Terminal Differentiation Of Hematopoietic Cells Research Articles

Pivotal role of DPYSL2A in KLF4-mediated monocytic differentiation of acute myeloid leukemia cells

Although the biological importance of Krüppel-like factor 4 (KLF4) transcription factor in the terminal differentiation of hematopoietic cells to the monocytes has been well established, the underlying mechanisms remain elusive. To clarify the molecular basis of KLF4-mediated monocytic differentiation, we performed detailed genetic studies in acute myeloid leukemia (AML) cells. Here, we report that dihydropyrimidinase like 2 (DPYSL2), also known as CRMP2, is a novel key differentiation mediator downstream of KLF4 in AML cells. Interestingly, we discovered that KLF4-mediated monocytic differentiation is selectively dependent on one specific isoform, DPYSL2A, but not on other DPYSL family genes. Terminal differentiation to the monocytes and proliferation arrest in AML cells induced by genetic or pharmacological upregulation of KLF4 were significantly reversed by short hairpin RNA (shRNA)-mediated selective depletion of DPYSL2A. Chromatin immunoprecipitation assay revealed that KLF4 associates with the proximal gene promoter of DPYSL2A and directly transactivates its expression. Together with the unique expression patterns of KLF4 and DPYSL2 limited to the differentiated monocytes in the hematopoietic system both in human and mouse, the identified KLF4-DPYSL2 axis in leukemia cells may serve as a potential therapeutic target for the development of novel differentiation therapies for patients with AML.

MTB, the murine homolog of condensin II subunit CAP-G2, represses transcription and promotes erythroid cell differentiation

Chromosome condensation is essential for proper segregation of duplicated sister chromatids in mitosis. Mammalian erythroid maturation is also associated with gradual nuclear condensation. However, few proteins that are directly involved in chromosome condensation during erythropoiesis have been identified. In this report, we show that MTB (more than blood), which was initially isolated in a yeast two-hybrid screen for proteins that interact with the basic helix-loop-helix (bHLH) protein stem cell leukemia (SCL), and later identified as the murine homolog of the condensin II subunit CAP-G2, participates in erythroid cell development. MTB interacts with SCL and another hematopoietic bHLH protein, E12, and is recruited to the nucleus by SCL and E12. In addition, MTB can repress SCL/E12-mediated transcriptional activation. Consistent with the model that MTB may function together with SCL/E12 heterodimer during erythroid cell development, MTB is highly expressed in the erythroid lineage and is upregulated upon erythroid differentiation. Moreover, overexpression of MTB promotes the terminal differentiation of the murine erythroleukemia erythroid cell line. Together, these findings demonstrate that the condensin II subunit MTB/mCAP-G2 plays a novel function during erythropoiesis and suggest that key hematopoietic transcription factors such as SCL and E12 may regulate the terminal differentiation of hematopoietic cells through the interaction with condensin complexes.

Activation and Interaction of ATF2 with the Coactivator ASC-2 Are Responsive for Granulocytic Differentiation by Retinoic Acid

Terminal differentiation of hematopoietic cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both lineage-specific and ubiquitous genes. Here we show that the transcription factor ATF2 is associated with the induction of granulocytic differentiation, and the molecular interaction of ATF2 with a tissue-specific coactivator activating signal cointegator-2 (ASC-2) potentiates the differentiation procedure. All-trans retinoic acid (RA) induced the phosphorylation and expression of ATF2 in the early and middle phase of granulocyte differentiation, respectively. The activation of granulocyte-specific gene expression is increased with the concerted action of another basic regionleucine zipper factor, CCAAT/enhancer-binding protein (C/EBPalpha), and ASC-2, which function in a cooperative manner. The interaction between ATF2 and C/EBPalpha in RA-treated cells was enhanced by the ectopic expression of ASC-2. ATF2-mediated transactivation was also increased by co-transfection of ASC-2. This resulted from the direct protein interaction that the N-terminal transactivation domain of ATF2 interacts with the central region of ASC-2. Furthermore, the molecular interaction of ATF2 and ASC-2 was stimulated by RA treatment and inhibited by p38beta kinase inhibitor. Taking these results together, these results suggest that the differentiation-dependent expression and phosphorylation of ATF2 protein physically and functionally interacts with C/EBPalpha and coativator ASC-2 and synergizes to induce target gene transcription during granulocytic differentiation.

