Differentiation Of Murine Erythroleukemia Cells Research Articles

4-Hydroxynonenal-Induced MEL Cell Differentiation Involves PKC Activity Translocation

4-Hydroxynonenal (HNE) is a highly reactive aldehyde, produced by cellular lipid peroxidation, able to inhibit proliferation and to induce differentiation in MEL cells at concentrations similar to those detected in several normal tissues. Inducer-mediated differentiation of murine erythroleukemia (MEL) cells is a multiple step process characterized by modulation of several genes as well as by a transient increase in the amount of membrane-associated protein kinase C (PKC) activity. Here we demonstrate that a rapid translocation of PKC activity from cytosol to the membranes occurs during the differentiation induced by HNE. When PKC is completely translocated by phorbol-12-myristate-13-acetate (TPA), the degree of HNE-induced MEL cells differentiation is highly decreased. However, if TPA is washed out from the culture medium before the exposition to the aldehyde, HNE gradually resumes its differentiative ability. The incubation of cells with a selective inhibitor of PKC activity, bisindolylmaleimide GF 109203X, partially prevents the HNE-induced differentiation in MEL cells. In conclusion, our results demonstrate that HNE-induced MEL cell differentiation is preceded by a rapid translocation of PKC activity, and that the inhibition of this phenomenon prevents the onset of terminal differentiation.

Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C β 1

Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type β 1 is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCβ 1 under different stimuli implicate PLCβ 1 as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCβ 1 in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35–36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCβ 1 cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCβ 1. By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCβ 1 on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35–36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCβ 1 could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCβ 1, which has been shown to behave as a key signalling intermediate in the control of the cell cycle.

MSin3A regulates murine erythroleukemia cell differentiation through association with the TAL1 (or SCL) transcription factor.

Activation of the TAL1 (or SCL) gene is the most frequent gain-of-function mutation in T-cell acute lymphoblastic leukemia (T-ALL). TAL1 belongs to the basic helix-loop-helix (HLH) family of transcription factors that bind as heterodimers with the E2A and HEB/HTF4 gene products to a nucleotide sequence motif termed the E-box. Reported to act both as an activator and as a repressor of transcription, the mechanisms underlying TAL1-regulated gene expression are poorly understood. We report here that the corepressor mSin3A is associated with TAL1 in murine erythroleukemia (MEL) and human T-ALL cells. Interaction mapping showed that the basic-HLH domain of TAL1 was both necessary and sufficient for TAL1-mSin3A interaction. TAL1 was found, in addition, to interact with the histone deacetylase HDAC1 in vitro and in vivo, and a specific histone deacetylase inhibitor, trichostatin A (TSA), relieved TAL1-mediated repression of an E-box-containing promoter and a GAL4 reporter linked to a thymidine kinase minimal promoter. Further, TAL1 association with mSin3A and HDAC1 declined during dimethyl sulfoxide-induced differentiation of MEL cells in parallel with a decrease in mSin3A abundance. Finally, TSA had a synergistic effect with enforced TAL1 expression in stimulating MEL cells to differentiate, while constitutive expression of mSin3A inhibited MEL cell differentiation. These results demonstrate that a corepressor complex containing mSin3A and HDAC1 interacts with TAL1 and restricts its function in erythroid differentiation. This also has implications for this transcription factor's actions in leukemogenesis.

Characterization of a mouse liminin receptor gene homologous to the human blood group Lutheran gene.

The human Lutheran (Lu) blood group antigens are carried by two glycoproteins (gps) that belong to the immunoglobulin (Ig) superfamily. These gps represent adhesion molecules that function as the unique erythroid receptors for laminin. We report here the cloning and functional expression of the orthologous mouse Lu mRNA as well as the genomic organization of the mouse Lu gene. The deduced human and mouse Lu gps share 72.5% identity and similar organization of the Ig-like domains. As in the human, the mouse Lu gene is organized in 15 exons. The proximal promoter showed consensus CACC-binding sites whereas the distal promoter exhibits a GATA-1-binding site and multiple E boxes. Like the human gene, the mouse Lu gene is also widely expressed among tissues but is transcribed as a unique 2.4-kb mRNA species. Expression of the mouse Lu mRNA is upregulated upon dimethyl sulfoxide-induced erythroid differentiation of murine erythroleukemia cells (MEL). During mouse embryonic development, the Lu transcript is detected as early as day 7 of gestation. Analysis of transfected human erythroleukemia K562 cells indicated that the adhesive properties of the Lu gps to laminin are conserved between human and mouse.

Transcriptional elongation of c-myb is regulated by NF-kappaB (p50/RelB).

