Murine Myeloid Leukemia Cell Line Research Articles

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

Gene therapy for Fabry disease, a deficiency in α-galactosidase A (α-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 α-gal Acan 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. Gene therapy for Fabry disease, a deficiency in α-galactosidase A (α-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 α-gal Acan 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. Gene therapy has been used successfully to treat a number of inherited disorders.1Aiuti A Slavin S Aker M Ficara F Deola S Mortellaro A et al.Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.Science. 2002; 296: 2410-2413Crossref PubMed Scopus (981) Google Scholar,2Gaspar HB Parsley KL Howe S King D Gilmour KC Sinclair J et al.Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.Lancet. 2004; 364: 2181-2187Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar Although many viral and non-viral gene delivery alternatives exist, retroviral vectors offer the advantages of stable integration into host genomes, the ability to infect a wide variety of cell types, and relatively high levels of transgene expression.3Robbins PD Ghivizzani SC Viral vectors for gene therapy.Pharmacol Ther. 1998; 80: 35-47Crossref PubMed Scopus (415) Google Scholar Concerns regarding the safety of integrating vectors have been prompted, however, by the development of leukemia in three X-linked severe combined immunodeficiency patients in a recent clinical trial using an oncoretroviral vector.4Hacein-Bey-Abina S Von Kalle C Schmidt M McCormack MP Wulffraat N Leboulch P et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (3001) Google Scholar A variety of explanations for this outcome have been proposed, but the exact mechanism of leukemogenesis has remained unresolved, as no other clinical trials have reported this type of adverse event.5McCormack MP Rabbitts TH Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2004; 350: 913-922Crossref PubMed Scopus (241) Google Scholar,6Baum C von Kalle C Staal FJ Li Z Fehse B Schmidt M et al.Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.Mol Ther. 2004; 9: 5-13Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Despite this outcome, retroviral gene therapy continues because of the conceptual effectiveness of the treatment and the fact that gene therapy is the only potential cure available for many disorders such as X-linked severe combined immunodeficiency. Therefore, the development of improved vectors and viable alternative safety strategies is exceedingly important and timely. We have been developing various retrovirus-based gene therapy approaches for Fabry disease, a lysosomal storage disorder resulting from a deficiency of α-galactosidase A (α-gal A) activity.7Brady RO Gal AE Bradley RM Martensson E Warshaw AL Laster L Enzymatic defect in Fabry's disease. Ceramidetrihexosidase deficiency.N Engl J Med. 1967; 276: 1163-1167Crossref PubMed Scopus (844) Google Scholar Fabry disease is a good candidate for gene therapy because there is reduced neurological involvement in contrast to many other lysosomal storage disorders, and supra-physiological levels of α-gal A are well-tolerated.8Kase R Shimmoto M Itoh K Utsumi K Kotani M Taya C et al.Immunohistochemical characterization of transgenic mice highly expressing human lysosomal alpha-galactosidase.Biochim Biophys Acta. 1998; 1406: 260-266Crossref PubMed Scopus (12) Google Scholar Using retroviral gene transfer strategies, we have achieved long-term enzymatic correction and corresponding lipid reduction in a mouse model of Fabry disease by bone marrow transplantation (BMT) of transduced cells9Medin JA Tudor M Simovitch R Quirk JM Jacobson S Murray GJ et al.Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.Proc Natl Acad Sci USA. 1996; 93: 7917-7922Crossref PubMed Scopus (65) Google Scholar,10Takenaka T Murray GJ Qin G Quirk JM Ohshima T Qasba P et al.Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells.Proc Natl Acad Sci USA. 2000; 97: 7515-7520Crossref PubMed Scopus (89) Google Scholar,11Yoshimitsu M Higuchi K Ramsubir S Nonaka T Rasaiah VI Siatskas C et al.Efficient correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem/progenitor cells.Gene Ther. 2007; 14: 256-265Crossref PubMed Scopus (40) Google Scholar and by direct delivery of lentivirus into neonates.