietic cells in a manner similar to cytokine stimulation. Based on these results, we sought to identify cell cycle-related proteins that might be regulated by BCR/ABL activity. The Ton.B.210 cell line was used to assure that changes were specifically due to BCR/ABL and not due to unrelated mutations in the cultured cell lines. Cells were left untreated or stimulated with doxycycline for 24 h and IL-3-deprived for 16 h. Immunoblotting of p27 on lysates from non-treated or doxycycline-treated Ton.B.210 cells demonstrated that resting, non-induced cells expressed a high amount of the CKI p27Kip1, whereas BCR/ABL-expressing cells displayed a very low amount of p27Kip1(Fig. 2 A). In addition, p21Cip1 expression levels were higher in proliferating cells.(Fig. 2 A, lower panel). Discordant expression levels of p21Cip1 and p27Kip1 has also been described in other proliferating cells (32Li Y. Jenkins C.W. Nichols M.A. Xiong Y. Oncogene. 1994; 9: 2261-2268PubMed Google Scholar). In some circumstances, CKIs can promote rather than inhibit the formation of active cyclin D-cdk4 complexes (33Cheng M. Olivier P. Diehl J.A. Fero M. Roussel M.F. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: 1571-1583Crossref PubMed Scopus (974) Google Scholar, 34LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. Sandhu C. Chou H.S. Fattaey A. Harlow E. Genes Dev. 1997; 11: 847-862Crossref PubMed Scopus (1222) Google Scholar). Our data would be consistent with a model in which the increased level of p21Cip1expression is enough to participate in the activation of cyclin D-cdk4 but is not high enough to inhibit cyclinE-cdk2. In contrast to p27Kip1, there was no variation of expression of cyclins A, E, D1, or D3 (data not shown). These results suggest that regulation of p27Kip1 expression by BCR/ABL might be an important mechanism in BCR/ABL-mediated proliferative signaling. To confirm that p27Kip1 down-regulation was directly due to BCR/ABL activity, doxycycline-induced Ton.B.210 cells were treated for 14 h with increasing concentrations of the small molecule Abl tyrosine kinase inhibitor STI571 (Fig. 2 B). At an optimal concentration of 1 × 10−6mSTI571, BCR/ABL tyrosine kinase activity was inhibited, and this was accompanied by a substantial increase in p27Kip1expression. In these experiments, the level of expression of the 85-kDa subunit of PI3K was used as a control for equal loading of gel lanes. The results described above indicated that p27Kip1 might be an important target for BCR/ABL in deregulating cell cycle control mechanisms, and therefore, we next sought to identify the signaling pathway responsible for p27Kip1 expression. As an initial screen, three drugs that inhibit different signaling pathways were studied: PD98059, LY-294002, and rapamycin, known to specifically inhibit mitogen-activated protein kinase, PI3K, and p70S6K pathways, respectively. Treatment of p210BCR/ABL-expressing Ba/F3 cells with carrier alone (Me2SO) or with PD98059 had no effect on cell cycle progression (Fig. 3 A, upper panels), whereas treatment for 14 h with either LY-294002 or rapamycin induced a G0/G1 arrest, without any significant effect on cell viability (note the absence of a sub-G1population in Fig. 3 A, lower panels). The effects of these three drugs on BCR/ABL-induced expression of p27Kip1 was then investigated (Fig. 3 B). Treatment of Ba/F3p210BCR/ABL with LY-294002 induced a dramatic increase of p27Kip1 expression, whereas a treatment with rapamycin and PD98059 had no effect. Since rapamycin effectively induces a G1 arrest in these cells, it is unlikely that the p27Kip1 up-regulation induced by LY-294002 is simply a consequence of cell cycle arrest. As p27Kip1 is a known inhibitor of the pRb kinase cdk2, we also sought to determine if pRb was found in a hyperphosphorylated form in BCR/ABL-expressing cells and if this phosphorylation was regulated by PI3K. As shown in Fig. 3 C, hyper- (ppRb) and hypo- (pRb) phosphorylated forms of Rb can be distinguished by an electrophoretic shift on a 6.5% SDS-PAGE. Ba/F3 p210 cells displayed almost exclusively a hyperphosphorylated form of Rb. Although PD98052, rapamycin, or Me2SO had no or little effect on Rb phosphorylation, treatment of Ba/F3 p210 cells with the PI3K inhibitor LY-294002 had a dramatic effect, with Rb reverting to an almost exclusively hypophosphorylated state. These results demonstrate that the PI3K pathway regulates p27Kip1 expression and Rb phosphorylation, most likely through the well known ability of p27Kip1 to regulate cdk2 activity. The serine/threonine kinase AKT/protein kinase B is a downstream mediator of PI3K activity. In an effort to determine if AKT activity was sufficient to mediate down-regulation of p27Kip1, an AKT mutant rendered constitutively active by membrane targeting through the fusion of a CAAX box (HA-AKT-CAAX), was transiently expressed in the BCR/ABL-inducible cell line, Ton-B-210. The