Adrian F. Gombart

Restoration of C/EBPα Expression in a BCR-ABL+ Cell Line Induces Terminal Granulocytic Differentiation

The transcription factor C/EBPα plays a critical role in the process of granulocytic differentiation. Recently, mutations that abrogated transcriptional activation of C/EBPα were detected in acute myeloid leukemia patient samples. Moreover, the progression of chronic myelogenous leukemia (CML) to blast crisis in patients was correlated with down-modulation of C/EBPα. The KCL22 cell line, derived from BCR-ABL+ CML in blast crisis, expressed wild-type C/EBPϵ protein but not a functional C/EBPα, -β, and -γ. Restoration of C/EBPα expression in KCL22 cells triggered a profound proliferative arrest, a block in the G2/M phase of the cell cycle and a gradual increase in apoptosis. Within 3 days of inducing expression of C/EBPα, a remarkable neutrophilic differentiation of the KCL22 blast cells occurred as shown by morphologic changes, induction of expression of CD11b, primary, secondary, and tertiary granule proteins, and granulocyte colony-stimulating factor receptor. Using high density oligonucleotide microarrays, the gene expression profile of KCL22 cells stably transfected with C/EBPα was compared with that of empty vector, and we identified genes not previously known to be regulated by C/EBPα. These included the up-regulation of those genes important for regulation of hematopoietic stem cell homing, granulocytic differentiation, and cell cycle, whereas down-regulation occurred for genes coding for signaling molecules and transcription factors that are implicated in regulation of proliferation and differentiation of hematopoietic cells. Our study showed that restoration of C/EBPα expression in BCR-ABL+ leukemic cells in blast crisis is sufficient for rapid neutrophil differentiation suggesting a potential therapeutic role for ectopic transfer of C/EBPα in acute phase of CML.

Identification of Transcriptional Activation and Repression Domains in Human CCAAT/Enhancer-binding Protein ε

Human CCAAT/enhancer-binding protein ε (C/EBPε), 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/EBPε in the transcriptional regulation of a subset of myeloid-specific genes. To elucidate the structure and function of C/EBPε in transcriptional activation, amino acid residues 1–115, 147–249, or 1–249 of C/EBPε 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/EBPε-(1–115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene. Sixteen deletion mutants of C/EBPε 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/EBPε 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/EBPε regulates transcription by utilizing both activation and repression functions.

Phenotypic and functional alterations of peripheral blood monocytes in neutrophil-specific granule deficiency

Neutrophil‐specific granule deficiency (SGD) is a rare, congenital disease characterized by atypical neutrophil structure and function, resulting in recurrent bacterial infections from early infancy. Homozygous recessive mutations in the CCAAT/enhancer‐binding protein ɛ (C/EBPɛ) gene were described in two of five SGD patients, indicating loss of C/EBPɛ function as the primary genetic defect in this disease. C/EBPɛ is expressed in murine and human macrophages. Macrophages from the C/EBPɛ‐deficient mice show impaired differentiation, phagocytic activity, and transcription of macrophage‐specific genes. To determine if monocyte/macrophage cells are impacted in SGD, we analyzed phenotypic features of peripheral blood (PB) monocytes in a SGD individual lacking functional C/EBPɛ. Flow cytometric analysis of PB leukocytes revealed aberrant expression of CD45, CD11b, CD14, CD15, and CD16 on cells from the SGD individual. Also, the PB CD14+ cells from this individual, weakly stained for the monocyte‐specific enzyme, nonspecific esterase, and electron microscopic examination, indicated morphologic differences between the SGD cells and those from normal controls. Serum interleukin (IL)‐6 levels in the SGD individual during a severe bacterial infection were lower compared with levels in other non‐SGD individuals with sepsis. In contrast, serum IL‐8 levels were markedly elevated in the SGD individual compared with those of non‐SGD individuals in sepsis. PB CD14+ cells from the SGD individual expressed higher IL‐8 mRNA levels compared with normal controls in response to lipopolysaccharide and interferon‐γ. These phenotypic and functional alterations of PB monocytes in the SGD individual suggest that C/EBPɛ plays a critical role in monocyte/macrophage development of humans and is consistent with observations in the murine system. This study implicates abnormalities in monocytes/macrophages and neutrophils in the onset and development of SGD.

Identifying progression-associated genes in adult T-cell leukemia/lymphoma by using oligonucleotide microarrays

Adult T‐cell leukemia/lymphoma (ATL) is associated with human T‐lymphotropic virus type‐1 (HTLV‐1). To understand the changes in expression that occur in the progression of chronic phase of ATL to acute crisis, the gene expression profiles of fresh ATL cells were compared in 4 pairs of samples (progression of chronic to acute phase in 3 patients, as well as 1 typical chronic phase sample vs. 1 typical acute phase sample) using high‐density oligonucleotide DNA arrays. We identified 203 genes that were commonly upregulated in acute vs. chronic phase samples including ribosomal proteins, proteosome subunits, eukaryotic translation factors, immunophilins, heat shock proteins and genes important for DNA replication. Additionally, we identified 91 commonly downregulated genes including immune molecules related to MHC and a phosphatase. Several of the genes were previously identified to be associated with the Tax protein of HTLV‐1. Some of the upregulated genes were located in amplified regions identified by comparative genomic hybridization in the corresponding chronic/acute ATL sample. Using real‐time quantitative PCR, we confirmed the array‐results in those specimens analyzed by microarray. These results demonstrated that distinct sets of genes that are known to be critical in cellular transformation and/or activation are up‐ or down‐regulated during the transition to the acute phase of ATL.

