C/EBPα and C/EBPɛ induce the monocytic differentiation of myelomonocytic cells with the MLL-chimeric fusion gene

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Abstract

CCAAT/enhancer binding proteins (C/EBPs) have an important function in granulocytic differentiation, and are also involved in the leukemogenesis of acute myeloid leukemia (AML). Their involvement in myelomonocytic leukemia, however, is still unclear. Therefore, the expression and function of C/EBPs in myelomonocytic cells with MLL-fusion genes were investigated. Retinoic acid (RA) induced monocytic differentiation in the myelomonocytic cell lines with MLL-fusion genes, THP-1, MOLM-14 and HF-6 cells, accompanied by monocytic differentiation with the upregulation of C/EBPα and C/EBPɛ. Monocytic differentiation by RA treatment was confirmed in primary AML cells using a clonogenic assay. When the activity of C/EBPα or C/EBPɛ was introduced into HF-6 cells, their cellular growth was arrested through differentiation into monocytes with the concomitant marked downregulation of Myc. Cebpe mRNA was upregulated by the induction of C/EBPα-ER, but not vice versa, thus suggesting that C/EBPɛ may have an important function in the differentiation process. Introduction of Myc isoforms into HF-6 cells partially antagonized the C/EBPs effects. These findings suggest that the ectopic expression of C/EBPɛ, as well as C/EBPα, can induce the monocytic differentiation of myelomonocytic leukemic cells with MLL-fusion gene through the downregulation of Myc, thus providing insight into the development of novel therapeutic approaches.

Introduction

Recurrent translocations involving the mixed lineage leukemia (MLL) gene located on chromosome 11q23 frequently occur in various hematological malignancies including acute myeloid leukemia (AML). In the translocations, truncated N-terminal MLL proteins with AT hooks and MT domain are fused in-frame to one of more than 40 translocation partners to produce proteins with novel properties. The translocation partners share little sequence homology, but the resultant chimeric proteins are thought to alter the transcription on MLL target genes (Li et al., 2005).

Rearrangements involving 11q23 or MLL are found in both AML and acute lymphoid leukemia (ALL) cases. In AML, they occur more frequently (5–30%) in the subtypes with monocytic components, such as M4, M5a and M5b, based on the French–American–British classification (Schoch et al., 2003; De Braekeleer et al., 2005). Consistent with this, in vivo studies in mice have revealed that MLL-fusion genes caused AML, which was arrested at the late myelomonocytic stage, regardless of the developmental level of the initiating cells (Cozzio et al., 2003). AML with MLL-fusion genes has been reported to have poorer prognosis with combination chemotherapy and stem cell transplantation (the median overall survival: 8.9 months) (Schoch et al., 2003). Therefore, the development of novel therapies against AML with MLL-fusion genes should be investigated.

CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that have an important function in the regulation of cellular proliferation and differentiation. In the hematopoietic system, C/EBPα and C/EBPɛ are required for granulocytic commitment of multipotent myeloid progenitors and terminal differentiation of granulocytes, respectively (Yamanaka et al., 1997; Zhang et al., 2004). The expression and function of C/EBPα and C/EBPɛ are often altered in AML leukemogenesis. Mutations in CEBPA are found in about 10% of AML cases (Pabst et al., 2001a; Gombart et al., 2002). The expression of C/EBPα in AML cells is inhibited by AML1-ETO or internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD), and the ectopic expression of C/EBPα induces granulocytic differentiation in the leukemic cells with these mutated genes (Pabst et al., 2001b; Zheng et al., 2004). PML-RARα in acute promyelocytic leukemia (APL) inhibits the expression of C/EBPɛ, and the ectopic expression of either C/EBPα or C/EBPɛ induces granulocytic differentiation in vivo (Truong et al., 2003). These studies suggest that the altered expression and function of C/EBPα and C/EBPɛ are involved in subsets of AML, and that the induction of C/EBPα and C/EBPɛ can thus induce the differentiation of leukemic cells.

Retinoic acid (RA) is known to induce terminal differentiation of normal myeloid progenitors, and is widely used to induce the differentiation of leukemic cells in APL patients. The application of the differentiation activity of RA to myelomonocytic cells with MLL-fusion genes has also been explored (Hemmi and Breitman, 1985; Iijima et al., 2004). However, the previous studies did not clarify the importance of the C/EBPs function in their RA-induced monocytic differentiation. This study demonstrated that the growth arrest and monocytic differentiation of myelomonocytic cells with MLL-fusion gene is induced by ectopic expression of either C/EBPα or C/EBPɛ alone, as well as by RA. These findings may lead to a novel therapeutic approach in AML with MLL-fusion genes by enhancing C/EBPs functions.

