ALBERT

Description: Targeted mutation of the myeloid transcription factor C/EBPɛ in mice results in gram-negative septic death at 3 to 5 months of age. This study defines the underlying molecular defects in their terminal granulocytic differentiation. The mRNA for the precursor protein of the cathelin-related antimicrobial peptides was almost completely absent in the bone marrow cells of C/EBPɛ−/− mice. This finding may help explain their susceptibility to gram-negative sepsis, because both are bacteriocidal peptides with potent activity against gram-negative bacteria. Superoxide production was found to be reduced in both granulocytes and monocytes of C/EBPɛ−/− mice. While gp91 phox protein levels were normal, p47phox protein levels were considerably reduced in C/EBPɛ −/− granulocytes/monocytes, possibly limiting the assembly of the NADPH oxidase. In addition, expression of mRNA of the secondary and tertiary granule proteins, lactoferrin and gelatinase, were not detected, and levels of neutrophil collagenase mRNA were reduced in bone marrow cells of the knock-out mice. The murine lactoferrin promoter has a putative C/EBP site close to the transcription start site. C/EBPɛ bound to this site in electromobility shift assay studies and mutation of this site abrogated binding to it. A mutation in the C/EBP site reduced the activity of the promoter by 35%. Furthermore, overexpression of C/EBPɛ in U937 cells increased the activity of the wild-type lactoferrin promoter by 3-fold. In summary, our data implicate C/EBPɛ as a critical factor of host antimicrobial defense and suggests that it has a direct role as a positive regulator of expression of lactoferrin in vivo.

Description: Targeted mutation of the myeloid transcription factor C/EBPɛ in mice results in gram-negative septic death at 3 to 5 months of age. This study defines the underlying molecular defects in their terminal granulocytic differentiation. The mRNA for the precursor protein of the cathelin-related antimicrobial peptides was almost completely absent in the bone marrow cells of C/EBPɛ−/− mice. This finding may help explain their susceptibility to gram-negative sepsis, because both are bacteriocidal peptides with potent activity against gram-negative bacteria. Superoxide production was found to be reduced in both granulocytes and monocytes of C/EBPɛ−/− mice. While gp91 phox protein levels were normal, p47phox protein levels were considerably reduced in C/EBPɛ −/− granulocytes/monocytes, possibly limiting the assembly of the NADPH oxidase. In addition, expression of mRNA of the secondary and tertiary granule proteins, lactoferrin and gelatinase, were not detected, and levels of neutrophil collagenase mRNA were reduced in bone marrow cells of the knock-out mice. The murine lactoferrin promoter has a putative C/EBP site close to the transcription start site. C/EBPɛ bound to this site in electromobility shift assay studies and mutation of this site abrogated binding to it. A mutation in the C/EBP site reduced the activity of the promoter by 35%. Furthermore, overexpression of C/EBPɛ in U937 cells increased the activity of the wild-type lactoferrin promoter by 3-fold. In summary, our data implicate C/EBPɛ as a critical factor of host antimicrobial defense and suggests that it has a direct role as a positive regulator of expression of lactoferrin in vivo.

Description: Cyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML–retinoic acid receptor alpha [PML-RARα] or PLZF-RARα) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RARα or PLZF-RARα in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RARα but not PLZF-RARα. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RARα. Reporter assays showed that PML-RARα led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RARα–induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RARα and PLZF-RARα cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia.

