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Prepublished online as a Blood First Edition Paper on October 3, 2002; DOI 10.1182/blood-2002-05-1374. This Article Abstract Full Text (PDF) Supplemental Figure All Versions of this Article: 2002-05-1374v1 101/3/1141    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Truong, B.-T. H. Articles by Kogan, S. C. Search for Related Content PubMed PubMed Citation Articles by Truong, B.-T. H. Articles by Kogan, S. C. Related Collections Neoplasia Oncogenes and Tumor Suppressors Gene Expression Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 February 2003, Vol. 101, No. 3, pp. 1141-1148 NEOPLASIA CCAAT/Enhancer binding proteins repress the leukemic phenotype of acute myeloid leukemia Bao-Tran H. Truong, Young-Jin Lee, Tracey A. Lodie, Dorothy J. Park, Danilo Perrotti, Naohide Watanabe, H. Phillip Koeffler, Hideaki Nakajima, Daniel G. Tenen, and Scott C. Kogan From the Comprehensive Cancer Center and Department of Laboratory Medicine, University of California, San Francisco; Harvard Institutes of Medicine, Harvard Medical School, Boston, MA; Division of Hematology/Oncology, Cedars-Sinai Medical Center, University of California, Los Angeles School of Medicine; Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA; and Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Japan.     Abstract Top Abstract Introduction Materials and methods Results Discussion References CCAAT/enhancer binding proteins (C/EBPs) are a family of factors that regulate cell growth and differentiation. These factors, particularly C/EBP and C/EBP, have important roles in normal myelopoiesis. In addition, loss of C/EBP activity appears to have a role in the pathogenesis of myeloid disorders including acute myeloid leukemia (AML). Acute promyelocytic leukemia (APL) is a subtype of AML in which a role for C/EBPs has been postulated. In almost all cases of APL, a promyelocytic leukemia-retinoic acid receptor  (PML-RAR) fusion protein is expressed as a result of a t(15;17)(q22;q12) chromosomal translocation. PML-RAR inhibits expression of C/EBP, whereas all-trans retinoic acid (tRA), a differentiating agent to which APL is particularly susceptible, induces C/EBP expression. PML-RAR may also inhibit C/EBP activity. Thus, the effects of PML-RAR on C/EBPs may contribute to both the development of leukemia and the unique sensitivity of APL to tRA. We tested the hypothesis that increasing the activity of C/EBPs would revert the leukemic phenotype. C/EBP and C/EBP were introduced into the FDC-P1 myeloid cell line and into leukemic cells from PML-RARA transgenic mice. C/EBP factors suppressed growth and induced partial differentiation in vitro. In vivo, enhanced expression of C/EBPs prolonged survival. By using a tamoxifen-responsive version of C/EBP, we observed that C/EBP could mimic the effect of tRA, driving neutrophilic differentiation in leukemic animals. Our results support the hypothesis that induction of C/EBP activity is a critical effect of tRA in APL. Furthermore, our findings suggest that targeted modulation of C/EBP activities could provide a new approach to therapy of AML. (Blood. 2003;101:1141-1148) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References CCAAT/enhancer binding proteins (C/EBPs) are a family of proteins that play important roles in the development and differentiation of many cell types, including granulocytes. C/EBP and C/EBP play central roles in normal granulopoiesis. Mice that lack C/EBP do not produce neutrophils and eosinophils,1 whereas mice that lack C/EBP generate neutrophils and eosinophils that exhibit abnormalities in morphology, gene expression, and function.2-5 Given the part that C/EBP and C/EBP play in myelopoiesis, we anticipated that their function might be disrupted in human diseases, including myeloid neoplasms. Point mutations in C/EBP have been identified in acute myeloid leukemias (AMLs)6,7; the AML with maturation-associated translocation fusion protein AML1-ETO inhibits C/EBP expression8 and activity9; and the chronic myelogenous leukemia (CML) fusion protein BCR-ABL inhibits C/EBP expression.10 Although C/EBP mutations were not identified in patients with AML,11 such mutations have been observed in human patients with neutrophil-specific granule deficiency.12,13 We have suggested that altered function of C/EBP and C/EBP might also be important in the pathogenesis of acute promyelocytic leukemia (APL).14,15 APL represents approximately 10% of human AMLs.16 This leukemia is now defined by the presence of a t(15;17)(q22;q12) chromosomal translocation, creating a PML-RARA fusion gene (or rare variants that also result in fusions to RARA).17 In 1987, all-trans retinoic acid (tRA) was discovered to be able to induce remissions in APL18 by causing the leukemic cells to differentiate into mature neutrophils.19 Subsequently, the combination of tRA with chemotherapy has led to a marked increase in long-term survival for patients with APL; tRA treatment represents a paradigm for molecularly targeted therapy that restores normal behavior to malignant cells.20 The promyelocytic leukemia-retinoic acid receptor  (PML-RAR) fusion protein is believed to contribute to APL pathogenesis by disrupting the function of PML and by repressing transcription, including genes regulated by RARs.21 The ability of the PML protein to function in apoptosis and growth suppression is blocked by PML-RAR, which disrupts the nuclear bodies of which PML is a part. At physiologic levels of retinoids, PML-RAR represses transcription at retinoic acid response elements (RAREs) by enhancing association with nuclear corepressors. A recent study suggested that recruitment of DNA methyltransferases can also be a mechanism by which PML-RAR inhibits gene expression.22 In addition to inhibiting PML and RARs, PML-RAR affects other proteins, including C/EBPs. PML-RAR blocks C/EBP activity14 and can inhibit expression of C/EBP.15 These observations raise the possibility that suppression of C/EBP activity has a role in the pathogenesis of APL. Treatment of APL cells with pharmacologic doses of tRA results in the restoration of PML nuclear bodies and transcriptional activation of retinoic acid (RA)-responsive genes. Of these effects, transcriptional activation appears central because differentiation takes place even when PML nuclear bodies are not reassembled.23 C/EBPs might be important targets of tRA. Consistent with this idea, C/EBPs can arrest cell growth and induce differentiation of a variety of cell types, including myeloid cell lines.8,24-28 Furthermore, PML-RAR actually enhances C/EBP induction in response to pharmacologic levels of tRA,15 tRA reverses the ability of PML-RAR to inhibit C/EBP,14 and tRA induces expression of another C/EBP family member, C/EBP.29 Together, these observations indicate that induction of C/EBP activity may underlie the unique responsiveness of APL to tRA therapy. We assessed whether either C/EBP or C/EBP could reverse the leukemic phenotype of a murine model of APL. In vitro, C/EBPs caused partial differentiation of the leukemic cells. In vivo, C/EBPs repressed the leukemias regardless of whether these leukemias were responsive or resistant to tRA. Use of a tamoxifen-responsive version of C/EBP demonstrated that C/EBP can, like tRA, induce differentiation of leukemic cells in an animal. These results are consistent with the idea that activation of C/EBPs is central to the tRA response and demonstrate that C/EBPs themselves exhibit antileukemic activity in vivo. Thus, developing therapeutic means to enhance C/EBP activity should permit differentiation therapy to be extended to non-APL subtypes of myeloid leukemia.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Plasmids A human C/EBP cDNA6 and a human cDNA encoding 32-kDa C/EBP15 were inserted into the mouse stem cell virus-internal ribosomal entry site-green fluorescent protein MSCV-IRES-GFP; [MIG]) retroviral vector30-32 with standard molecular cloning techniques. MIG containing rat C/EBP with the wild-type upstream open reading frame (rC/EBP-WT) and rat C/EBP with deletion of the upstream open reading frame and spacer (rC/EBP-uORF) have been described previously.10 A tamoxifen-responsive C/EBP was created by fusing a human C/EBP cDNA (gift of K. G. Xanthopoulos, designated hCEBP-ERTM) to the hormone-binding domain of a tamoxifen-responsive mouse estrogen receptor33; this construct, designated hCEBP-ERTM, was also inserted into MIG. Cell culture FDC-P1 cells34 were grown in Dulbecco modified Eagle medium with 10% fetal bovine serum, 100 U/mL penicillin G, 100 µg/mL streptomycin, 2 mM L-glutamine, and 5% X63Ag8-mouse interleukin-3 (mIL-3)-conditioned media.35 Freshly harvested cells from bone marrow and spleens of leukemic mice were cultured in Myelocult M5300 (StemCell Technologies, Vancouver, BC, Canada) with 100 