Murine Erythroleukemia Cell Line Research Articles

Sunitinib selectively targets leukemogenic signaling of mutant SHP2 in juvenile myelomonocytic leukemia

Leukemogenic SHP2 mutations occur in 35% of patients with juvenile myelomonocytic leukemia (JMML), a hematopoietic malignancy with poor response to cytotoxic chemotherapy. Novel therapeutic strategies are urgently needed for patients with JMML. Previously, we established a novel cell model of JMML with HCD-57, a murine erythroleukemia cell line that depends on EPO for survival. SHP2-D61Y or -E76K drove the survival and proliferation of HCD-57 in absence of EPO. In this study, we identified sunitinib as a potent compound to inhibit SHP2-mutant cells by screening a kinase inhibitor library with our model. We used cell viability assay, colony formation assay, flow cytometry, immunoblotting, and a xenograft model to evaluate the effect of sunitinib against SHP2-mutant leukemia cells in vitro and in vivo. The treatment of sunitinib selectively induced apoptosis and cell cycle arrest in mutant SHP2-transformed HCD-57, but not parental cells. It also inhibited cell viability and colony formation of primary JMML cells with mutant SHP2, but not bone marrow mononuclear cells from healthy donors. Immunoblotting showed that the treatment of sunitinib blocked the aberrantly activated signals of mutant SHP2 with deceased phosphorylation levels of SHP2, ERK, and AKT. Furthermore, sunitinib effectively reduced tumor burdens of immune-deficient mice engrafted with mutant-SHP2 transformed HCD-57. Our data demonstrated that sunitinib selectively inhibited SHP2-mutant leukemia cells, which could serve as an effective therapeutic strategy for SHP2-mutant JMML in the future.

Effect of Nicotinamide, 1-Methylnicotinamide, and N’-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p<0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p<0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Differential gene expression analysis by RNA-seq reveals the importance of actin cytoskeletal proteins in erythroleukemia cells.

Development of drug resistance limits the effectiveness of anticancer treatments. Understanding the molecular mechanisms triggering this event in tumor cells may lead to improved therapeutic strategies. Here we used RNA-seq to compare the transcriptomes of a murine erythroleukemia cell line (MEL) and a derived cell line with induced resistance to differentiation (MEL-R). RNA-seq analysis identified a total of 596 genes (Benjamini–Hochberg adjusted p-value < 0.05) that were differentially expressed by more than two-fold, of which 81.5% (486/596) of genes were up-regulated in MEL cells and 110 up-regulated in MEL-R cells. These observations revealed that for some genes the relative expression of mRNA amount in the MEL cell line has decreased as the cells acquired the resistant phenotype. Clustering analysis of a group of genes showing the highest differential expression allowed identification of a sub-group among genes up-regulated in MEL cells. These genes are related to the organization of the actin cytoskeleton network. Moreover, the majority of these genes are preferentially expressed in the hematopoietic lineage and at least three of them, Was (Wiskott Aldrich syndrome), Btk (Bruton’s tyrosine kinase) and Rac2, when mutated in humans, give rise to severe hematopoietic deficiencies. Among the group of genes that were up-regulated in MEL-R cells, 16% of genes code for histone proteins, both canonical and variants. A potential implication of these results on the blockade of differentiation in resistant cells is discussed.