Results

RA inhibited the proliferation and induced the differentiation of myelomonocytic cells with MLL-fusion genes

To see the potential commitment of cell lineage in myelomonocytic cells with MLL-fusion genes, human myelomonocytic cell lines with MLL-AF9, THP-1 and MOLM-14 cells, were treated with RA. HF-6 cells, a murine cell line established by the introduction of MLL-SEPT6 derived from t(X;11)(q24;q23) into murine hematopoietic cells were also used (Ono et al., 2005). HF-6 cells were assumed to be arrested at the myelomonocytic stage, based on their pale cytoplasm occasionally accompanied by a few vacuoles and azurophilic granules, weakly-positive for myelomonocytic markers Mac-1, Gr-1 and c-Kit, and negative reactions for Sca-1 and CD34. When all of these cell lines were treated with all-trans RA (ATRA) or 9-cis RA, their proliferation was suppressed in a dose-dependent manner (Figure 1a). HF-6 and THP-1 cells completely died within 7 days after 10−6M of RA (either ATRA or 9-cis RA) treatment. However, the inhibitory effect of RA on the proliferation of MOLM-14 cells was partial. The cells gradually increased in number, and repeated exposure to fresh media with 10−6M of RA completely inhibited their growth (data not shown). Morphologically, monocytic differentiation was observed in all the cell lines following RA treatment (10−6M) (Figure 1b). HF-6 cells differentiated by RA were positive for α-naphthyl butyrate staining, which was specific for the monocytic lineage (Figure 1c), and negative for naphthol AS-D chloracetate and myeloperoxidase stainings, which were specific for the myeloid lineage (data not shown). The expression of the monocytic differentiation markers CD11b and CD36 were upregulated with RA treatment in THP-1 and MOLM-14 cells (Figure 1d).

Figure 1
figure1

Effects of retinoic acid (RA) on myelomonocytic cells with MLL-fusion genes. (a) RA-induced growth inhibition of myelomonocytic cells with MLL-fusion genes. Kasumi-1 cells were used as a negative control. (b) RA-induced monocytic differentiation of myelomonocytic cells with MLL-fusion genes. HF-6 cells were cultured with RA for 2 days, and THP-1 and MOLM-14 cells for 5 days. All these cells exhibited an extended, less basophilic cytoplasm with few granules and an irregular shaped-nucleus with indentation after RA treatment. A number of vacuoles were also recognized in RA-treated HF-6 cells. May–Giemsa staining. (c) α-naphthyl butyrate (α-NB) staining and inhibition with sodium fluoride (NaF) in HF-6 cells treated with RA. Magnification: × 400 in (b) and (c). (d) The induction of CD11b and CD36 in THP-1 and MOLM-14 cells by 5-day treatment with 10−6M RA. The analyses were performed using flow cytometry.

The effects of ATRA or 9-cis RA treatment on primary AML cells harboring MLL-fusion were also analysed. Three cases of primary AML cells were sorted out according to their surface markers before clonogenic assay (Supplementary Table 1). Small colony-forming units or clusters from leukemic cells (L-CFU as the abbreviation including both of them) were observed on days 11–14. L-CFU was classified in the following three types, granulocyte/macrophage-L-CFU (L-CFU-GM), granulocyte-L-CFU (L-CFU-G) and macrophage-L-CFU (L-CFU-M), according to the cellular contents (Figure 2a). ATRA or 9-cis RA induced L-CFU-M formation, mainly small monocytic clusters, in place of L-CFU-GM and L-CFU-G (Figure 2b). An immunophenotypic analysis of L-CFU was performed in case 1, and uncovered the decrease of total cellular number and the increase of CD36-positive cells (Figure 2c). Taken together, these findings suggest that ATRA and 9-cis RA treatment inhibited the proliferation of AML cells with MLL-fusion genes, accompanied by monocytic differentiation.

Figure 2
figure2

Retinoic acid (RA) inhibited the colony formation and induced monocytic differentiation of primary acute myeloid leukemia (AML) cells with MLL-fusion genes. Primary AML cells with MLL-fusion genes were sorted out before clonogenic assay, according to the expression of their surface markers (Supplementary Table 1). Colony-formation units from AML samples (L-CFU) with or without 10−6M of all-trans RA (ATRA) or 9-cis RA were analysed on days 11–14 in triplicate, except for case 2 (one plate per group). (a) Representative pictures of macrophage-L-CFU (L-CFU-M), granulocyte/macrophage-L-CFU (L-CFU-GM) and granulocyte-L-CFU (L-CFU-G). Magnification: × 100 (L-CFU-M), × 40 (L-CFU-GM and L-CFU-G) (b) ATRA- and 9-cis RA-induced monocytic differentiation of primary AML cells with MLL-fusion genes. The results of the average number of total L-CFU without RA treatment are shown as 100% in each case. Note that the percentage of L-CFU-M markedly increased after treatment with ATRA or 9-cis RA, whereas those of L-CFU-GM and L-CFU-G decreased. (c) RA inhibited the proliferation of AML-derived cells and induced CD36-positive cells in case 1. All the cells in the plates for clonogenic assay were collected and counted. Their expression of CD36 was analysed by flow cytometry, and the absolute number of CD36-positive cells was calculated.

