Block In Terminal Differentiation Research Articles

Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation

CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins.Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE.Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp).To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice.In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation. DisclosuresNo relevant conflicts of interest to declare.

Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis.

Ageing results in loss of tissue homeostasis across taxa. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinaldiseases.

Diffron C, a Novel Oligonucleotide Based Gene-Silencing Agent, Induces Terminal Differentiation in Primary Murine Cells

Abstract Myelodysplastic syndrome (MDS) and acute myelogenous leukaemia (AML) are closely related diseases of haematopoietic stem cells (HSCs), characterized by ineffective terminal differentiation, effacement of normal blood cell production and accumulation of neoplastic cells, known as blasts, in the bone marrow, peripheral blood, and other tissues. In the past ten years it has become apparent that cancers are not composed of uniform populations of cancer cells. Instead, the great majority of the cells making up a cancer have very limited ability to undergo cell division, or have completely lost the ability to divide - only a minority of cells, known as cancer stem cells, retain the ability to divide in an unlimited way. To identify potential therapeutic targets, we studied the molecular signature of leukemia stem cells identifying the orphan nuclear receptor NR2F6 as a potential therapeutic target for differentiation therapy for MDS and AML. We've previously shown that NR2F6 is over expressed in patients with myelodysplastic syndromes and acute myeloid leukemia. In vitro, overexpression of NR2F6 inhibits differentiation of cell lines and primary bone marrow cells. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model lead to a condition that resembled myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. While overexpression of NR2F6 in 100% transduced bone marrow grafts that were sorted by flow cytometry prior to transplantation lead to a rapidly fatal erythroleukemia that was characterized by a block in terminal differentiation, pancytopenia and profound anemia. Furthermore, silencing of NR2F6 expression in human and mouse-leukemia cell lines causes terminal differentiation and death by apoptosis. This forms the basis of the therapeutic concept that we wished to develop further, by identifying gene-silencing methods that could be advanced to preclinical development for the treatment of myelodysplastic syndrome and acute leukemia. The discovery of the role of NR2F6 in leukemogenesis and the induction of differentiation of leukemia cell lines following silencing of NR2F6 expression suggests that this is a logical therapeutic target for differentiation therapy using gene silencing technology. Hence, we wished to show that silencing of NR2F6 using DiffronC, an oligonucleotide based gene-silencing technology, in primary mouse hematopoietic cells promoted terminal differentiation. Herein, we show that while DiffronC did not significantly reduce the number of colony forming units, it did significantly increase colony size. This was especially striking in cultures grown under erythroid conditions, where we observed a 3.21 increase in the number of cells per colony. We then showed that DiffronC reduced the clonal longevity of bone marrow cells: In replating experiments we observed a significant decrease in secondary colonies in cells treated with NR2F6 shRNA. Furthermore, DiffronC resulted in a drastic reduction in KSL stem and progenitor cells after six days of ex vivo culture, and caused a dramatic decline in lineage negative cells, and a concurrent increase in cells expressing the myeloid markers CD11b and Gr-1, suggesting that they had differentiated into cells of the neutrophil lineage. This was confirmed by examination of the cytomorphology of the bone marrow cultures. Taken together, these results establish that NR2F6 is a negative regulator of terminal differentiation of the hematopoietic lineage and lays the foundation for use of gene silencing technology using DiffronC as differentiation therapy for diseases with blocked differentiation such as leukemia and myelodysplastic syndromes. Disclosures Ichim: Entest BioMedical Inc: Employment, Equity Ownership, Research Funding; Regen BioPharma Inc: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Regen BioPharma Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wells:Alexion: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Regulation of NK cell maturation by the intramembrane protease SPPL3 (HEM5P.224)