U/mL penicillin G, 100 µg/mL streptomycin, 5% X63Ag8-mIL-3-conditioned media, and 5% Sp2-IL-6-conditioned media.36 Retroviral transduction BOSC23 cells were transfected with retroviral constructs as previously described.37 Retroviral supernatants were filtered through 0.45-µm filters and stored at 80°C. FDC-P1 cells were plated at 50 000 cells per well in 24-well tissue culture plates. On each of 2 consecutive days, the cells were transduced by incubation with 2 mL viral-containing supernatant with 4 µg/mL polybrene and centrifugation at 1100 g for 1.5 hours at room temperature. Transduction of leukemic cells was similarly performed, except that leukemic cells were plated at 2 × 106 cells per well. Western blot analysis Whole-cell lysates were prepared by lysing 2 × 107 cells in 600 µL 2 × sample buffer, heating at 90°C to 95°C for 5 minutes, and shearing through a 20-gauge needle. Western blot analysis was performed as previously described38 with rabbit polyclonal antibodies to C/EBP or C/EBP (Santa Cruz Biotechnology, Santa Cruz, CA). Analysis of growth of FDC-P1 cells After 2 rounds of infection, transduced cells were pooled and washed once with buffered saline, and GFP+ cells were sorted using the FACSVantage cell sorter (Becton Dickinson, San Jose, CA). Culture of sorted cells started at 30 000 cells. At 48-hour intervals, the medium was changed, live cells were counted with trypan blue exclusion, and the percentage of GFP+ cells was assessed by flow cytometry. Analysis of differentiation For flow immunophenotyping, anti-Gr-1-phycoerythrin and anti-Mac-1-phycoerythrin (BD Pharmingen, San Diego, CA) were used. Cells were resuspended in 100 µL buffered saline and incubated with indicated antibodies for 20 to 30 minutes on ice in the dark, then washed and resuspended in buffered saline. Stained cells were analyzed on a FACScan, and at least 10 000 events were collected for each sample. Fluorescence-activated cell sorter (FACS) data were analyzed with CELLQUEST (Becton Dickinson). Differential cell counts of Wright Giemsa-stained bone marrow smears were performed according to published guidelines.39 The guidelines for the differential counts were modified for FDC-P1 cells as described in the legend to Table 1. The guidelines were also modified for leukemic cells cultured in vitro because an atypical cell type was present; myeloid cells with atypical nuclear segmentation and basophilic cytoplasm were enumerated separately from the intermediate forms and neutrophils of normal appearance.                                View this table: [in this window] [in a new window]   Table 1. Differential counts of FDC-P1 cells transduced with control, hC/EBP, or hC/EBP retroviral constructs Mice Mice were bred and maintained at the University of California at San Francisco, and their care was in accordance with University of California at San Francisco guidelines. Leukemias from MRP8 PML-RARA40 and MRP8 PML-RARAm441 transgenic mice were maintained by serial transplantation. Female FVB/N mice, 6 to 8 weeks old, received sublethal irradiation (4.5 Gy) and intravenous injection into the lateral tail vein with either leukemic cells (1 × 106 cells per animal for serial passaging) or sorted GFP+- transduced leukemic cells (25 000 or 50 000 cells per animal). Tamoxifen treatment As previously described,42 4-hydroxytamoxifen (4-HT; Sigma, St Louis, MO) was dissolved in ethanol at 100 mg/mL, then diluted in autoclaved sunflower seed oil (Sigma) at 10 mg/mL, sonicated for approximately 20 minutes, and stored at 20°C. One milligram 4-HT per mouse was injected intraperitoneally on consecutive days. Statistical analysis Statistical analyses were performed with Excel 2000 using the Student t test, one-tailed distribution, and unequal variance.     