C/EBPalpha and the G-CSF receptor gene--partners in granulopoiesis?
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Animals↗
Biomedical subjects
A D Friedman
Publications and source records attributed to A D Friedman.
Intensive timed sequential remission induction chemotherapy with high-dose cytarabine for childhood acute myeloid leukemia.
BACKGROUND: Timed sequential chemotherapy and high-dose cytarabine (cytosine arabinoside, Ara-C; HDAC) are both effective treatments for acute myeloid leukemia (AML). We review our institutional experience with timed sequential induction chemotherapy consisting of daunorubicin/Ara-C/-thioguanine (DAT) or idarubicin/Ara-C/-thioguanine (IAT) followed on day 14 by HDAC regardless of the degree of marrow aplasia for children with newly diagnosed AML. PROCEDURE: Children presenting with newly diagnosed AML were treated with induction chemotherapy consisting of idarubicin (12 mg/m/day on days 1-3 or daunorubicin at 45 mg/m(2)/day for the first five patients), Ara-C (100 mg/m(2)/day by continuous infusion on days 1-7), and thioguanine (100 mg/m(2)/day on days 1-7). HDAC (1 g/m(2)/dose every 12 hr for 10 doses) was administered beginning on day 14, regardless of the results of bone marrow examination. RESULTS: Thirteen children received timed sequential HDAC. Only one child received HDAC later than Day 18. Eleven of the children achieved a complete remission. All patients experienced grade 4 hematologic toxicity, and all had fever as well. There were 11 children with documented infections. Ten had grade 3 or 4 GI toxicity. One patient died of sepsis. CONCLUSIONS: HDAC administered as a part of timed sequential therapy yields an excellent remission induction rate with manageable toxicity.
Adolescent↗
A novel variant three-way translocation of inversion 16 in a case of AML-M4eo following low dose methotrexate therapy.
We report a case of acute myelomonocytic leukemia with eosinophilia (AML-M4eo) in a 65-year-old man following low dose methotrexate treatment for pemphigus vulgaris. Cytogenetic studies at diagnosis revealed a complex karyotype including a reciprocal translocation between 11q14.2 and 16q22, an inversion of chromosome 16(p13.1q22), and an apparently terminal deletion of 7q31. The presence of inv(16) was confirmed by reverse transcription-polymerase chain reaction which demonstrated a Type A fusion transcript derived from the core binding factor (CBF) beta and the smooth muscle myosin heavy chain (MYH11) genes. The patient was in complete hematologic and cytogenetic remission 6 months following intensive chemotherapy. Because AML-M4eo with inv(16) has a favorable prognosis, molecular studies should be performed in case the identification of inv(16) by conventional cytogenetics is difficult due to a complex karyotype.
Aged↗
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.
Animals↗
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).
Animals↗
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.
Cytokines↗
C/EBPalpha bypasses granulocyte colony-stimulating factor signals to rapidly induce PU.1 gene expression, stimulate granulocytic differentiation, and limit proliferation in 32D cl3 myeloblasts.
Within hematopoiesis, C/EBPalpha is expressed only in myeloid cells, and PU.1 is expressed mainly in myeloid and B-lymphoid cells. C/EBPalpha-deficient mice lack the neutrophil lineage and retain monocytes, whereas PU.1-deficient mice lack monocytes and have severely reduced neutrophils. We expressed a C/EBPalpha-estrogen receptor ligand-binding domain fusion protein, C/EBPalphaWT-ER, in 32D cl3 myeloblasts. 32D cl3 cells proliferate in interleukin-3 (IL-3) and differentiate to neutrophils in granulocyte colony-stimulating factor (G-CSF). In the presence of estradiol, C/EBPalphaWT-ER induced morphologic differentiation and the expression of the myeloperoxidase, lactoferrin, and G-CSF receptor mRNAs. C/EBPalphaWT-ER also induced a G1/S cell cycle block, with induction of p27 and Rb hypophosphorylation. bcr-ablp210 prevented 32D cl3 cell differentiation. Activation of C/EBPalpha-ER in 32D-bcr-ablp210 or Ba/F3 B-lymphoid cells induced cell cycle arrest independent of terminal differentiation. C/EBPalphaWT-ER induced endogenous PU.1 mRNA within 8 hours in both 32D cl3 and Ba/F3 cells, even in the presence of cycloheximide, indicating that C/EBPalpha directly activates the PU.1 gene. However, activation of a PU.1-ER fusion protein in 32D cl3 cells induced myeloperoxidase (MPO) RNA but not terminal differentiation. Thus, C/EBPalpha acts downstream of G-CSF and upstream of PU.1, p27, and potentially other factors to induce myeloblasts to undergo granulocytic differentiation and cell cycle arrest.