Abstract Natural killer (NK) cells play an essential role in tumor surveillance through direct tumor cell lysis. Signal peptide peptidase-like 3 (SPPL3) is a highly conserved intramembrane aspartyl protease, but very little is known about its functions in vivo. We developed an SPPL3 conditional knockout mouse and found that deletion of SPPL3 within the hematopoietic system using Vav1-iCre resulted in a peripheral defect in NK cell number. The reduced NK cell population was hypofunctional in tumor clearance in vivo and direct tumor lysis in vitro. Analysis of SPPL3-deficient NK cells revealed a substantial block in terminal maturation, at the CD27+CD11b- stage. Deletion of SPPL3 in NK cells by NKp46-iCre confirmed a cell-autonomous requirement for SPPL3 in NK development and function. CRISPR/Cas9 genome editing was used to create a knockin mouse expressing a protease-dead SPPL3 knockin allele, D271A. NK cells expressing SPPL3 D271A exhibited the same block in terminal differentiation as SPPL3-deficient NK cells. Our results reveal that the proteolytic function of the SPPL3 intramembrane protease is critical for normal NK cell maturation at a checkpoint that regulates the transition from CD11b- to CD11b+ cells.

Epidermal Snail expression drives skin cancer initiation and progression through enhanced cytoprotection, epidermal stem/progenitor cell expansion and enhanced metastatic potential

Expression of the EMT-inducing transcription factor Snail is enhanced in different human cancers. To investigate the in vivo role of Snail during progression of epithelial cancer, we used a mouse model with skin-specific overexpression of Snail. Snail transgenic mice spontaneously developed distinct histological subtypes of skin cancer, such as basal cell carcinoma, squamous cell carcinoma and sebaceous gland carcinoma. Development of sebaceous gland carcinomas strongly correlated with the direct and complete repression of Blimp-1, a central regulator of sebocyte homeostasis. Snail expression in keratinocyte stem cells significantly promotes their proliferation associated with an activated FoxM1 gene expression signature, resulting in a larger pool of Mts24-marked progenitor cells. Furthermore, primary keratinocytes expressing Snail showed increased survival and strong resistance to genotoxic stress. Snail expression in a skin-specific p53-null background resulted in accelerated formation of spontaneous tumours and enhanced metastasis. Our data demonstrate that in vivo expression of Snail results in de novo epithelial carcinogenesis by allowing enhanced survival, expansion of the cancer stem cell pool with accumulated DNA damage, a block in terminal differentiation and increased proliferation rates of tumour-initiating cells.

Abstract 3491: Differential antileukemic activity of plant polyphenol combinations in acute myeloid leukemia (AML) cells

Abstract AML is the most common acute leukemia in adults with high mortality rate. This malignancy results from the block of terminal differentiation of the multipotent hematopoietic progenitors at early stages of myelopoiesis. The standard approach for AML treatment is cytotoxic chemotherapy with cytarabine and anthracyclines which has low efficiency and is accompanied by severe side effects. Therefore, AML represents a largely unsolved challenge for chemotherapy of malignant disease. Plant polyphenols, which are abundant in diet rich in vegetables and fruits, have been shown to act as chemopreventive and anticancer agents. In this study we demonstrate the in-vitro antileukemic activity of curcumin (CUR), carnosic acid (CA) and silibinin (SIL), alone and in combination, in several human AML cell lines representing different stages of myeloid differentiation: KG-1a (FAB: M1; WHO: AML with minimal differentiation), HL60 (FAB: M2; WHO: AML with maturation), and U937 (FAB: M4; WHO: acute myelomonocytic leukemia). All polyphenols tested inhibited cell proliferation in a concentration-dependent manner. Remarkably, when administered together even at non-cytotoxic concentrations, CUR (2.5-5 µM) and CA (5-10 µM) induced robust cell death in all cell lines tested. This effect was due to the induction of apoptosis concomitant with activation of caspase −8, −9 and −3, and was associated with cell cycle arrest in the S-phase. Interestingly, the induction of cell death was not accompanied either by generation of the intracellular reactive oxygen species or by a decrease in the glutathione content. In addition, the combined treatment induced caspase-8-dependent cleavage of the BH3-only protein Bid, but had no effect on the other Bcl-2 family pro- and anti- apoptotic proteins tested, including Bcl-2, Bcl-xL, Mcl-1, Bax and Bak. In contrast to the CUR/CA combination, cell treatment with SIL (30-60 µM) combined with either CUR or CA resulted in an enhanced inhibition of proliferation with a minor decrease in cell viability. This antiproliferative effect was accompanied by a marked G0/G1 cell cycle arrest with a concomitant decrease in the protein levels of cyclin E and cyclin-dependent kinase-4, and elevation of the cyclin-dependent kinase inhibitor p27Kip1 levels. Importantly, the polyphenol combinations tested here did not affect the viability of human normal skin fibroblasts as well as of both proliferating and non-proliferating peripheral blood mononuclear cells. Our findings indicate that distinct combinations of plant polyphenols exert strong but differential in vitro antileukemic effects by selectively inducing synergistic growth arrest and/or apoptotic cell death. These results suggest that such combinations may have potential for the treatment of AML. Supported by RO1-CA117942-02 grant from the NIH-NCI (to both G.P.S. and M.D.) and by Israel Science Foundation grant 778/07 (to M.D.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3491.

