Murine Myeloid Leukemia Cell Line Research Articles

MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells

MLL-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce mixed lineage leukemia (MLL) through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II (Pol II) in HEL, a human cell line without MLL1 arrangement (MLLr). MLL1 and AF9 only co-regulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia (AML) cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely-believed elongation.

Alternative Macrophage Programming After Clot Phagocytosis

Abstract Repair program(s) in "alternatively activated" macrophages can help with short-term wound responses after sterile damage events. Clot resolution by fibrinolysis and phagocytosis by tissue macrophages can activate anti-inflammatory and pro-fibrotic signals like Transforming Growth Factor- β. It has been hypothesized by the late Andrew Tager and Rachel Chambers that some forms of human pulmonary fibrosis may involve chronic alveolar damage, leakage of blood components, clots, and subsequent pro-fibrotic events that then fail to remodel back to normal alveolar structure. We found evidence of chronic bleeds and hemosiderin-positive macrophages in the lungs of Platelet Endothelial Cell Adhesion Molecule-1 deficient mice that developed a progressive and fatal pulmonary fibrosis. We are testing this hypothesis in vitro using murine broncho-alveolar macrophages exposed to aged clots. Interestingly, fresh blood and clots were not efficiently phagocytosed by the murine myeloid leukemia cell line RAW264 nor by broncho-alveolar macrophages. Clots needed to be aged for two days first. Ingestion also causes the macrophages to become fluorescent, which is a useful way to assess phagocytosis. We are now developing mRNA sequencing protocols to assess the gene programs of macrophages over time.

Sub-clonal analysis of the murine C1498 acute myeloid leukaemia cell line reveals genomic and immunogenic diversity

BackgroundIn acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. MethodologyThe murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and – competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). ResultsGenomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. ConclusionWe showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML.

Abstract 3216: Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line

Abstract In acute myeloid leukemia (AML), clonal heterogeneity at diagnosis and the presence of minor sub-clones were shown to be responsible for minimal residual disease and relapses in patients. Several studies have proposed that their persistence could be due to their dormant state (i.e. quiescence and/or control of their growth by immune cells). As contribution of immunity to clonal persistence could not be evaluated in xenografts models, we assessed for the clonal diversity of a murine AML cell line. Genomic analyses revealed heterogeneity among the parental cell line and its derived clones. Injected individually into immune-deficient mice, all clones induced AML with different kinetics. However, when administered into immune-competent animals, some clones triggered AML with no mice survival, whereas others elicited reduced lethality rates. The leukemia-surviving mice presented an efficient anti-tumoral CTL activity and reduced lymphopenia. Investigating for residual leukemic cells in the organs of these mice, elimination rather than persistence was observed. Thus, we showed that genetic heterogeneity among the clones led to differential immunogenicity resulting either in their immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients. Citation Format: Carine Brinster, Virginie DRISS, Bruno Quesnel, Martin Figeac, Frédéric Leprêtre, Céline Villenet, Isabelle Briche. Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3216. doi:10.1158/1538-7445.AM2015-3216

Transformation by Mutant IDH and (R)-2HG Is Reversible.

