Abstract

Article1 September 1999free access STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells Tetsuya Nosaka Tetsuya Nosaka Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Toshiyuki Kawashima Toshiyuki Kawashima Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Kazuhide Misawa Kazuhide Misawa Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Koichi Ikuta Koichi Ikuta Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8501 Japan Search for more papers by this author Alice L.-F. Mui Alice L.-F. Mui Department of Surgery, University of British Columbia, Jack Bell Research Centre, Vancouver Hospital and Health Sciences Centre, 2660 Oak Street, Vancouver, BC, Canada, V6H 3Z6 Search for more papers by this author Toshio Kitamura Corresponding Author Toshio Kitamura Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Tetsuya Nosaka Tetsuya Nosaka Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Toshiyuki Kawashima Toshiyuki Kawashima Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Kazuhide Misawa Kazuhide Misawa Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Koichi Ikuta Koichi Ikuta Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8501 Japan Search for more papers by this author Alice L.-F. Mui Alice L.-F. Mui Department of Surgery, University of British Columbia, Jack Bell Research Centre, Vancouver Hospital and Health Sciences Centre, 2660 Oak Street, Vancouver, BC, Canada, V6H 3Z6 Search for more papers by this author Toshio Kitamura Corresponding Author Toshio Kitamura Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan Search for more papers by this author Author Information Tetsuya Nosaka1, Toshiyuki Kawashima1, Kazuhide Misawa1, Koichi Ikuta2, Alice L.-F. Mui3 and Toshio Kitamura 1 1Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108-8639 Japan 2Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8501 Japan 3Department of Surgery, University of British Columbia, Jack Bell Research Centre, Vancouver Hospital and Health Sciences Centre, 2660 Oak Street, Vancouver, BC, Canada, V6H 3Z6 *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4754-4765https://doi.org/10.1093/emboj/18.17.4754 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signal transducers and activators of transcription (STATs) play key roles in growth factor-mediated intracellular signal transduction. In the present study using a constitutively active STAT5 mutant, we show that STAT5 has pleiotropic functions regulating cell proliferation, differentiation and apoptosis in an IL-3-dependent Ba/F3 cell line. The mutant STAT5 possessed constitutive tyrosine phosphorylation and DNA binding activity, induced expression of bcl-xL and pim-1 in the absence of IL-3 in Ba/F3 cells, and rendered Ba/F3 cells factor-independent. Unexpectedly, IL-3 treatment of the factor-independent Ba/F3 cells expressing the constitutively active STAT5 resulted in apoptosis within 24 h, or differentiation followed by cell death. In these cells, mRNA expression of growth inhibitory genes downstream of STAT5 such as CIS, JAB/SOCS-1/SSI-1, and p21WAF1/Cip1 was highly induced, correlating with prolonged hyper-phosphorylation of the mutant STAT5 after IL-3 stimulation. Of the STAT5-regulated genes, we found that constitutive expression of JAB/SOCS-1/SSI-1 was sufficient to induce apoptosis of Ba/F3 cells, while p21WAF1/Cip1 could induce differentiation of these cells. In contrast, constitutive expression of pim-1 was sufficient to induce IL-3-independent growth of Ba/F3 cells. These findings suggest that a single transcription factor regulates cell fate by varying the intensity and duration of the expression of a set of target genes. Introduction Signal transducers and activators of transcription (STATs) are transcription factors indispensable for intracellular signaling after stimulation with cytokines, growth factors and hormones (Darnell et al., 1994; Ihle, 1995, 1996; O'Shea, 1997). STAT proteins form homo- or heterodimers upon phosphorylation of tyrosine