Animals↗
Long-term complete remission and immune tolerance after intensive chemotherapy for lymphoproliferative disorders complicating liver transplant.
BACKGROUND: B cell lymphoproliferative disorders (LPD) and liver rejection are major lethal complications after hepatic transplantation. Reduction in immunosuppression is the treatment for the former, but is a risk factor for the latter. METHODS: Here, we report three consecutive children with monoclonal LPD complicating orthotopic liver transplantation. All of them were treated with brief (<4 months) but intensive chemotherapy. RESULTS: These three patients have remained in complete remission for LPD for 18 months to more than 3 years. Aggressive antimicrobial prophylaxis was successful in preventing life-threatening infections. The patient who received the highest cumulative doses of chemotherapy may have also developed relative immune tolerance to the allograft. CONCLUSIONS: High-dose-intensity chemotherapy may be effective in the treatment of monoclonal LPD, as well as in the induction of immune tolerance for the prevention of allograft rejection and LPD recurrence.
Acyclovir↗
C/EBPepsilon directly interacts with the DNA binding domain of c-myb and cooperatively activates transcription of myeloid promoters.
C/EBPepsilon is essential for granulocytic differentiation. We investigated the role of C/EBPepsilon in the transcriptional activation of various myeloid-specific genes. We found that two C/EBPepsilon isoforms, p32 and p30, possessing transcriptional activation domains were coexpressed in myeloid cells. Interestingly, isoform C/EBPepsilon p30 but not p32 was differentially upregulated in NB-4 promyelocytic leukemia cells treated with retinoids. Both isoforms bound specifically to C/EBP sites in myeloid promoters. The kd for C/EBPepsilon binding to the C/EBP site of the neutrophil elastase promoter was 4.2 nmol/L. In transfection assays using the nonhematopoietic cell line, CV-1, the p32 isoform activated promoters from the myeloid-specific mim-1, neutrophil elastase, and granulocyte colony-stimulating factor (G-CSF) receptor genes by 2.5-, 1.8-, and 1.6-fold, respectively. The p30 isoform lacked significant transcriptional activity, suggesting that other hematopoietic-specific factors were required for its function. Consistent with this prediction, transfections into the hematopoietic cell line Jurkat showed a 9.0- and 2.5-fold activation of the mim-1 promoter by the p32 and p30 isoforms, respectively. The additional 32 NH2-terminal residues made p32 a significantly more potent transcriptional activator than p30. T lymphoblasts (Jurkat cells) and immature myeloid cells (eg, Kcl22 cells) expressed high levels of the c-myb hematopoietic transcription factor. Cotransfection of c-myb with either the p32 or p30 isoform of C/EBPepsilon in CV-1 cells cooperatively transactivated the mim-1 promoter by 20- and 16-fold, respectively, and the neutrophil elastase promoter by 10-and 7-fold, respectively. Pulldown assays showed that each C/EBPepsilon isoform interacted directly with the DNA binding domain of the c-myb protein. Further studies showed that Kcl22 myeloid cells only contained active C/EBPepsilon, but not C/EBPalpha, C/EBPbeta, or C/EBPdelta. A mutation of the C/EBP site in the neutrophil elastase promoter markedly decreased the transactivation of the promoter in Kcl22 myeloblasts. These results demonstrate a role for C/EBPepsilon in regulating myeloid promoters, such as neutrophil elastase, probably through a direct interaction with c-myb.
Animals↗
An enhancer located between the neutrophil elastase and proteinase 3 promoters is activated by Sp1 and an Ets factor.