C/EBPs were upregulated in RA-induced monocytic differentiation of myelomonocytic cells with MLL-fusion genes

To gain insight into the molecular mechanisms of RA-induced growth arrest and monocytic differentiation of myelomonocytic cells with MLL-fusion genes, the expression of C/EBPs was analysed. HF-6 cells weakly expressed the N-terminally truncated (30 kDa isoform: p30) isoforms of C/EBPα, and lesser extent, the functional isoform (42 kDa isoform: p42) of C/EBPα, and did not express C/EBPɛ. RA upregulated C/EBPα; 9-cis RA induced them more than ATRA, and 10−6M was more effective, when compared with 10−8M. The p42 isoform of C/EBPα was upregulated more intensely than the p30 isoform, which was reported to antagonize the p42 isoform (Pabst et al., 2001a) (Figure 3a). RA also upregulated the p32 and p30 isoforms of C/EBPɛ, and lesser extent, the p27 isoform (Figure 3b). The upregulation of Cebpa and Cebpe mRNAs in HF-6 cells by 10−6M RA was detected in an analysis using quantitative reverse transcription (RT)–PCR (Figure 3c). The induction of the p42 isoform of C/EBPα and the p32 isoform of C/EBPɛ by RA was also observed in THP-1 and MOLM-14 cells (Figures 3d and e) (Lee et al., 2002). However, the induction of the p30 isoform of C/EBPα, and the p30 and p27 isoforms of C/EBPɛ were not apparent in either of these two cell lines.

Figure 3
figure3

Retinoic acid (RA) induced the expression of CCAAT/enhancer binding proteins (C/EBPs) in myelomonocytic cells with MLL-fusion genes. Immunoblotting revealed the induction of (a) the p42 isoform of C/EBPα, and (b) p32 and p30 isoforms of C/EBPɛ in HF-6 cells with RA. (c) Quantitative reverse transcription (RT)–PCR also revealed the upregulation of C/EBPα and C/EBPɛ mRNAs in HF-6 cells with RA (10−6M). The mRNA content was measured relative to that of murine Gapdh. The relative ratios in regard to the data before RA treatment (at 0 h) are presented in percentages. Upregulation of the p42 isoform of C/EBPα and p32/p30 isoform of C/EBPɛ by RA was also recognized in (d) THP-1 and (e) MOLM-14 cells using immunoblotting. C: control, A: treatment with 10−6M of ATRA, 9: treatment with 10−6M of 9-cis RA, d: days.

Although only two kinds of MLL-fusion genes were examined, the findings indicated that myelomonocytic cells with MLL-fusion genes were committed to the monocytic lineage and differentiated by RA treatment, with the concomitant upregulation of C/EBPs.

Induction of C/EBPs activity inhibited the cellular growth and promoted the monocytic differentiation in HF-6 cells

To investigate whether the induction of C/EBPs activity could promote the monocytic differentiation and maturation in myelomonocytic cells with MLL-fusion genes, inducible forms of C/EBPα or C/EBPɛ were retrovirally introduced into HF-6 cells. Because C/EBPα and C/EBPɛ were fused to the estrogen receptor (C/EBPα-ER and C/EBPɛ-ER, respectively), 4-Hydroxytamoxifen (4-HT) could induce C/EBPs activity (Fukuchi et al., 2006; Nakajima et al., 2006). Their ectopic expression in HF-6 cells was confirmed by immunoblotting (Figure 4a). HF-6 cells with empty vector (HF-6/pMY) proliferated, but the cellular growth was completely inhibited by the expression of C/EBPα-ER or C/EBPɛ-ER (Figure 4b). HF-6/pMY cells had morphologically blastic features, whereas HF-6/C/EBPα-ER and HF-6/C/EBPɛ-ER cells showed monocytic differentiation after 1 day with or without 4-HT stimulation as they did with RA treatment (Figure 4c). To quantify the monocytic differentiation of HF-6 cells by inducing C/EBPs activity, the cells were counted by classifying them into three forms; an immature form is monoblastic cells with a basophilic cytoplasm and a round nucleus composed of fine chromatin formation. A mature form is similar to mature macrophages, which has an extended, clear cytoplasm with vacuolation and an indented or lobulated nucleus with coarse chromatin formation. An intermediate form has morphological features between those of an immature and mature form: a basophilic cytoplasm and an indented nucleus with rather fine chromatin formation. The mature forms were not induced in HF-6/pMY cells, but were induced in HF-6/C/EBPα-ER and HF-6/C/EBPɛ-ER cells, regardless of 4-HT treatment (Figure 4d). Analysis by flow cytometry showed marked induction of Mac-1 and Gr-1 in HF-6/C/EBPα-ER and HF-6/C/EBPɛ-ER cells on day 2 (Figure 4e). Their intensities were higher in the cells expressing C/EBPα-ER than C/EBPɛ-ER, and enhanced with 4-HT treatment (data not shown). Because the empty vector did not lead to any changes in HF-6 cells as mentioned above, the growth inhibition and monocytic differentiation were thought to be specific for the expression of C/EBPα-ER or C/EBPɛ-ER.