Targeting KLF1 for the Treatment of Sickle Cell Disease Using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein in the fetus during the last months of embryonic development and the first few months of life after birth. HbF has a slightly greater oxygen binding affinity than adult hemoglobin (HbA, α2β2) and inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD.Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of KLF1, we can promote production of HbF through the upregulation of γ-globin expression.Since rodents don’t express γ-globin, we have employed both human and engineered mouse cell lines to demonstrate upregulation of γ-globin mRNA expression in vitro. We used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse KLF1, and treated with antisense oligonucleotides (ASOs) targeting mouse KLF1. After 7 days of free uptake with the ASOs, we observed a 6-fold increase of human γ-globin mRNA expression after achieving 65% mRNA reduction of mouse KLF1 compared to the untreated control. We were also able to demonstrate significant upregulation of human γ-globin protein expression in these cells by western blot. We have shown similar results in a human erythroleukemia cell line, K562, using ASOs targeting human KLF1. K562 cells were electroporated with the KLF1 ASOs and 4 days later, we observed a 5-fold increase of human γ-globin mRNA expression after achieving 40% mRNA reduction of human KLF1 compared to the untreated control. These data indicate that targeting mouse or human KLF1 with ASO treatment can cause an increase in human γ-globin expression in vitrothat is necessary for the upregulation of fetal hemoglobin.We have also shown that we are able to target the bone marrow in both mice and rats through subcutaneous administration of our KLF1 ASOs. In wild type mice, at a dose of 100 mpk/wk for 4 weeks, we observed KLF1 target reduction of 88% and a β-globin reduction of 58% compared to the saline control in whole bone marrow. In Sprague-Dawley rats, at a dose of 50 mpk/wk for 4 weeks, we observed KLF1 target reduction of 83% and a β-globin reduction of 77% compared to the saline control in whole bone marrow. Therefore, we are able to achieve significant β-globin mRNA reduction in the bone marrow in both mice and rats after subcutaneous administration of KLF ASOs. These data indicate that reducing KLF1 with antisense oligonucleotides is a viable option for the treatment of sickle cell anemia. DisclosuresPeralta:Isis Pharmaceuticals, Inc.: Employment. Low:Isis Pharmaceuticals, Inc.: Employment. Booten:Isis Pharmaceuticals, Inc.: Employment. Zhou:Univeristy of Alabama at Birmingham: Employment. Kim:Isis Pharmaceuticals, Inc.: Employment. Freier:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Murray:Isis Pharmaceuticals, Inc.: Employment. Townes:University of Alabama at Birmingham: Employment. Hung:Isis Pharmaceuticals, Inc.: Employment.

Mechanism of action for the cytotoxic effects of the nitric oxide prodrug JS-K in murine erythroleukemia cells.

The nitric oxide (NO) prodrug JS-K, a promising anti-cancer agent, consists of a diazeniumdiolate group necessary for the release of NO as well as an arylating ring. In this study, we research the mechanism by which JS-K kills a murine erythroleukemia cell line and determine the roles of NO and arylation in the process. Our studies indicate that JS-K inhibits the PI 3-kinase/Akt and MAP kinase pathways. This correlates with the activation of the tumor suppressor FoxO3a and increased expression of various caspases, leading to apoptosis. The arylating capability of JS-K appears to be sufficient for inducing these biological effects. Overall, these data suggest that JS-K kills tumor cells by arylating and inactivating signaling molecules that block the activation of a tumor suppressor.

Targeting BCL11A and KLF1 For The Treatment Of Sickle Cell Disease and β-Thalassemia In Vitro using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis.B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression.To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures:Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

Abstract 2984: A novel role for survivin, a potential target for cancer drugs, in vesicle trafficking