High levels of c-myb expression are necessary for the proliferation of hematopoietic precursor cells whereas down-regulation of c-myb is required for terminal differentiation; this down-regulation occurs through a conditional block to transcriptional elongation in intron I. We previously observed that cAMP analogs prevented the late down-regulation of c-myb during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells and blocked differentiation; this correlated with the induction of NF-kappaB (p50/RelB) complexes which were shown to bind to NF-kappaB recognition sites flanking the transcriptional pause site of c-myb. We now selected stably-transfected MEL cells which overexpressed p50, RelB or both at levels similar to those induced by cAMP to determine whether these NF-kappaB proteins regulate c-myb expression in intact cells. We demonstrate that transcriptionally active NF-kappaB (p50/RelB) complexes, but not p50 or RelB alone, prevented the early and late down-regulation of c-myb mRNA and increased c-myb transcriptional elongation in HMBA-induced MEL cells. The increase in c-myb expression was sufficient to block erythroid differentiation and allow continuous proliferation of cells in the presence of HMBA. Steady-state c-myb mRNA levels in untreated cells were not affected by overexpression of NF-kappaB, suggesting that p50/RelB specifically modulated the efficiency of transcriptional attenuation during MEL cell differentiation.

Induction of Erythrocyte Protein 4.2 Gene Expression during Differentiation of Murine Erythroleukemia Cells

Protein 4.2 (P4.2) is an important component in the erythrocyte membrane skeletal network that regulates the stability and flexibility of erythrocytes. Recently, we provided the evidence for specific P4.2 expression in erythroid cells during development (L. Zhu et al., 1998, Blood 91, 695–705). Using dimethyl sulfoxide (DMSO)-induced differentiation of murine erythroleukemia (MEL) cells as a model, transcription of the P4.2 gene was found to be induced during erythroid differentiation. To examine the mechanism for this induction, we isolated the mouse P4.2 genomic DNA containing the 5′ flanking sequence and defined the location of the P4.2 promoter. Transcription of the mouse P4.2 gene initiates at multiple sites, with the major initiation site mapped at 174 nucleotides upstream of the ATG start codon. The mouse P4.2 promoter is TATA-less and contains multiple potential binding sites for erythroid transcription factors GATA-1, NF-E2, EKLF, and tal-1/SCL. Transient transfection experiments demonstrated that a 1.7-kb mouse P4.2 promoter fused with the luciferase coding regions was induced in DMSO-treated MEL cells. Deletion analysis showed that a 259-bp P4.2 promoter DNA (nucleotide position −88 to +171 relative to the major transcription initiation site designated +1), containing a GATA-binding site at position −29 to −24, could still respond to the induction in differentiated MEL cells. Importantly, mutations in the −29/−24 GATA motif rendered the promoter unresponsive to DMSO induction. Electrophoretic mobility shift assay revealed that GATA-1 could bind to the −29/−24 GATA motif and this was confirmed by the observation that the nuclear protein bound to the motif was supershifted by an anti-GATA-1 monoclonal antibody. Taken together, these results suggest that the erythroid transcription factor GATA-1 plays an important role in the induction of P4.2 gene expression during erythroid cell differentiation.

D,L-α-DIFLUOROMETHYLORNITHINE, AN IRREVERSIBLE INHIBITOR OF ORNITHINE DECARBOXYLASE, INDUCES DIFFERENTIATION IN MEL CELLS

In the present study, the effect of D,L-α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), on Friend's murine erythroleukemia (MEL) cell differentiation is investigated. DFMO was able to induce differentiation of MEL cells in culture as determined by haemoglobin (Hb) content and percentage of cells synthesizing Hb detected by benzidine staining. DFMO at a concentration of 2m m resulted in about 70% benzidine-positive cells on the fifth day. There was a time-dependent increase in the percentage of benzidine-positive cells starting from day three. However, only a 24h presence of DFMO in the medium was required to induce differentiation suggesting that DFMO switches on a pathway during this period leading to terminal differentiation of MEL cells. DFMO induced differentiation of MEL cells was sensitive to dexamethasone and 5-bromo-2′-deoxyuridine.