residues, usually by Janus kinases (JAKs). Dimerized STAT proteins immediately enter the nucleus and bind to the specific DNA sequences in the promoter regions of various genes, resulting in gene activation or repression. Recent gene-targeted mice experiments revealed important biological functions of STATs (Kaplan et al., 1996a,b; Meraz et al., 1996; Shimoda et al., 1996; Takeda et al., 1996, 1997; Thierfelder et al., 1996; Liu et al., 1997; Udy et al., 1997; Teglund et al., 1998). Among seven members of the STAT family, STAT5A and STAT5B are known to be activated by a wide variety of cytokines (O'Shea, 1997). STAT5A-deficient mice showed incomplete mammopoiesis and failure of lactogenesis based on defective signaling to prolactin stimulation (Liu et al., 1997), and decreased proliferation of splenocytes to interleukin (IL)-2 stimulation, which was reported to result from defective induction of IL-2 receptor α chain (Nakajima et al., 1997). On the other hand, STAT5B-deficient mice showed a phenotype similar to that of Laron-type dwarfism, which is a human growth hormone (GH)-resistance disease generally associated with a defective GH receptor, and STAT5B was suggested to play a major role in mediating the sexually dimorphic effects of GH pulses in the liver (Udy et al., 1997). Recently, STAT5A and -B doubly disrupted mice have been generated (Teglund et al., 1998). In addition to the phenotypes of each STAT5-deficient mice, the mice showed mild lymphocytopenia, profound deficiency in peripheral T cell proliferation, and a detectable reduction of colony forming efficiency of bone marrow cells in response to IL-3, IL-5, IL-7 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Interestingly, profound deficiency in peripheral T cell proliferation was not rescued by high concentrations of IL-2, which would bypass the requirement for the expression of the IL-2 receptor α chain, suggesting a direct role of STAT5 for IL-2-induced cell cycle progression of peripheral T cells (Moriggl et al., 1999). However, development of thymus and hematopoiesis of myeloid and erythroid lineages were not significantly impaired in the STAT5A and -B doubly deficient mice. This is in sharp contrast to the phenotypes of JAK1-, JAK2- or JAK3-deficient mice in which hematopoiesis was severely affected (Nosaka et al., 1995; Park et al., 1995; Thomis et al., 1995; Neubauer et al., 1998; Parganas et al., 1998; Rodig et al., 1998). On the contrary, in vitro experiments suggested that STAT5 is involved in cell proliferation in hematopoietic cell lines (Damen et al., 1995; Mui et al., 1996), and more than half the cases of freshly isolated human lymphoid leukemic cells were found to show constitutive activation of STAT5 (Weber-Nordt et al., 1996). It is, therefore, still controversial whether STAT5 plays critical roles in proliferation and differentiation of hematopoietic cells (Fujii et al., 1995; Quelle et al., 1996; Matsumura et al., 1997; Welte et al., 1999). Although generation of null mutation is a powerful approach to study the biological function of the molecule, phenotype is occasionally masked if biologically alternative pathways complement the functional defects. Another approach to unveiling the function of a particular molecule is to activate a single molecule and examine events directly downstream. The results of this strategy are less affected by the other molecules or pathways compared with the analyses by gene disruption or expression of dominant-negative mutants. We have recently identified a constitutively active STAT5A which renders IL-3-dependent cell lines IL-3-independent by screening randomly mutated STAT5As (Onishi et al., 1998). Here we report that in addition to inducing IL-3-independent proliferation, the constitutively active STAT5 induces apoptosis and differentiation in the same cell line after IL-3 stimulation. Results Constitutive activation of STAT5 in the absence of JAK2 activation The mutant STAT5A, designated 1*6, which harbors two mutations of