Abstract Survivin is a 16.5 kD protein that is highly expressed in most cancer cells. Although it is also expressed in embryonic tissues and proliferating adult cells, it may be a promising target for cancer therapy if it has distinct functions in normal versus malignant cells. It is well established that survivin has an essential role in dividing cells as a member of the chromosome passenger complex (CPC). We recently showed that survivin is highly expressed in orthochromatic erythroblasts, cells that have exited the cell cycle and undergoing enucleation. Based on this observation, we predicted that survivin would play a novel role in maturation of these late erythroblasts. To discover the function of survivin in these post-mitotic cells, we first analyzed the localization of survivin and other members of the CPC by immunofluorescence. We discovered that survivin is expressed in cytoplasm, but did not colocalize with aurora B kinase or INCENP in primary enucleating erythroblasts. Next, we overexpressed survivin in the murine erythroleukemia (MEL) cell line, which undergoes terminal differentiation but does not enucleate, and quantified the effect on maturation by flow cytometry. In contrast to control MEL cells, which down-regulate survivin and fail to enucleate, nearly 15% of the survivin overexpressing cells showed nuclear extrusion. To obtain insight into this novel effect of survivin, we purified survivin protein complexes from MEL cell lysates and performed mass spectrometry. We found that EPS-15 (EGFR pathway substrate clone 15) and clathrin, two proteins that are involved in endocytic vesicle trafficking, interact with survivin. We confirmed these interactions by immunoprecipitation and immunofluorescence studies in primary human erythroblasts. Next, we individually knocked down survivin and EPS-15 in primary human erythroblast cultures and demonstrated that both proteins are required for optimal enucleation. Moreover, deletion of the survivin gene in fetal liver erythroblasts isolated from survivin floxed embryos, reduced the enucleation efficiency without affecting cell survival or differentiation. In order to determine how survivin and EPS15 contribute to enucleation, we performed light microscopic and ultrastructural examination of primary murine red cells undergoing terminal maturation. We observed that loss of survivin was associated with a decrease in the number of large cellular vacuoles within erythroblasts. Furthermore, by immunogold-EM, we found that survivin co-localized with EPS-15 within LAMP1-positive vacuoles in late erythroblasts. Finally, we observed that vacuolin-1, a small molecule that increases the number and size of vacuoles, rescued the enucleation defect of survivin-/- erythroblasts. Together, these results suggest that survivin plays an important and novel role in vesicle trafficking within maturing red blood cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2984. doi:10.1158/1538-7445.AM2011-2984

Studies on Heme Oxygenase 1 During Erythroid Differentiation

AbstractAbstract 4254Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures:No relevant conflicts of interest to declare.

Introducing undergraduate students to real‐time PCR

An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis, can be induced to progress further through the process by 72 h exposure to 1.8% DMSO. This gives a control cell sample and a DMSO-treated preparation. Students isolate RNA from both cell cultures, check its purity, yield and integrity by UV spectrophotometry and denaturing gel electrophoresis, then synthesized cDNA. The relative levels of three sequences: β globin, amino levulinate synthase, and carbonic anhydrase-1 are estimated by real-time PCR, using 18S rRNA as the reference sequence. The changes in gene expression are robust and reproducible, enabling students to experience a "cutting edge" research technique in an undergraduate lab setting. While the undergraduate student experience in practical classes with such sensitive techniques is often mixed, the changes in gene expression in this model are sufficiently great that students can gain the satisfaction of consistent results. In addition they gain experience at setting up checks and controls at stages throughout a multistep process and an appreciation of the difference between a reaction which has gone to completion with one that is measured as a rate. This experiment would also complement cell biology projects involving red cell development. It could also be extended to more thoroughly investigate the technique of real-time PCR.

Of mice and men: Human RNA polymerase III promoter U6 is more efficient than its murine homologue for shRNA expression from a lentiviral vector in both human and murine progenitor cells

RNA interference mediated by transcription of short hairpin RNAs (shRNAs) from lentiviral expression vectors has emerged as an efficient method to effectively and specifically silence gene expression in a vast variety of mammalian cells. shRNA expression is routinely driven by a RNA polymerase III promoter, most often by the U6 promoter. Here we demonstrate that U6 promoter activity-and consequently gene silencing success-differs significantly among species. We have modified pLeGO-G, an HIV-based third-generation lentivector, to express a 19nt shRNA sequence against the human transcription factor nuclear factor erythroid 2 or against its murine homologue, as well as an shRNA against murine JAK2, from either the human or the murine U6 promoter. Gene silencing efficiency was analyzed in a human erythroleukemic cell line, in primary human CD34(+) cells, as well as in a murine erythroleukemic cell line and in primary murine bone marrow. ShRNA expression from the human U6 promoter resulted in a fourfold increase in knockdown efficiency compared to expression from the murine U6 promoter in both human and murine cells. The U6 promoter constitutes an important determinant for efficient gene silencing by shRNAs.

