Development Of Leukemia In Mice Research Articles

PAX5-PML Induces Pro B Acute Lymphoblastic Leukemia in Mice

PAX5 is a transcription factor required for B-cell development and maintenance. We previously showed that PAX5-PML, a fusion gene found in acute lymphoblastic leukemia (ALL), dominant negatively inhibited PAX5 transcriptional activity. Reported data including ours revealed that PAX5 fusion proteins had possible oncogenic ability; however, leukemogenicity of PAX5 fusion genes and other PAX5 mutations in mice model has not been clarified, yet. Here we demonstrated leukemia development in mice by introducing PAX5-PML. Pro B cells derived from mouse fetal liver were transfected with PAX5-PML expression vector and transplanted into mice. All 8 transplanted mice died with pro B ALL from day 63 to 158. Leukemic cells could be serially transplanted and mice died more rapidly with repetition (Figure A). Among the target genes transcriptionally activated by PAX5, expressions of BLNK, Fcer2a, and CD72 were significantly repressed in leukemia cells but repression of CD19 and CD79a were mild, suggesting the importance of down regulation of these genes for differentiation block. We compared mRNA expression profile between leukemia cells and normal pro B cells and gene set enrichement analysis (GSEA) identified candidates for second hits for development of leukemia. We analyzed the mechanism of the selective repression of CD19, Fcer2a, and BLNK and the significance of the second hit candidates, using a cell line established from leukemia cells of the third transplanted mouse. The results will show the meeting. [Display omitted] DisclosuresSugimoto:Otsuka Pharmaceutical Co., Ltd: Employment. Naoe:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.

Phospholipase c beta 1 (PLCb1) in acute myeloid leukemia (AML): a novel potential therapeutic target

Acute myeloid leukemia (AML) is the most common type of leukemia in adults in which leukemic myeloid derived cells replace normal blood cells leading to a loss in systemic function. Once initiated the disease develops rapidly and is typically fatal within weeks or months if left untreated. AML is a complex disease and although, the exact causes of the development of AML are unknown, risk factors include age, pre-leukemic diseases such as myelodysplastic syndrome, exposure to chemicals and radiation and genetics. The mainstay treatment is still chemotherapy together with stem cell replacement therapy and while life expectancy has increased slowly, the 5 year survival rates range between 12 and 70% with relapse rates as high as 70% depending on the subtype (canceruk). These statistics illustrate the urgent requirement for the development of novel targeted therapeutics. Phospholipases C (PLC) are critical intracellular signaling enzymes that control a wide range of cellular functions including proliferation and apoptosis that have been implicated in myelodysplastic diseases and in leukemia (Faenza et al., 2013; Shah et al.). Importantly they constitute a highly druggable family of enzymes distinct from other well established drug development targets such as protein kinases. Using the human leukemic cell line THP1, we carried out a small targeted RNAi screen to establish a role of all known PLCs in cell growth, differentiation and maintenance of the transformed phenotype. We discovered that silencing of PLCb1 or PLCH2 resulted in a strong growth arrest. PLCb1 knockdown also initiated apoptosis and attenuated growth of THP1 cells in semisolid culture, which is known to reflect the ability of cells to induce leukemia in vivo. Accordingly, we found that knockdown of PLCb1 strongly attenuated THP1-mediated development of leukemia in mice. These growth inhibitory effects of PLCb1 knockdown were extended to a mouse model of human leukaemia induced by the MLL-AF9 translocation and to human primary leukemia cells. Of direct importance to the consideration for drug development we observed that PLCb1 knockdown selectively attenuated the growth of primary human AML cells, without effecting cell growth and differentiation of normal CD34+ hematopoietic stem and progenitor cells from healthy donors. We therefore propose PLCb1 as a novel candidate for a therapeutic target in AML.