H298 to R and S710 to F (Figure 1A), showed constitutive tyrosine phosphorylation, nuclear localization and DNA binding activity in the absence of IL-3 (Onishi et al., 1998). The 1*6 mutant exhibited prolonged hyper-tyrosylphosphorylation after IL-3 treatment (Onishi et al., 1998), which is a probable molecular basis of its constitutive activity. While stable transfectants expressing high levels of the wild-type STAT5A and those of the single point mutants were easily obtained and maintained, we frequently failed to maintain those of the 1*6 mutant in the presence of IL-3. We therefore suspected a cytotoxic effect of the 1*6 mutant. Interestingly, the 1*6 mutant was cytotoxic in the presence of IL-3, and Ba/F3 cells expressing high levels of the 1*6 mutant could be maintained for a long period only in the absence of IL-3. To obtain the clones expressing higher levels of the mutant STAT5A than those isolated in the presence of IL-3, we selected the Ba/F3 cells by the ability to grow in the absence of IL-3 after retroviral transduction in which the infection efficiency was 30–60%. These cells expressed higher levels of the 1*6 mutant (data not shown) and DNA binding activity (Figure 1B) compared with the cells selected in the presence of IL-3 (Onishi et al., 1998). We do not think that particular clones with secondary mutations were chosen in the absence of IL-3 because 20–50% of the original cell population grew within 48 h of IL-3 deprivation (data not shown). JAK2 was not activated beyond the basal level in the 1*6 cells in the absence of IL-3 (Figure 1C), suggesting that basal level of JAK activity is sufficient for accumulation of phosphorylated form of the mutant STAT5A, or that a previously unrecognized tyrosine kinase is involved in activating the mutant STAT5A. Figure 1.The mutant STAT5 induces IL-3-independent DNA binding activity without activation of JAK2. (A) Structure of the constitutively active mutant STAT5. The 1*6 mutant STAT5 has two mutations; one in the activation domain (designated mutation 1) and the other in the DNA binding domain (mutation 6). Flag was added as an epitope tag to the C-terminal end of the mouse STAT5A to distinguish it from endogenous STAT5A (Wang et al., 1996). (B) Electrophoresis mobility shift analysis (EMSA) with STAT5A binding sequence in Ba/F3 stable transfectants. Cell lysates of Ba/F3 cells, Ba/F3 expressing wild-type STAT5A-Flag, and Ba/F3 expressing the 1*6 mutant STAT5A-Flag before and after IL-3 stimulation were subjected to EMSA. −, removal of IL-3 for 11 h (parental Ba/F3 cells and the wild-type transfectant) and continuous culture without IL-3 (1*6 transfectant); +, cultured with 2 ng/ml of IL-3; ++, stimulated with 10 ng/ml of IL-3 for 30 min after IL-3 deprivation. Lower arrowhead, STAT5A–DNA complex; upper arrowhead, supershifted complex with an anti-Flag antibody. (C) Immunoprecipitation followed by Western blot analysis for phosphotyrosine of JAK2. − and +, same as in (B); ++, stimulated with 20 ng/ml of IL-3 for 10 min after IL-3 deprivation. WT, wild type. Download figure Download PowerPoint IL-3-induced apoptosis or differentiation in the cells expressing the mutant STAT5A Stable transfectants of Ba/F3 cells expressing high levels of the 1*6 mutant grew well without IL-3, albeit more slowly than IL-3-driven parental Ba/F3 cells (Figure 2A and B; Table II). While the transfectants expressing the wild-type STAT5A grew well even in the presence of 10 μg/ml of IL-3, most of the 1*6 cells that express comparable levels of the mutant STAT5A to the levels of the transduced STAT5A in the wild-type expressor died within 24 h, showing apoptotic appearance after addition of 2–3 ng/ml of IL-3 (Figures 2B, 3B and 4B), and DNA ladder was observed in the 1*6 cells with IL-3 stimulation (Figure 4A). The 1*6 cells which escaped apoptosis were larger in size than the cells before IL-3 treatment and exhibited round morphology (Figure 3B and D). These cells did not divide further and died within 2 weeks. We