Results Top Abstract Introduction Materials and methods Results Discussion References Expression of C/EBP or C/EBP represses growth and induces partial differentiation of FDC-P1 cells CCAAT/enhancer binding proteins, including C/EBP and C/EBP, have important roles in myeloid differentiation. We wished to assess whether increasing the activity of either C/EBP or C/EBP could suppress the leukemic phenotype of murine AML cells. We initially examined whether the C/EBPs would suppress growth and/or induce differentiation of the FDC-P1 cell line, a nonleukemic factor-dependent line of immature myeloid cells. FDC-P1 cells were transduced with retroviruses designed to express only GFP (the MIG control virus), hC/EBP, or hC/EBP (Figure 1A). Immunoblots of FDC-P1 whole-cell lysates indicated that these retroviruses drove expression of the C/EBP proteins (Figure 1B). Compared with control, overexpression of C/EBPs in FDC-P1 cells repressed proliferation (Figure 2A). In addition, C/EBP and C/EBP increased expression of both Ly-6G (Gr-1) and CD11b (Mac-1), which are surface markers of myeloid differentiation (Figure 2B). Although C/EBP and C/EBP both induced FDC-P1 cells to partially differentiate, C/EBP-transduced cells showed greater morphologic change, including the appearance of cells with nuclear segmentation (Figure 2C; Table 1). The growth suppression and partial differentiation observed in transduced FDC-P1 cells showed that C/EBP and C/EBP proteins were expressed and functional. View larger version (43K): [in this window] [in a new window]   Figure 1. C/EBP retroviral constructs. (A) Expression vectors containing a bicistronic system expressing the gene of interest and GFP under the control of the MSCV promoter. MSCV-IRES-GFP (MIG) is an expression vector with a multiple cloning site (MCS), at which either a human C/EBP (42 kDa) or human C/EBP (32 kDa) cDNA was inserted. The human C/EBP includes an open reading frame (uORF) upstream to the start codon for C/EBP. rC/EBP-WT is similar to the hC/EBP construct, but contains rat C/EBP. rC/EBP-uORF is rat C/EBP without the upstream open reading frame (uORF). The hC/EBP-ERTM retroviral construct contains human C/EBP fused at the C-terminus to a tamoxifen-responsive mouse estrogen receptor hormone-binding domain. (B) Western blots of whole-cell lysates of transduced FDC-P1 cells. Lysates were prepared at 24 hours after the second round of transduction. (i) FDC-P1 cells were transduced with retroviruses expressing MIG (control), hC/EBP, rC/EBP-WT, and rC/EBP-uORF. Lysates were blotted with rabbit polyclonal antibodies raised to an internal region of rat C/EBP. Arrow indicates the position of C/EBP. (ii) Lysates of MIG-, hC/EBP-, and hC/EBP-ERTM-transduced FDC-P1 cells were blotted with rabbit polyclonal antibodies raised to the carboxy terminus of rat C/EBP. Arrows indicate the positions of hC/EBP (32 kDa) and hC/EBP-ERTM (70 kDa). The identities of the cross-reacting bands in the hC/EBP lane are uncertain. The bands in the hC/EBP-ERTM lane below the full-length hC/EBP-ERTM may represent smaller hC/EBP-ER translation products initiated from downstream AUG codons.43 View larger version (33K): [in this window] [in a new window]   Figure 2. C/EBPs suppress growth and induce partial differentiation of FDC-P1 cells. FDC-P1 cells grown continuously in IL-3 were transduced on 2 sequential days with control, hC/EBP, or hC/EBP retroviruses. For these experiments, day 0 is considered to begin 24 hours after the second round of retroviral transduction. (A) The growth of C/EBP- and C/EBP-infected FDC-P1 cells was suppressed. Equal numbers of cells were plated on day 0. The growth curves represent the number of transduced FDC-P1 cells at days 0, 2, 4, and 6. At each time point, cells were counted and the percentage of cells with GFP expression was assessed. The number of GFP+ cells is shown on this graph. Results shown are means ± SDs from 3 independent experiments. (B) Both C/EBP and C/EBP induced expression of myeloid markers by FDC-P1 cells. Transduced FDC-P1 cells were stained for Gr-1 (Ly-6G) and Mac-1 (CD11b). Results shown in histograms are of live (based on forward scatter versus side scatter) GFP+ cell populations. These data are representative of 2 independent experiments. (C) C/EBP-transduced FDC-P1 cells were partially differentiated toward more mature forms. GFP+ FDC-P1 cells were sorted at day 4 (6 days after the first round of transduction) with the FACSVantage cell sorter. Cytospins of 25 000 sorted cells of each transduction were stained with Wright Giemsa. Images were captured with × 40 objectives. Expression of C/EBP or C/EBP induces differentiation of RA-sensitive and RA-resistant murine leukemias We next examined the ability of C/EBP and C/EBP to induce differentiation of leukemias derived from transgenic mice that expressed either PML-RAR (RA-sensitive leukemia no. 1111) or PML-RARm4 (RA-resistant leukemia no. 4048.2). Freshly harvested leukemic cells were transduced with control, hC/EBP-, or hC/EBP-containing retroviruses. In vitro, the morphology of transduced