Absence Of The E3 Ubiquitin Ligase SKP2 Attenuates Notch-Induced T-Cell Leukemogenesis

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood, accounting for almost 30% of pediatric cancers. In pediatric T-cell acute lymphoblastic leukemia (T-ALL), around 30% of the children still undergo disease relapse, which is associated with poor prognosis. Hence, novel therapeutic strategies for the treatment of T-ALL are necessary, since conventional therapies still fail to cure a significant number of patients. Notch signaling contributes to the regulation of normal T-cells homeostasis, whereas oncogenic, activated form of Notch is present in the majority of the T-ALL cases.Previous studies in our laboratory demonstrated that Notch1 signaling induces transcriptional activation of SKP2, the F-box protein of the SCF E3-ubiquitin ligase complex. SKP2 targets the major cell cycle inhibitors, the CKIs p21Cip1, p27Kip1, p57Kip2 and p130, for proteasome-mediated degradation. Increased Skp2 expression accelerates cell cycle progression in hematopoietic cells, and its overexpression is frequently found in cancers, in particular lymphomas and leukemias, in which is associated with poor prognosis.We found that Skp2 expression is dynamically regulated during T-cell differentiation coinciding with the Notch expression pattern. Moreover, primary thymocytes cultured in vitro responded to Notch stimulation by the Delta1 ligand increasing their Skp2 expression and their cell cycle activity. As anticipated, we found that Skp2 expression is increased in T-ALL patient samples. Our hypothesis is that Notch activation promotes T-cell leukemogenesis by altering the cell cycle control through upregulation of SKP2. To better define the role of SKP2 in T-ALL, we analyzed the Skp2 expression in a murine model of Notch-induced T-cell leukemia. Mice transplanted with stem/progenitor cells transduced with the constitutive active form of Notch (ICN), developed T-ALL and died by week 12 after transplant. Analysis of T-ALL cells revealed a 5 fold upregulation of Skp2 expression compared to controls. Next, we tested whether SKP2 was required for T-ALL development. To this end, we evaluated leukemia development in mice transplanted with SKP2 deficient cells overexpressing ICN. Stem/progenitor cells derived from Skp2+/+, Skp2+/- or Skp2-/- mice were transduced with ICN and then transplanted into WT recipients. Loss of SKP2 significantly delayed the development of T-cell leukemia in transplanted mice and increased their survival by 60% at 12 weeks.Taken together, these results demonstrate a previously unrecognized role for SKP2 in the initiation and progression of T-ALL and its potential role as a therapeutic target. Disclosures:No relevant conflicts of interest to declare.

New Design Of Human T-ALL Transplantation In NSG Mice Uncovers The Major Role Of CD31/PECAM1 In The Central Nervous System Infiltration

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is mainly a child and adolescent blood malignancy. T-ALL patients present an increased risk of Central Nervous System (CNS) relapse defined by leukemic cell infiltration in cerebrospinal fluid and brain. Using transgenic mice and T-ALL cell lines previous works have shown that T-ALL migration in CNS depends on CCR7 chemokine receptor expression (S. Buonamici et al., Nature, 2009). VE-cadherin and CD31/PECAM1 also seem implicated, as it has been shown in vitro (S. M. Akers et al., Exp Hematol, 2010). In patients, high level of IL-15 at diagnosis predicts current CNS invasion and sometimes at relapse (G. Cario et al., J Clin Oncol, 2007). So far no study has investigated mechanisms involved in CNS infiltration using T-ALL patient samples in vivo. In the present study we developed a mouse model of CNS infiltration using leukemic cells isolated from patients and transplanted into NOD/SCID IL2Rуc-/- (NSG) mice. Proper conditioning of NSG mice and different routes of injection were tested to define a protocol avoiding non-specific CNS infiltration of leukemic cells. Also bone marrow (BM) engraftment levels of leukemia between 60 to 100% were used to set up the excision time of hematopoietic tissues and brain. Leukemic blasts from 8 patients with or 9 patients without CNS invasion were grafted and brain infiltration was followed up using standard histology and immunohistochemistry techniques. Our data indicate that (1) under specific experimental procedures, leukemic cells from patients with CNS invasion did infiltrate mouse CNS (8/8 samples) whereas the majority of cells from “non-infiltrated” patients did not (7/9 samples), (2) leukemic cells recovered from NSG brain and BM were similar in terms of brain and/or BM infiltration in secondary transplant experiments. Moreover, T-Leukemia Initiating Cell frequency was the same whatever the BM or CNS origin of blasts in the primary recipient. Interestingly, analysis of blasts at diagnosis showed that surface expression of adhesion molecules can not discriminate CNS+ or CNS- leukemic cells. However, blocking of CD31 decreased in vitro migration of blasts from CNS+ compared to CNS- patients through endothelial layer derived from blood brain barrier cells. Pioneered in vivo experiments show that CNS+ blasts pre-treated with CD31 antibody and injected in NSG are less prone to colonize mouse brain. Moreover, knocking down CD31 in CNS+ T-ALL by lentiviral shRNA strategy impairs leukemia development in mice, further decreasing CNS infiltration, whatever injection routes is used including intrafemoral injection. In conclusion, T-ALL xenografts in NSG mice mimic CNS invasion in patients. CD31 is a major player in blast cells migration in vitro and brain infiltration in vivo. This new model opens a new area of investigation to improve our knowledge of the molecular mechanisms of CNS infiltration in T-ALL. Disclosures: No relevant conflicts of interest to declare.