Figure 4
figure4

CCAAT/enhancer binding proteins (C/EBPs) inhibited the proliferation and induced the monocytic differentiation of HF-6 cells. (a) Detection of C/EBPα-ER and C/EBPɛ-ER in HF-6 cells. They were recognized by anti-ER antibody. Anti-GFP antibody was used for detection of viral integration in HF-6 cells. *: the specific bands, **: nonspecific bands. (b) Growth inhibition of HF-6 cells by the induction of C/EBPs activity. The cells were sorted with green fluorescent protein on day 0, and sequentially analysed the proliferation with or without 1 μM of 4-Hydroxytamoxifen (4-HT). pMY: pMY-IRES-GFP, used as a control vector. (c) Morphological changes of HF-6 cells by induction of C/EBPs activity. Cytospin slides were prepared on day 1. As a result of C/EBPs expression, the cytoplasm became less basophilic and obtained a number of vacuoles, and the nucleus became indented or lobulated. May–Giemsa staining, magnification: × 400. (d) The differentiation count of HF-6 cells expressing pMY, C/EBPα-ER or C/EBPɛ-ER on day1. They were cultured with or without 4-HT for 24 h. (e) The expression of Mac-1 and Gr-1 in HF-6 cells induced by C/EBPs activity. Evaluation was done at sorting (day 0) and 2-day incubation with 1 μM of 4-HT (day 2). Dotted lines: with isotype controls. (f) Induction of apoptosis in HF-6 cells by C/EBPs activation. Annexin-positive/propidium iodine (PI)-negative cells were counted as apoptotic cells at sorting and after 18-h incubation with 1 μM of 4-HT.

To evaluate the contribution of apoptosis in growth inhibition of HF-6 cells by induction of C/EBPs activity, the percentage of annexin V-positive/propidium iodide-negative cells was determined in HF-6/C/EBPα-ER or HF-6/C/EBPɛ-ER cells cultured with 4-HT. After 18 h-incubation with 1 μM of 4-HT, this cell fraction was not increased in HF-6/pMY cells (6.59%), but was induced by C/EBPα or C/EBPɛ activation in HF-6 cells (44.81 and 26.46%, respectively, Figure 4f).

Taken together, these findings suggested that the induction of either C/EBPα or C/EBPɛ activity by itself could induce the monocytic differentiation and apoptosis in HF-6 cells.

Changes of mRNA expression in HF-6 cells induced by C/EBPs activation

To observe the expression of the genes associated with cellular proliferation and differentiation in HF-6/C/EBPα-ER or HF-6/C/EBPɛ-ER cells, their expression levels were determined using quantitative RT–PCR (Figure 5).

Figure 5
figure5

Changes in the mRNA expression of HF-6 cells induced by CCAAT/enhancer binding proteins (C/EBPs) activation. The expression levels of Myc, Sfpi1, Cebpa and Cebpe mRNA in HF-6/C/EBPα-ER or HF-6/C/EBPɛ-ER cells were analysed by quantitative reverse transcription (RT)–PCR. Total RNA was extracted before or after 4 and 12-h incubation with 1 μM of 4-HT. The mRNA content was measured relative to that of murine Gapdh. The relative ratios in regard to the data from the HF6/empty vector (pMY) before 4-HT treatment are presented in percentages.

We found that the ectopic expression of either C/EBPα-ER or C/EBPɛ-ER activity markedly decreased Myc expression (21±3 and 13±3%, at 12-h incubation with 4-HT, respectively) and upregulated Sfpi1, the murine homolog of PU.1 (454±151 and 345±134% at 4-h incubation with 4-HT, respectively). Although the gene expression changes were mild, the ectopic expression of C/EBPα and C/EBPɛ similarly downregulated Hoxa7 and Hoxa9, which are known to be related to chimeric MLL-induced leukemogenesis, (Ayton and Cleary, 2003), and upregulated Cdkn1α encoding p21WAF1 (data not shown). Cebpe was upregulated by the induction of C/EBPα-ER with 4-HT (593±69% at 4-h incubation with 4-HT), but the induction of C/EBPɛ-ER with 4-HT could not upregulate Cebpa (102±2 and 134±7% at 4- and 12-h incubation with 4-HT, respectively).