also characterized four single clones of the 1*6 cells, and found that two out of four clones underwent morphological differentiation into macrophage-like cells as well as apoptosis upon IL-3 stimulation (Figures 3D and 5C). In these cells, IL-3 stimulation induced expression of CD11b, a macrophage differentiation marker, while the same clone in the absence of IL-3 as well as the parental Ba/F3 cells did not express CD11b (Figure 6). Differentiated cells died within 1 week. As a whole population, IL-3-treated 1*6 cells never gave rise to long-term growth (data not shown). Cell cycle analysis of the 1*6 cells after IL-3 stimulation revealed G1 arrest and apoptosis with profound reduction of the number of the cells in S phase, which is similar to the cell cycle profile of the parental Ba/F3 cells after IL-3 deprivation (Figure 7; Table I). Figure 2.IL-3 induces growth inhibition of the cells expressing the 1*6 mutant STAT5A-Flag. Growth curves of stable transfectants of Ba/F3 cells expressing the wild-type or the mutant STAT5A-Flag construct either without (A) or with (B) IL-3 are shown. Live cells of triplicate cultures were counted and mean values together with standard errors were plotted against time. WT, wild type; 1, STAT5 with mutation 1 alone; 6, mutation 6 alone; 1*6, double mutations. IL-3 concentration was 2 ng/ml. Cells were diluted appropriately before reaching confluence. Download figure Download PowerPoint Figure 3.Phase contrast microscopy of the cells expressing the 1*6 mutant STAT5A-Flag. (A)(no IL-3) and (B) (after stimulation with 3 ng/ml of IL-3 for 48 h), bulk culture; (C) (no IL-3) and (D) (3 ng/ml of IL-3 for 41 h), one of the clones showing apoptosis and differentiation followed by cell death upon IL-3 stimulation. Download figure Download PowerPoint Figure 4.IL-3-induced apoptosis in Ba/F3 cells expressing the 1*6 mutant STAT5A-Flag. (A) DNA ladder formation in Ba/F3 cells expressing the 1*6 mutant STAT5A after IL-3 treatment. The cells expressing the wild-type STAT5A-Flag was deprived of IL-3 for 40.5 h and those expressing the 1*6 mutant STAT5A-Flag were treated with 3 ng/ml of IL-3 for 24 h. −, no IL-3; +, culture with IL-3; Marker, DNA ladder marker (Bio-Rad, 50, 100, 200, 300, 400, 500, 700, 1000, 1500, 2000 bp). (B) The rate of apoptotic events in Ba/F3 cells expressing the mutant STAT5A-Flag after stimulation with IL-3. Bulk culture of the stable transfectant expressing the 1*6 mutant STAT5A-Flag was treated with 3 ng/ml of IL-3, and morphologically apoptotic cells were enumerated by trypan blue exclusion test in triplicate. Download figure Download PowerPoint Figure 5.IL-3-induced differentiation in Ba/F3 cells expressing the 1*6 mutant STAT5A-Flag. May–Giemsa staining of cytospins of parental Ba/F3 cells with IL-3 (A) and of one of the clones expressing the mutant STAT5A without (B) and with (C) IL-3 (3 ng/ml, 45 h) are shown. Original magnification was ×400. Download figure Download PowerPoint Figure 6.FACS analysis of the same samples as in Figure 5. The cells were stained with phycoerythrin (PE)-conjugated rat anti-mouse CD11b antibody or PE-conjugated isotype-matched control rat IgG2b. −, no IL-3; +, with IL-3. Download figure Download PowerPoint Figure 7.Cell cycle analysis of the cells expressing the 1*6 mutant STAT5A-Flag. Parental Ba/F3 cells were deprived of IL-3 and the 1*6 cells were treated with 3 ng/ml of IL-3, followed by DNA staining at the indicated time points. Download figure Download PowerPoint Table 1. Cell cycle analysis of the cells expressing the 1*6 mutant STAT5A-Flag Time Ba/F3 with IL-3 deprivation 1*6 with IL-3 stimulation G0–G1 S G2–M apoptosis G0–G1 S G2–M apoptosis 0 h 32.5 52.6 14.9 1.0 37.6 50.2 12.2 1.1 6 h 46.7 39.0 14.3 5.7 55.7 37.0 7.3 7.2 12 h 74.8 20.0 5.2 21.4 67.2 24.6 8.2 33.2 24 h 72.6 22.6 4.8 82.8 66.7 24.8 8.5 72.4 Percentages of each phase of cell cycle in Figure 7 are shown. Table 2. Ability to confer IL-3-independence on Ba/F3 cells and doubling time of Ba/F3 