C-Myc Is Essential but Not Sufficient for c-Myb-mediated Block of Granulocytic Differentiation

The c-myb proto-oncogene plays a central role in hematopoiesis and encodes a major translational product of 75 kDa. c-Myb is highly expressed in immature hematopoietic cells, and its expression is down-regulated during terminal differentiation. Deregulated expression of c-Myb has been shown to block terminal differentiation of hematopoietic cells. Here we have studied the mechanism of action and the nature of target genes through which c-Myb mediates the block in differentiation of 32Dcl3 murine myeloid cells. We show that the ectopic overexpression of c-Myb in 32Dcl3 cells results in the overexpression of c-Myc. However, enforced expression of c-Myc in 32Dcl3 cells did not alter the normal pattern of differentiation. In addition, expression of dominant-negative mutants of c-Myc relieved c-Myb-mediated block in differentiation. These results led us to conclude that c-myc is a target gene of c-Myb and activation of the c-myc gene is a necessary event in Myb-mediated transformation. However, c-Myc expression alone is inadequate to elicit the phenotypic effects seen with Myb-mediated block in differentiation of myeloid cells, suggesting that activation of additional transcriptional targets by c-Myb plays a critical role in this process.

Deregulated E2F-1 blocks terminal differentiation and loss of leukemogenicity of M1 myeloblastic leukemia cells without abrogating induction of p15INK4B and p16INK4A

AbstractThe transcription factor E2F-1 has been postulated to play a crucial role in the control of cell cycle progression because of its ability to be bound and regulated by the retinoblastoma gene product (pRb). Exogenous expression of E2F-1, under growth restrictive conditions, was shown to result in p53-dependent programmed cell death. The consequences of deregulated expression of E2F-1 on terminal differentiation of hematopoietic cells in the absence of E2F-1–mediated apoptosis, as well as mechanistic insights into how deregulated E2F-1 may affect terminal differentiation, have not been established. The autonomously proliferating M1 myeloblastic leukemia cell line, which is null for p53 expression and can be induced by interleukin-6 (IL-6) to undergo terminal macrophage differentiation with concomitant loss of leukemogenicity, provides a particularly attractive model system to address these issues. Deregulated and continued expression of E2F-1 blocked the IL-6–induced terminal differentiation program at an early blast stage, giving rise to immature cells, which continued to proliferate without undergoing apoptosis and retained their leukemogenic phenotype. Although E2F-1 blocked IL-6–mediated terminal differentiation and its associated growth arrest, it did not prevent the rapid induction of both p15INK4B and p16INK4A, inhibition of cdk4 kinase activity, and subsequent hypophosphorylation of pRb. The results obtained imply that genetic alterations that both impair p53 function and deregulate E2F-1 expression may render hematopoietic cells refractory to the induction of differentiation and are, thereby, likely to play a major role in the progression of leukemias.

Deregulated E2F-1 blocks terminal differentiation and loss of leukemogenicity of M1 myeloblastic leukemia cells without abrogating induction of p15INK4B and p16INK4A