Overexpression of the ETS-family transcription factor ERG and dysregulated cytokine signaling drive erythroid leukemia development in mice

Overexpression of the ETS-family transcription factor ERG and dysregulated cytokine signaling drive erythroid leukemia development in mice

PML-RAR co-operates with Sox4 in acute myeloid leukemia development in mice

Acute promyelocytic leukemia is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. To identify co-operating pathways to leukemogenesis, we crossed MRP8-PML/RARA transgenic mice with BXH-2 mice which harbor an endogenous murine leukemia virus that causes acute myeloid leukemia. Approximately half of the leukemias that arose in this cross showed features of acute promyelocytic leukemia. We identified 22 proviral insertion sites in acute promyelocytic-like leukemias and focused our analysis on insertion at Sox4, a HMG box transcription factor. Using a transplant model, co-operation between PML-RARα and Sox4 was confirmed with increased penetrance and reduced latency of disease. Interestingly, karyotypic analysis revealed cytogenetic changes suggesting that the factors combined to initiate but not complete leukemic transformation. The cooperation between these transcription factors is consistent with the paradigm of multiple routes to the disease and reinforces the concept that transcription factor networks are important therapeutic targets in myeloid leukemias.

The transcription factor PlagL2 activates Mpl transcription and signaling in hematopoietic progenitor and leukemia cells

Cytokine signaling pathways are frequent targets of oncogenic mutations in acute myeloid leukemia, promoting proliferation and survival. We have previously shown that the transcription factor PLAGL2 promotes proliferation and cooperates with the leukemia fusion protein Cbfβ-SMMHC in acute myeloid leukemia development. Here we show that PLAGL2 upregulates expression of the thrombopoietin receptor Mpl, using 2 consensus sites in its proximal promoter. We also show that Mpl overexpression efficiently cooperates with Cbfβ-SMMHC in development of leukemia in mice. Finally, we demonstrate that PlagL2-expressing leukemic cells show hyper-activation of Jak2 and downstream STAT5, Akt and Erk1/2 pathways in response to Tpo ligand. These results show that PlagL2 expression activates expression of Mpl in hematopoietic progenitors, and that upregulation of wild type Mpl provides an oncogenic signal in cooperation with CBFβ-SMMHC in mice.

MicroRNA miR-125b causes leukemia

MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover, miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm, suggesting an important role for miR-125b in early hematopoiesis. Furthermore, coexpression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared with control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, we show that overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL-induced leukemia and to independently induce leukemia in a mouse model.

MicroRNA Mir-125b Causes Leukemia

AbstractAbstract 3158MicroRNA miR-125b has been shown to be involved in different kind of leukemia. Indeed, the chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b up to 90 fold. Moreover, miR-125b is also upregulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All of the mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half of them died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative disorder, suggesting an important role of miR-125b in myeloid and lymphoid lineages. Co-expression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus we showed that the overexpression of miR-125b is sufficient to induce leukemia in vivo and decrease the latency of BCR-ABL -induced leukemia. Disclosures:No relevant conflicts of interest to declare.