Abstract 2413Somatic mutations in IDH1 and IDH2 are common in normal karyotype AML as well as in other clonal myeloid disorders and several solid tumors. Mutant IDH overproduces the R-enantiomer of 2-hydroxyglutarate, (R)-2HG (Dang, et al Nature 462: 739, 2009), which is hypothesized to alter the epigenetic landscape of cancer cells by inhibiting the activity of α-ketoglutarate-dependent enzymes, including the TET family of 5-methylcytosine hydroxylases and the jumonji-domain-containing family of histone demethylases. There is considerable interest in developing inhibitors of mutant IDH in the hope that, by decreasing (R)-2HG production in cancer cells, their epigenetic regulation can be restored. However, there is, to date, no evidence that transformation by mutant IDH is reversible or that inhibiting production of (R)-2HG has any effect on cancers harboring IDH mutations. Herein we report that in two different myeloid transformation assays, transformation by (R)-2HG and mutant IDH1 is reversible by removal of (R)-2HG.We previously reported that stable expression of a tumor-derived mutant IDH1 (IDH1R132H) induces growth factor independence and blocks EPO-induced differentiation in the human TF-1 erythroleukemia cell line, and that treatment of TF-1 cells with a cell-permeable form of (R)-2HG, TFMB-(R)-2HG, is sufficient to recapitulate this phenotype (Late Breaking Abstract #LBA-4, ASH 2011). We have extended these studies and found that transformation by TFMB-(R)-2HG is reversible and that this reversibility is influenced by the duration and intensity (dose) of (R)-2HG exposure. We developed a second myeloid transformation assay using a murine myeloid leukemia cell line that is transformed by expression of a HoxB8-ER fusion protein when cultured in the presence of estrogen. Upon estrogen withdrawal, the cells undergo monocytic differentiation and apoptosis. We expressed wild-type IDH1 or IDH1R132H in the cells and found that cells expressing wild-type IDH1 differentiate normally, but cells expressing IDH1R132H do not upregulate monocytic markers CD11b/Mac1 and Gr1 upon estrogen withdrawal. Furthermore, treatment of the IDH1R132H-expressing cells with an inhibitor of mutant IDH1 restores their ability to undergo monocytic differentiation upon estrogen withdrawal. Our findings suggest that continued exposure to (R)-2HG is required to maintain the cellular changes induced by mutant IDH, and further suggest that targeting (R)-2HG production may have therapeutic efficacy in the treatment of cancers harboring IDH mutations. Disclosures:No relevant conflicts of interest to declare.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Gene therapy for Fabry disease, a deficiency in alpha-galactosidase A (alpha-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as alpha-gal A can provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Ex vivoCulture with Interleukin (IL)-12 Improves CD8+ T-Cell Adoptive Immunotherapy for Murine Leukemia Independent of IL-18 or IFN-γ but Requires Perforin

In animal models and clinical trials, adoptive transfer of activated, antigen-specific CD8(+) T cells mediates tumor regression in a cell dose-dependent manner. The cytokine interleukin (IL)-12 promotes CD8(+) T-cell cytotoxicity and, with IL-18, synergistically up-regulates IFN-gamma release. We have shown that culturing CD8(+) T cells ex vivo with IL-12 and IL-18 enhanced antitumor responses in vivo and in vitro using a model of C1498/ovalbumin, a murine acute myeloid leukemia cell line expressing the antigen ovalbumin. Activated ovalbumin-specific CD8(+) T cells cultured with IL-12, IL-18, both, or neither were assayed for antigen-specific cytokine production and cytolytic activity and adoptively transferred to C57BL/6 mice with established tumors. Maximal IFN-gamma release occurred after T-cell culture with IL-12 and IL-18. Tumor-specific in vitro cytotoxicity was enhanced by IL-12, unaffected by addition of IL-18, and abrogated in perforin-deficient T cells irrespective of cytokine exposure. T cells cultured with IL-12 more effectively eliminated tumors, and addition of IL-18 did not further augment responses. IFN-gamma-deficient CD8(+) T cells showed effective antitumor activity that was enhanced by IL-12 with or without IL-18. Perforin-deficient CD8(+) T cells were poor mediators of antitumor activity, though, and showed no improvement after culture with IL-12 and/or IL-18. Thus, ex vivo culture with IL-12 was sufficient to augment antigen-specific in vitro cytotoxicity and antitumor activity in vivo in an IFN-gamma-independent but perforin-dependent manner. Ex vivo culture with IL-12 may improve CD8(+) T-cell immunotherapy of cancer in the absence of donor cell-derived IFN-gamma via perforin-mediated cytolysis.

G-CSF Induces Stabilization of ETS Protein Fli-1 during Myeloid Cell Development

Granulocyte colony-stimulating factor (G-CSF) is a growth factor that regulates the production and function of neutrophils. G-CSF has been used to treat neutropenia in neonates, pediatric cancer patients, and patients undergoing stem cell transplantation. The regulation of transcription factors mediating G-CSF activity has not been well characterized. The goal of this study was to examine the regulation of the ETS binding protein, Friend leukemia integration site 1 (Fli-1), in myeloid cells treated with G-CSF. Fli-1 has oncogenic properties in humans and mice, and plays a role in vascular and hematopoietic cell development. We previously reported that Fli-1 and the serum response factor bind at adjacent sites within the serum response element-1 of the early growth response gene-1 promoter in the murine myeloid leukemic cell line, NFS60. We also identified that Fli-1 DNA binding increased in G-CSF-treated cells compared with untreated cells. To determine whether the change in binding activity is due to increased Fli-1 transcription or protein stability, we examined endogenous Fli-1 expression in G-CSF-treated or -untreated NFS60 cells. Our results demonstrated that levels of Fli-1 protein, but not RNA, were higher in extracts from cells treated with G-CSF. The increase in Fli-1 protein was also dependent on protein synthesis. Finally, we showed that the half-life of Fli-1 is prolonged in G-CSF-treated cells compared with control-treated cells. These results suggest that G-CSF induces stabilization of Fli-1 protein in myeloid cells, thus proposing a novel mechanism by which hematopoietic growth factors regulate transcription factors.