Inositides in the nucleus: further developments on phospholipase Cβ1 signalling during erythroid differentiation and IGF-I induced mitogenesis

Inositol lipids originally shown to be metabolized in the cytosol have been detected also in the nucleus, where they are both synthesized and hydrolyzed. In the case of erythroid differentiation of murine erythroleukemia cells (Friend cells) it has been previously shown that PLC beta 1, which is the major nuclear PLC, undergoes down-regulation upon treatment with DMSO or tiazofurin which act as differentiative agents. On the contrary, i.e., during IGF-I induced mitogenesis, it has been shown that PLC beta 1 is rapidly activated and this event is essential for the onset of DNA synthesis. Even though its key role in cell growth has been shown, both the mechanism by which nuclear PLC beta 1 is activated and the direct relationship with erythroid differentiation are still unknown. We have addressed the question if PLC beta 1 expression and activity in the nucleus are directly related or not to the establishment of the differentiated state and we have checked the two main ways of activation, i.e., via G-protein or via phosphorylation, in order to establish whether nuclear PLC beta 1 is regulated the same way as the one at the plasma membrane or not. The data reported here show that nuclear PLC beta 1 is responsible for a continuous recycling of Friend cells, acting as a negative regulator of differentiation and that its activation is dependent on the phosphorylation state.

The role of Ets family transcription factor PU.1 in hematopoietic cell differentiation, proliferation and apoptosis.

The PU.1 gene encodes an Ets family transcription factor which controls expression of many B cell- and macrophage-specific genes. Expression of the gene is critical for development of lymphoid and myeloid cell lineages, since PU.1-deficient mice exhibit defects in the development of these cell lineages. The PU.1 gene is identical to the Spi-1 gene isolated from common proviral integration sites in Friend virus-induced murine erythroleukemia (MEL), and deregulated expression of the gene is believed to be an essential step of the disease. We recently demonstrated that overexpression of PU.1 inhibits erythroid differentiation of MEL cells induced with the differentiating agent DMSO. We also noticed unexpectedly that overexpression of PU.1 together with DMSO induces marked growth arrest and apoptosis in MEL cells, supporting the notion that some oncogenes induce growth inhibition and apoptosis rather than cell proliferation and transformation under specific circumstances as shown with the c-myc gene. In this review, the role of PU.1 in hematopoietic cell differentiation, proliferation and apoptosis is described and the possible molecular mechanisms of PU.1-induced effects in MEL cells are discussed.

Cloning of the cDNA encoding phenylalanyl tRNA synthetase regulatory alpha-subunit-like protein whose expression is down-regulated during differentiation.

Hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA), induce differentiation of transformed cells. Differential display of RNA was used to identify genes whose expression is changed during SAHA-induced differentiation of murine erythroleukemia (MEL) cells. One such cDNA was identified whose mRNA level decreased by 50% after 8h of SAHA treatment as determined by Northern blot analysis. The full-length cDNA (1944bp in length) was cloned by sequencing of an EST clone and rapid amplification of 5' cDNA ends (5'-RACE). The predicted amino acid sequence is 589 amino acids and shares 45% identity with the yeast cytoplasmic phenylalanyl tRNA synthetase (PheRS) regulatory alpha-subunit. Human EST clones which share over 90% identity of predicted amino acid sequence with this cDNA map to chromosome 2 near the paired box homeotic gene 3 (PAX3) locus, a region syngenic to mouse chromosome 1. This is the first report of the cloning of the full-length cDNA for the murine PheRS regulatory alpha-subunit-like protein. The level of PheRS alpha-subunit-like mRNA is regulated during differentiation but not during cell cycle progression.

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells.

Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.

Cytodifferentiating Agents Affect the Replication of Herpes Simplex Virus Type 1 in the Absence of Functional VP16

The herpes simplex virus type 1 (HSV-1) mutantin1814 encodes an altered form of the virion protein VP16 that is unable to transactivate immediate-early (IE) transcription. As a consequence of the mutation,in1814 initiates productive replication inefficiently after infection of tissue culture cells. Previous studies showed that this defect could be overcome by the inclusion in the culture medium of hexamethylene bisacetamide (HMBA), a compound that promotes the differentiation of murine erythroleukemia cells (MELCs). The effects of additional agents known to induce differentiation of MELCs were investigated.N′-Methylnicotinamide, at concentrations optimal for the induction of MELCs, complemented the replication ofin1814 and stimulated IE gene expression. Suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid, which induce differentiation of MELCs at micromolar concentrations, did not complementin1814 but specifically blocked the action of HMBA. The histone deacetylase inhibitor trichostatin A, which also induces differentiation of MELCs, antagonized the effect of HMBA in a manner similar to that of suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid. The results demonstrate that the requirement for VP16 activity is dependent on the metabolic state of the host cell and that the pathways leading to complementation ofin1814 and differentiation of MELCs are overlapping but not identical.