NKX3.1 is a direct TAL1 target gene that mediates proliferation of TAL1-expressing human T cell acute lymphoblastic leukemia

TAL1 (also known as SCL) is expressed in >40% of human T cell acute lymphoblastic leukemias (T-ALLs). TAL1 encodes a basic helix-loop-helix transcription factor that can interfere with the transcriptional activity of E2A and HEB during T cell leukemogenesis; however, the oncogenic pathways directly activated by TAL1 are not characterized. In this study, we show that, in human TAL1–expressing T-ALL cell lines, TAL1 directly activates NKX3.1, a tumor suppressor gene required for prostate stem cell maintenance. In human T-ALL cell lines, NKX3.1 gene activation is mediated by a TAL1–LMO–Ldb1 complex that is recruited by GATA-3 bound to an NKX3.1 gene promoter regulatory sequence. TAL1-induced NKX3.1 activation is associated with suppression of HP1-α (heterochromatin protein 1 α) binding and opening of chromatin on the NKX3.1 gene promoter. NKX3.1 is necessary for T-ALL proliferation, can partially restore proliferation in TAL1 knockdown cells, and directly regulates miR-17-92. In primary human TAL1-expressing leukemic cells, the NKX3.1 gene is expressed independently of the Notch pathway, and its inactivation impairs proliferation. Finally, TAL1 or NKX3.1 knockdown abrogates the ability of human T-ALL cells to efficiently induce leukemia development in mice. These results suggest that tumor suppressor or oncogenic activity of NKX3.1 depends on tissue expression.

760. LMO2 Overexpression and Loss of p19ARF Genetically Cooperate in the Development of T-Cell Acute Leukemia in Mice

760. LMO2 Overexpression and Loss of p19ARF Genetically Cooperate in the Development of T-Cell Acute Leukemia in Mice

Haploinsufficiency of Ebf1 Collaborates with BCR-ABL1 to Decrease the Latency of Acute Lymphoblastic Leukemia.

In previously published genome-wide copy number analysis of leukemic samples from 242 pediatric acute lymphoblastic leukemia (ALL) patients, we reported that mutations in genes regulating B lymphoid development are the most common lesion in B-progenitor ALL, and these include PAX5, IKZF1, and EBF1. Mono-allelic deletion of EBF1 was observed in 8/200 B-progenitor leukemia samples, including a BCR-ABL1 ALL. EBF1 encodes a transcription factor that is required for the development of B cells, and with E2A regulates the expression of B-lineage specific genes. Mice null for Ebf1 arrest B cell development at the pro-B cell stage, whereas Ebf1+/− mice have a 50% reduction in the number of immature and mature B cells but a normal number of pro-B cells. Importantly, neither haploinsufficiency nor the complete loss of Ebf1 results in the development of leukemia in mice. To examine the role of genetic alterations targeting B-lymphoid differentiation in the pathogenesis in BCR-ABL1 ALL, we transduced Ebf1+/+ and Ebf1+/− bone marrow cells with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus and transplanted the resultant cells into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 Ebf1+/− cells developed B lineage ALLs at a shorter latency than observed with BCR-ABL1 Ebf1+/+ cells (median overall survival of 17 days in Ebf1+/− vs 42 days in Ebf1+/+, P<0.0001). All leukemias had a B220+Cd19+Bp1+ pre-B cell immunophenotype; however, the leukemias that developed from the Ebf1+/− cells aberrantly expressed high levels of the stem cell marker Sca1 (mean fluorescence level for Sca1 of 69.6 in Ebf1+/− (n=22) vs 16.8 in Ebf1+/+ (n=14), p<0.0001). To begin to understand how a decrease in the copy number of Ebf1 may contribute to leukemogenesis, we examined early B cell development in bone marrow (BM) cells from two week-old C57BL6 Ebf1+/− and Ebf1+/+ mice. Our analysis confirmed previous reports indicating a 2-fold reduction of B220+CD43− B cells in Ebf1+/− compared to Ebf1+/+ mice. Interestingly, however, we also detected an approximately 6-fold increase in a transitional population of B220loIL-7R+cKitlo Pre-pro B cells that also expressed Sca1 (2194 mean number of Ebf1+/− cells per 100,000 BM cells (n=10) vs 372 mean number of Ebf1+/+ cells per 100,000 BM cells (n=8), p<0.0001), an observation that raises the possibility that Ebf1 haploinsufficiency expands the pool of cells that are susceptible to transformation by BCR-ABL expression. It will be important to examine whether the accelerated tumorigenesis resulting from Ebf1 haploinsufficiency is a consequence of a subtle shift in differentiation, or some alternative mechanism of oncogenic cooperativity. Studies are underway to directly assess the role of B220loIL-7R+cKitlo Sca1+ cells in BCR-ABL1 driven ALL.

