CCAAT-enhancer binding protein: a component of a differentiation switch.

The CCAAT-enhancer binding protein (C/EBP) has now been found to promote the terminal differentiation of adipocytes. During the normal course of adipogenesis, C/EBP expression is restricted to a terminal phase wherein proliferative growth is arrested, and specialized cell phenotype is first manifested. A conditional form of C/EBP was developed, making it feasible to test its capacity to regulate the differentiation of cultured adipocytes. Premature expression of C/EBP in adipoblasts caused a direct cessation of mitotic growth. Moreover, when abetted by the effects of three adipogenic hormones, C/EBP promoted terminal cell differentiation. Since C/EBP is expressed in a variety of tissues, it may have a fundamental role in regulating the balance between cell growth and differentiation in higher animals.

Transcriptional control of granulocyte and monocyte development.

PU.1 directs the hematopoietic stem cell to the lymphoid-myeloid progenitor (LMP) and interacts with GATA-binding protein 1 to inhibit commitment to the megakaryocyte-erythroid progenitor. The CCAAT/enhancer-binding protein (C/EBP)alpha then directs the LMP to the granulocyte-monocyte progenitor (GMP) stage, while inhibiting lymphoid development via cross-inhibition of Pax5 and potentially other regulators. Increased PU.1 activity favors monocytic commitment of the GMP. Induction of PU.1 by C/EBPalpha and interaction of PU.1 with c-Jun elevates PU.1 activity. Zippering of C/EBPalpha with c-Jun or c-Fos also contributes to monocyte lineage specification. An additional factor, potentially an Id1-regulated basic helix-loop-helix protein, may be required for the GMP to commit to the granulocyte lineage. Egr-1, Egr-2, Vitamin D Receptor, MafB/c: Fos and PU.1:interferon regulatory factor 8 complexes direct further monocytic maturation, while retinoic acid receptor (RAR) and C/EBPepsilon direct granulopoiesis. Both C/EBPalpha and RARs induce C/EBPepsilon, and PU.1 is also required, albeit at lower levels, for granulocytic maturation. HoxA10 and CAAT displacement protein act as transcriptional repressors to delay expression of terminal differentiation. Gfi-1 and Egr-1,2/Nab2 complexes repress each other to maintain myeloid lineage fidelity. NF-kappaB directly binds and cooperates with C/EBPbeta to induce the inflammatory response in mature myeloid cells and potentially also cooperates with C/EBPalpha to regulate early myelopoiesis.

Identification of a region on the outer surface of the CBFbeta-SMMHC myeloid oncoprotein assembly competence domain critical for multimerization.

In the core binding factor (CBF)beta-smooth muscle myosin heavy chain (SMMHC) acute myeloid leukemia (AML) oncoprotein, CBFbeta lies N-terminal to the alpha-helical rod domain of SMMHC. Deletion of the SMMHC assembly competence domain (ACD), conserved among skeletal, smooth and nonmuscle myosins, prevents multimerization, inhibition of CBF and inhibition of cell proliferation. To define the amino acids critical for ACD function, three outer surface residues of ACD helices A-D, the subsequent helices E-H or the more N-terminal X or Z helices were now mutated. Variants were assessed for multimerization in low ionic strength in vitro and for nuclear localization as a measure of in vivo multimerization. Mutation of individual helices C-H reduced multimerization, with alteration of the outer surface of helices D or E having the greatest effect. The ability of these SMMHC variants to slow murine myeloid progenitor proliferation largely paralleled their effects on multimerization. Divergence at the boundaries of the ACD may reflect quantitative differences between in vitro and in vivo filament assembly. Each helix mutant retained the ability to bind the mSin3A corepressor. Agents interacting with the outer surface of the CBFbeta-SMMHC ACD that prevent multimerization may be effective as novel therapeutics in AML.

Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation.

The transcription factor hypoxia inducible factor 1 (HIF1), an HIF1alpha-aryl hydrocarbon receptor nuclear translocator (ARNT) dimeric factor, is essential to the cellular response to hypoxia. We described a t(1;12)(q21;p13) chromosomal translocation in human acute myeloblastic leukemia that involves the translocated Ets leukemia (TEL/ETV6) and the ARNT genes and results in the expression of a TEL-ARNT fusion protein. Functional studies show that TEL-ARNT interacts with HIF1alpha and the complex binds to consensus hypoxia response element. In low oxygen tension conditions, the HIF1alpha/TEL-ARNT complex does not activate transcription but exerts a dominant-negative effect on normal HIF1 activity. Differentiation of normal human CD34+ progenitors cells along all the erythrocytic, megakaryocytic and granulocytic pathways was accelerated in low versus high oxygen tension conditions. Murine 32Dcl3 myeloid cells also show accelerated granulocytic differentiation in low oxygen tension in response to granulocyte colony-stimulating factor. Interestingly, stable expression of the TEL-ARNT in 32Dcl3 subclones resulted in impaired HIF1-mediated transcriptional response and inhibition of differentiation enhancement in hypoxic conditions. Taken together, our results underscore the role of oxygen tension in the modulation of normal hematopoietic differentiation, whose targeting can participate in human malignancies.