Description: Human C/EBPε is a newly cloned gene coding for a CCAAT/enhancer binding protein that may be involved in the regulation of myeloid differentiation. Our studies showed that levels of C/EBPε mRNA were markedly increased in NB4 cells (promyelocytic leukemia line), because they were induced by 9-cis retinoic acid (9-cis RA) to differentiate towards granulocytes. Accumulation of C/EBPε mRNA occurred as early as 1 hour after exposure of NB4 cells to 9-cis RA (5 × 10−7 mol/L); and at 48 hours, levels were increased by 5.1-fold. Dose-response studies showed that 10−7 to 10−6 mol/L 9-cis RA (12 hours) resulted in peak levels of C/EBPε mRNA; but even 10−10 mol/L 9-cis RA increased levels of these transcripts. NB4 cells pulse-exposed (30 minutes) to all-trans retinoic acid (ATRA), washed, and cultured (3 days) with either dimethylsulfoxide (DMSO) or hexamethylene bisacetamide (HMBA) had a prominent increase in levels of C/EBPε mRNA and an increase in granulocytic differentiation, but exposure to either DMSO or HMBA alone had no effect on base levels of C/EBPε and did not induce differentiation. Macrophage-differentiation of NB4 reduced levels of C/EBPε mRNA. Nuclear run-off assays and half-life studies showed that accumulation of C/EBPε mRNA by 9-cis RA was due to enhanced transcription. Furthermore, this C/EBPε mRNA accumulation did not require synthesis of new protein factors because 9-cis RA induced C/EBPε mRNA accumulation in the absence of new protein synthesis. ATRA also induced expression of C/EBPε protein in NB4 cells, as shown by Western blotting. In contrast to the increase of C/EBPε in 9-cis RA–mediated granulocytic differentiation, the DMSO-induced differentiation of HL-60 cells down the granulocytic pathway was associated with an initial reduction of C/EBPε mRNA levels. In summary, we have discovered that expression of C/EBPε mRNA is markedly enhanced as the NB4 promyelocytes are induced by retinoids to differentiate towards granulocytes. This induction of C/EBPε mRNA expression is transcriptionally mediated and occurs in the absence of synthesis of additional protein factors. We suspect that the C/EBPε promoter/enhancer contains a retinoic acid-response element that is directly stimulated by retinoids.

Description: Cyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML–retinoic acid receptor alpha [PML-RARα] or PLZF-RARα) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RARα or PLZF-RARα in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RARα but not PLZF-RARα. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RARα. Reporter assays showed that PML-RARα led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RARα–induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RARα and PLZF-RARα cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia.

Description: Acute promyelocytic leukemia (APL) is associated with chromosomal translocations involving retinoic acid receptor α (RARα) and its fusion partners including promyelocytic leukemia (PML) and promyelocytic leukemia zinc finger (PLZF). Using oligonucleotide arrays, we examined changes in global gene expression mediated by the ectopic expression of either PML/RARα (retinoid-sensitive) or PLZF/RARα (retinoid-resistant) in U937 cells. Of more than 5000 genes analyzed, 16 genes were commonly up-regulated, and 57 genes were down-regulated by both fusion proteins suggesting their role in the APL phenotype. In our APL model, for example, TNFAIP2, TNFR2, ELF4, RARγ, and HoxA1 were down-regulated by both fusion proteins in the absence of retinoic acid (RA). RA strongly up-regulated these genes in PML/RARα, but not in PLZF/RARα expressing U937 cells. Expression studies in NB4, retinoid-resistant NB4-R2, normal human CD34+ cells, and APL patient samples strongly suggest their role in the regulation of granulocytic differentiation. Furthermore, combined treatment with tumor necrosis factor α (TNFα) and RA synergistically enhanced granulocytic differentiation in NB4 cells but not in NB4-R2 cells. Our data indicate that APL pathogenesis and retinoid-induced granulocytic differentiation of APL cells involve genes in the cell death pathway, and that cooperation between the RA and TNFα signaling pathways exists. Targeting both the retinoid-dependent differentiation and the cell death pathways may improve leukemic therapy, especially in retinoid-resistant acute myeloid leukemia. (Blood. 2003;102:3727-3736)