Molecular Mechanisms of Mouse Skin Tumor Promotion

Multiple molecular mechanisms are involved in the promotion of skin carcinogenesis. Induction of sustained proliferation and epidermal hyperplasia by direct activation of mitotic signaling pathways or indirectly in response to chronic wounding and/or inflammation, or due to a block in terminal differentiation or resistance to apoptosis is necessary to allow clonal expansion of initiated cells with DNA mutations to form skin tumors. The mitotic pathways include activation of epidermal growth factor receptor and Ras/Raf/mitogen-activated protein kinase signaling. Chronic inflammation results in inflammatory cell secretion of growth factors and cytokines such as tumor necrosis factor-α and interleukins, as well as production of reactive oxygen species, all of which can stimulate proliferation. Persistent activation of these pathways leads to tumor promotion.

Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced microRNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these pro-differentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.

Tumor Suppressor Genes in Myeloid Differentiation and Leukemogenesis

Tumor suppressor genes, such as p53, RB, the INK4-ARF family and PML, suppress malignant transformation by regulating cell cycle progression, ensuring the fidelity of DNA replication and chromosomal segregation, or by inducing apoptosis in response to potentially deleterious events. In myeloid leukemia, hematopoietic differentiation resulting from highly coordinated, stage-wise expression of myeloid transcription and soluble signaling factors is disrupted leading to a block in terminal differentiation and uncontrolled proliferation. This virtually always involves functional inactivation or genetic disruption of one or several tumor suppressor genes in order to circumvent their checkpoint control and apoptosis-inducing functions. Hence, reactivation of tumor suppressor gene function has therapeutic potential and can possibly enhance conventional cytotoxic chemotherapy. In this review, we focus on the role of different tumor suppressor genes in myeloid differentiation and leukemogenesis, and discuss implications for therapy.

Down-Regulation of CEBPA Expression Has a Stronger Impact on Myeloid Differentiation in Human Versus Mouse Cells: Mechanistic Implications for CEBPA Mutations in AML.