These results suggested that (i) C/EBPα or C/EBPɛ-induced monocytic differentiation may be related to the downregulation of Myc and upregulation of Sfpi1, and (ii) the expression profiles with the ectopic expression of C/EBPα or C/EBPɛ closely resembled each other, except for upregulation of Cebpe mRNA by C/EBPα.

Overexpression of Myc partially antagonized the C/EBPs functions in HF6 cells

As Myc was markedly downregulated in HF-6 cells by C/EBPs induction, the ability of Myc to inhibit C/EBPs-induced monocytic differentiation of HF-6 cells was examined. Retroviral vectors expressing the two major isoforms Myc1 and Myc2 (pMYpuro-Myc1 and pMYpuro-Myc2, respectively) were constructed and infected to HF-6 cells to generate HF-6/Myc1 and HF-6/Myc2 cells. Overexpression of Myc isoforms in these cells was confirmed by immunoblotting (Figure 6a). HF-6/Myc1 and HF-6/Myc2 cells had morphologically blastic features similar to the original HF-6 cells (Figure 6b). The expression intensity of Mac-1 and Gr-1 was slightly decreased in these cells, when compared with original HF-6 cells and those with the empty vector (HF-6/pMYpuro) (Figure 6c). The proliferation rates of HF-6/Myc1 and HF-6/Myc2 cells were similar to those of HF-6 cells (data not shown).

Figure 6
figure6

The overexpression of Myc rescued the growth arrest and partially inhibited differentiation of HF6 cell induced by CCAAT/enhancer binding proteins (C/EBPs) activation. (a) The detection of the overexpressed Myc1 and Myc2 in HF-6 cells by immunoblotting. (b) Morphology of HF-6 cells with indicated viruses. May–Giemsa staining, magnification: × 600. (c) Flow cytometric analysis of HF-6/Myc1 and HF-6/Myc2. The intensity of Mac-1 and Gr-1 was slightly decreased in HF-6/Myc1 and HF-6/Myc2. (d) Detection of the overexpressed C/EBPs in HF-6/Myc1 and HF-6/Myc2. *: C/EBPα-ER, **: C/EBPɛ-ER. (e) The differentiation count of HF-6/Myc1 and HF-6/Myc2 cells expressing C/EBPα-ER or C/EBPɛ-ER on day 2. They were cultured with 1 μM of 4-HT. (f) Flow cytometric analysis of Mac-1 and Gr-1 in HF-6/pMYpuro, HF-6/Myc1 and HF-6/Myc2 cells expressing C/EBPα-ER or C/EBPɛ-ER. The analyses were performed on day 2. Dotted lines: with isotype controls. (g) The proliferation of HF-6/Myc1 and HF-6/Myc2 expressing C/EBPα-ER or C/EBPɛ-ER. The cells were cultured with 1 μM of 4-HT.

C/EBPα-ER or C/EBPɛ-ER was then infected into HF-6/Myc1 and HF-6/Myc2 cells, and their expression was confirmed with immunoblotting (Figure 6d). Although the intermediate forms of monocytic lineage cells were increased in HF-6/Myc1 cells expressing C/EBPα-ER or C/EBPɛ-ER, no mature forms were induced in all these cells (Figure 6e). C/EBPα-ER or C/EBPɛ-ER slightly upregulated the intensities of Mac-1 and Gr-1 in HF-6/Myc1 and HF-6/Myc2 cells, but the intensities were not as high as those observed in HF-6/C/EBPα-ER or HF-6/C/EBPɛ-ER cells (Figures 4e and 6f). Both of the cells expressing C/EBPα-ER or C/EBPɛ-ER could proliferate under 4-HT treatment, although the partial growth inhibition was still stronger in HF-6/Myc1 than in HF-6/Myc2 (Figure 6g).

These assays were also performed using proliferating HF-6/Myc1 and HF-6/Myc2 cells introduced with C/EBPα-ER or C/EBPɛ-ER after 1-week culture. The sustained expression of C/EBPα-ER or C/EBPɛ-ER in these cells was confirmed using quantitative RT–PCR. These cells showed a blastic appearance, lower intensities of Mac-1 and Gr-1 and increased proliferation rates, in comparison to the cells immediately after infection. The expression levels of Myc and Sfpi1 mRNA in HF-6/Myc1 and HF-6/Myc2 cells were equal, regardless of the ectopic expression of C/EBPs. The induction of Cebpe mRNA was not observed in HF-6/Myc1 or HF-6/Myc2 cells expressing C/EBPα-ER (data not shown).