Determination of the hemoglobin F program in human progenitor-derived erythroid cells.

The absolute adult and fetal hemoglobin (HbF) contents of the erythroid cells derived from the differentiation of normal human and simian erythroid progenitors and of the peripheral blood erythroid burst-forming units (BFU-E) of patients with nondeletion hemoglobinopathies have been measured with a sensitive radioligand immunoassay. The HbF content varied between 0.13 and 2.96 pg/cell, representing between 0.7% and 19.6% of the total hemoglobin with a mean value of 7.0%. The absolute content of HbF was indistinguishable in the well-hemoglobinized progeny of marrow erythroid colony-forming units, marrow or blood BFU-E, or of mixed colony-forming units. The term HbF program refers to this inherent capacity to produce fetal hemoglobin (HbF) in the erythroid cells derived from these progenitors in vitro. The HbF content of marrow erythroblasts as determined by the same radioligand immunoassay was similar to that found in the peripheral blood, suggesting that the switch off of gamma-chain production occurs after the erythroid colony-forming unit stage of maturation. Increasing concentrations of a crude erythropoietin-containing preparation induced higher numbers of erythroid colonies, which were larger in size, but the HbF program was unaffected. In contrast to the hemoglobin accumulation in human progenitor-derived colonies, simian progenitor-derived colonies produced considerably more HbF, and the amount of HbF was strongly influenced by progenitor maturity. Assays of the HbF content of erythroblasts derived from culture of the peripheral blood BFU-E of patients with nondeletion hemoglobinopathies and their parents showed that the HbF program in the progenitors of such patients is highly variable. Some produce only a slight excess of HbF in progenitor-derived erythroblasts, whereas others have extraordinarily high HbF programs. The molecular basis of this variability is presently unknown.

The murine myeloperoxidase promoter contains several functional elements, one of which binds a cell type-restricted transcription factor, myeloid nuclear factor 1 (MyNF1).

The myeloperoxidase (MPO) gene is expressed specifically in myeloid cells. There is significant homology between the murine and human MPO genes in the 1.6-kb region located upstream of the murine MPO transcription initiation sites. 5',3', and internal deletions of this DNA segment localized several cis-acting DNA elements in the murine MPO promoter which are functional in 32D cl3 cells, a murine myeloblast cell line which expresses MPO. These DNA elements did not function well in mouse L-cell fibroblasts. Additional mutagenesis of the most active promoter region allowed the delimitation of a functional 20-bp segment. Mutation of the enhancer core motif within this segment was functionally deleterious, and an oligonucleotide containing these base pairs increased the activity of a minimal promoter. This same oligonucleotide, but not a mutant variant, could bind a set of nuclear proteins, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -1 beta), present in 32D cl3 cells but absent from L cells, murine erythroleukemia cells, and SP2 lymphoid cells.

Core binding factor cannot synergistically activate the myeloperoxidase proximal enhancer in immature myeloid cells without c-Myb.

The myeloperoxidase (MPO) gene is transcribed specifically in immature myeloid cells and is regulated in part by a 414-bp proximal enhancer. Mutation of a core binding factor (CBF)-binding site at -288 decreased enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granulocyte colony-stimulating factor (G-CSF). A novel functional analysis, linking the CBF-binding site to an enhancer deletion series, located at -147 an evolutionarily conserved c-Myb-binding site which was required for optimal enhancer activity and synergy with CBF in 32D cells. These sites cooperated in isolation and independent of a precise spacing. Deletional analysis carried out in the absence of the c-Myb-binding site at -147 located at -301 a second c-Myb-binding site which also synergized with CBF to activate the enhancer. A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding site was intact. Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 32D cells. Similarly, activation by c-Myb was impaired in constructs lacking the CBF-binding site. CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in response to G-CSF. A fusion protein containing the Gal4 DNA-binding domain and the AML-1B activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 cells. Determining how CBF and c-Myb synergize in myeloid cells might contribute to our understanding of leukemogenesis by the AML1-ETO, AML1-MDS1, CBFbeta-SMMHC, and v-Myb oncoproteins.