The adjacent neutrophil elastase, proteinase 3, and azurocidin genes encode serine proteases expressed specifically in immature myeloid cells. Subclones of a 17-kilobase (kb) murine neutrophil elastase genomic clone were assessed for their ability to stimulate the neutrophil elastase promoter in 32D cl3 myeloid cells. Region -9.3 to -7.3 kb stimulated transcription 7-fold, whereas other genomic segments were inactive. This enhancer is located in the second intron of the proteinase-3 gene and so may regulate more than one gene in the myeloid protease cluster. Deletional analysis of the enhancer identified several segments which activated the neutrophil elastase and thymidine kinase promoters 3-6-fold. The most active segment was a 220-base pair region centered at -8.6 kb, which activated transcription 31-fold. This segment contains an Sp1 consensus site, which bound Sp1, flanked by two Ets family consensus sequences, which bound PU.1, GABP, and an Ets factor present in myeloid cell extracts. Mutation of the Sp1-binding site reduced enhancer activity 8-fold in 32D cl3 cells, and mutation of either or both Ets-binding sites reduced activity 3-4-fold. Sp1 activated the distal enhancer 5-fold, GABP 3-fold, and the combination 8-fold in Schneider cells.
Animals↗
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.
Apoptosis↗
CBFbeta-SMMHC, expressed in M4eo acute myeloid leukemia, reduces p53 induction and slows apoptosis in hematopoietic cells exposed to DNA-damaging agents.
CBFbeta-SMMHC is expressed in M4Eo acute myeloid leukemia (AML) as a result of inv(16), but how it contributes to leukemogenesis is unknown. p53 mutations are rare in de novo AML, but they are common in many malignancies. Expression of CBFbeta-SMMHC in Ba/F3 cells reduced p53 induction in response to ionizing radiation or etoposide 3- to 4-fold. However, p53 induction was normal in Ba/F3 cells expressing a CBFbeta-SMMHC variant that does not interfere with DNA binding by CBF, indicating that a CBF genetic target regulates p53 induction. The p53 gene may be regulated by CBF, because p53 mRNA levels were reduced by CBFbeta-SMMHC. Reduced p53 induction was not caused by slowed cell proliferation, a consequence of CBFbeta-SMMHC expression, because p53 was induced similarly in control cultures and in cultures propagated in 10-fold less interleukin-3 (IL-3). CBFbeta-SMMHC did not slow apoptosis resulting from IL-3 withdrawal, where p53 induction is minimal, but slowed apoptosis in Ba/F3 cells exposed to 10 Gy of ionizing radiation or 3 to 8 microgram/mL etoposide, providing 2-fold protection at 6 or 18 hours. Inhibition of apoptosis was temporary, because all the cells exposed to these doses ultimately died, and clonal survival assays performed using 0. 04 microgram/mL etoposide did not show protection by CBFbeta-SMMHC. p21 levels were increased in cells subjected to DNA damage, regardless of CBFbeta-SMMHC expression and attenuated p53 induction. Bcl-2, bcl-xL, bcl-xS, and bax levels were unaffected by CBFbeta-SMMHC. Attenuated p53 induction may contribute to leukemogenesis by CBFbeta-SMMHC by slowing apoptosis via a p21-independent mechanism.
Acute Disease↗
TLE, the human homolog of groucho, interacts with AML1 and acts as a repressor of AML1-induced transactivation.
The AML1 gene encodes DNA-binding proteins that contain the Runt domain and is found at the breakpoints of some translocations associated with leukemias. It has been reported that AML1 plays pivotal roles in myeloid differentiation, probably through the transcriptional regulation of various hematopoietic genes. Here we demonstrate the physical and the functional interaction between AML1 and TLE1 (transducin-like Enhancer of split) human homolog of Groucho that is known to be a corepressor of Hairy-related proteins. TLE1 binds to AML1 through the Runt domain and the C terminus of AML1 that includes the VWRPY motif. The interaction is mainly mediated by the SP domain of TLE1. Moreover, TLE1 inhibits AML1-induced transactivation of the target promoters through the C terminus of AML1. These results suggest that TLE1 acts as a repressor of AML1 and provide important insights into the mechanism of the negative regulation of the AML1 functions in hematopoiesis and leukemogenesis.
Animals↗
The core binding factor (CBF) alpha interaction domain and the smooth muscle myosin heavy chain (SMMHC) segment of CBFbeta-SMMHC are both required to slow cell proliferation.