Taken together, these data suggested that the ectopic expression of Myc overcame the growth arrest, while also partially inhibiting the monocytic differentiation of HF-6 cells induced by the C/EBPα or C/EBPɛ activity.

Discussion

This study demonstrated that the induction of C/EBPα or C/EBPɛ activity by itself could inhibit the cellular growth and induce the monocytic differentiation of myelomonocytic cells with MLL-fusion genes. In addition, the downregulation of Myc induced by C/EBPs activity appears to have an important function in their monocytic differentiation.

Many previous studies have shown that C/EBPα and C/EBPɛ have an important function in granulocytic differentiation. However, this study showed that human and murine myelomonocytic cells with MLL-fusion genes ceased their proliferation and were differentiated into the monocytic lineage following treatment with RAs, potent differentiation inducers for the granulocytic lineage, accompanied by the upregulation of C/EBPα and C/EBPɛ. In addition, the ectopic expression of C/EBPs induced HF-6 cells into monocytes. Consistent with this, C/EBPα and C/EBPɛ are also reported to induce monocytic differentiation: C/EBPα regulates the monocyte-colony-stimulating factor receptor gene and CD14 (Zhang et al., 1996; Pan et al., 1999), and contributes to monocytic commitment of primary myeloid progenitors by directly activating PU.1 (Wang et al., 2006). C/EBPɛ also induces the gene expression of monocyte-colony-stimulating factor receptor (Williams et al., 1998), and is required for the development and function of mature macrophages (Tavor et al., 2002). The expression of chemokines MIP-1γ and MCP-3 is defective in macrophages from C/EBPɛ-deficient mice (Kubota et al., 2000). This study indicates that HF-6 cells are presumably arrested at the monoblastic stage based on both morphological and immunophenotypical analysis. It may be presumed that the differentiation inducers including RAs and C/EBPs are shared in both monocytic and granulocytic differentiation, and that the cell lineage commitment and differentiation is dependent on the developmental level of the cell, rather than the types of differentiation inducers.

This study showed that the induction of Sfpi1, the mouse homolog of PU.1, was commonly observed in the C/EBPs-induced monocytic differentiation of HF-6 cells. This may be a key step in the differentiation, because PU.1 is crucial for the monocytic development. Although C/EBPα functions against PU.1 and induces granulocytic differentiation in granulocyte–macrophage progenitors (Dahl et al., 2003), there have been several reports that show positive regulation of PU.1 by C/EBPα in other cell components. C/EBPα can bind and activate the PU.1 enhancer (Kummalue and Friedman, 2003). PU.1 mRNA can be upregulated by C/EBPα in the granulocytic differentiation of 32Dcl3 cells (Wang et al., 1999). C/EBPs and PU.1 cooperatively regulate neutrophil esterase promoter and eosinophilic granule gene expression (Oelgeschlager et al., 1996; Gombart et al., 2003). Moreover, C/EBPs, namely C/EBPβ, are upregulated and bind to PU.1 promoter to induce the expression of PU.1 mRNA in ATRA-induced granulocytic differentiation of APL cells (Mueller et al., 2006). These reports as well as the data presented here suggest that C/EBPs induce PU.1, and that C/EBPs and PU.1 function cooperatively in late granulocytic and monocytic differentiation after the granulocyte–macrophage progenitor stage, including myelomonocytic cells with MLL-fusion genes.

The gene expression profiles were similar between C/EBPα- and C/EBPɛ-induced HF-6 cells in this study. This implies a common mechanism in monocytic differentiation of HF-6 cells by C/EBPs. C/EBPɛ induction by the ectopic expression of C/EBPα-ER suggests that C/EBPɛ probably have an important function in monocytic differentiation of HF-6 cells. Another possible explanation is that C/EBPα and C/EBPɛ share the common targets in growth arrest and differentiation. C/EBPα is not only a transcriptional factor, but functions through protein–protein interaction. The target molecules in this interaction include the proteins related to cell cycle progression, such as cyclin-dependent kinase 2 (CDK2) inhibitor p21, pRB, E2F, CDK2 and CDK4 (Schuster and Porse, 2006). C/EBPɛ downregulates CDK4/6, cyclin D2/A/E, Bcl-2 and Bcl-x (Nakajima et al., 2006), and has a direct interaction with E2F (Gery et al., 2004; Walkley et al., 2004). These interaction and downregulation are supposed to cause cellular growth arrest, thus resulting in the induction of apoptosis as well as monocytic differentiation, as a common pathway. These effects on cellular growth may be more potent than the induction of monocytic differentiation as observed in HF-6 cells, when the reinforcement of C/EBPs function is applied as an antileukemic therapy. However, mutated C/EBPα without transcriptional activity induces differentiation of APL cells in vivo but mutated C/EBPɛ cannot (Lee et al., 2006), suggesting that C/EBPα and C/EBPɛ do not actually share the same molecular pathway of differentiation.