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar We have developed and utilized retroviral vectors that engineer expression of both α-gal A and human CD25 (huCD25) in a bicistronic format.13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar CD25, also known as the T-cell activation antigen (Tac) and the interleukin (IL)-2 receptor alpha chain-α,14Taniguchi T Minami Y The IL-2/IL-2 receptor system: a current overview.Cell. 1993; 73: 5-8Abstract Full Text PDF PubMed Scopus (739) Google Scholar is incapable of mediating IL-2 internalization or signaling by itself; however, in tandem with the β-chain of the receptor and the γc-chain, it forms the “high-affinity” receptor for IL-2.15Gaffen SL Signaling domains of the interleukin 2 receptor.Cytokine. 2001; 14: 63-77Crossref PubMed Scopus (152) Google Scholar Though it can be induced upon activation, expression of CD25 is absent on resting T cells, B cells, monocytes, and CD34+-enriched cells.16Hanisch UK Quirion R Interleukin-2 as a neuroregulatory cytokine.Brain Res Brain Res Rev. 1995; 21: 246-284Crossref PubMed Scopus (181) Google Scholar,17David D Bani L Moreau JL Demaison C Sun K Salvucci O et al.Further analysis of interleukin-2 receptor subunit expression on the different human peripheral blood mononuclear cell subsets.Blood. 1998; 91: 165-172PubMed Google Scholar Thus, its limited expression pattern and lack of ability to mediate signaling make it a good choice as a cell surface marking protein in bicistronic vectors. In our previous studies, huCD25 expression was used functionally to assess viral titers, for the enrichment of transgene-positive cells before BMT, and for tracking transduced cells after BMT.13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar As it is cleaved from the IL-2 receptor complex on the cell surface and can be detected as soluble CD25 (sCD25) in the plasma,18Rubin LA Kurman CC Fritz ME Biddison WE Boutin B Yarchoan R et al.Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro.J Immunol. 1985; 135: 3172-3177PubMed Google Scholar we have also used sCD25 as a surrogate marker to evaluate the level of transgene expression in an experimental setting.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar In this study, we extend the use of huCD25 expression from our bicistronic retroviral vector constructs into the development and application of a built-in safety mechanism within the gene therapy context. We propose that if an unwanted proliferative abnormality occurs following retroviral gene transfer, huCD25 can act as a target antigen to eliminate transduced cells selectively using either clinically approved anti-CD25 antibodies or newer, highly potent antibody–toxin conjugates (immunotoxins). We chose to use the murine anti-Tac (AT) monoclonal antibody19Uchiyama T Broder S Waldmann TA A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells.J Immunol. 1981; 126: 1393-1397Crossref PubMed Google Scholar fused to saporin (SAP),20Lappi DA Esch FS Barbieri L Stirpe F Soria M Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies.Biochem Biophys Res Commun. 1985; 129: 934-942Crossref PubMed Scopus (71) Google Scholar a toxin that irreversibly damages ribosomes by cleaving adenine molecules from ribosomal RNA.21Barbieri L Valbonesi P Bonora E Gorini P Bolognesi A Stirpe F Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A).Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (265) Google Scholar We have demonstrated both in vitro and in vivo that the AT–SAP (ATS) complex can specifically target and kill retrovirally transduced cells that express huCD25. Importantly, we have achieved enzymatic correction of a mouse model of Fabry disease using our bicistronic vector and were then able to remove transduced cells using both ATS and AT. Thus, we propose this model of using a cell surface antigen such as huCD25 in a bicistronic gene expression cassette as a novel safety mechanism for retroviral vectors. We first wanted to determine the specificity and efficacy of the huCD25-targeted immunotoxin ATS. A murine myeloid leukemia cell line, C1498, was infected with a lentiviral vector pHR′cPPT-EF-α-gal A-IRES-huCD25-W-SIN (LV/α-gal A/huCD25) that is engineered to express both human α-gal A and huCD25.