1α,25-dihydroxyvitamin D3 displays divergent growth effects in both normal and malignant cells

Abstract Induction of growth arrest and differentiation of some cancer cells by 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ], and its potent analogs, is well characterized. However, aggressive cancer cell lines are often either insensitive to the antiproliferative effects of 1α,25(OH) 2 D 3 or require toxic concentrations to recapitulate them which has, to-date, precluded its use in anticancer therapy. Therefore we are interested in mechanisms by which 1α,25(OH) 2 D 3 signaling has become deregulated in malignant cells in order to identify novel therapeutic targets. We observed previously that 1α,25(OH) 2 D 3 and its metabolites, generated via the C-24 oxidation pathway, drive simultaneous differentiation and hyper-proliferation within the same cell population. Thus we have proposed that metabolism of 1α,25(OH) 2 D 3 via the C-24 oxidation pathway represents a novel-signaling pathway, which integrates proliferation with differentiation. In the current study we examined further the role of this pathway and demonstrated that these effects are not restricted to leukemic cells but are observed also in both normal myeloid progenitors and breast cancer cell lines. Intriguingly, stable transfection of MCF-7 breast cancer cells with antisence vitamin D 3 receptor (VDR) reduced antiproliferative sensitivity to 1α,25(OH) 2 D 3 but significantly enhanced growth stimulation, which, in turn, was blocked by inhibiting metabolism of 1α,25(OH) 2 D 3 via C-24 oxidation pathway with ketoconazole. Taken together, these studies indicate that metabolism of 1α,25(OH) 2 D 3 via C-24 oxidation pathway gives rise to ligands with different biologic effects. We propose that this mechanism may allow the co-ordination of population expansion and cell maturation during differentiation. Cancer cells appear to corrupt this process during malignant transformation, by only responding to the pro-proliferative signals, thereby deriving a clonal advantage.

Establishment of the acute myeloid leukemia cell line Kasumi‐6 from a patient with a dominant‐negative mutation in the DNA‐binding region of the C/EBPα gene

A myeloid leukemia cell line designated Kasumi‐6 was established from the bone marrow cells of an individual with acute myeloid leukemia, subtype M2. Both the original leukemic cells and the Kasumi‐6 cell line harbor a hemizygous point mutation in the gene encoding the CCAAT/enhancer binding protein alpha (C/EBPα), a critical myeloid transcriptional factor. The C to G transition at nucleotide 1063 of the C/EBPα gene results in amino acid transition R305P in the fork or hinge region between the DNA‐binding basic region and the leucine zipper dimerization domain of the C/EBPα protein. The Kasumi‐6 cells expressed both the p42 and p30 isoforms of the C/EBPα protein endogenously, but electrophoretic mobility shift assays demonstrated an absence of C/EBPα binding to its respective site. Exogenous expression of the mutant form of C/EBPα demonstrated that it was unable to bind DNA and activate transcription from a G‐CSF receptor–luciferase reporter construct. Furthermore, coexpression of the wild‐type and mutant forms revealed that the mutant form repressed reporter gene activation by the wild type in a dose‐responsive manner. This was concomitant with a dose‐responsive decrease in wild‐type protein binding to the G‐CSF receptor C/EBP site. The data suggest that the R305P alteration confers a dominant‐negative property on the mutant C/EBPα protein whereby the mutant polypeptide heterodimerizes with the wild‐type polypeptide and prevents it from binding to DNA, thus blocking transcriptional activation. The Kasumi‐6 cell line can serve as a model to study the cellular and molecular biology of the non‐t(8;21) M2 type of myeloid leukemia and can elucidate the role of mutated C/EBPα in leukemogenesis.

Expression of bactericidal/permeability-increasing protein requires C/EBPɛ

Bactericidal/permeability-increasing protein (BPI) is a 55-kd cationic protein found mainly in neutrophil primary granules. BPI shows cytotoxicity against Gram-negative bacteria. In this study, we studied the role of a myeloid-specific transcription factor, CCAAT/enhancer binding protein ɛ (C/EBPɛ), in the regulation of BPI gene expression. A patient with neutrophil-specific granule deficiency with a homozygous inactivating mutation in the CEBPɛ gene showed severely impaired expression of both BPI messenger RNA (mRNA) and BPI protein. Both U937 and NB4 cells treated with 10?7 M all -trans retinoic acid (ATRA) for 6 days displayed increased levels of BPI protein and accompanying up-regulated C/EBPɛ expression. Chromatinimmunoprecipitation analysis and electrophoretic mobility shift assays revealed binding of the C/EBPɛ protein to the C/EBP-binding site in the BPI gene promoter. U937 cells stably transfected with a zinc-inducible C/EBPɛ expression vector showed a 30-fold increase in BPI mRNA levels compared with cells transfected with control empty vector after culturing for 48 hours with 100 μM ZnSO4. BPI mRNA expression was severely reduced in the bone marrow of C/EBPɛ-deficient mice compared with wild-type mice. Expression of BPI in human cord blood cells was increased by incubation with 10?7 MATRA for 48 hours. These results demonstrate the requirement for C/EBPɛ in mediating BPI gene expression in myeloid cells in vitro and in vivo.