Abstract 3580: The role of CDNB in understanding the mechanism of action of JS-K, a promising anti-leukemia compound

Abstract Previous studies have shown that JS-K (O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate), which belongs to the diazeniumdiolate group of compounds, is a promising anti-cancer agent which is active against human leukemia cells but not normal hematopoietic cells. The uniqueness of JS-K is due to its ability to exploit the defense system of cancer cells against xenobiotics that leads to chemotherapy failure. JS-K consists of two components - a diazeniamdiolate group necessary for the release of nitric oxide (NO) and an arylating ring - and it is unknown which is responsible for the biological effects of the compound. Because treatment with pure NO is impractical, studying the activity of the arylating ring of JS-K in comparison to the entire molecule should help us understand the biological mechanisms of this compound. CDNB (1-chloro-2,4-dinitrobenzene), which contains an arylating ring analogous to that of JS-K without the diazeniumdiolate group necessary to release NO, was used to investigate the role of arylation due to the similarity in electrophilicity between CDNB and JS-K. In this study, we used a murine erythroleukemia (MEL) cell line from mice infected with the Friend spleen focus-forming virus. These cells grow as tumors when injected subcutaneously into mice and cause meningeal leukemia when injected intraveneously, providing a unique and convenient small animal model to study the effects of anti-leukemic agents. To determine the in vitro effects of JS-K and CDNB on MEL cells, experiments were performed using several methods, including proliferation and apoptotic assays, DNA-laddering, Western blotting and cell cycle analysis. Our studies indicate that both JS-K and CDNB inhibit proliferation of MEL cells with low IC50 values and induce necrosis and apoptosis by a caspase-dependent mechanism (e.g. PARP and caspase-3 activation). Further studies indicate that both compounds cause DNA damage and disturb the cell cycle. Cell cycle arrest after treatment appears to be due to a block in the activation of the serine kinase Akt, which results in the activation of the transcription factor FoxO3a, which in turn upregulates the cyclin-dependent kinase inhibitor p27, blocking the cell cycle. Although both tested compounds activate apoptosis, cause DNA damage and alter the cell cycle, JS-K is slightly more effective than CDNB. We conclude from these data that the arylating capability of JS-K, in addition to its ability to release NO, is a major contributor to the anticancer effects of this compound in MEL cells. Studies are in progress to determine if JS-K can block the growth of MEL cells in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3580.

Targeting of EZH2 to a defined genomic site is sufficient for recruitment of Dnmt3a but not de novo DNA methylation

Polycomb-mediated gene silencing and DNA methylation underlie many epigenetic processes important in normal development as well as in cancer. An interaction between EZH2 of the Polycomb repressive complex 2 (PRC2), which trimethylates lysine 27 on Histone 3 (H3K27me3), and all three DNA methyltransferases (DNMTs) has been demonstrated, implicating a role for PRC2 in directing DNA methylation. Interestingly, however, the majority of H3K27me3 marked genes lack DNA methylation in ES cells, indicating that EZH2 recruitment may not be sufficient to promote DNA methylation. Here, we employed a Gal4DBD/gal4UAS-based system to directly test if EZH2 binding at a defined genomic site is sufficient to promote de novo DNA methylation in a murine erythroleukaemia cell line. Targeting of a Gal4DBD-EZH2 fusion to an intergenic transgene bearing a gal4 binding-site array promoted localized recruitment of SUZ12 and BMI1, subunits of PRC2 and PRC1, respectively, and deposition of H3K27me3. Further analysis of the H3K27me3-marked site revealed the persistence of H3K4me2, a mark inversely correlated with DNA methylation. Strikingly, while DNMT3a was also recruited in an EZH2-dependent manner, de novo DNA methylation of the transgene was not observed. Thus, while targeting of EZH2 to a specific genomic site is sufficient for recruitment of DNMT3a, additional events are required for de novo DNA methylation.