Chronic lymphocytic leukaemia and acute myeloid leukaemia are not associated with AKT1 pleckstrin homology domain (E17K) mutations

The serine/threonine kinase AKT1 (v-akt murine thymoma viral oncogene homologue 1) has a central role in the signaling of growth factors and other stimuli, leading to diverse cellular functions including cell survival, proliferation, growth and metabolism (reviewed in Vivanco & Sawyers, 2002). Furthermore, the deregulation of the phosphoinositide-3-kinase (PI3K)/AKT pathway is a common mechanism for transformation and by far the most frequently mutated component of this pathway is the tumour suppressor PTEN (Manning & Cantley, 2007). The original description of AKT1 involved virally induced carcinogenesis resulting from a constitutively active form of v-akt (Manning & Cantley, 2007). A recent report identified a recurrent mutation in the pleckstrin homology (PH) domain of AKT1 that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in breast cancer (8%), ovarian cancer (2%) and colorectal cancer (6%) (Carpten et al, 2007). The mutation was shown to activate AKT1 and lead to cellular transformation and the development of leukaemia in mice. In addition, the PI3K/AKT pathway plays an important role in both chronic and acute leukaemia pathogenesis, and a growing number of compounds have been developed to target the PI3K/AKT. Therefore, we investigated a possible role of AKT1 E17K mutation in a large cohort of patients with chronic lymphocytic leukaemia (CLL) and acute myeloid leukaemia (AML). We studied the AKT1 E17K genotype in 106 CLL patients and 95 patients with AML (Table I). DNA was extracted using Trizol or Qiagen and the following primer sequences were used to generate the DNA product (AKT1 x3 FWD ACATCTGTCCTGGCACAC; AKT1 x3 REV GCCAGTGCTTGTTGCTTG) (Carpten et al, 2007). Polymerase chain reaction (PCR) amplifications were performed using the Gene Amp PCR core reagent kit (Applied Biosystems, Foster City, CA, USA). The final concentrations of the PCR reagents were 1× PCR Gold buffer, 2 mmol/l MgCl2, 200 μmol/l of each dNTP, 400 nmol/l each forward and reverse primer, 1·25 U of AmpliTaq Gold DNA polymerase enzyme, and 200 ng of genomic DNA in a 50-μl reaction volume. Thermal cycler conditions were: 95°C for 5 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were analysed by automated fluorescent sequencing using Big Dye Terminator Kit and ABI 3100 sequencer (Applied Biosystems). The sequences were analysed visually and with the Staden package (available at http://staden.sourceforge.net). The AKT1 E17K mutation was not found in any of the 204 leukaemia patients, suggesting that the mutation is unlikely to play a significant role in transformation or disease modification in CLL and AML. Similar to the results reported here, a very recent report did not identify the AKT1 E17K mutation in 49 AML patients (Tibes et al, 2008). While the signalling molecules upstream of AKT1 are commonly mutated in AML (FLT3, RAS, and KIT), AKT1 appears to be activated by other causes than a mutation of the PH domain. AKT1 has recently been shown to be up-regulated in CLL cases with 13q deletion (Kienle et al, 2007), and AKT1 has been suggested to be an important component of the survival pathway that can be activated in CLL B cells by antigen stimulation (Longo et al, 2007). PTEN, the negative regulator of AKT1, has been implicated in a number of epithelial cancers where it is inactivated by mutation (Vivanco & Sawyers, 2002). Inactivation of PTEN by mutations were not found in a small series of patient material from CLL, but PTEN protein expression was reduced or not detected in 48% of B-CLL patients (Leupin et al, 2003). Similarly, PTEN mutations seem to be rare events in AML (Liu et al, 2000). While it is currently unclear how AKT1 is deregulated in these leukaemias, one could speculate that AKT1 may be active in CLL because of BCR stimulation. In AML, AKT1 may be activated by mutated kinases (e.g. FLT3 activated by either an internal tandem duplication or a tyrosine kinase domain mutation) that could render the mutational activation of AKT1 itself dispensable. This work was supported in part by grants from the Else-Kröner-Fresenius-Stiftung (P32/2004) and German José Carreras Leukaemia Foundation grants (DJCLS R06/28v).