The crucial role of Myc in C/EBPs-induced monocytic differentiation of HF-6 cells was also demonstrated in this study. Myc induces transformation of hematopoietic cells cooperatively with MLL-ENL in vitro (Schreiner et al., 2001). Myc inhibits the expression of C/EBPα by binding its promoter region (Freytag and Geddes, 1992). In contrast, C/EBPα negatively regulates c-Myc through the c-Myc promoter (Johansen et al., 2001). C/EBPɛ also upregulates the Myc-antagonist Mad1 and downregulates c-Myc through repression of E2F1-mediated transcription (Gery et al., 2004; Walkley et al., 2004). Partial inhibition of C/EBPɛ-induced differentiation by c-Myc is also observed in 32D cells (Nakajima et al., 2006). These observations suggest that Myc contributes to leukemogenesis by MLL-fusion genes through inhibition of C/EBPα expression, and that overexpression of C/EBPs downregulates Myc and induces differentiation. The present findings that C/EBPs induced monocytic differentiation of HF-6 cells are consistent with the underlying mechanism reported in previous studies.

However, in contrast to Myc, ectopic expression of internal tandem duplication of FLT3 (FLT3-ITD) cannot inhibit C/EBPs-induced monocytic differentiation in HF-6 cells (H Matsushita et al., unpublished data). FLT3-ITD contributes to the cellular proliferation of AML cells through several signal pathways including the JAK/STAT, phosphoinositol 3-kinase and MAPK pathways. These observations suggest that growth arrest and differentiation induction by C/EBPα and C/EBPɛ are achieved through molecules functioning in the cell cycle rather than signals for cellular proliferation related to FLT3-ITD.

Human AML cells with MLL-fusion genes, MV4;11 and MOLM-14 cells, have also been reported to be differentiated by the induction of C/EBPα, however, the committed cell lineage was granulocytic, but not monocytic (Radomska et al., 2006). This difference of the committed cell lineage could be dependent on the methods used for determination of the lineage. They evaluated only nitroblue tetrazolium reduction activity in MV4;11 with C/EBPα, and their granulocytic changes were not typical and definite. It is sometimes difficult to determine whether the lineage is monocytic or granulocytic in the differentiated cells from leukemic cells with conventional methods including cytochemical studies and immunophenotyping, because the differentiation processes share the same mechanisms and phenotypes in various degrees. Especially, these cells are neither normal granulocytes nor monocytes, and may not have the typical features of either of them. The other possibility is that this discrepancy could be due to differences in species or the developmental level, that is, the murine cell line HF-6 cells might be more strictly committed to the monocytic lineage than the human cell lines MV4;11 and MOLM-14 cells.

In summary, myelomonocytic cells with MLL-fusion gene were observed to cease the cellular growth and differentiate into monocytes by RA treatment concomitantly with upregulation of C/EBPα and C/EBPɛ or by induction of C/EBPα or C/EBPɛ activity. The downregulation of Myc is crucial, and C/EBPɛ may therefore have an important function in C/EBPs-induced differentiation. This is the first report to show the monocytic differentiation of myelomonocytic cells with MLL-fusion gene induced by ectopic expression of C/EBPɛ by itself, as well as C/EBPα. These findings may lead to the development of novel C/EBPs-modulating therapeutic approaches against AML with MLL-fusion genes.

Materials and methods

Reagents

All-trans RA, 9-cis RA and 4-HT (Sigma, St Louis, MO, USA) were resuspended in ethanol. Puromycin and blasticidin-S (Sigma) were diluted in distilled water. All the aliquots were stored at −20 °C.

Cell lines

The THP-1 and Kasumi cells were obtained from RIKEN BioResource Center (Ibaragi, Japan) and American Type Culture Collection (ATCC) (Manassas, VA, USA), respectively. MOLM-14 cells were provided from Cell Biology Institute, Research Center, Hayashibara Biochemical Laboratories (Okayama, Japan). They were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 1 U/ml penicillin G, 1 μg/ml streptomycin. HF-6 cells were maintained in the same growth medium supplemented with murine interleukin-3 (10 ng/ml final concentration). The packaging cell line PLAT-E, a generous gift from Dr Toshio Kitamura (Institute of Medical Science, University of Tokyo, Tokyo, Japan), was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 μg/ml puromycin, 10 μg/ml blasticidin-S, 1 U/ml penicillin G and 1 μg/ml streptomycin. All cells were cultured at 37 °C and 5% CO2.