Granulocyte-macrophage colony-stimulating factor induces the differentiation of murine erythroleukaemia cells into dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells (APC) within the immune system and antigen-pulsed DC can be used as an effective vaccine for active immunotherapy of cancer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the generation of DC. We previously showed that GM-CSF can induce murine erythroleukaemia cells (FBL-3) to differentiate into monocyte-like cells. To develop a new vaccinating method to stimulate the host immune response to leukaemia, we further investigate whether FBL-3 cells induced by GM-CSF can differentiate into DC in the present study. After being treated with GM-CSF, FBL-3 cells expressed high levels of 33D1 and NLDC-145, which are the specific markers of DC. The expression of MHC-II, B7-1, B7-2 and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated markedly; the typical morphology of DC were also observed by electron microscopy. Functionally, the GM-CSF-induced FBL-3 cells could apparently stimulate the proliferation of naive allogeneic and autologous T lymphocytes and induce the generation of specific CTL more efficiently than the wild-type FBL-3 cells. Mice immunized with GM-CSF-induced FBL-3 cells could resist the subsequent challenge with the wild-type FBL-3 cells. Collectively, these data indicate that GM-CSF differentiates murine erythroleukaemia cells into DC phenotypically, morphologically and functionally. FBL-3-derived DC can be used as a new type of vaccine. Our results may have important implications for the immunotherapy of leukaemia.

Direct association of YY-1 with c-Myc and the E-box binding protein in regulation of glycophorin gene expression.

We previously reported that YY-1, a versatile transcription factor, regulates expression of glycophorin gene by binding to its locus control region-like region (Gp-LCR) in combination with E-box binding protein during murine erythroleukemia (MEL) cell differentiation. In the present work, we demonstrated that YY-1 and c-Myc, a nuclear oncoprotein, were physically associated in vivo and that down regulation of c-Myc liberated free YY-1 from its complex, resulting in the functional binding of YY-1 to the Gp-LCR. We also showed that the E-box binding protein (EBP) which bound to E-box was physically associated with YY-1, facilitated binding of YY-1 to the neighboring site and their combinatorial binding may stimulate the GpLCR mediated enhancement of erythroid-specific transcription of glycophorin gene in MEL cells.

Increased Expression of Acetylcholinesterase T and R Transcripts during Hematopoietic Differentiation Is Accompanied by Parallel Elevations in the Levels of Their Respective Molecular Forms

Differentiation of hematopoietic cells is known to be accompanied by profound changes in acetylcholinesterase (AChE) enzyme activity, yet the basic mechanisms underlying this developmental regulation remain unknown. We initiated a series of experiments to examine the molecular mechanisms involved in regulating AChE expression during hematopoiesis. Differentiation of murine erythroleukemia (MEL) cells using dimethyl sulfoxide resulted in a 5- and 10-fold increase in intracellular and secreted AChE enzyme activity, respectively. Interestingly, these increases resulted from a preferential induction of the globular molecular form G1 and a slight increase in G4 instead of an increase in the levels of the G2 membrane-bound form, a molecular form expressed in mature erythrocytes. Concomitantly, expression of the two predominant AChE transcripts (R and T, for read-through and tail, respectively) in MEL cells was induced to a similar extent with differentiation. Nuclear run-on assays performed with nuclei isolated from induced versus uninduced MEL cells revealed that in contrast to the large increases seen in the transcription of the beta-globin gene, the transcriptional activity of the AChE gene remained largely unaffected after differentiation. Determination of the half-lives of the R and T transcripts demonstrated that they both exhibited an increase in stability in induced MEL cells. Taken together, results from these studies indicate that post-transcriptional regulatory mechanisms account for the increased expression of AChE in differentiated hematopoietic cells.

Dimethylsulfoxide-Induced Cell Death of Murine Erythroleukemia Cells Exposed to Ionising Radiation

The present investigation was aimed at studying the effects of dimethylsulfoxide (DMSO) in combination with high dose (15 and 60 Gy) ionising radiation on the growth and differentiation of murine erythroleukemia cells (MEL). The incubation with DMSO was performed for 96 h starting immediately after exposure to radiation and resulted only in a slight inhibition of cell growth and in a high increase in cell death with the induction of both necrosis and apoptosis. The enhancement of radiation cytotoxicity was directly related to dose, time in culture and degree of differentiation as demonstrated by the severe and multiple aberrations observed in light and electron microscopy. Of interest was the observation in induced cells of a marked rearrangement of the plasma membrane architecture as well as that of the nuclear envelope, with a massive translocation and/or decrease in the nuclear pore complexes.