The transcription factor E2F-1 has been postulated to play a crucial role in the control of cell cycle progression because of its ability to be bound and regulated by the retinoblastoma gene product (pRb). Exogenous expression of E2F-1, under growth restrictive conditions, was shown to result in p53-dependent programmed cell death. The consequences of deregulated expression of E2F-1 on terminal differentiation of hematopoietic cells in the absence of E2F-1–mediated apoptosis, as well as mechanistic insights into how deregulated E2F-1 may affect terminal differentiation, have not been established. The autonomously proliferating M1 myeloblastic leukemia cell line, which is null for p53 expression and can be induced by interleukin-6 (IL-6) to undergo terminal macrophage differentiation with concomitant loss of leukemogenicity, provides a particularly attractive model system to address these issues. Deregulated and continued expression of E2F-1 blocked the IL-6–induced terminal differentiation program at an early blast stage, giving rise to immature cells, which continued to proliferate without undergoing apoptosis and retained their leukemogenic phenotype. Although E2F-1 blocked IL-6–mediated terminal differentiation and its associated growth arrest, it did not prevent the rapid induction of both p15INK4B and p16INK4A, inhibition of cdk4 kinase activity, and subsequent hypophosphorylation of pRb. The results obtained imply that genetic alterations that both impair p53 function and deregulate E2F-1 expression may render hematopoietic cells refractory to the induction of differentiation and are, thereby, likely to play a major role in the progression of leukemias.

The v-myc oncogene.

v-myc is the viral homolog of c-myc transduced by several acute transforming retroviruses, many of which encode this gene as a Gag-Myc fusion protein. The v-myc oncogene can transform several lineages of mammalian and avian cells either alone or in cooperation with other oncogenes. While the Gag portion of the Gag-Myc fusion protein and the nuclear localization signal each appear to be dispensable for transformation, the N- and C-termini of the Myc sequence have been found to be essential for transformation. All v-myc genes contain point mutations which seem to confer a greater potency to v-myc in the process of transformation, proliferation, and apoptosis. In v-myc-transformed myelomonocytic cells, secondary events occur, such as the expression of colony stimulating factor-1 (CSF-1) which play a critical role in immortalization and subsequent tumor progression. Inhibition of the autocrine loop of CSF-1 was found to induce apoptosis in the immortalized cells. While overexpression of v-Myc blocks terminal differentiation of hematopoietic cells, this is not sufficient to block the differentiation of certain neural and skeletal muscle cells. Recent developments on the effects of v-myc on cell growth, transformation, differentiation and apoptosis are discussed in this review.

Growth arrest associated with 12- o-tetradecanoylphorbol-13-acetate-induced hematopoietic differentiation with a defective retinoblastoma tumor suppressor-mediated pathway

The retinoblastoma tumor suppressor (Rb) gene product plays an essential role in cell-cycle regulation. However, its role in terminal differentiation of hematopoietic cells is speculative. Here we show a model of 12- o-tetradecanoylphorbol-13-acetate (TPA)-induced hematopoietic differentiation and growth arrest with a defective Rb-mediated pathway. TPA treatment arrested the cell cycle of a human hematopoietic cell line, MEG-01s, at the G1-S boundary and induced expression of p21/SDI1/WAF1/CIP1 and p27/KIP1. Both of these proteins were present in cyclin E-associated complexes, the histone H1 and Rb kinase activities of which were then inactivated. However, MEG-01s cells lacked the intact Rb protein and the Rb-mediated pathway was defective. This model raises a question about the role for Rb in terminal differentiation of hematopoietic cells.

Role of histone deacetylases in acute leukemia.

Accumulating evidence points to a connection between cancer and transcriptional control by histone acetylation and deacetylation. This is particularly true with regard to the acute leukemias, many of which are caused by fusion proteins that have been created by chromosomal translocations. Genetic rearrangements that disrupt the retinoic acid receptor-α and acute myeloid leukemia-1 genes create fusion proteins that block terminal differentiation of hematopoietic cells by repressing transcription. These fusion proteins interact with nuclear hormone co-repressors, which recruit histone deacetylases to promoters to repress transcription. This finding suggests that proteins within the histone deacetylase complexes may be potential targets for pharmaceutical intervention in many leukemia patients. J. Cell. Biochem. Suppls. 30/31:194-202, 1998. © 1998 Wiley-Liss, Inc.