leukemia cells was assessed by differential counts of Wright Giemsa-stained cytospins. Overexpression of C/EBP proteins induced partial morphologic differentiation of both RA-sensitive and RA-resistant leukemic cells (Figure 3). Certain aspects of these results should be noted. First, in these experiments, very few mature neutrophils were induced by the C/EBPs. Instead, the C/EBPs led to the appearance in culture of partially differentiated cells, including many with nuclear segmentation. Second, a greater number of differentiated cells was seen with the PML-RARm4 leukemia than with the PML-RAR leukemia, including the control transduced cells. This finding is consistent with our previous observation that leukemias induced by an MRP8 PML-RARAm4 transgene showed somewhat more differentiation in vivo than leukemias induced by MRP8 PML-RARA.41 View larger version (28K): [in this window] [in a new window]   Figure 3. C/EBPs induce partial morphologic differentiation of murine APL cells. Freshly harvested bone marrow and spleen cells from leukemic mice were transduced on 2 consecutive days with the retroviral constructs. GFP+ cells were sorted 5 days after the first transduction. Cytospins of sorted cells were stained with Wright Giemsa, and differential counts were performed (100-300 cells). (A) RA-responsive leukemia no. 1111. Results of 3 independent experiments expressed as means ± SDs are shown. Compared with MIG-infected control, C/EBP-infected cells showed significantly fewer immature forms or blasts (P = .03) and significantly more segmented forms (P = .01). C/EBP-infected cells showed significantly more intermediate-stage cells without segmentation (P = .04), with the changes in immature forms or blasts and segmented forms nearing statistical significance (P = .06). (B) RA-resistant leukemia no. 4048.2. Results of 2 independent experiments expressed as means ± SDs are shown. Decreases in immature forms or blasts and increases in intermediate forms and segmented forms are apparent. Because some variation occurred between the 2 experiments, only the increase in segmented forms induced by C/EBP reached statistical significance (P = .05). Myeloid cells with atypical nuclear segmentation and basophilic cytoplasm were enumerated separately from intermediate forms and from neutrophils of normal appearance. C/EBPs suppress the leukemic phenotype of PML-RAR and PML-RARm4 leukemias On the basis of the results with FDC-P1 cells and mouse leukemic cells in vitro, we proceeded to use retroviral transduction of 3 independent leukemias to test the ability of C/EBP and C/EBP to limit leukemic cell growth in vivo. In addition to the RA-sensitive and RA-resistant leukemias described in the previous paragraph, a second RA-responsive PML-RAR leukemia was studied, no. 935. Leukemic cells were harvested and then transduced on 2 consecutive days with MIG, hC/EBP, or h/CEBP retroviruses. GFP+ cells were sorted, and 25 000 or 50 000 cells were injected intravenously into sublethally irradiated histocompatible recipient animals. Recipients of PML-RAR leukemias transduced with C/EBP survived longer than recipients of leukemias transduced with the MIG control (Figure 4A). C/EBP modestly prolonged survival (mean increase in survival of 3 days, median 2.5 days, maximum 10 days; P = .01), whereas C/EBP had a more substantial impact on survival (mean increase in survival of 13 days, median 6 days, maximum 59 days; P = .004). Results in Figure 4A are pooled data from 3 independent experiments. The results of the individual experiments are shown in a supplemental figure on the Blood website; see the Supplemental Figure link at the top of the online article. Of note, RA treatment of RA-sensitive leukemias prolonged survival more than did transduction by C/EBPs (mean increase in survival of 40 days [median 42 days] with a 5-mg 21-day tRA pellet; mean increase in survival of 74 days [median 41 days] with a 10-mg 21-day tRA pellet).41,44 The impact of C/EBP transduction was, however, likely to be attenuated by the presence of untransduced cells, loss of retroviral expression, and/or down-regulation of protein expression (see below). Despite these limitations, 2 recipients of cells transduced with C/EBP had their survival prolonged to a degree comparable to that of RA treatment. View larger version (27K): [in this window] [in a new window]   Figure 4. C/EBPs suppress growth of murine myeloid leukemia in