Acute myeloid leukemia (AML) is often associated with gene mutations that affect transcription factors regulating differentiation. CEBPA mutations occur in 5–10% of AML cases. About half of those result in the translation of an N-terminal truncated C/EBPα protein, termed p30. While C/EBPα is not required for adult hematopoiesis, its ablation leads to a block in granulocytic differentiation at the blast stage. Earlier studies showed that p30 dominantly inhibits the transcriptional function of wtC/EBPα. We have previously shown that expression of p30 in human but not murine hematopoietic progenitors leads to the accumulation of myeloblasts. Here, we investigated the role of p30 C/EBPα in AML induction by using different dimerizing mutants of C/EBPα. In addition, we sought to determine if down-regulation of CEBPA expression mimicked the effect of p30 in human hematopoietic progenitors. To evaluate the requirement for dimerization and transcriptional activity of the p30 C/EBPα, a mutant was generated in which the first two leucines of the leucine zipper were changed to valine (C/EBPα L12V), which has been previously shown to disrupt C/EBPα function. CD34+ cells isolated from human cord blood were transduced with retroviral vectors expressing either GFP alone, human or murine p30 C/EBPα, or murine p30-L12V and plated into methylcellulose. While murine mutC/EBPα and human mutC/EBPα showed the expected block in granulocytic differentiation and inhibition of erythrocyte lineage commitment (mean number of 0 ± 2 CFU-GM), mutC/EBPα L12V had no effect as shown by the same colony distribution as the control vector (mean number of 20 CFU-GM). This observation implies that mutC/EBPα requires dimerization and/or transcriptional activity for the inhibition of human hematopoietic differentiation. To predict if p30 homodimerization or heterodimerization between p30 C/EBPα and wtC/EBPα is required for this activity, we replaced the leucine zipper of the p30 C/EBPα with the GCN4 exchange mutant to form p30-GZ, which allows homodimerization but not heterodimerization with wtC/EBPα. We demonstrate here that expression of p30-GZ showes an almost identical phenotype as mutC/EBPα arguing that p30 homodimerization is important for its activity while heterodimerization with wtC/EBPα is not necessary. To test the effects of down-regulation of C/EBPα and to compare the induced phenotype with the one induced by p30 C/EBPα, we generated a short-hairpin (sh) RNA against human C/EBPα and cloned it into a retroviral expression vector. Human CD34+ cells from cord blood were transduced with these vectors and evaluated for the down-regulation of C/EBPα mRNA. Expression of C/EBPα shRNA reduced C/EBPα mRNA levels to 25%. Examination of liquid cultures (with GM-CSF, SCF, IL3) by using FACS analysis demonstrated a relative increase in erythroid cells: 32% CD71+/CD13− C/EBPα siRNA versus 7% CD71+/CD13− control vector, and substantial defect in proliferation of myeloid cells (0% CD14 and 4% CD15 C/EBPα siRNA compared with 10% CD14 and 12% CD15 control vector). Most striking were results in methylcellulose cultures, where a dramatic reduction in CFU-GM colonies (mean number of 8 ± 2 CFU-GM C/EBPα siRNA versus 37 ± 4 CFU-GM control vector), but also BFU-E (mean number of 18 ± 3 BFU-E C/EBPα siRNA versus 42 ± 4 BFU-E control vector) was observed in cultures expressing shRNA against C/EBPα. In addition, an almost complete block in terminal differentiation of both monocytic and granulocytic differentiation was observed in cells dispersed from the colonies. Using shRNA technology, we show that C/EBPα is required at two stages of human myeloid differentiation:during early stages of myeloid/erythroid commitment andfor terminal granulocytic and monocytic maturation.In contrast to mouse studies, the block to terminal differentiation could not be relieved by GM-CSF stimulation. This shows that C/EBPα activity is not readily substituted by alternative pathways in human myeloid differentiation. We thus propose that p30 induces myeloid differentiation at early stages of differentiation but cannot promote terminal differentiation. This coupled with reduced absolute levels of C/EBPα leads to accumulation of myeloblasts, a hallmark of AML.

Leukemia suppressor function of Egr-1 is dependent on transforming oncogene

We have shown that deregulated expression of either c-Myb or E2F-1 blocks terminal differentiation of M1 myeloid leukemia cells at the blast stage, whereas deregulated c-Myc blocks differentiation at the intermediate stage. Each of these oncogenes potentiates M1 leukemia in vivo. The zinc-finger transcription factor Egr-1 abrogates the block in M1 terminal differentiation imparted by oncogenic c-Myc or E2F-1, suppressing their leukemia-promoting function in nude mice. In this study, we asked whether Egr-1 also abrogates the block in terminal differentiation and suppresses leukemia imparted by deregulated c-Myb. Interestingly, the ectopic expression of Egr-1 in M1 cells expressing deregulated c-Myb only partially abrogated the block in terminal differentiation and did not suppress the leukemic phenotype. Two important implications from these data are that the leukemia suppressor function of Egr-1 is not directly related to how early the transforming oncogene blocks the differentiation program and that the tumor suppressor function of Egr-1 is dependent on the specific oncogene. Egr-1 is dominant to c-Myc- and E2F-1-, but not to c-Myb-, driven leukemia. These findings extend the notion that the molecular nature of genetic lesions responsible for leukemia determines the effectiveness of any given tumor suppressor.