Patient samples

Three cases of AML samples with 11q23 abnormalities were used (Supplementary Table 1). The AML cells were purified from bone marrow or peripheral blood using Ficoll as mononuclear cells, and were stored in a liquid nitrogen tank. The studies were conducted according to the guidelines of the revised Helsinki protocol, after informed consent from all patients’ parents was obtained.

Myeloperoxidase and esterase staining

The Esterase staining kit, Esterase AS-D staining kit and New PO-K staining kit (Muto Pure Chemicals, Tokyo, Japan) were used for α-naphthyl butyrate, naphthol AS-D chloracetate and myeloperoxidase staining, respectively, according to the manufacturer’s protocol.

Plasmids

pMY-IRES-GFP/C/EBPα-ER and pMY-IRES-GFP/C/EBPɛ-ER were generated by ligating either the C/EBPα-ER or C/EBPɛ-ER fragment into pMY-IRES-GFP, respectively (Kitamura et al., 2003; Fukuchi et al., 2006; Nakajima et al., 2006). Two murine Myc isoforms (582–1946 for Myc1 and 627–1946 for Myc2 in NM_010849, respectively) were amplified by RT–PCR and ligated into pMYpuro to generate pMYpuro-Myc1 and pMYpuro-Myc2 (Kitamura et al., 2003).

Transfection, retroviral production and infection

These procedures were described previously (Nakajima and Ihle, 2001).

Assay for cell proliferation and apoptosis

Cellular proliferation and apoptosis were analysed using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) and Annexin V-Biotin Kit (Beckman Coulter, Fullerton, CA, USA), respectively. Streptavidin-allophycocyanin (APC) (BD Pharmingen, San Diego, CA, USA) was used to detect annexin V-biotin in an analysis of apoptosis.

Flow cytometry

The cells were preincubated in phosphate-buffered saline supplemented with 0.2% human γ-globulins (Sigma) for 15 min at 4 °C. They were then incubated with the monoclonal antibodies for 30 min at 4 °C. The applied monoclonal antibodies included anti-PE-conjugated anti-mouse CD117 (c-Kit), CD11b (Mac-1), and Ly-6C (Gr-1), FITC-conjugated anti-mouse CD34, Ly-6A/E (Sca-I) and Gr-1, APC-conjugated anti-human CD34, PE-conjugated anti-human CD11b and FITC-conjugated anti-human CD33 and CD36 (BD Pharmingen). An analysis was performed using FACSCaliber (Becton Dickinson, Franklin Lakes, NJ, USA).

Clonogenic assay

To delete normal hematopoietic stem cells and increase the purity of AML cells, AML samples were sorted out using FACS/Vantage (Becton Dickinson) according to the expression of their surface marker. All of the primary AML samples did not express CD34 (Supplementary Table 1). The purity of AML progenitors was evaluated by a FISH analysis using 5′- and 3′-MLL probe (Supplementary Table 1). A clonogenic assay was performed as described previously (Kawada et al., 1999), with or without 10−6M of ATRA or 9-cis RA. L-CFU-G, L-CFU-GM and L-CFU-M from primary AML cells were counted on days 11–14.

Quantitative RT–PCR

Total RNA was extracted with Isogen (Nippon Gene, Tokyo, Japan). cDNA was synthesized with SuperScript First-Strand Synthesis System for RT–PCR (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed with SYBR Premix Ex Taq (Perfect Real Time) (Takara Bio, Shiga, Japan) in LightCycler ST300 (Roche Diagnostics, Indianapolis, IN, USA). The relative levels of gene expression were calculated using standard curves generated by the serial dilution of the PCR products. The mRNA content was measured relative to that of murine Gapdh. All the samples were independently analysed at least three times for each gene. The primer pairs are shown in Supplementary Table 2.

Immunoblotting

The procedures were described previously (Nakajima and Ihle, 2001). The applied primary antibodies included rabbit serum against C/EBPα (14AA), C/EBPɛ (C-22), estrogen receptor-α (ER) (MC-20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), green fluorescent protein (Invitrogen) and Myc (Upstate, Lake Placid, NY, USA), and monoclonal antibodies against β-actin (AC-15) (Sigma).

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Acknowledgements

We thank Dr Toshio Kitamura (Institute of Medical Science, University of Tokyo, Tokyo, Japan) for providing PLAT-E cells. This study was supported in part by Research and Study Program of Tokai University Educational System General Research Organization and a Grant-in Aid for Scientific Research (C) No. 19591139 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author information

Correspondence to H Matsushita.

Additional information

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Supplementary information

Supplementary Table 1 (PPT 149 kb)

Supplementary Table 2 (PPT 151 kb)

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Keywords

  • acute myeloid leukemia
  • mixed lineage leukemia gene
  • monocytic differentiation
  • CCAAT/enhancer binding protein-α
  • CCAAT/enhancer binding protein-ɛ

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