vivo. Bone marrow and spleen cells of leukemic mice were harvested and transduced with retroviruses expressing MIG (control), hC/EBP, or hC/EBP. At 24 hours after the second round of infection, GFP+ cells were sorted and injected intravenously into sublethally irradiated (4.5 Gy) healthy FVB/N females. (A) PML-RAR leukemias no. 935 and no. 1111, with 25 000 to 50 000 cells per animal. Survival curves include combined data from 3 independent experiments. See data from individual experiments by clicking on the Supplemental Data Set link at the top of the online article on the Blood website. Control mice, n = 15; C/EBP mice, n = 16; C/EBP mice, n = 15. (B) PML-RARm4 leukemia no. 4048.2, with 25 000 cells per animal. Control mice, n = 6; C/EBP mice, n = 4; C/EBP mice, n = 4. Consistent with previous observations, recipients of a PML-RARm4 leukemia survived longer than recipients of PML-RAR leukemias, and both C/EBP and C/EBP substantially lengthened the lives of PML-RARm4 recipient mice (Figure 4B). For C/EBP, mean increase in survival was 63 days, median was 56 days, and maximum was 98 days (P = .003); for C/EBP, mean increase in survival was 54 days, median was 49 days, and maximum was 98 days (P = .002). These results in RA-sensitive and RA-resistant leukemias show that expression of retroviruses containing CEBPA or CEBPE cDNAs can suppress the leukemic phenotype of AML. When possible, leukemic cells were harvested from moribund recipients, and the cells were assessed for the expression of GFP (Table 2A). There appeared to be selection against cells that expressed the MSCV-hC/EBP-IRES-GFP retrovirus: In the 2 experiments in which 50 000 cells were used, GFP expression was essentially absent in leukemic cells analyzed (suggesting that untransduced cells present in the sorted population may have given rise to the leukemias). In the 2 experiments in which 25 000 cells were used, GFP expression from the C/EBP retrovirus was seen in some animals; persistent expression of GFP was observed in mice that survived longest. Compared with MIG-transduced leukemias, no selection against persistent expression of the MSCV-hC/EBP-IRES-GFP retrovirus was apparent.                                View this table: [in this window] [in a new window]   Table 2. Expression of GFP in recipients of transduced leukemias PML-RAR leukemia tolerates expression of a C/EBP retrovirus only when an upstream open reading frame is present Although GFP was expressed by a high percentage of leukemic cells in most C/EBP recipients and in some C/EBP recipients, it was important to assess whether C/EBP proteins were in fact present in the leukemic cells. Neither C/EBP nor C/EBP proteins were detected by Western blot analysis (data not shown). Because the C/EBPs and GFP are encoded by a bi-cistronic transcript, the lack of C/EBP protein expression might be due to decreased translation. In fact, initiation of C/EBP translation can be inhibited by a short open reading frame and spacer (uORF) upstream of the initiation codon for C/EBP. We hypothesized that deletion of the uORF might create additional selective pressure against the C/EBP retrovirus. Constructs expressing rat C/EBP either with or without the uORF (rC/EBP-WT and rC/EBP-uORF; Figure 1A) were used to test this idea. Both constructs were able to produce C/EBP protein in FDC-P1 cells (Figure 1B). PML-RAR leukemic cells (no. 935) were transduced with control, rC/EBP-WT, and rC/EBP-uORF retroviruses, and 25 000 GFP+ cells were injected into sublethally irradiated recipients. Although deletion of the uORF did not significantly influence survival (control, n = 4, mean survival 37 days; rC/EBP-WT, n = 6, mean survival 40 days; rC/EBP-uORF, n = 6, mean survival 38 days), deletion of the uORF led to markedly diminished GFP expression in the leukemias that arose (Table 2). A tamoxifen-inducible version of C/EBP mimics retinoic acid in a mouse model of APL Induction of C/EBP has been proposed to be a central mechanism by which tRA causes the differentiation of APL cells. To address whether C/EBP can induce neutrophilic maturation of leukemic cells in vivo, we used a tamoxifen-inducible form of C/EBP, hC/EBP-ERTM. PML-RAR leukemic cells (no. 1111) were transduced with control and C/EBP-ERTM retroviruses, then transplanted into sublethally irradiated histocompatible mice. Leukemias were allowed to develop in recipients of either control or C/EBP-ERTM-transduced