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar Infected pools were enriched for expression of huCD25 by magnetic activated cell sorting. We tested two populations of cells that have a broad spectrum of huCD25 expression with a 5 nM concentration of each reagent: ATS, AT, control immunogloblin (Ig)G Ab conjugated to SAP (IgG-SAP), or SAP only. These populations, shown in Figure 1a and b, were 93% and 45% positive for huCD25 expression, respectively. MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assays confirmed that both populations of cells treated with ATS showed reduced proliferation (Figure 1c and d) and increased cell death as measured by lactate dehydrogenase release (Figure 1e and f) compared with cells treated with other reagents. Non-transduced cells did not show any inhibition of proliferation or increased cytotoxicity when treated with ATS (data not shown). Next, we tested the ability of ATS to clear a clonal population of transduced cells. A single-cell clone expressing huCD25 (C1498/huCD25) was isolated from the infected pool of cells by flow cytometry–based sorting (Figure 2a). Both C1498/huCD25 and C1498 non-transduced (C1498 NT) cells were incubated with increasing concentrations of each reagent. We then measured the effects on cellular proliferation and cell killing. As shown in Figure 2b, inhibition of cellular proliferation was significantly higher (P < 0.001) when cells were treated with ATS than when cells were treated with control reagents. This effect was specific to cells expressing huCD25, as C1498 NT cells treated with ATS did not show the same level of impaired growth. Similar results were obtained from a lactate dehydrogenase release assay. At low doses (<1 nM), cell killing was higher in cells treated with ATS than in cells treated with control reagents (P < 0.001) (Figure 2c). Leukemia model. As an initial step toward determining whether treatment with a CD25 antibody or immunotoxin could clear huCD25-expressing leukemic cells in our mouse model of Fabry disease, we first optimized the dose of C1498 leukemia cells to use in this strain. Increasing doses (1 × 103 to 1 × 106) of C1498 NT cells were injected into Fabry mice and the effects were monitored. Although leukemic cells were not present in the peripheral blood, we found systemic subcutaneous invasion, splenomegaly, and lymphoadenopathy, which mimics some leukemic phenotypes (data not shown). For cell doses of 1 × 103 and 1 × 104 cells/mouse, it was found that 100% and 70% of mice, respectively, survived the challenge (data not shown). For higher cell doses of 1 × 105 and 1 × 106 cells/mouse, 100% of the mice succumbed to the leukemia within 60 days and 30 days, respectively (data not shown). To obtain a more clinically relevant leukemia model, we chose to use a cell dose of 1 × 106 cells/mouse for future studies as at this higher cell dose the phenotype of the transplanted mice progressed to the disease state more quickly and aggressively. As no previous in vivo studies have been carried out with murine ATS and most studies using other AT derivatives use receptor-saturating doses of antibody,22Zhang Z Zhang M Garmestani K Talanov VS Plascjak PS Beck B et al.Effective treatment of a murine model of adult T-cell leukemia using 211At-7G7/B6 and its combination with unmodified anti-Tac (daclizumab) directed toward CD25.Blood. 2006; 108: 1007-1012Crossref PubMed Scopus (28) Google Scholar it was next necessary to determine an effective dose of immunotoxin. We tested the ability of two different doses of ATS to eliminate huCD25-expressing cells in Fabry mice challenged with C1498/huCD25 leukemia. Mice were lethally irradiated and injected with 1 × 106 C1498/huCD25 cells and supportive syngeneic BM cells. At days 2, 4, and 6 after leukemic transplant, animals were injected with either 5 μg ATS or 20 μg ATS, injected with SAP only, or left untreated (n = 3 per group). Eleven days after challenge, blood was sampled and plasma analyzed for levels of sCD25 by enzyme-linked immunosorbent assay (ELISA). Evaluation of sCD25 levels is a common method used in the clinical setting to monitor tumor burden and treatment response in patients with CD25-expressing lymphoma and leukemia.23Perez-Encinas M Villamayor M Campos A Gonzalez S Bello JL Tumor burden and serum level of soluble CD25, CD8, CD23, CD54 and CD44 in non-Hodgkin's lymphoma.Haematologica. 