Woodchuck interleukin-6 gene: structure, characterization, and biologic activity

Woodchuck is an important animal model for studying human hepatitis B virus (HBV) infection. Within the cytokine network, interleukin-6 (IL-6) plays an important role in immune responses that may lead to viral clearance. To further understand woodchuck IL-6 biology, we cloned and characterized the IL-6 gene from white blood cells. The complete woodchuck IL-6 gene is about 7 kb and consists of five exons and four introns. The IL-6 gene organization of the woodchuck is similar to those of the human, rat, and mouse. Also several elements are highly conserved in the 300 bp promoter region of the IL-6 gene, including a nuclear factor kappa B (NF-κB) binding site. The woodchuck IL-6 gene encodes a polypeptide of 207 amino acids in a precursor form and 189 amino acids in the mature form. The expressed protein was 23 kDa according to SDS–PAGE. To demonstrate biologic activity, we expressed woodchuck IL-6 and showed that the purified recombinant protein induced terminal differentiation, as reflected by upregulation of Fcγ receptor expression, and substantially inhibited proliferation of M1 cells, a murine myeloid leukemia cell line. The inhibitory effect of woodchuck IL-6 on M1 cells was blocked by an anti-gp130 monoclonal antibody, suggesting that woodchuck IL-6 activity is specifically mediated by signaling through the IL-6 receptor complex. Cloning of the woodchuck IL-6 gene and demonstrating biologic activity of the gene product will facilitate studies of human hepatitis B virus using the woodchuck model.

Functional evidence from microcell-mediated chromosome transfer of myeloid leukemia suppressor genes on human chromosomes 7 and 11.

The long arm of human chromosome 7 between 7q22 and 7q36 has been identified as a region harboring one or more tumor-suppressor genes (TSGs) inactivated in acute myeloid leukemia (AML). Additional TSGs mapping to other chromosomes may well be involved in the etiology of this disease. For example, experiments using a mouse model system have indicated the possible presence of an AML TSG at 11p11-12. Microcell-mediated chromosome transfer (MMCT) has been used to introduce human chromosomes 7 and 11 into a murine myeloid leukemia cell line. A proportion of MMCT hybrid clones containing either whole chromosome 7 or fragments of chromosome 11 showed a significant delay in leukemogenic onset when injected into syngeneic mice. Screening of hybrid clones did not associate any human microsatellite markers with decreased leukemogenic potential in vivo. However, preliminary evidence was obtained of allelic loss at chromosomal regions homologous with human 7q22 in murine F1 hybrid AMLs. Our data provide functional evidence of AML-associated TSGs localized to human chromosomes 7 and 11 in support of previously published studies on cytogenetic and allelic losses associated with AML development.

Interleukin-12 (IL-12) gene therapy of leukemia: immune and anti-leukemic effects of IL-12-transduced hematopoietic progenitor cells.

Recombinant interleukin-12 (rlL-12) is a potent immunomodulatory cytokine that has been shown to exert strong antitumoral and antimetastatic activity against several mouse tumors grown as solid lesions. The therapeutic efficacy of rIL-12 against hematological tumors and the transfer of IL-12 genes into hematopoietic progenitor cells to deliver IL-12 to the bone marrow (BM) to treat residual leukemia has not been studied adequately. We have investigated the retroviral-mediated transduction of hematopoietic progenitor cells with IL-12 genes and the in vivo anti-leukemic activity of transduced cells against the murine myeloid leukemia cell line 32Dp210. We were able to efficiently transduce the IL-3-dependent 32Dc13 myeloid progenitor cell line and primary hematopoietic progenitor cells using an MFG-based polycistronic retroviral vector containing the cDNAs of p35 and p40 murine IL-12 genes. 32Dc13 myeloid progenitor cells expressing IL-12 genes (32DIL-12 cells) have stably secreted biologically active murine IL-12 for >9 months. Mice transplanted with 32DIL-12 cells transiently express the transgene in the BM and spleen, which is associated with a rapid elevation of interferon-gamma (IFN-gamma) in the circulation and with secretion of IFN-gamma by spleen cells in vitro. In addition, spleen and BM cells of mice injected with 32DIL-12 cells readily acquire the capacity to lyse natural killer cell-sensitive YAC-1 target cells and 32Dp210 myeloid leukemia cells. Furthermore, whereas mice challenged with leukemia cells suffered 100% mortality within 14 days, approximately 40% of mice coinjected with 32Dp210 leukemia cells and 32DIL-12 progenitor cells exhibited long-term, leukemia-free survival (>60 days). This study demonstrates that IL-12 can be stably expressed in hematopoietic cells; in addition, when transplanted, transduced cells induce IFN-gamma production and activation of natural killer cells, both of which may be involved in inhibiting the progression of leukemia in vivo.