EKLF/KLF1 Controls Cell Cycle Entry via Direct Regulation of E2f2

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

249. Regulated expression of KIT protein in juvenile and adult germ cells of the rodent testis

KIT receptor is an established marker of differentiating spermatogonia and its activation is required to trigger spermatogonial maturation. KIT mRNA, however, can be detected in undifferentiated spermatogonia in the absence of protein expression, as previously established by us in the irradiated adult rat testes [1]. This differential regulation of mRNA and protein is presumably modulated by either local hormone action or by cues from the adult testicular microenvironment. Endogenous regulatory factors known to stimulate KIT synthesis in juvenile male germ cells in vitro are bone morphogenetic protein 4 (BMP4) and retinoic acid (RA), while factors known to suppress KIT at the onset of spermatogenesis have not yet been identified. Activin A, implicated in KIT downregulation in a murine erythroleukemia cell line [2], is produced within the juvenile mammalian testis and influences activities of spermatogonia and Sertoli cells. We hypothesised that activin acts to repress KIT expression in spermatogonia and therefore modulate spermatogonial behaviour. Evidence for this was first derived from Sertoli and germ cell co-cultures of day 8 wild type mouse testes in which exogenous activin addition caused a dose-dependent reduction of KIT mRNA. Whole testes mRNA analyses of two activin transgenic mouse models, the newborn Inhba−/− (lacking activin A) and postnatal InhbaBK/BK (decreased bioactive activin), revealed a significant elevation in KIT expression relative to wild type littermates. In the postnatal day 7 InhbaBK/BK testes, an elevated proportion of differentiated spermatogonia, increased cell surface KIT protein levels, enhanced mRNA levels of a known downstream target of KIT signalling pathway, cyclind3 and a meiotic marker, Sycp3, were observed. These data provide the first comprehensive evidence for activin modulation of KIT expression at spermatogenesis onset, in germ cells of the juvenile testis. This finding is of fundamental importance to other KIT-dependent processes. (1) Prabhu, S.M., et al. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis. Reproduction, 2006. 131(3): p. 489–99. (2) Hino, M., et al. Down-modulation of c-kit mRNA and protein expression by erythroid differentiation factor/activin A. FEBS Lett, 1995. 374(1): p. 69–71.

PU.1 is dispensable to block erythroid differentiation in Friend erythroleukemia cells

Friend murine erythroleukemia cell lines derive from erythroblasts transformed with the Friend complex where the spleen-focus forming virus integrated in the vicinity of the Sfpi-1 locus. Erythroleukemia cells do not differentiate and grow indefinitely in the absence of erythropoietin. Activation of the transcription factor PU.1, encoded by the Sfpi-1 gene, is thought to be responsible for the transformed phenotype. These cells can overcome the blockage and reinitiate their differentiation program when exposed to some chemical inducers such as hexamethylene bisacetamide. In this study, we established cell cultures that were capable to proliferate unconstrained in the presence of the inducer. Resistant cell lines restart erythroid differentiation, though, if forced to exit the cell cycle or by overexpressing the transcription factor GATA-1. Unexpectedly, expression of PU.1 was suppressed in the resistant clones albeit the spleen-focus forming virus was still integrated in the proximity of the Sfpi-1 locus. Exposure to 5-Aza-2′-deoxycytidine activates PU.1 expression suggesting that the PU.1 coding gene is highly methylated in the resistant cells. Altogether these results suggest that PU.1 is dispensable to block erythroid differentiation.

Exogenous Wild‐Type p53 Gene Improved Survival of Nude Mice Injected with Murine Erythroleukemia Cell Line Through Amelioration of Hemorheological Changes

Previous investigations have shown that human wild-type p53 gene (WTp53) inhibits the growth of leukemia and tumor cells in vitro. In the present study, the authors used nude mice and examined the therapeutic role of p53 gene for erythroleukemia in vivo in the absence of MHC effects. The nude mice were injected with murine erythroleukemia cells (MEL), MEL cells transfected with wild-type p53 gene (MEL-W), and MEL cells transfected with mutated p53 gene (MEL-M). Abnormalities were found in the hemorheological and biophysical properties of red blood cells in all 3 groups of animals, but the abnormalities were lesser in degree and later in appearance in MEL-W group than in MEL and MEL-M groups. Furthermore, the nude mice in MEL-W group lived longer than those in MEL and MEL-M groups. The results showed that WTp53 restrained the growth of erythroleukemia cells in vivo and reduced the erythroleukemia tumorigenesis in the microcirculation by improving the hemorheological and biophysical properties of MEL cells, which helped to prolong the life span of nude mice suffering from erythroleukemia. These results contribute to our knowledge on the use of wild-type p53 gene for the treatment of erythroleukemia disease.