We have expressed several variants of core binding factor beta (CBFbeta)-smooth muscle myosin heavy chain (SMMHC) from the metallothionein promoter in Ba/F3 cells. Deletion of amino acids 2-11 from the CBFbeta segment, required for interaction with CBFalpha, prevented CBFbeta-SMMHC from inhibiting CBF DNA binding and cell cycle progression. Deletion of 283 carboxyl-terminal residues from the SMMHC domain, required for multimerization, also inactivated CBFbeta-SMMHC. Nuclear expression of CBFbeta(Delta2-11)-SMMHC was decreased relative to CBFbeta-SMMHC. CBFbeta(Delta2-11)-SMMHC linked to a nuclear localization signal still did not slow cell growth. The ability of each CBFbeta-SMMHC variant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation by an AML1-VP16 fusion protein. Thus, CBFbeta-SMMHC slows cell cycle progression from G1 to S phase by inhibiting CBF DNA binding and transactivation.
Bone Marrow Cells↗
Identification of transcriptional activation and repression domains in human CCAAT/enhancer-binding protein epsilon.
Human CCAAT/enhancer-binding protein epsilon (C/EBPepsilon), a new member of the C/EBP family, significantly up-regulates both the mim-1 and human myeloperoxidase promoters, suggesting an important role for C/EBPepsilon in the transcriptional regulation of a subset of myeloid-specific genes. To elucidate the structure and function of C/EBPepsilon in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBPepsilon were fused to the yeast GAL4 DNA binding domain. These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the GAL-C/EBPepsilon-(1-115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene. Sixteen deletion mutants of C/EBPepsilon mapped the transactivation domain to amino acids 1-18 at the N terminus and revealed the presence of a transcription repression element between amino acid residues 116 and 162. Expression vectors containing the repression domain of C/EBPepsilon strongly inhibited gene transcription from TK, SV40, and adenoviral major late promoters bearing GAL4 binding sites. Fusion of this repression domain to the VP16 activation domain inhibited the transactivation function of VP16. Deletion of this repression domain increased gene transcription from a neutrophil elastase promoter-luciferase reporter. Taken together, these data suggest that C/EBPepsilon regulates transcription by utilizing both activation and repression functions.
CCAAT-Enhancer-Binding Proteins↗
Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing's sarcoma.
Many poor-risk neuroblastomas and tumours of the Ewing's sarcoma family (ET) recur despite autologous transplants. Recurrence may be due to tumor cells contained in the BM harvests or PBSC harvests. The objectives of this prospective study were to: (1) determine the incidence and degree of tumor cell contamination in paired BM and PBSC harvests; and (2) determine the efficacy of tumor cell purging by immunomagnetic CD34+ cell selection. 198 samples from 11 consecutive patients with neuroblastoma or Ewing's sarcoma were analyzed. We assayed tumor contamination by RT-PCR assay for PGP 9.5, plus immunohistochemistry for neuroblastoma-specific antigens (the latter in neuroblastoma only). None of these patients had tumor cells detected in their BM by clinical histology immediately before BM or PBSC harvests. However, 82% of PBSC and 89% of backup BM harvests were contaminated with tumor by RT-PCR and/or immunocytochemistry assays. Unselected PBSC and BM harvests contained similar quantities of tumor cells (median, approximately 200000 cells). Cyclophosphamide plus G-CSF mobilization did not affect the incidence or level of contamination in PBSC harvests, as compared to blood obtained before mobilization. Immunomagnetic CD34+ cell selection depleted tumor cells by a median of 3.0 logs for PBSC, and 2.6 logs for BM harvests.
Adolescent↗
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.
Animals↗
The myeloperoxidase gene proximal enhancer directs hematopoietic-specific expression in transgenic mice.
The myeloperoxidase (MPO) gene is expressed specifically in immature myeloid cells. The MPO gene includes a promoter proximal enhancer which is coincident with DNaseI hypersensitive chromatin sites and is specifically active in myeloid cell lines. We developed transgenic murine lines in which 1.3 kb of murine MPO proximal 5' flanking region DNA was linked to a TATAA homology and RNA initiation site derived from the HSV-TK promoter and to a luciferase reporter (MPOTKLUC). In each of six founder lines, high-level luciferase activity was evident in marrow, thymus and spleen. Modest- to high-level luciferase expression was also evident in brain and in the heart in several of the lines, and luciferase activity was at or near background levels in lung, liver, kidney, stomach, colon, bladder, skeletal muscle, skin and small intestine in all of the MPOTKLUC transgenic mice. Within marrow cells, luciferase activity was evident in myeloid (GR-1+), B lymphoid (B220+) and T-lymphoid (CD4+) cells. Additional regulatory regions, thus, may be required to further restrict MPO gene expression to immature myeloid cells.
Animals↗