TNF-alpha gene therapy with myeloid progenitor cells lacks the toxicities of systemic TNF-alpha therapy.

We examined the antileukemic activity and the toxicity of HPC transduced with human tumor necrosis factor (TNF) cDNA. Both clonal (32Dcl3) and BM-derived primary hematopoietic progenitors (BM-Prog) expressing hTNF-alpha gene (32DTNF-alpha and BMTNF-alpha cells, respectively) inhibited the development of leukemia in mice with a small dose of 32Dp210 cells, a myeloid leukemia cell line. Whether the trans-gene expressing 32DTNF-alpha cells produce toxicities commonly associated with systemic TNF-alpha therapy was determined by examining the effect of TNF-alpha-secreting progenitor cells on body weight, tissue histology, growth of HPC, and engraftment of BMT. Administration of a low or high dose of TNF-alpha-secreting 32DTNF-alpha cells to mice failed to produce loss in body weight, a measure of TNF-alpha-related cachexia. There was also no evidence of tissue necrosis or mononuclear cell (MNC) infiltration in lung, liver, kidney, or intestine of mice injected with transduced progenitor cells. Furthermore, 32DTNF-alpha cells showed no effect on the clonal growth of HPC in colony-forming assays or loss of cellularity in BM, spleen, or blood. Finally, TNF-alpha-secreting cells were found not to interfere with the engraftment of BM transplant and hematopoietic reconstitution thereafter. We conclude from these findings that unlike systemic administration of TNF-alpha, TNF-alpha gene therapy with transduced HPC is nontoxic and may have a role in eradicating residual leukemia after BMT.

Accelerated entry into S phase associated with up-regulation of cyclin D1 as a mechanism for granulocyte colony-stimulating factor (G-CSF)-induced apoptosis of murine myeloid leukemia cells

We previously reported that injection of recombinant granulocyte colony-stimulating factor (G-CSF) suppressed the development of leukemia in mice transplanted with C2M-A5 (C2M) myeloid leukemia cells and that the anti-leukemic effect of G-CSF was ascribed to the induction of apoptosis of C2M cells. These observations make a striking contrast with other previous reports on the biological activities of G-CSF. In the present study, in order to further clarify the G-CSF-induced apoptosis of C2M cells, we studied the effects of G-CSF on the cell cycle as well as the molecular events involving D-type cyclines and their cyclin-dependent kinases (cdk) in G-CSF-treated C2M cells. Cell cycle analysis revealed that G-CSF treatment of C2M cells resulted in accelerated entry from the first gap (G 1) phase into the DNA synthesis (S) phase. Western blotting disclosed that G-CSF treatment resulted in down-regulation of cyclin D2 and cdk2 and up-regulation of cyclin D1 and cdk4. The reciprocal relationship between the up-regulation of cyclin D1 and down-regulation of cyclin D2 was closely associated with accelerated entry into S phase and subsequent apoptosis of C2M cells. These results suggest that G-CSF-induced apoptosis of C2M cells might be ascribed to imbalanced cell cycle progression due to deregulated expression of d-type cyclins and their cdks.

Folate Deficiency Delays the Onset But Increases the Incidence of Leukemia in Friend Virus-Infected Mice

AbstractClinical studies have indicated that folate deficiency may enhance the development of various malignancies. In animal studies that examined the effect of folate deficiency on malignancies, conflicting results have been reported. In some studies, folate deficiency increased the development and growth of malignant tumors; in others, it decreased the development and growth of malignancies. We examined the effect of transient folate deficiency on the development of leukemia in mice infected with the anemia-inducing strain of Friend leukemia virus. Friend virus disease can be considered as a model for human acute leukemias that are preceded by a preleukemic period. These include leukemias that develop in patients who received previous chemotherapy and/or radiation therapy, as well as patients with chronic granulocytic leukemia or myelodysplasia. Folate deficiency around the time of Friend virus-infection delayed the onset but increased the incidence of leukemia. The rates of rearrangement of the Spi-1 (PU.1 ) oncogene by provirus integration and alteration of the p53 tumor-suppressor gene were the same in leukemia cell lines derived from folate-deficient mice as they were in cell lines from control mice. These results indicate that folate deficiency did not exert its enhancement of leukemogenesis through changes in either Spi-1 or p53, even though these two genes have been found to be the most frequently altered ones in Friend virus-induced leukemias. Our results suggest that folate deficiency may enhance the development of acute leukemia in patients who are at high risk for this disease.