Egr-1 abrogates the block imparted by c-Myc on terminal M1 myeloid differentiation

AbstractBoth deregulated growth and blocks in differentiation cooperate in the multistage process of leukemogenesis. Thus, understanding functional interactions between genes that regulate normal blood cell development, including cell growth and differentiation, and how their altered expression contributes to leukemia, is important for rational drug design. Previously, we have shown that the zinc finger transcription factor Egr-1 plays a role in monocytic differentiation. Ectopic expression of Egr-1 in M1 myeloblastic leukemia cells was observed to activate the macrophage differentiation program in the absence of the differentiation inducer interleukin 6 (IL-39) and to promote terminal differentiation in its presence. In addition, we have shown that deregulated expression of the proto-oncogene c-myc blocks the myeloid terminal differentiation program. Here we show that restoring expression of Egr-1 in M1 cells that express deregulated c-Myc abrogates the c-Myc block in terminal differentiation, resulting in cells that undergo functional macrophage maturation. However, there is an absence of both growth arrest and cell adhesion. In addition, Egr-1 expression diminished M1myc leukemogenicity in vivo. These findings indicate that Egr-1 can act as a tumor suppressor gene and suggest that Egr-1 or Egr-1 targets may provide important tools for differentiation therapy in certain leukemic phenotypes.

Abortive myogenesis in denervated skeletal muscle: differentiative properties of satellite cells, their migration, and block of terminal differentiation

Little is known about the biological properties of myogenic satellite cells during post-denervation muscle atrophy. The present study investigated the differentiative capacity of satellite cells and their involvement in the compensatory regenerative process in long-term denervated rat muscle. Electron microscopy and immunocytochemical labeling of muscle tissue 1-18 months following denervation demonstrated that despite activation of satellite cells, myogenesis in denervated muscle is abortive and does not lead to the formation of normal muscle fibers. Small sizes, poor development of the contractile system in newly formed denervated myotubes, and the absence of satellite cells on the surface indicate that their differentiation typically does not progress to terminal stages. Many immature myotubes degenerate, and others survive but are embedded in a collagen lattice near their parent fibers. Interestingly, newly formed myotubes located on the surface of parent muscle fibers beneath the basal lamina typically did not contain developed myofibrils. This suggests that the contacts of daughter and parent muscle fibers block myofibrillogenesis. Assembly of sarcomeres in most cases occurs following complete spatial separation of daughter and parent muscle fibers. Another manifestation of the involvement of myogenic precursors in abortive myogenesis is the formation of clusters of underdeveloped branching myotubes surrounded by a common basal lamina. We found that myoblasts can also fuse directly with differentiated muscle fibers. The presence of satellite cells near the openings in the basal lamina and in the interstitial space indicates that myogenic precursors can migrate through the basal lamina and form myotubes at a distance from parent fibers. Our data may explain why long-term denervated skeletal muscle has a poor capacity for regeneration and functional restoration.

Egr-1 Abrogates the Block Imparted by c-Myc on Terminal M1 Myeloid Differentiation.