The transcription factor KLF11 can induce γ‐globin gene expression in the setting of in vivo adult erythropoiesis

Previous studies in a fetal erythroid cell line demonstrated that the transcription factor, Krüppel-like factor 11 (KLF11), could specifically induce transcription from a gamma-globin gene promoter, and that this induction was mediated through a specific canonical CACCC cis-DNA binding motif. We report here that ectopic expression of KLF11 can also induce fetal gamma-globin gene expression in the setting of adult erythropoiesis both in vitro and in vivo. Studies in an adult-stage murine erythroleukemia (MEL) cell line demonstrated that retrovirus vector-mediated transduction of KLF11 could increase both the amount of expression from a basally active, but not from a overtly silenced, recombinant gamma-globin transgene, as well as the frequency of cells expressing this transgene. A similar pattern of gamma-globin gene induction was also observed both in vitro and in vivo following KLF11 transduction of bone marrow from mice containing a basally active gamma-globin transgene. These studies provide the first evidence that ectopic expression of a transcription factor can induce gamma-globin gene expression in vivo during adult erythropoiesis.

Microarrays for undergraduate classes

A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by erythropoietic agents such as DMSO over a 72-h time period. Students isolate total RNA from control (0 h) and 72-h DMSO-treated murine erythroleukemia cells. From this, they synthesize a cDNA copy incorporating amino-allyl dUTP, which is then coupled to either a Cy5 or a Cy3 dye. Equal amounts of the two labeled cDNA samples are then applied to a standard cDNA microarray, which is then hybridized, washed, and scanned. Up- and down-regulated genes are selected using an "in-house" user-friendly data base program. Quality control checks are included at various stages throughout the procedure and, as the process of erythropoiesis is well characterized, a number of erythroid sequences serve as internal controls on the validity of the array data. Through this experiment, students gain experience in a wide range of molecular biology techniques, the use of controls to check a multistep process, validation of results, and strategies to manage the large amount of data generated. Most importantly, it provides undergraduate students with an opportunity to carry out experiments using cutting edge techniques normally found only in research laboratories.

Erythroblast Transformation by the Friend Spleen Focus-Forming Virus Is Associated with a Block in Erythropoietin-Induced STAT1 Phosphorylation and DNA Binding and Correlates with High Expression of the Hematopoietic Phosphatase SHP-1

Infection of mice with Friend spleen focus-forming virus (SFFV) results in a multistage erythroleukemia. In the first stage, the SFFV envelope glycoprotein interacts with the erythropoietin receptor and a short form of the receptor tyrosine kinase sf-Stk, resulting in constitutive activation of signal transducing molecules and the development of erythropoietin (Epo)-independent erythroid hyperplasia and polycythemia. The second stage results from the outgrowth of a rare virus-infected erythroid cell that expresses nonphysiological levels of the myeloid transcription factor PU.1. These cells exhibit a differentiation block and can be grown as murine erythroleukemia (MEL) cell lines. In this study, we examined SFFV MEL cells to determine whether their transformed phenotype was associated with a block in the activation of any Epo signal-transducing molecules. Our studies indicate that Epo- or SFFV-induced activation of STAT1/3 DNA binding activity is blocked in SFFV MEL cells. The block is at the level of tyrosine phosphorylation of STAT1, although Jak2 phosphorylation is not blocked in these cells. In contrast to Epo, alpha interferon can induce STAT1 phosphorylation and DNA binding in SFFV MEL cells. The SFFV-transformed cells were shown to express elevated levels of the hematopoietic phosphatase SHP-1, and treatment of the cells with a phosphatase inhibitor restored STAT1 tyrosine phosphorylation. MEL cells derived from Friend murine leukemia virus (MuLV) or ME26 MuLV-infected mice, which do not express PU.1, express lower levels of SHP-1 and are not blocked in STAT1/3 DNA-binding activity. Our studies suggest that SFFV-infected erythroid cells become transformed when differentiation signals activated by STAT1/3 are blocked due to high SHP-1 levels induced by inappropriate expression of the PU.1 protein.