Description: Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

Description: Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

Description: Primary effusion lymphoma (PEL) is a distinct clinicopathologic entity associated with Kaposi's sarcoma-associated herpes virus (KSHV). Several cytokines, including interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) may be important for survival of KS cells. However, little is known about the interaction of cytokines with KSHV-infected lymphocytes from PEL. Therefore, we investigated what cytokines were produced by KSHV-infected PEL cell lines (KS-1, BC-1, BC-2), what cytokine receptors were expressed by these cells, what response these cells had to selected cytokines, and what was the effect of IL-6 antisense phosphorothioated oligonucleotides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and protein studies showed that these three cell lines produced IL-10, IL-6, and the receptors for IL-6. The granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, IL-12, bFGF, PDGF, and c-kit transcripts were not detected in the cell lines. High levels (0.7 to 5 ng/mL/106cells/48 hours) of IL-6 protein were consistently detected in supernatants of the cell lines by enzyme-linked immunosorbent assay (ELISA) tests. In clonogenic assays, interferon-α (IFN-α) and IFN-γ suppressed the clonal growth of the PEL cells, but GM-CSF, IL-4, IL-6, IL-8, IL-10, and oncostatin M did not change it. We examined for several autocrine loops that have been suggested to occur in KS. Experiments using antisense oligonucleotides showed that the clonal growth of KS-1 and BC-1 was nearly 100% inhibited by IL-6 antisense oligonucleotides (10 μmol/L), but not at all by either oligonucleotides (≤10 μmol/L) to IL-6 sense, IL-6 scrambled, viral IL-6 (vIL-6) antisense, or IL-10 antisense. Furthermore, the IL-6 antisense oligonucleotides had no effect on two B-cell lymphoma cell lines, which were not infected with KSHV. Addition of IL-6 antibody did not inhibit clonal growth of any of the cell lines. Taken together, we have defined the cytokines and their receptors expressed on PEL cells and have found that these cells synthesized IL-6 and IL-6 receptors; interruption of this pathway by IL-6 antisense oligonucleotides specifically prevented the growth of these cells. These findings will offer potential new therapeutic strategies for PEL.

Description: Primary effusion lymphoma (PEL) is a distinct clinicopathologic entity associated with Kaposi's sarcoma-associated herpes virus (KSHV). Several cytokines, including interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) may be important for survival of KS cells. However, little is known about the interaction of cytokines with KSHV-infected lymphocytes from PEL. Therefore, we investigated what cytokines were produced by KSHV-infected PEL cell lines (KS-1, BC-1, BC-2), what cytokine receptors were expressed by these cells, what response these cells had to selected cytokines, and what was the effect of IL-6 antisense phosphorothioated oligonucleotides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and protein studies showed that these three cell lines produced IL-10, IL-6, and the receptors for IL-6. The granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, IL-12, bFGF, PDGF, and c-kit transcripts were not detected in the cell lines. High levels (0.7 to 5 ng/mL/106cells/48 hours) of IL-6 protein were consistently detected in supernatants of the cell lines by enzyme-linked immunosorbent assay (ELISA) tests. In clonogenic assays, interferon-α (IFN-α) and IFN-γ suppressed the clonal growth of the PEL cells, but GM-CSF, IL-4, IL-6, IL-8, IL-10, and oncostatin M did not change it. We examined for several autocrine loops that have been suggested to occur in KS. Experiments using antisense oligonucleotides showed that the clonal growth of KS-1 and BC-1 was nearly 100% inhibited by IL-6 antisense oligonucleotides (10 μmol/L), but not at all by either oligonucleotides (≤10 μmol/L) to IL-6 sense, IL-6 scrambled, viral IL-6 (vIL-6) antisense, or IL-10 antisense. Furthermore, the IL-6 antisense oligonucleotides had no effect on two B-cell lymphoma cell lines, which were not infected with KSHV. Addition of IL-6 antibody did not inhibit clonal growth of any of the cell lines. Taken together, we have defined the cytokines and their receptors expressed on PEL cells and have found that these cells synthesized IL-6 and IL-6 receptors; interruption of this pathway by IL-6 antisense oligonucleotides specifically prevented the growth of these cells. These findings will offer potential new therapeutic strategies for PEL.