C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter.

The murine neutrophil elastase (NE) gene is expressed specifically in immature myeloid cells. A 91-bp NE promoter region contains three cis elements which are conserved evolutionarily and are essential for activation of the promoter in differentiating 32D cl3 myeloid cells. These elements bound c-Myb (at -49), C/EBPalpha (at -57), and PU.1 (at -82). In NIH 3T3 cells, the NE promoter was activated by c-Myb, C/EBPalpha, and PU.1, via their respective binding sites. Cooperative activation was seen by any combination of c-Myb, C/EBPalpha, and PU.1, including all three together, again via their DNA-binding sites. In CV-1 cells, but not in NIH 3T3 cells, cooperation between Myb and C/EBPalpha depended on the integrity of the PU.1-binding site. In addition to C/EBPalpha, C/EBPdelta strongly activated the NE promoter, alone or with c-Myb, but C/EBPbeta was less active. Either of C/EBPalpha's two transactivation domains cooperatively activated the promoter with c-Myb, in both NIH 3T3 and 32D c13 cells. Synergistic binding to DNA in a gel shift assay between C/EBPalpha, c-Myb, and PU.1 could not be demonstrated. Also, separation of the C/EBP- and c-Myb-binding sites by 5 or 10 bp did not prevent cooperativity. These results suggest that a coactivator protein mediates cooperative activation of the NE promoter by a C/EBP and c-Myb. These factors, together with PU.1, direct restricted expression of the NE promoter to immature myeloid cells.

PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells.

The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.

Multiple functional domains of AML1: PU.1 and C/EBPalpha synergize with different regions of AML1.

Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which we can study synergistic, tissue-specific activation for two reasons. First, myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors which are crucial for normal hematopoiesis: PU.1, AML1, and C/EBPalpha. Second, these proteins interact in such a way as to demonstrate at least two examples of synergistic activation. We have shown that AML1 and C/EBPalpha activate the M-CSF receptor promoter in a synergistic manner. As we report here, AML1 also synergizes, and interacts physically, with PU. 1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPalpha has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPalpha but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPalpha. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPalpha, and PU.1.

CBFbeta-SMMHC slows proliferation of primary murine and human myeloid progenitors.

CBFbeta-SMMHC is expressed in 8% of acute myeloid leukemias and inhibits AML1/RUNX1. In this study, murine marrow or human CD34(+) cells were transduced with retroviral or lentiviral vectors expressing CBFbeta-SMMHC or two mutant variants. CBFbeta-SMMHC reduced murine or human myeloid cell proliferation three- to four-fold in liquid culture relative to empty vector-transduced cells, during a period when vector-transduced cells accumulated five-fold and human cells 20-fold. CBFbeta-SMMHC decreased the formation of myeloid, but not erythroid, colonies two- to four-fold, and myeloid colonies expressing CBFbeta-SMMHC were markedly reduced in size. However, CBFbeta-SMMHC did not slow differentiation to granulocytes or monocytes. Neither CBFbeta-SMMHC(Delta2-11), which does not bind AML1, nor CBFbeta-SMMHC(DeltaACD), which does not multimerize or efficiently bind corepressors, slowed proliferation or reduced myeloid colonies. CBFbeta-SMMHC increased the G1/S ratio 1.4-fold. AML1 had an effect opposite to CBFbeta-SMMHC, stimulating proliferation of murine myeloid progenitors 2.0-fold in liquid culture. Thus, CBFbeta-SMMHC directly inhibits the proliferation of normal myeloid progenitors via inhibition of AML1 and dependent upon the integrity of its assembly competence domain. These findings support the development of therapeutics that target the ability of CBFbeta-SMMHC to interact with AML1 or to multimerize via its assembly competence domain.

GADD153/CHOP, a DNA damage-inducible protein, reduced CAAT/enhancer binding protein activities and increased apoptosis in 32D c13 myeloid cells.

