Dan A. Liebermann

The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage

We have isolated cDNA clones of myeloid differentiation primary response (MyD) genes, activated in the absence of de novo protein synthesis following induction for differentiation along either the macrophage or granulocyte lineage in human myeloblastic leukemia HL-60 cells. One cDNA clone of a primary response gene, expressed upon macrophage differentiation, encoded for Egr-1, a zinc finger transcription factor. The Egr-1 gene was observed to be transcriptionally silent in HL-60 cells, but active in U-937 and M1 cells, the latter two being predetermined for macrophage differentiation. Egr-1 antisense oligomers in the culture media blocked macrophage differentiation in both myeloid leukemia cell lines and normal myeloblasts. HL-60 cells constitutively expressing an Egr-1 transgene (HL-60Egr-1) could be induced for macrophage, but not granulocyte, differentiation. These observations indicate that expression of Egr-1 is essential for and restricts differentiation of myeloblasts along the macrophage lineage.

Genotoxic-stress-response genes and growth-arrest genes. gadd, MyD, and other genes induced by treatments eliciting growth arrest.

As discussed throughout this paper, many mammalian DDI genes are associated with growth responses, including both positive responses to growth stimulation and negative responses involving transient growth arrest and terminal differentiation. It is interesting that several immediate-early genes encoding transcription factors, the jun genes, are DDI, are induced by terminal differentiation, and also are associated with positive growth responses. In negative growth-response genes, their control is complex and almost certainly involves multiple regulatory mechanisms. The role of growth-arrest genes after exposure to DNA-damaging agents is currently not known, but as growth arrest can have a protective effect on cells exposed to DNA-damaging agents in both bacteria and eukaryotes, some protective role(s) for the gadd genes may exist. Whatever the roles are for the individual gadd genes, the response of the gadd genes to DNA-damaging agents and other growth-arrest signals has been highly conserved during mammalian evolution, and it is likely that this stress response, as reflected by induction of one or more gadd genes, is present in most or perhaps all mammalian cells. Our findings that the gadd group overlaps with another group of growth-arrest genes, the MyD, indicate that these two groups combined define a new class of genes whose protein products are likely to play a role in cell growth cessation.

Proto-oncogenes of the fos/jun family of transcription factors are positive regulators of myeloid differentiation.

The proto-oncogenes c-jun, junB, junD, and c-fos recently have been shown to encode for transcription factors with a leucine zipper that mediates dimerization to constitute active transcription factors; juns were shown to dimerize with each other and with c-fos, whereas fos was shown to dimerize only with juns. After birth, hematopoietic cells of the myeloid lineage, and some other terminally differentiated cell types, express high levels of c-fos. Still, the role of fos/jun transcription factors in normal myelopoiesis or in leukemogenesis has not been established. Recently, c-jun, junB, and junD were identified as myeloid differentiation primary response genes stably expressed following induction of terminal differentiation of myeloblastic leukemia M1 cells. Intriguingly, c-fos, though induced during normal myelopoiesis, was not induced upon M1 differentiation. To gain further insights into the role of fos/jun in normal myelopoiesis and leukemogenicity, M1fos and M1junB cell lines, which constitutively express c-fos and junB, respectively, were established. It was shown that enforced expression of c-fos, and to a lesser extent junB, in M1 cells results in both an increased propensity to differentiate and a reduction in the aggressiveness of the M1 leukemic phenotype. M1fos cells constitutively expressed immediate-early and late genetic markers of differentiated M1 cells. The in vitro differentiation of normal myeloblasts into mature macrophages and granulocytes, as well as the increased propensity of M1fos leukemic myeloblasts to be induced for terminal differentiation, was dramatically impaired with use of c-fos antisense oligomers in the culture media. Taken together, these observations show that the proto-oncogenes which encode for fos/jun transcription factors play important roles in promoting myeloid differentiation. The ability of the M1 leukemic myeloblasts to be induced for terminal differentiation in the absence of apparent fos expression indicates that there is some redundancy among the fos/jun family of transcription factors in promoting myeloid differentiation; however, juns alone cannot completely compensate for the lack of fos. Thus, genetic lesions affecting fos/jun expression may play a role in the development of preleukemic myelodysplastic syndromes and their further progression to leukemias.

The novel primary response gene MyD118 and the proto-oncogenes myb, myc, and bcl-2 modulate transforming growth factor beta 1-induced apoptosis of myeloid leukemia cells.

Cell numbers are regulated by a balance among proliferation, growth arrest, and programmed cell death. A profound example of cell homeostasis, controlled throughout life, is the complex process of blood cell development, yet little is understood about the intracellular mechanisms that regulate blood cell growth arrest and programmed cell death. In this work, using transforming growth factor beta 1 (TGF beta 1)-treated M1 myeloid leukemia cells and genetically engineered M1 cell variants, the regulation of growth arrest and apoptosis was dissected. Blocking of early expression of MyD118, a novel differentiation primary response gene also shown to be a primary response gene induced by TGF beta 1, delayed TGF beta 1-induced apoptosis, demonstrating that MyD118 is a positive modulator of TGF beta 1-mediated cell death. Elevated expression of bcl-2 blocked the TGF beta 1-induced apoptotic pathway but not growth arrest induced by TGF beta 1. Deregulated expression of either c-myc or c-myb inhibited growth arrest and accelerated apoptosis, demonstrating for the first time that c-myb plays a role in regulating apoptosis. In all cases, the apoptotic response was correlated with the level of MyD118 expression. Taken together, these findings demonstrate that the primary response gene MyD118 and the c-myc, c-myb, and bcl-2 proto-oncogenes interact to modulate growth arrest and apoptosis of myeloid cells.

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.

Interleukin-6 and Leukemia Inhibitory Factor Induction of JunB Is Regulated by Distinct Cell Type-specific Cis-acting Elements

Interleukin (IL)-6 plays an important role in a wide range of biological activities, including differentiation of murine M1 myeloid leukemic cells into mature macrophages. At the onset of M1 differentiation, a set of myeloid differentiation primary response (MyD) genes are induced, including the proto-oncogene for JunB. In order to examine the molecular nature of the mechanisms by which IL-6 activates the immediate early expression of MyD genes, JunB was used as a paradigm. A novel IL-6 response element, −65/−52 IL-6RE, to which a 100-kDa protein complex is bound, has been identified on the JunB promoter. Leukemia inhibitory factor (LIF)-induced activation of JunB in M1 cells was also mediated via the −65/−52 IL-6RE. The STAT3 and CRE-like binding sites of the JunB promoter, identified as IL-6-responsive elements in HepG2 liver cells were found, however, to play no role in JunB inducibility by IL-6 in M1 myeloid cells. Conversely, the −65/−52 IL-6RE is shown not to be necessary for JunB inducibility by IL-6 or LIF in liver cells. It appears, therefore, that immediate early activation of JunB is regulated differently in M1 myeloid cells than in HepG2 liver cells. This indicates that distinct cis-acting control elements participate in cell type-specific induction of JunB by members of the IL-6 cytokine superfamily.