1998; 83: 752-754PubMed Google Scholar This method also allows sensitive detection of the presence of CD25-positive cells for such studies as it can reflect contributions from concealed populations. As shown in Figure 3, treatment with ATS significantly reduced sCD25 (P < 0.05) levels compared with animals treated with the control reagent, SAP, and with those animals left untreated. As treatment with the lower dose of 57 μg of ATS had a similar effect to treatment with the 20 μg dose (Figure 3), we chose to use the lower dose of ATS in future experiments because this was more cost-effective and may lower the risk of secondary or non-specific toxicities. To test the efficacy of our CD25-targeting approach further, a larger experiment using 5 μg doses of ATS was then performed. Mice were lethally irradiated and injected with 1 × 106 C1498/huCD25 or C1498 NT cells along with supportive syngeneic BM cells via the tail vein. Mice transplanted with C1498/huCD25 cells were then treated with equimolar amounts of ATS, AT, or IgG-SAP. Mice transplanted with C1498 NT cells were treated with 57 μg ATS as a control. All animals were bled on days 7, 11, and 18 after transplantation, and levels of sCD25 in the plasma were measured by ELISA. As shown in Figure 4a, in mice challenged with C1498/huCD25 cells, at 18 days after transplantation average plasma sCD25 levels were significantly lower in animals treated with ATS (474 pg/ml) and AT (848 pg/ml) than in mice treated with IgG-SAP (4,762 pg/ml; P < 0.01) or not treated (15,450 pg/ml; P < 0.05). This indicates a lower tumor burden in mice treated with both CD25-targeted reagents, ATS and AT. The inherent α-gal A deficiency of Fabry mice and the fact that the transplanted tumor cells were engineered to express α-gal A meant that differences in α-gal A activity itself could be used as another surrogate marker of tumor burden. Therefore, plasma α-gal A activity was measured and was found to be lowest in mice treated with ATS (16 nmol/hour/ml) and AT (21 nmol/hour/ml) (Figure 4b). These levels were significantly lower than those in mice that received IgG-SAP (47 nmol/hour/ml; P < 0.001 and P < 0.01, versus ATS and AT, respectively) or that were left untreated (77 nmol/hour/ml; P < 0.05). Therefore, both ATS and AT are able to de-bulk tumor burden in this huCD25-expressing leukemia model. To determine the ability of anti-CD25 antibodies to affect survival, animals were monitored daily; a Kaplan–Meier representation of survival is shown in Figure 4c. In mice treated with ATS, the median survival duration was 29 days. This was significantly higher (P < 0.01) than that seen in mice that were not treated (median survival = 23 days). Increased survival was also seen in mice treated with AT (median survival of 30 days, P <0.05 versus untreated mice). Therefore, even in the context of a very high leukemic burden, treatment with CD25-targeted antibodies increased survival compared with control treatments. Note that these results are representative of two independent experiments. BMT model. We next wanted to test our clearance strategy in the context of a therapeutic BMT model. BMT is a common gene therapy approach,24Boelens JJ Trends in haematopoietic cell transplantation for inborn errors of metabolism.J Inherit Metab Dis. 2006; 29: 413-420Crossref PubMed Scopus (95) Google Scholar and incorporation of a cell surface protein that can be targeted can improve the safety of the system. Murine bone marrow mononuclear cells (BMMNCs) were isolated and infected twice with one of two ecotropic oncoretroviral vectors, either E86/pMFG/α-gal A/IRES/huCD25 clone 21 (RV/α-gal A/huCD25) or E86/pUMFG/enYFP (RV/enYFP).13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar Flow cytometry analysis of these transduced BMMNCs showed that cells infected with RV/α-gal A/huCD25 were approximately 20% positive for expression of huCD25 (Figure 5a) and cells infected with RV/enYFP were approximately 20% positive for enhanced yellow fluorescent protein (enYFP) expression (Figure 5b). Cells were then injected into lethally irradiated Fabry mice, which were monitored monthly for engraftment. At 8 weeks after transplantation, plasma from recipient Fabry mice was analyzed for α-gal A activity and for levels of sCD25. Average plasma α-gal A activity in mice transplanted with BMMNCs infected with RV/α-gal A/huCD25 was 65 nmol/hour/ml, approximately sixfold higher than in both control Fabry mice and mice transplanted with RV/enYFP-infected BMMNCs (Figure 5c). This indicates that we achieved therapeutic correction of α-gal A activity in Fabry animals at levels approximately twofold higher than in wild-type C57BL/6 mice (Figure 5c). At this time, the average level of sCD25 in the