leukemic cells. Leukemic mice were then treated with either vehicle or 4-HT. In contrast to findings in the other mice, bone marrow examination revealed that 4-HT caused neutrophilic differentiation of the leukemic cells that had been transduced with C/EBP-ER. Morphologically, only the 4-HT-treated C/EBP-ERTM samples showed numerous mature neutrophils (Figure 5A). In addition, the cells exhibited increased CD11b (Mac-1) expression (Figure 5B). Differential counts of bone marrow smears are presented in Figure 5C. 4-HT induced substantial neutrophilic differentiation and allowed nonmyeloid hematopoietic cells (erythroid cells and lymphocytes) to repopulate the bone marrow. These findings parallel the effects of RA on this mouse model of human APL.40 View larger version (60K): [in this window] [in a new window]   Figure 5. Effect of C/EBP-ER on differentiation of myeloid leukemia in vivo. PML-RAR leukemia no. 1111 was transduced with retroviruses expressing only GFP (MIG) or hC/EBP-ERTM, and 25 000 sorted GFP+ cells were injected intravenously into sublethally irradiated FVB/N mice. The leukemic cells were allowed to engraft and expand. Beginning on day 27, 4-HT or vehicle was administered daily by intraperitoneal injection. The MIG recipient animals became moribund and were killed on day 30. The hC/EBP-ERTM recipient animals were killed on day 31. (A) Bone marrow smears of recipients of MIG- and hC/EBP-ERTM-transduced cells either with or without 4-HT treatment. Wright Giemsa stain was used, and images were captured with × 40 objectives. (B) Surface-marker expression of CD11b (Mac-1) in MIG or C/EBP-ERTM recipients. Histograms were gated on live (based on FSC and SSC) GFP+ cells. The solid graph represents vehicle treatment, and the black line represents 4-HT treatment. (C) Differential counts (200 cells) of bone marrow smears of individual recipients of control or C/EBP-ERTM-transduced cells, treated as indicated. Immature indicates immature forms or blasts; intermediate, intermediate forms; mature, mature neutrophilic cells; other, erythroid cells, lymphocytes, and eosinophils.     Discussion Top Abstract Introduction Materials and methods Results Discussion References Differentiation therapy of APL with tRA has transformed this leukemia into a highly curable illness. This remarkable impact of differentiation therapy on one subtype of AML has created hope that molecularly-targeted therapies can be developed for other types of AML, including those with complex karyotypic abnormalities and poor prognosis. C/EBPs are central mediators of neutrophil maturation. Our results show that C/EBPs induce differentiation of retinoic acid responsive and resistant leukemias. Moreover, in an animal model of AML, C/EBPs suppressed the leukemic phenotype. Thus, developing therapeutics that stimulate C/EBP expression and activity should extend the range of leukemias that can be treated with differentiation therapy. In U937 and 32Dcl3 cell lines, overexpression of either C/EBP or C/EBP can block cell growth and induce differentiation.8,25-28 Similarly, we observed that C/EBP and C/EBP impaired growth and induced differentiation of FDC-P1 cells and mouse leukemias. However, we saw differences between our hC/EBP and hC/EBP constructs. hC/EBP caused a greater degree of morphologic differentiation of FDC-P1 cells than did hC/EBP. In contrast, the hC/EBP vector seemed to have a greater impact on overall survival than did C/EBP. Therefore, expression of CEBPA and CEBPE can cause different effects. These dissimilarities could reflect intrinsic differences in the biologic activities of C/EBP and C/EBP or instead could be a result of differences in expression from the retroviral vector, posttranscriptional regulation of translation, or posttranslational modification. Perrotti et al10 have shown that BCR-ABL inhibits C/EBP expression by inducing hnRNP E2 to bind the uORF/spacer region of C/EBP transcripts in 32Dcl3 BCR-ABL cells and in cells from human patients with chronic myelogenous leukemia in blast phase. Our observation that the presence of the uORF/spacer tolerizes a PML-RAR leukemia to the presence of C/EBP mRNA extends the cell types in which this mechanism of posttranscriptional regulation has been seen. Posttranscriptional regulation of C/EBP might be important in both normal and leukemic hematopoiesis. As a consequence, the factors regulating translation could represent an additional