Both deregulated growth and blocks in differentiation cooperate in the multistage process of leukemogenesis. Thus, understanding functional interactions between genes that regulate normal blood cell development, including cell growth and differentiation, and how their altered expression contributes to leukemia, is important for rational drug design. Previously, we have shown that the zinc finger transcription factor Egr-1 plays a role in monocytic differentiation. Ectopic expression of Egr-1 in M1 myeloblastic leukemia cells was observed to activate the macrophage differentiation program in the absence of the differentiation inducer interleukin 6 (IL-6), and to promote terminal differentiation in its presence. In addition, we have shown that deregulated expression of the proto-oncogene c-myc blocks the myeloid terminal differentiation program. Here we show that restoring expression of Egr-1 in M1 cells that express deregulated c-Myc abrogates the c-Myc block in terminal differentiation, resulting in cells that undergo functional macrophage maturation. Interestingly, it was shown that the ability of Egr-1 to override the c-Myc block in terminal differentiation occurred in the absence of growth arrest and cell adhesion, resulting in cells with the characteristics of functionally mature macrophages. As with normal macrophages, the cells also underwent programmed cell death. It was also observed that Egr-1 diminished the aggressiveness of M1myc leukemias, and abrogated the leukemic potential of M1myc cells treated with IL-6. Taken together, these observations demonstrate that Egr-1 behaves like a tumor suppressor, suggesting that, on the one hand, genetic lesions which alter Egr-1 expression can cooperate with deregulated c-Myc in exacerbating the leukemic phenotype and, on the other hand, that enhancement of Egr-1 expression, or its downstream targets, can overcome blocks in differentiation due to deregulated c-Myc, as well as its associated leukemias These findings suggest that Egr-1 and/or Egr-1 targets may provide important tools for differentiation therapy in certain leukemic phenotypes

Differential Requirements for Survivin in Hematopoietic Cell Development.

The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.

Effects on differentiation by the promyelocytic leukemia PML/RARalpha protein depend on the fusion of the PML protein dimerization and RARalpha DNA binding domains.

The block of terminal differentiation is a prominent feature of acute promyelocytic leukemia (APL) and its release by retinoic acid correlates with disease remission. Expression of the APL-specific PML/RARalpha fusion protein in hematopoietic precursor cell lines blocks terminal differentiation, suggesting that PML/ RARalpha may have the same activity in APL blasts. We expressed different PML/RARalpha mutants in U937 and TF-1 cells and demonstrated that the integrity of the PML protein dimerization and RARalpha DNA binding domains is crucial for the differentiation block induced by PML/RARalpha, and that these domains exert their functions only within the context of the fusion protein. Analysis of the in vivo dimerization and cell localization properties of the PML/RARalpha mutants revealed that PML/RARalpha--PML and PML/RARalpha--RXR heterodimers are not necessary for PML/RARalpha activity on differentiation. We propose that a crucial mechanism underlying PML/RARalpha oncogenic activity is the deregulation of a transcription factor, RARalpha, through its fusion with the dimerization interface of another nuclear protein, PML.

Decreased expression of fibronectin and the α5β 1 integrin during terminal differentiation of human keratinocytes

We have examined the expression of fibronectin and the alpha 5 beta 1 fibronectin receptor during terminal differentiation of human epidermal keratinocytes, using involucrin as a terminal differentiation marker. The levels of mRNAs encoding fibronectin and the alpha 5 and beta 1 integrin subunits were measured in keratinocyte populations that had been enriched for involucrin-negative or -positive cells by unit gravity sedimentation or suspension-induced terminal differentiation. All three mRNAs decreased in abundance during terminal differentiation, and the corresponding proteins were localised by immunofluorescence to the basal layer in stratified colonies. We also examined expression in ndk, a strain of epidermal cells with a complete block in terminal differentiation, which, as a result, do not express involucrin. Messenger RNA levels for fibronectin and the alpha 5 and beta 1 subunits were higher in ndk, than in unfractionated keratinocytes and the corresponding proteins were expressed by all ndk, consistent with a basal keratinocyte phenotype. We conclude that expression of fibronectin and the alpha 5 beta 1 fibronectin receptor decreases during terminal differentiation and that such changes are likely to play a role in the selective migration of terminally differentiating cells from the basal epidermal layer.