cells in the absence of IL-3 after transduction IL-3-independence Doubling time (h) Vector − NA pim-1 + 21.3 ± 1.1 c-myc − NA bcl-xL − NA pim-1 and c-myc + 18.0 ± 0.9 pim-1 and bcl-xL + 17.7 ± 1.1 c-myc and bcl-xL + 14.6 ± 0.7 pim-1, c-myc and bcl-xL + 15.5 ± 0.8 1*6STAT5A-Flag + 14.0 ± 0.6 Parental Ba/F3 cells − 9.3 ± 0.3a a Doubling time in the presence of IL-3. NA, not applicable. Up-regulation of positive regulators for cell growth in the absence of IL-3 and of negative regulators in the presence of IL-3 The 1*6 mutant of STAT5A transactivated the gene expression from the β-casein promoter containing STAT5 binding sites by 20- and 50-fold compared with the wild type in the presence and absence of IL-3, respectively, in a transient transfection assay using Ba/F3 cells (Onishi et al., 1998). We tested whether the wild-type and the mutant STAT5As can induce expression of known STAT5-target genes including oncostatin M (Yoshimura et al., 1996), pim-1 (Selten et al., 1986), bcl-x (Boise et al., 1993), c-fos, JAB/SOCS-1/SSI-1 (a CIS family gene) (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997), CIS (Yoshimura et al., 1995), and cyclin-dependent kinase inhibitor p21WAF1/Cip1 (El-Deiry et al., 1993; Harper et al., 1993) after IL-3 stimulation. Northern blot analysis demonstrated that the same set of target genes was induced in response to IL-3 stimulation in parental Ba/F3 cells, Ba/F3 transfectant expressing the wild-type STAT5A, and that expressing the 1*6 mutant STAT5A (Figure 8A). It is, however, noticeable that induction of all these genes except for c-fos was much stronger and sustained for a longer time period in the 1*6 cells than in the wild-type transfectant or parental cells. pim-1, bcl-xL and c-myc, which are positive regulators for cell survival or proliferation, were expressed in the 1*6 cells in the absence of IL-3, while the wild-type transfectant or parental cells showed little or no expression of these genes in the absence of IL-3. bcl-2 was not up-regulated in the 1*6 cells in the absence of IL-3 (data not shown). Reverse transcription (RT)–PCR analysis confirmed that virtually all the mRNAs hybridized with the bcl-x probe in Northern blot analysis were bcl-xL, whose product is an inhibitor of apoptosis, and an alternatively spliced bcl-xS transcript encoding a promoter of apoptosis, was not detected (Figure 8B). Induction of bcl-xL and c-myc in 1*6 cells is reminiscent of the previous study showing that activation of any two of the three distinct pathways inducing c-fos/c-jun, c-myc and bcl-2 is sufficient to confer cytokine-independent growth of Ba/F3 cells (Miyazaki et al., 1995). The mechanism of c-myc induction in the 1*6 cells is currently unknown. pim-1 was also shown to cooperate with c-myc to induce lymphoma (Breuer et al., 1989; van Lohuizen et al., 1989). In addition, it was reported that enforced expression of pim-1 in mouse myeloid cells resulted in enhancement of factor-independent survival and inhibition of apoptosis (Lilly and Kraft, 1997). Expression of cyclin D1 and p27Kip1 was under the level of detection in these cells (data not shown). Other genes, cyclin A, D2, E, and interleukin-1β converting enzyme (ICE), were not transactivated in the 1*6 cells. Figure 8.Analysis of the downstream gene expression in Ba/F3 cells stably expressing the wild type or the 1*6 mutant STAT5A-Flag. (A) Northern blot analysis. Ba/F3 cells and a Ba/F3 transfectant with the wild-type STAT5A-Flag, which had been starved of IL-3 for 6.3 h, and that with the 1*6 mutant STAT5A-Flag which had been maintained without IL-3, were stimulated with 10 ng/ml of IL-3 for the indicated periods. OSM, oncostatin M; ICE, interleukin-1β converting enzyme (caspase 1); GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B)RT–PCR analysis. Total RNAs which were used for the Northern blot analysis were subjected to RT–PCR analysis to distinguish between bcl-xL and bcl-xS. M, HaeIII-digested φX174 DNA marker; D, PCR with genomic DNA extracted