GADD153/CHOP is a DNA damage-inducible, nuclear leucine zipper protein that is capable of producing a G1-S arrest in fibroblastic cells and of dimerizing with and inhibiting CAAT/enhancer binding protein (C/EBP) activities. CHOP was induced in 32D cl3 myeloid cells exposed to methylmethane sulfonate (MMS), a DNA alkylating agent. The degree of induction was dependent upon the dose of MMS to which the cells were exposed. CHOP was not expressed, at least at similar levels, during 32D cl3 cell granulocytic differentiation or during their apoptosis upon growth factor withdrawal. High-level expression of exogenous CHOP in 32D cl3 cells markedly inhibited the trans-activation activities of endogenous C/EBPs. These cells proliferated in IL-3, although low-level ongoing apoptosis not observed with control cells was detected. When these cultures were transferred to granulocyte colony-stimulating factor (G-CSF), the majority of the cells underwent apoptosis, although the levels of CHOP did not increase. Similarly, 32D cl3 cells treated with doses of MMS sufficient to induce endogenous CHOP underwent apoptosis more rapidly when placed in G-CSF-containing, compared with interleukin 3 (IL-3)-containing, medium. However, induction of CHOP by MMS was similar in IL-3 and in G-CSF. The heightened sensitivity of 32D l13 cells to CHOP in G-CSF could result either from the loss of IL-3-specific signals or from increased expression of C/EBPs. Because myeloid leukemias express C/EBPalpha, induction of CHOP might contribute to their chemotherapy-induced apoptosis, and alterations in CHOP expression could contribute to their development of chemotherapy resistance.

Exogenous cdk4 overcomes reduced cdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF - a model for overcoming inhibition of proliferation by CBF oncoproteins.

Core Binding Factor (CBF) is required for the development of definitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFbeta-SMMHC are commonly expressed in subsets of acute leukemia. CBFbeta-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this effect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is sufficient to slow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFbeta-SMMHC-induced inhibition of proliferation. Representational difference analysis (RDA) identified cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFbeta-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16INK4a, overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These findings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins. Oncogene (2000).

CBF beta-SMMHC, expressed in M4Eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells.

CBF beta-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBF beta-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBF beta-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBF beta-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBF beta. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBF beta-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBF beta-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBF beta-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor alpha or beta subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBF beta-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBF beta-SMMHC by allowing its increased expression.

Leukemogenesis by CBF oncoproteins.

The AML1 and CBFbeta subunits of core binding factor (CBF) are involved in several chromosomal abnormalities frequently associated with acute leukemias. As a result, the CBFbeta-SMMHC, AML1-ETO and AML1-MDS1/EVI1 fusion proteins are expressed in subsets of acute myeloid leukemia, and TEL-AML1 is expressed in B-lineage acute lymphocytic leukemia. These CBF oncoproteins likely contribute to leukemogenesis in part by inhibiting endogenous CBF. As a result they are expected to inhibit differentiation and perhaps apoptosis. In addition, the domains unique to each fusion protein may also contribute to leukemogenesis via unique mechanisms.

DNA-binding domain of AML1, expressed in t(8;21) and t(3;21) myeloid leukemias, inhibits PEBP2/CBF DNA-binding but is not sufficient to transform 32D cl3 myeloid cells.

Truncated AML1 proteins are predicted to be expressed from out-of-frame AML1 transcripts present in myeloid leukemia cells harboring t(8;21) and t(3;21). To test whether these proteins, consisting of almost exclusively an N-terminal AML1 DNA-binding domain, interfere with myeloid differentiation we expressed a similar truncated AML1 protein in 32D cl3 myeloid cells. In all clones examined, the ectopically expressed truncated AML1 protein prevented binding of endogenous PEBP2/CBFs to DNA, possibly by interacting with all available CBF beta subunits. However, compared to control clones, the 32D cl3 clones expressing truncated AML1 remained IL-3 dependent for survival, proliferated similarly in low and high concentrations of IL-3, and differentiated similarly upon transfer to G-CSF. Thus, truncated AML1 proteins may contribute to myeloid leukemogeneis by inhibiting PEBP2/CBF activities, although contributions from other oncoproteins are likely required as well.

An isoform of the Wilms' tumor suppressor gene potentiates granulocytic differentiation.

WT1 is expressed in hematopoietic progenitor cells and in acute leukemia, but its role in normal and malignant hematopoiesis has not been clearly defined. Alternative splicing of the WT1 mRNA yields several protein isoforms with distinct DNA binding and transcriptional regulatory activities. In this study, we investigated the effect of the WT1 isoform lacking two alternatively spliced sequences (WT1 (-/-)) in 32D cl3 cells, a murine myeloid progenitor cell line. The expression of WT1 (-/-) accelerated the granulocyte-colony stimulating factor (G-CSF)-mediated differentiation of these cells, as judged by morphology and by the expression of differentiation-associated genes and cell surface antigens. WT1 (-/-) inhibited G1/S progression in G-CSF but not in interleukin-3, potentially accounting for its ability to accelerate differentiation. It is likely that dominant-negative mutants previously reported in leukemia patients participate in leukemogenesis by inhibiting this function of the wild-type protein.