Combination of Granulocyte Colony-Stimulating Factor and Low-Dose Cytosine Arabinoside Further Enhances Myeloid Differentiation in Leukemia Cells in Vitro

We examined the differentiation-inducing effect on freshly isolated myeloid leukemia cells in liquid suspension culture by combined treatment with granulocyte colony-stimulating factor (G-CSF) plus low-dose cytosine arabinoside (Ara-C; 5-10 ng/ml) in 25 patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in leukemic transformation. Culture with G-CSF alone showed leukemic cell growth stimulation in 15 out of the 25 cases (60%) and induction of cell differentiation in 19 out of the 25 cases (76%), respectively. In 23 cases (92%), either growth stimulation and/or differentiation induction of leukemia cells was observed in response to G-CSF. This suggests that most myeloid leukemia cells are able to respond to G-CSF stimulation. In addition, treatment of cells with low-dose Ara-C alone resulted in the enhancement of myeloid specific antigens expression in 16 cases (64%). Treatment of leukemia cells with higher concentrations of Ara-C (over 50 ng/ml) alone resulted in cytocidal effects but not in the induction of differentiation. Furthermore, 15 cases (60%) showed pronounced myeloid differentiation of leukemia cells after combined exposure to G-CSF plus low-dose Ara-C as compared with cells treated with either G-CSF or Ara-C alone. The enhanced effect of differentiation induction by combining G-CSF plus low-dose Ara-C was also observed in a murine myeloid leukemia cell line WEHI-3B in vitro. These data suggest that treatment with G-CSF plus low-dose Ara-C is capable of inducing differentiation of leukemic cells in vitro, and also appears to be useful for the differentiation-based therapy of patients with AML and MDS.

Analysis with flow cytometry of green fluorescent protein expression in leukemic cells.

The measurement of DNA content with propidium iodide (PI) in cells transfected with expression vectors encoding the green fluorescent protein (GFP) is a useful tool in studying a variety of biological functions of proteins within cells. The purpose of this study was to determine conditions of formaldehyde fixation that permit intracellular GFP fluorescence and adequate DNA histograms to be generated following transient transfection of cells with a GFP-encoding plasmid. Cell cycle analysis was also performed in GFP-positive cells. The murine myeloid leukemic cell line, 32Dcl3, was used as the model system. Cells were transfected with a GFP-encoding plasmid (pEGFPC1). Following fixation in different formaldehyde concentrations and permeabilization with 70% ethanol, cells were stained with PI and analyzed by flow cytometry for GFP fluorescence and DNA content. Transfected cells were also analyzed for GFP fluorescence and DNA content following release from nocodazole block. Fixing cells in 0.51-1.75% formaldehyde concentrations prior to ethanol permeabilization resulted in 14-19% of transfected cells being GFP-positive, with acceptable coefficients of variation on the G(1) peak of DNA histograms. Analysis of cells synchronized to and released from the G(2)-M phase by nocodazole suggested that GFP-positive cells, when compared to GFP-negative cells, did not appear to progress out of G(2)-M following release from nocodazole block. Simultaneous detection of GFP fluorescence and DNA content by PI staining is possible following transient transfection of cells with a single expression vector encoding GFP. Our results demonstrate that GFP expression can be detected, using flow cytometry to perform cell cycle analysis in murine leukemic cells.