from Ba/F3 cells. (C) p21, but not p53 was overexpressed in the cells expressing the mutant STAT5A. A Ba/F3 transfectant with the wild-type STAT5A-Flag starved of IL-3 for 8.5 h and that with the 1*6 mutant STAT5A-Flag which had been maintained without IL-3 were treated with 3 ng/ml of IL-3, followed by sequential immunoprecipitation (IP) and Western blot analysis with the same antibody as in IP, except that anti-Flag antibody was used for IP and anti-STAT5A antibody for Western blot analysis to detect transduced STAT5A. Download figure Download PowerPoint The mutant STAT5A alone is sufficient to induce autonomous cell growth To identify STAT5-regulated genes that are responsible for autonomous growth of the 1*6 cells, we carried out retrovirus-mediated cDNA expression screening by transduction of a cDNA library from the 1*6 cells maintained in the absence of IL-3, into the parental Ba/F3 cell line. In this strategy, the downstream genes can be isolated only if expression of the single gene was sufficient to confer IL-3-independency on Ba/F3 cells. After the cDNA library transduction, 22 Ba/F3 clones which became IL-3-independent were isolated, and all the clones were found to harbor the cDNA of the 1*6 mutant. This result indicates that the 1*6 gene itself is a prerequisite and sufficient to confer IL-3 independency, and suggests that the 1*6 mutant up-regulated the expression of at least two genes to induce IL-3-independent growth. Interestingly, 11 out of 22 factor-independent clones were found to possess the cDNAs encoding truncated 1*6 STAT5A which is devoid of N-terminal 137 amino acids. The other 11 clones harbored the full-length 1*6 STAT5A. Both forms of 1*6 STAT5A conferred IL-3-independency after re-introduction into Ba/F3 cells and possessed transactivational ability on the β-casein promoter in Ba/F3 cells although the truncated form had less transactivation activity than the full-length form (data not shown). These findings suggest that N-terminal 137 amino acids of the 1*6 STAT5A is not necessary either to confer factor-independent cell growth or to the basal mechanism of transactivation. IL-3 induced expression of p21WAF1/Cip1, but not of p53 and any soluble factors inducing apoptosis/differentiation in cells expressing the mutant STAT5A High expression of p21 protein induced by IL-3 in the 1*6 cells (Figure 8C) may explain G1 arrest, differentiation (Matsumura et al., 1997) and escape from apoptosis (Polyak et al., 1996) in some of the 1*6 cells. p53 was not overexpressed in the 1*6 cells undergoing apoptosis (Figure 8C). To examine whether IL-3-induced apoptosis and differentiation in 1*6 cells were mediated by soluble factors produced by the 1*6 cells, supernatant of the bulk culture of the 1*6 cells undergoing apoptosis and differentiation in the presence of IL-3 was tested for the ability to induce the same phenotypic changes in parental Ba/F3 cells. Ba/F3 cells grew well in the supernatant of the IL-3-treated 1*6 cells without undergoing apoptosis and differentiation, suggesting that no soluble factor was involved in IL-3-induced apoptosis and differentiation of 1*6 cells (data not shown). Although Fas was inducibly expressed on fluorescence-activated cell sorting (FACS) analysis, anti-mouse Fas ligand neutralizing antibody (gift from Dr T.Suda) did not block the IL-3-induced apoptosis of the 1*6 cells, and Fas ligand was not induced in Northern blotting and FACS analyses in 1*6 cells with IL-3 treatment (data not shown), indicating that the Fas/Fas ligand system is not involved in the process. Enforced expression of pim-1 induces factor-independent growth of Ba/F3 cells Since activation of at least two genes was thought to be required for IL-3-independent cell growth, we tested combinations of several known genes for the ability to induce cell proliferation. Of the up-regulated genes in the 1*6 cells, c-myc, pim-1 and bcl-xL have been implicated in cell proliferation or survival. Therefore, Ba/F3 cells were retrovirally