B-cell dysfunction following human bone marrow transplantation: functional-phenotypic dissociation in the early posttransplant period

We investigated the defect in humoral immunity that occurs following bone marrow transplantation (BMT). B cells from BMT recipients were tested for their ability to undergo the sequential steps of activation (RNA synthesis on stimulation with anti-mu or PMA), proliferation (DNA synthesis on stimulation with anti-mu plus B cell growth factor [BCGF], phorbol myristate acetate [PMA], or Staphylococcus aureus Cowan I [SAC] strain bacteria) and differentiation (Ig synthesis stimulated by T cell replacing factor [TRF]). B-cell maturation-associated cell surface markers were simultaneously investigated. “Early” (less than 10 months) posttransplant patients demonstrated defective B-cell activation and also failed to undergo normal proliferation and differentiation. Despite their functional impairment, the early patients' B cells displayed an “activated” phenotype with increased proportions of B cells displaying CD23 (a BCGF receptor) and decreased proportions of Leu 8+ B cells. Furthermore, these patients were uniquely distinguished by the fact that their B cells only weakly (if at all) expressed the CD19 antigen. In contrast, B cells from “late” patients (greater than or equal to 10 months post-BMT) activated and proliferated normally and displayed a normal cell surface phenotype, yet were unable to differentiate to high rate Ig secretion with TRF. Our results suggest a phenotype/function dissociation in early posttransplant period. With time, B cells in BMT patients acquire a normal surface phenotype and can activate and proliferate normally, yet still demonstrate a block in terminal differentiation.

B-Cell Dysfunction Following Human Bone Marrow Transplantation: Functional-Phenotypic Dissociation in the Early Posttransplant Period

We investigated the defect in humoral immunity that occurs following bone marrow transplantation (BMT). B cells from BMT recipients were tested for their ability to undergo the sequential steps of activation (RNA synthesis on stimulation with anti-mu or PMA), proliferation (DNA synthesis on stimulation with anti-mu plus B cell growth factor [BCGF], phorbol myristate acetate [PMA], or Staphylococcus aureus Cowan I [SAC] strain bacteria) and differentiation (Ig synthesis stimulated by T cell replacing factor [TRF]). B-cell maturation-associated cell surface markers were simultaneously investigated. "Early" (less than 10 months) posttransplant patients demonstrated defective B-cell activation and also failed to undergo normal proliferation and differentiation. Despite their functional impairment, the early patients' B cells displayed an "activated" phenotype with increased proportions of B cells displaying CD23 (a BCGF receptor) and decreased proportions of Leu 8+ B cells. Furthermore, these patients were uniquely distinguished by the fact that their B cells only weakly (if at all) expressed the CD19 antigen. In contrast, B cells from "late" patients (greater than or equal to 10 months post-BMT) activated and proliferated normally and displayed a normal cell surface phenotype, yet were unable to differentiate to high rate Ig secretion with TRF. Our results suggest a phenotype/function dissociation in early posttransplant period. With time, B cells in BMT patients acquire a normal surface phenotype and can activate and proliferate normally, yet still demonstrate a block in terminal differentiation.

An unusual strain of human keratinocytes which do not stratify or undergo terminal differentiation in culture.

We have characterized an unusual cell phenotype in third passage cultures of a human keratinocyte strain derived from newborn foreskin epidermis. The cells had the same DNA fingerprint pattern as the second passage, morphologically normal, keratinocytes; they formed desmosomes and expressed the keratin profile characteristic of normal keratinocytes in culture. However, unlike normal keratinocytes, the cells did not grow as compact colonies and did not stratify or undergo terminal differentiation, even after TPA treatment or suspension culture. For these reasons we named them ndk for "nondifferentiating keratinocytes." The ndk cells also differed from normal keratinocytes in that they did not require a feeder layer and were not stimulated by cholera toxin to proliferate. The ndk cells had an absolute requirement for hydrocortisone and their growth rate was increased when epidermal growth factor was added to the medium. Although ndk failed to undergo terminal differentiation in culture, they were not transformed, since they were still sensitive to contact inhibition of growth, did not proliferate in soft agar, and had a limited lifespan in culture. The appearance of the ndk phenotype was correlated with a doubling of chromosome number and the presence of a lp marker chromosome. We suggest that these cells are a useful experimental adjunct to cultures of normal keratinocytes, in which proliferation and terminal differentiation are tightly coordinated, because in ndk cells there appears to be a block in terminal differentiation.