Molecular cloning and functional analysis of cDNA encoding a rat leukemia inhibitory factor: towards generation of pluripotent rat embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo. Maintenance of the stem-cell phenotype of ES cells in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) such as leukemia inhibitory factor (LIF). Here we report the cloning of complete rat LIF cDNA and its nucleotide sequence so as to facilitate studies of rat ES cell technologies on tumor biology. The nucleotide sequence of the rat LIF cDNA indicated that the rat LIF has 91% amino acid sequence identity with murine LIF. The cloned rat LIF cDNA has a putative biological activity as a differentiation-inducing factor on the murine myeloid leukemia cell line M1 cells. Culture supernatant of the rat LIF cDNA-transduced rat fibroblast cell line could maintain the stem-cell phenotype of rat ES cells which showed alkaline phosphatase activity, and this effect was much stronger than that by murine LIF. The availability of rat LIF which shows DIA will assist the in vitro analysis of rat ES cells, and culture of these cells is a route for the generation of gene targeting in rat.

Forced GATA-1 Expression in the Murine Myeloid Cell Line M1: Induction of c-Mpl Expression and Megakaryocytic/Erythroid Differentiation

AbstractThe “zinc-finger” transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and β-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation.

Forced GATA-1 Expression in the Murine Myeloid Cell Line M1: Induction of c-Mpl Expression and Megakaryocytic/Erythroid Differentiation

The "zinc-finger" transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and beta-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation.

Differential involvement of caspases in apoptosis of myeloid leukemic cells induced by chemotherapy versus growth factor withdrawal

To assess the potential involvement of the caspase family in the IL-3-dependent murine myeloid leukemic cell line 32D, we studied the effect of bcl-2, crmA and three synthetic caspase inhibitors on apoptosis induced by chemotherapy or IL-3 withdrawal. Apoptosis induced by IL-3 deprivation or by ActD appears to be mediated by a crmA-insensitive pathway. Cell death by IL-3 withdrawal is inhibited by the caspase-inhibitor ZVAD-fmk, but not DEVD-fmk or YVAD-cmk. In contrast, DEVD-fmk as well as ZVAD-fmk protect 32D cells from ActD-induced apoptosis. These results indicate that different caspases are involved in apoptosis induced by growth factor withdrawal and by chemotherapy.

A Possible Correlation Between Growth-Factor Sensitivity and Response to Mitoxantrone Treatment in a Murine Myeloid Leukaemia (SA7HD) Cell Line

SA7 murine myeloid leukaemia cells usually respond to stimulation in vitro by WEHI-conditioned medium by displaying increased dose-dependent proliferation. However, at recurrence following in vivo treatment of the leukaemia with mitoxantrone, the leukaemia cells developed significant insensitivity (p - 0.04) to stimulation by WEHI-conditioned medium. This altered growth-factor sensitivity was detected when two different assays were used. The recurrent leukaemic cells were morphologically indistinguishable from untreated leukaemic cells, but in normal mice they regained sensitivity to growth factors after a single transplant. The recurrent leukaemic cells were significantly resistant to some concentrations of mitoxantrone in vitro (p = 0.012). The magnitude of this resistance was mainly a function of the dose of mitoxantrone used in the initial treatment of the leukaemia. These data suggest an association between growth-factor sensitivity and response to mitoxantrone treatment including the development of resistance in the SA7HD murine myeloid leukaemia cell line.

Leukemia Inhibitory Factor Binds to Human Breast Cancer Cells and Stimulates Their Proliferation

Leukemia inhibitory factor (LIF) is a cytokine that was originally described as a differentiation factor of a murine myeloid leukemia cell line and subsequently found to be an important mediator of embryonic development. Although extensively studied in the hematopoietic system, its effects on solid tumors are generally unknown. In the present study we investigated the role of LIF in human breast cancer cells. Using the reverse transcriptase-polymerase chain reaction, we found that the human breast carcinoma MCF-7 cell line expressed the message for both LIF receptor and its signal-transducing protein gp130, suggesting that these receptors might be biologically active. Binding studies with radiolabeled LIF demonstrated that MCF-7 cells interacted with this cytokine, and the ligand binding was specific and time, dose, and temperature dependent. In addition, a Scatchard analysis of the data revealed a single class of high-affinity (Kd 0.27 nM) receptors with a density of approximately 430 sites per cell. MCF-7 cells exposed to LIF internalized and degraded the ligand. LIF stimulated the growth of MCF-7 as well as other estrogen-dependent and independent breast cancer cell lines, but the effect on normal breast epithelial lines was less significant. Likewise, it stimulated colony formation by breast cancer cells obtained from five different breast cancer patients in a dose-dependent fashion. These results overall suggest that human breast tumor cells express functional LIF receptors that play a role in breast cancer cell proliferation.