Ton-B-210 cells were co-transfected with a plasmid encoding green fluorescent protein (GFP) and either a wild-type AKT construct (HA-AKT-WT), as control, or HA-AKT-CAAX construct. Twenty-four hours after transfection, GFP-positive cells were isolated by flow cytometry and then maintained in culture for an additional 24 h either in the absence or presence of doxycycline in order to induce BCR/ABL expression. The cells were then IL-3-deprived for 18 h and lysed. As shown in Fig. 4 A, IL-3 deprivation of cells without BCR/ABL leads to a dramatic increase of p27Kip1expression in cells transfected either with an empty vector or with a vector encoding for a wild-type form of AKT. Expression of an activated form of AKT or BCR/ABL, however, led to a significant and equivalent decrease of p27Kip1 expression. These results suggest that activation of AKT, a known consequence of BCR/ABL signaling, is sufficient in these cells to regulate p27Kip1expression. To determine if AKT functions downstream of PI3K in the regulation of p27Kip1 expression in Ba/F3 cells, activated AKT was expressed in BCR/ABL-transformed cells in which PI3K had been inhibited by LY-294002 (Fig. 4 B). Ba/F3-p210 cells were co-transfected as above with plasmids encoding GFP and either wild type or the activated form of AKT. After isolation of GFP-positive cells by flow sorting, the positive cells were allowed to recover for an additional 18 h and then either left untreated or were treated with LY-294002 or rapamycin. The expression of the constitutively active form of AKT (HA-AKT-CAAX) completely inhibited the increase of p27Kip1 expression induced by LY-294002, whereas the expression of a wild type form (HA-AKT-WT) had no detectable effect. These results indicate that AKT can regulate p27Kip1expression in hematopoietic cells and that it is likely downstream of PI3K activity, because an activated AKT mutant can override the effect of PI3K inhibition on p27Kip1 expression. In other cell systems, p27Kip1 regulation of expression has been shown to occur at both transcriptional and postranscriptional levels (35Servant M.J. Coulombe P. Turgeon B. Meloche S. J. Cell Biol. 2000; 148: 543-556Crossref PubMed Scopus (124) Google Scholar). In some cells, p27Kip1 has been shown to be ubiquitinated and thereby targeted for proteasome-mediated degradation (36Podust V.N. Brownell J.E. Gladysheva T.B. Luo R.S. Wang C. Coggins M.B. Pierce J.W. Lightcap E.S. Chau V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4579-4584Crossref PubMed Scopus (224) Google Scholar, 37Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1735) Google Scholar, 38Alessandrini A. Chiaur D.S. Pagano M. Leukemia ( Baltimore ). 1997; 11: 342-345Crossref PubMed Scopus (145) Google Scholar). To determine if BCR/ABL-induced p27Kip1 down-regulation is mediated by a protein degradation pathway, we studied several protease inhibitors and two different proteasome inhibitors, lactacystine andN-acetyl-leucyl-leucine norleucinal. As shown in Fig. 5 A, after a 5-h treatment, both proteasome inhibitors specifically induced an increase of p27Kip1 expression in Ba/F3-p210 cells, whereas the protease inhibitors had little or no effect. To further investigate possible degradation of p27Kip1, Ton.B.210 cells, induced or not with doxycycline, were treated with 10 μmribosomal complex inhibitor cycloheximide. By blocking translation with cycloheximide, the role of post-translational events such as degradation in regulating protein levels can be specifically evaluated. Lysates of the cells were prepared at different time points of treatment. Equivalent amounts of protein were loaded on SDS-PAGE and probed with an anti- p27Kip1 antibody. As shown in Fig. 5 B, we clearly show that the disappearance of p27Kip1 protein is faster in BCR/ABL-expressing cells (Fig. 5 B, lower panel) than in non-induced cells (Fig. 5 B, upper panel), suggesting a faster degradation of p27Kip1 in BCR/ABL-expressing cells. After densitometry analysis of the bands, the half-life of p27Kip1 was estimated to be longer than 8 h in non-induced cells and 2 h in BCR/ABL- expressing cells. These results suggest that p27Kip1 protein down-regulation in BCR/ABL-transformed cells is likely to be due predominantly to proteasome-dependent degradation and that this process is regulated by PI3-kinase and AKT. In other cells, p27Kip1 has also been shown to be regulated at the level of transcription (35Servant M.J. Coulombe P. Turgeon B. Meloche S. J. Cell Biol. 2000; 148: 543-556Crossref PubMed Scopus (124) Google Scholar, 39Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1231) Google Scholar). Therefore, p27Kip1RNA levels were compared before and after induction of BCR/ABL in Ton.B.210 cells using semiquantitative real time PCR, as described under “Materials and Methods.” Treatment of Ton.B.210 cells with doxycycline in presence or in absence of LY-294002 resulted in a les