Structure-activity relationship of 17 structural analogues of n-butyric acid upon c-myc expression.

Terminal differentiation of hematopoietic cells in vivo and in vitro is almost invariably accompanied by down-regulated expression of the c-myc proto-oncogene. Constitutive expression of c-myc in tumor cells inhibits terminal differentiation and maintains proliferation. In Burkitt's lymphoma, chromosomal translocations cause a deregulation of the c-myc gene through fusion of this locus with one of the immunoglobulin gene loci. However, the down-regulation of c-myc by n-butyric acid, a potent inducer of differentiation, is also observed in BL cells. Unlike other inducers of differentiation such as dimethylsulfoxide or hexamethylenebisacetamide, which down-regulate c-myc expression, albeit transiently, n-butyric acid causes a continuous, transcriptional shut-off. Because of the possible therapeutic implication of this finding, we have assayed structural analogues of n-butyric acid for their effect on c-myc expression in Burkitt's lymphoma cells. Of the analogues tested, 12 were active and 5 were inactive. Only unbranched fatty acids with 4 and 5 carbon atoms showed activity, a 4-carbon chain being optimal. 3-chloropropionic acid had maximal activity at a 3-fold lower concentration than n-butyric acid (1 mM versus 3 mM). The corresponding ester-analogues were equally effective. Those analogues found capable of down-regulating c-myc in Burkitt's lymphoma cells were similarly effective in their ability to induce terminal differentiation in murine erythroleukemia cells.

Interferon gamma abrogates the differentiation block in v-myc-expressing U-937 monoblasts.

Extensive studies suggest a role for the myc protooncogene family in the control of cell proliferation and differentiation in vertebrates. Previously, deregulated expression of exogenous myc genes has been shown to inhibit induced differentiation in Friend erythroleukemia (MEL) cells and in human monoblastic U-937 cells. To examine the nature of the block of phorbol 12-myristate 13-acetate-induced differentiation in v-myc-expressing U-937 cells, we have studied the effect of other inducers utilizing signal pathways distinct from phorbol 12-myristate 13-acetate (i.e., 1 alpha, 25-dihydroxycholecalciferol and retinoic acid). We show that v-myc also inhibits differentiation associated with these inducers. However, the v-myc-associated block of phorbol 12-myristate 13-acetate-, 1 alpha,25-dihydroxycholecalciferol-, and retinoic acid-induced differentiation retinoic acid-induced differentiation can be overcome by adding interferon gamma as a costimulatory factor. Costimulation with interferon gamma restores terminal differentiation, as shown by acquisition of a macrophage phenotype and an irreversible growth arrest in the G0/G1 phase of the cell cycle, but induces only limited differentiation on its own. The differentiation is accomplished without altering the expression or nuclear localization of the v-myc protein. These results argue against the widely held view that down-regulation of myc expression is a general prerequisite for terminal differentiation of hematopoietic cells and suggests that interferon gamma induces a signal(s) that circumvents the v-myc activity.

Autocrine β-related interferon controls c- myc suppression and growth arrest during hematopoietic cell differentiation

Different hematopoietic cells produce minute amounts of β-related interferon (IFN) following induction of differentiation by chemical or natural inducers. The endogenous IFN binds to type I cell surface receptors and modulates gene expression in the producer ceils. We show that self-induction of two members of the IFN-induced gene family differs in the dose response sensitivity and the prolonged kinetics of mRNA accumulation from the response to exogenous IFN- β 1. Production and response to endogenous IFN are also detected when bone marrow precursor cells differentiate to macrophages after exposure to colony stimulating factor 1. In M1 myeloid cells induced to differentiate by lung-conditioned medium, addition of antibodies against IFN-β partially abrogates the reduction of c- myc mRNA and the loss in cell proliferative activity, which both occur during differentiation. The endogenous IFN therefore functions as an autocrine growth inhibitor that participates in controlling c- myc suppression and the specific G0 G1 arrest during terminal differentiation of hematopoietic cells.