Control of in vivo differentiation of myeloid leukemic cells. IV. Inhibition of leukemia development by myeloid differentiation-inducing protein.

It is shown that a 5-day schedule of two injections per day of the myeloid differentiation-inducing protein MGI-2 inhibited the in vivo development of leukemia in SL and SJL/J mice with different syngeneic MGI+D+ clones of myeloid leukemic cells. With this schedule of treatment high levels of MGI-2 were maintained in the serum for long periods. In contrast to these results with MGI-2, the same schedule of injections of the myeloid growth-inducing protein MGI-I did not affect the in vivo development of leukemia in mice with MGI+D+ myeloid leukemic cells, but stimulated normal myelopoiesis in the bone marrow. Different forms of MGI-I including MGI-IM and MGI-IG had different serum half-lives, and the form of MGI-I with the shortest serum half-life showed the smallest in vivo effect on normal myelopoiesis. MGI-2 injections did not inhibit the in vivo development of differentiation-defective WEHI-3B myelomonocytic leukemic cells or YAC lymphoma cells. The results indicate that the in vivo inhibitory effect of MGI-2 on the development of myeloid leukemia correlated with its differentiation-inducing potential on the leukemic cells. It is concluded that this approach of inhibiting leukemia development by inducing differentiation should also be applied to human leukemic patients, whose cells have been shown to be inducible for differentiation in culture by human MGI-2 or by other differentiation-inducing compounds.

Virus protein p30 in blood predicts development of leukaemia in mice injected with MuLV

ATTEMPTS to monitor by bioassays the fate of murine leukaemia virus (MuLV) inoculated into animals have consistently detected virus in various organs and blood plasma of apparently healthy carrier hosts1. A correlation of early MuLV titre with spontaneous leukaemia has been demonstrated in mice of the BALB/c × AKR cross2. Detection of C-type viral proteins in organs from mice3–5 has demonstrated a correlation between levels of endogenous viral proteins and the incidence of spontaneous leukaemia. These findings have been confirmed and expanded by the demonstration6 that the concentration of the major core protein, p30, of mouse C-type viruses in blood from mice with a high incidence of spontaneous leukaemia (AKR and C58) is a hundred times greater at 2 months of age than in blood of 10 strains with a low incidence of the disease. During a study of the oncogenic potential of an ecotropic MuLV, D2BAL-10, we have found that high concentrations of p30 measured early in life in the blood of animals inoculated at birth with MuLV correlated with subsequent development of leukaemia.

Studies on the humoral regulation of granulopoiesis in leukaemic RFM mice.

Intraperitoneal diffusion chambers have been used to investigate changes in humoral factors during the development of myeloid leukaemia in mice. Normal mouse bone marrow cells form colonies of granulocytes and macrophages when cultured in semi-solid agar medium within intraperitoneal diffusion chambers. The use of mice bearing transplanted myeloid leukaemia as Agar Diffusion Chamber (ADC) hosts enhances colony formation from normal marrow. The humoral basis for this stimulation has been shown by the colony stimulating activity of the fluid entering the diffusion chambers when assayed against normal mouse bone marrow cells in agar culture in vitro. The stimulus to colony growth in ADCs and the in vitro colony stimulating activity depend on the phase in the development of the leukaemia investigated, and the stimulation was abolished by splenectomy. There was no apparent relationship between the growth of the leukaemic cell population in vivo and the level of the stimulating factor detected in leukaemic mice.

Erythropoiesis in murine myeloid leukaemia.

Erythropoiesis during the development of myeloid leukaemia in mice was studied, using assay of radio-iron incorporation in blood, exorepopulation and autorepopulation techniques. These tests indicated a certain tendency of decreasing erythropoiesis during the leukaemic process due to declining numbers of the normal erthropoietic cell precursors.