Regulation of granulopoiesis by transcription factors and cytokine signals.

The development of mature granulocytes from hematopoietic precursor cells is controlled by a myriad of transcription factors which regulate the expression of essential genes, including those encoding growth factors and their receptors, enzymes, adhesion molecules, and transcription factors themselves. In particular, C/EBPalpha, PU.1, CBF, and c-Myb have emerged as critical players during early granulopoiesis. These transcription factors interact with one another as well as other factors to regulate the expression of a variety of genes important in granulocytic lineage commitment. An important goal remains to understand in greater detail how these various factors act in concert with signals emanating from cytokine receptors to influence the various steps of maturation, from the pluripotent hematopoietic stem cell, to a committed myeloid progenitor, to myeloid precursors, and ultimately to mature granulocytes.

C/EBPalpha and G-CSF receptor signals cooperate to induce the myeloperoxidase and neutrophil elastase genes.

To assess cooperation between G-CSF signals and C/EBPalpha, we characterized Ba/F3 pro-B cell lines expressing C/EBPalphaWT-ER and the G-CSF receptor (GCSFR). In these lines, GCSFR signals can be evaluated independent of their effect on C/EBPalpha levels. G-CSF alone did not induce the MPO, NE, LF, or PU.1 RNAs, and C/EBPalphaWT-ER alone stimulated low-level MPO and high-level PU.1 expression. Simultaneous activation of the GCSFR and C/EBPalphaWT-ER markedly increased MPO and NE induction at 24 h, and LF mRNA was detected at 48 h. G-CSF did not increase endogenous GCSFR, endogenous C/EBPalpha or exogenous C/EBPalphaWT-ER levels, and C/EBPalphaWT-ER did not induce endogenous or exogenous GCSFR. Several GCSFR mutants were also co-expressed with C/EBPalphaYWT-ER. Mutation of all four cytoplasmic tyrosines prevented NE induction but enhanced MPO induction. Mutation of Y704 was required for increased MPO induction. Consistent with this finding, removing IL-3 without G-CSF addition enabled MPO, but not NE, induction by C/EBPalphaWT-ER. GCSFR signals or related signals from other receptors may cooperate with C/EBPalpha to direct differentiation of normal myeloid stem cells.

CCAAT/Enhancer binding protein ß controls androgen-deprivation-induced senescence in prostate cancer cells.

The processes associated with transition to castration-resistant prostate cancer (PC) growth are not well understood. Cellular senescence is a stable cell cycle arrest that occurs in response to sublethal stress. It is often overcome in malignant transformation to confer a survival advantage. CCAAT/Enhancer Binding Protein (C/EBP) ß function is frequently deregulated in human malignancies and interestingly, androgen-sensitive PC cells express primarily the liver-enriched inhibitory protein isoform. We found that C/EBPß expression is negatively regulated by androgen receptor (AR) activity and that treatment of androgen-sensitive cell lines with anti-androgens increases C/EBPß mRNA and protein levels. Accordingly, we also find that C/EBPß levels are significantly elevated in primary PC samples from castration-resistant compared with therapy-naive patients. Chromatin immunoprecipitation demonstrated enhanced binding of the AR to the proximal promoter of the CEBPB gene in the presence of dihydroxytestosterone. Upon androgen deprivation, induction of C/EBPß is facilitated by active transcription as evident by increased histone 3 acetylation at the C/EBPß promoter. Also, the androgen agonist R1881 suppresses the activity of a CEBPB promoter reporter. Loss of C/EBPß expression prevents growth arrest following androgen deprivation or anti-androgen challenge. Accordingly, suppression of C/EBPß under low androgen conditions results in reduced expression of senescence-associated secretory genes, significantly decreased number of cells displaying heterochromatin foci and increased numbers of Ki67-positive cells. Ectopic expression of C/EBPß caused pronounced morphological changes, reduced PC cell growth and increased the number of senescent LNCaP cells. Lastly, we found that senescence contributes to PC cell survival under androgen deprivation, and C/EBPß-deficient cells were significantly more susceptible to killing by cytotoxic chemotherapy following androgen deprivation. Our data demonstrate that upregulation of C/EBPß is critical for complete maintenance of androgen deprivation-induced senescence and that targeting C/EBPß expression may synergize with anti-androgen or chemotherapy in eradicating PC.