transduced with these genes in various combinations. Unexpectedly, pim-1 alone was sufficient to induce IL-3-independency, and co-expression of c-myc or bcl-xL enhanced the phenotype (Table II), suggesting that 1*6 STAT5A conferred IL-3-independency on Ba/F3 cells through up-regulation of pim-1. This finding is somehow inconsistent with our former results in which no gene other than 1*6 STAT5A was identified as an inducer of IL-3-independency after transduction with a cDNA expression library derived from the 1*6 cells. However, a previous study disclosed the dose-dependency of pim-1 function as an oncogene (van der Houven van Oordt et al., 1998). Transduction of pim-1 gene via retrovirus infection would result in multiple integrations of the pim-1 retrovirus in each cell, while in the case of cDNA library trasnduction, each cell should not harbor more than one integration of pim-1. Thus, the difference in the expression level may explain this inconsistency. Ability of bcl-xL together with c-myc to confer IL-3-independency on Ba/F3 cells is consistent with a previous study in which co-expression of bcl-2 and c-myc induced IL-3-independent proliferation of Ba/F3 cells (Miyazaki et al., 1995). JAB is responsible for inducing apoptosis and p21 for differentiation In an attempt to identify the genes to induce apoptosis or differentiation, JAB, CIS, and p21 were transduced individually into Ba/F3 cells together with an enhanced green fluorescent protein (EGFP) using a bicistronic retrovirus vector pMX-IRES-EGFP. After selecting green fluorescent cells by FACS, the fate of those cells was monitored in the presence of IL-3. Within 24 h after sorting, virtually all of the cells expressing JAB were found to have undergone apoptosis (Figure 9B), while control cells expressing EGFP alone and the cells expressing CIS continued to grow (Figure 9A and C). Identical results to those in Ba/F3 cells were also obtained in the 1*6 cells in the absence of IL-3 (data not shown). These findings indicate that JAB is one of the genes responsible for IL-3-driven apoptosis in the 1*6 cells. In similar experiments performed with p21 in Ba/F3 cells, the total number of the sorted cells expressing p21 was one-tenth of the control cells expressing EGFP alone 5 days after sorting (Figure 10). Ten to 20% of the cells expressing p21 showed large and round morphology and some of them became adherent (Figure 11B), while only 1–2% of the control cells showed such morphology and never became adherent (Figure 11A). This finding indicates that p21 is one of the genes involved in differentiation and escape from apoptosis in Ba/F3 cells. Figure 9.Enforced expression of JAB induces apoptosis of Ba/F3 cells. Ba/F3 cells were transduced with the bicistronic retroviral vector pMX-IRES-EGFP (A), pMX-JAB-IRES-EGFP (B) or pMX-CIS-IRES-EGFP (C), followed by sorting based on EGFP expression 24 h after infection. Phase-contrast microscopies of the sorted cells 46 h after infection which were cultured in a medium containing 2 ng/ml of IL-3 are shown. Download figure Download PowerPoint Figure 10.Enforced expression of p21WAF1/Cip1 results in slower rate of cell growth. Growth curves of Ba/F3 cells transduced with either pMX-IRES-EGFP (Vector) or pMX-p21-IRES-EGFP (p21) are shown. The cells were sorted into a medium containing 2 ng/ml of IL-3, based on EGFP expression 24 h after infection. Average values of two independent experiments are shown. Download figure Download PowerPoint Figure 11.Enforced expression of p21WAF1/Cip1 induces morphological differentiation of Ba/F3 cells. Ba/F3 cells were transduced with pMX-IRES-EGFP (A and C) or pMX-p21-IRES-EGFP (B and D), sorted and cultured as in Figure 10. Phase contrast (A, B) and fluorescent (C, D) microscopies 5 days after sorting are shown. Download figure Download PowerPoint Expression of pim-1 is elevated in factor-independent human leukemic cells harboring constitutively activated STAT5 Finally, to see whether Pim-1 is involved in autonomous gr