Regulation of NF-E2 Activity in Erythroleukemia Cell Differentiation

The erythroid transcription factor NF-E2 is an obligate heterodimer composed of two different subunits (p45 and p18), each containing a basic region-leucine zipper DNA binding domain, and it plays a critical role in erythroid differentiation as an enhancer-binding protein for expression of the beta-globin gene. We show here that dimethyl sulfoxide treatment of wild-type murine erythroleukemia cells, but not a mutant clone of dimethyl sulfoxide-resistant cells, increases NF-E2 activity significantly, which involves both up-regulation of DNA binding and transactivation activities. Both activities were reduced markedly by treatment of cells with 2-aminopurine but not by genistein. Activation of the Ras-Raf-MAP kinase signaling cascade increased NF-E2 activity significantly, but this was suppressed when MafK was overexpressed. Domain analysis revealed an activation domain in the NH2-terminal region of p45 and a suppression domain in the basic region-leucine zipper of MafK. These findings indicate that induction of NF-E2 activity is essential for erythroid differentiation of murine erythroleukemia cells, and serine/threonine phosphorylation may be involved in this process. In addition, they also suggest that a MafK homodimer can suppress transcription, not only by competition for the DNA binding site, but also by direct inhibition of transcription. Hence, MafK may function as an active transcription repressor.

CAMP-induced NF-kappaB (p50/relB) binding to a c-myb intronic enhancer correlates with c-myb up-regulation and inhibition of erythroleukemia cell differentiation.

During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-kappaB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappaB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of c-myb. The cAMP-induced NF-kappaB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-kappaB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-kappaB. We conclude that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kappaB p50/RelB heterodimers.

Inhibition by 1α,25-Dihydroxyvitamin D3of Activin A-Induced Differentiation of Murine Erythroleukemic F5-5 Cells

1α,25-Dihydroxyvitamin D3(1α,25-(OH)2D3) and other vitamin D3(VD3) analogs enhanced the inhibitory effect of Activin A on murine erythroleukemia (MEL) cell proliferation and differentiation in a dose-dependent manner. 1α,25-(OH)2D3inhibited differentiation more potently than proliferation by one order of magnitude. The VD3analog study demonstrated either effect of VD3on MEL cells via vitamin D receptor (VDR), as evidenced from the close relationship with the reported affinities for VDR. The effects of 1α,25-(OH)2D3were preceded by the suppression of ornithine decarboxylase (ODC) activity, a rate-limiting enzyme in polyamine metabolism. Difluoromethylornithine (DFMO), an inhibitor of ODC, inhibited MEL cell proliferation, which was reversed by the simultaneous addition of putrescine, a product of ODC, but did not affect differentiation. 1α,25-(OH)2D3inhibited cell differentiation during the phenotype-expression stage as reflected by the inhibition of β-globin gene expression, while it inhibited proliferation in the commitment stage. Furthermore, it seems unlikely that the different effects of VD3on proliferation and differentiation may be a result of up-regulation of VDR or nongenomic action. In summary, it was suggested that 1α,25-(OH)2D3inhibited Activin A-induced MEL cell proliferation and differentiation by distinct mechanisms and inhibited the proliferation by inhibiting ODC activity. We demonstrated the presence of 1α,25-(OH)2D3action on leukemic cells at physiological concentration, which was distinct from the pharmacological effect of VD3reported thus far.

Function of the c-Myc antagonist Mad1 during a molecular switch from proliferation to differentiation.

Mad-Max heterodimers have been shown to antagonize Myc transforming activity by a mechanism requiring multiple protein-protein and protein-DNA interactions. However, the mechanism by which Mad functions in differentiation is unknown. Here, we present evidence that Mad functions by an active repression mechanism to antagonize the growth-promoting function(s) of Myc and bring about a transition from cellular proliferation to differentiation. We demonstrate that exogenously expressed c-Myc blocks inducer-mediated differentiation of murine erythroleukemia cells without disrupting the induction of endogenous Mad; rather, high levels of c-Myc prevent a heterocomplex switch from growth-promoting Myc-Max to growth-inhibitory Mad-Max. Cotransfection of a constitutive c-myc with a zinc-inducible mad1 results in clones expressing both genes, whereby a switch from proliferation to differentiation can be modulated. Whereas cells grown in N'N'-hexamethylene bisacetamide in the absence of zinc fail to differentiate, addition of zinc up-regulates Mad expression by severalfold and differentiation proceeds normally. Coimmunoprecipitation analysis reveals that Mad-Max complexes are in excess of Myc-Max in these cotransfectants. Moreover, we show that the Sin-binding, basic region, and leucine zipper motifs are required for Mad to function during a molecular switch from proliferation to differentiation.