point for therapeutic intervention: inhibition of hnRNP E2 may be possible, resulting in the stimulation of C/EBP activity, growth arrest, and differentiation. The relationships among C/EBPs, RARs, mutations that contribute to AML, and the therapeutic effects of retinoids are complex. In APL, PML-RAR is able to inhibit RARs and may inhibit C/EBP activity and C/EBP expression. Activation of transcription from RAREs and induction of C/EBP activity, including , , and , may combine to underlie the therapeutic effects of tRA. C/EBPs and RARs can both induce granulocytic differentiation. Therefore, the particular sensitivity of APL to tRA may in fact be due to the ability of pharmacologic levels of tRA to reverse PML-RAR inhibition of both RAR and C/EBPs and thence to activate these factors. The ability of C/EBPs to suppress the leukemic phenotype indicates that C/EBP induction may be critical to the tRA response. However, overexpression of these proteins may provide a more powerful stimulus to growth suppression and differentiation than the effect of pharmacologic tRA on C/EBPs. In other words, induction of C/EBP activity may be central to the response of APL to tRA, but may not represent the only important effect of tRA: it may be that the combination of C/EBP induction along with additional effects causes differentiation. Overexpression of C/EBPs could obviate the requirement for these additional effects of tRA by causing supraphysiologic effects on C/EBP targets. In some settings, C/EBP activity appears sufficient to induce complete neutrophil differentiation whereas in other contexts C/EBPs induce only partial differentiation. Expression of C/EBP, , , or  in 32Dcl3 cells largely recapitulates the effect of granulocyte colony-stimulating factor (G-CSF) on this cell line.26-28,45 Expression of C/EBP and C/EBP in FDC-P1 cells inhibited growth and induced expression of myeloid antigens, however these factors were insufficient to induce the production of mature neutrophils from this cell line. Likewise, C/EBP and C/EBP induced partial morphologic differentiation of leukemias from MRP8 PML-RARA and MRP8 PML-RARAm4 transgenic mice in vitro, but again few mature neutrophils were generated in these cultures. The character of the cells as well as the context in which these experiments were performed, in vitro culture in the presence of IL-3 (and, for the leukemic cells, IL-6), might have limited the ability of the cells to differentiate fully. Remarkably, in vivo, activation of C/EBP-ERTM resulted in the generation of fully differentiated neutrophils. These observations support the concept that neutrophil differentiation is the result of integrating multiple external and internal signals. C/EBPs likely suppress the leukemic phenotype through a combination of induction of transcriptional targets and direct inhibition of cell cycle progression. Target genes of C/EBP include G-CSF receptor, myeloperoxidase, and PU.1; targets of C/EBP include specific granule proteins such as lactoferrin.1,4,26,46-48 Induction of these targets contributes to the phenotype of neutrophilic cells. In addition, C/EBP is able to block growth by enhancing the activity of the cyclin-dependent kinase inhibitor p21, inhibiting E2F transactivation, and blocking cdk2 and cdk4 activity.49-56 C/EBP and C/EBP are probably part of a self-reinforcing pathway that causes myeloid cells to become postmitotic and to assume the characteristics of mature neutrophils. New therapeutics that stimulate C/EBP activity or that mimic their dual effects should extend the reach of differentiation therapy in AML.     Acknowledgments We thank H. Jeffrey Lawrence and Nancy Berliner for helpful discussions and Adam Olshen for advice on statistical analyses. We also acknowledge the continuing support of J. Michael Bishop, Frank McCormick, Kevin Shannon, and Daphne Haas-Kogan.     Footnotes Submitted May 10, 2002; accepted September 14, 2002. Prepublished online as Blood First Edition Paper, October 3, 2002; DOI 10.1182/blood-2002-05-1374. Supported by the 32nd Edward Mallinckrodt Junior Scholar award (S.C.K.) and by a Burroughs Wellcome Fund Career Award (S.C.K.). Additional support was provided by grants K08-CA75986, U01-CA84221, and R01-CA88046 from the National Institutes of Health and by the Parker Hughes Trust Fund. The online version of the article contains a data supplement. 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