plasma of Fabry mice transplanted with BMMNCs infected with RV/α-gal A/huCD25 was 1212 ± 370 pg/ml. In contrast, sCD25 was undetectable in mice transplanted with RV/enYFP-infected cells, in wild-type C57BL/6 mice, and in untreated Fabry mice (data not shown). Mice were then treated with either ATS, AT, or IgG-SAP, as in our leukemia model (see above). Seven days after the third dose of immunotoxin, plasma was sampled to determine the effect of treatment. Comparisons were made with pre-treatment values collected for each mouse at 8 weeks after transplantation. As shown in Figure 6a, treatment with ATS resulted in lower plasma sCD25 levels than in mice that were treated with IgG-SAP or mice that were not treated (P < 0.05). In addition, analysis of huCD25 expression on peripheral blood (PB)mononuclear cells by flow cytometry showed that mice treated with ATS had significantly reduced numbers of huCD25-expressing PB mononuclear cells than mice treated with IgG-SAP (P < 0.01) or untreated mice (P < 0.05) (Figure 6b). Similar effects were observed in mice treated with AT, further supporting the conceptual ability of targeted anti-CD25 antibodies to eliminate retrovirally transduced donor hematopoietic cells in vivo. Expression of enYFP was monitored before and after treatment with ATS and it was found that levels remained stable over the course of the experiment (Figure 6c), demonstrating the specificity of the immunotoxin for cells expressing huCD25. To examine the effect of a later administration of antibody or immunotoxin, one final dose was administered and then mice were killed. Enzyme activity was measured in various tissues to determine the systemic effect of each reagent. PB mononuclear cells from mice that were treated with ATS showed significantly lower (P < 0.05) α-gal A activity than mice treated with IgG-SAP (Figure 7a). Similarly, α-gal A activity in the livers of mice treated with ATS was significantly lower (P < 0.05) than enzyme activity in the livers of mice treated with AT or IgG-SAP or untreated mice (Figure 7b). Likewise, in the spleens of mice treated with ATS, there was significantly lower (P< 0.01) α-gal A activity than in IgG-SAP-treated or untreated mice (Figure 7c). Gene therapy is the most promising curative treatment for monogenetic diseases such as lysosomal storage disorders.25Futerman AH van Meer G The cell biology of lysosomal storage disorders.Nat Rev Mol Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (628) Google Scholar Although our laboratory has made considerable advances toward the development of retrovirus-based gene therapy strategies for Fabry disease, concerns remain regarding the safety of integrating vectors. To address this issue, we propose that a cell surface marker such as huCD25 can act as an effective built-in safety mechanism in the event of insertional genotoxicity by facilitating the clearance of transduced cells with a specifically targeted immunotoxin. Our laboratory has previously used huCD25 in combination with α-gal A in studies evaluating the efficacy of retroviral gene therapy for Fabry disease.12Yoshimitsu M Sato T Tao K Walia JS Rasaiah VI Sleep GT et al.Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.Proc Natl Acad Sci USA. 2004; 101: 16909-16914Crossref PubMed Scopus (78) Google Scholar,13Qin G Takenaka T Telsch K Kelley L Howard T Levade T et al.Preselective gene therapy for Fabry disease.Proc Natl Acad Sci USA. 2001; 98: 3428-3433Crossref PubMed Scopus (66) Google Scholar We have not observed any untoward effects of exogenously expressing this protein nor have we observed altered therapeutic effects of this surface antigen on α-gal A–mediated correction in vivo. Monoclonal antibodies have been successfully used in the clinic for many years to treat hematological malignancies, with minimal toxicity.26Dillman RO Monoclonal antibody therapy for lymphoma: an update.Cancer Pract. 2001; 9: 71-80Crossref PubMed Scopus (40) Google Scholar,27Gokbuget N Hoelzer D Treatment with monoclonal antibodies in acute lymphoblastic leukemia: current knowledge and future prospects.Ann Hematol. 2004; 83: 201-205Crossref PubMed Scopus (72) Google Scholar For instance, rituximab, an anti-CD20 antibody, has been used to treat a variety of lymphoid malignancies.26Dillman RO Monoclonal antibody therapy for lymphoma: an update.Cancer Pract. 2001; 9: 71-80Crossref PubMed Scopus (40) Google Scholar, 28McLaughlin P Grillo-Lopez AJ Link BK Levy R Czuczman MS Williams ME et al.Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program.J Clin Oncol. 1998; 16: 2825-2833Crossref PubMed Scopus (2570) Google Scholar, 29Grillo-Lopez AJ Hedrick E Rashford M Benyunes M Rituximab: ongoing and future clinical development.Semin Oncol. 2002; 29: 105-112Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 30Cvetkovic RS Perry CM Rituximab: a review of its use in non-Hodgkin's lymphoma and chronic lymphocytic leukaemia.Drugs. 2006; 66: 791-820Crossref PubMed Scopus (95) Google Scholar In addition, a strategy for clearing transduced hematopoietic cells in vivo using an anti-CD20 Ab was proposed for the treatment of graft-versus-host disease.31Introna M Barbui AM Bambacioni F Casati C Gaipa G Borleri G et al.Genetic modification of human T cells with CD20: a strategy to purify and lyse transduced cells with anti-CD20 antibodies.Hum Gene Ther. 2000; 11: 611-620Crossref PubMed Scopus (114) Google Scholar The premise is that T cells can be transduced with a viral vector carrying the complementary DNA for CD20 before BMT, and if graft-versus-host disease occurs, then anti-CD20 antibodies can be used to eliminate the donor T cells. These studies have shown promising results in vitro; however, no studies have been carried out to demonstrate efficacy in vivo.32van Meerten T Claessen MJ Hagenbeek A Ebeling SB The CD20/alphaCD20 'suicide' system: novel vectors with improved safety and expression profiles and efficient elimination of CD20-transgenic T cells.Gene Ther. 2006; 13: 789-797Crossref PubMed Scopus (30) Google Scholar,33Serafini M Manganini M Borleri G Bonamino M Imberti L Biondi A et al.Characterization of CD20-transduced T lymphocytes as an alternative suicide gene therapy approach for the treatment of graft-versus-host disease.Hum Gene Ther. 2004; 15: 63-76Crossref PubMed Scopus (79) Google Scholar Aberrant levels of CD25 expression characterize numerous disorders such as adult T-cell leukemia/lymphoma, Hodgkin's lymphoma, hairy cell leukemias, and true histiocytic lymphomas.34Kreitman RJ Wilson WH White JD Stetler-Stevenson M Jaffe ES Giardina S et al.Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies.J Clin Oncol. 2000; 18: 1622-1636Crossref PubMed Scopus (391) Google Scholar Treatment of these diseases using antibodies against CD25, as well as newer recombinant immunotoxins, has resulted in complete and partial remissions in patients.34Kreitman RJ Wilson WH White JD Stetler-Stevenson M Jaffe ES Giardina S et al.Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies.J Clin Oncol. 2000; 18: 1622-1636Crossref PubMed Scopus (391) Google Scholar,35Kreitman RJ Wilson WH Robbins D Margulies I Stetler-Stevenson M Waldmann TA et al.Responses in refractory hairy cell leukemia to a recombinant immunotoxin.Blood. 1999; 94: 3340-3348Crossref PubMed Google Scholar Currently, anti-CD25 antibodies are widely used for the prevention of renal graft rejection and in some cases for prophylactic treatment against graft-versus-host disease.36Lin M Ming A Zhao M Two-dose basiliximab compared with two-dose daclizumab in renal transplantation: a clinical study.Clin

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.

Mitoxantrone treatment of murine myeloid leukaemia sublines

The low-cell-dose (LD) and the high-cell-dose (HD) transplant variants of the SA7 murine myeloid leukaemia cell line have different growth characteristics and clinical presentations. In addition, the low-cell-dose transplant subline (SA7LD) was more responsive than the high-cell-dose variant (SA7HD) to mitoxantrone treatment in vivo. Bone-marrow cells of mice cured of SA7LD leukaemia, as well as bone-marrow cells of normal mice treated with priming doses of mitoxantrone in vivo became significantly (p = 0.012) less sensitive to subsequent treatment with mitoxantrone in vitro. This effect was detected by both the colony assay and the tritiated thymidine uptake assay. There appears to be a correlation between the ability of normal bone-marrow cells present in leukaemic mice to develop this protective effect and their ability to survive chemotherapy with mitoxantrone. The protective effect was "lost" by bone-marrow cells of mice dying while in remission. Doses of mitoxantrone that resulted in the loss of protective effect by bone-marrow cells of normal mice were found to be fatal to SA7HD leukaemia-bearing mice. However, these doses were not toxic to normal mice.