Laboratory and wild-derived mice with multiple loci for production of xenotropic murine leukemia virus.

PubMed

Kozak, C A; Hartley, J W; Morse, H C

1984-07-01

Mendelian segregation analysis was used to define genetic loci for the induction of infectious xenotropic murine leukemia virus in several laboratory and wild-derived mice. MA/My mice contain two loci for xenotropic virus inducibility, one of which, Bxv -1, is the only induction locus carried by five other inbred strains. The second, novel MA/My locus, designated Mxv -1, is unlinked to Bxv -1 and shows a lower efficiency of virus induction. The NZB mouse carries two induction loci; both are distinct from Bxv -1 since neither is linked to the Pep-3 locus on chromosome 1. Finally, one partially inbred strain derived from the wild Japanese mouse, Mus musculus molossinus, carries multiple (at least three) unlinked loci for induction of xenotropic virus. Although it is probable that inbred strains inherited xenotropic virus inducibility from Japanese mice, our data suggest that none of the induction loci carried by this particular M. m. molossinus strain are allelic with Bxv -1.

Laboratory and wild-derived mice with multiple loci for production of xenotropic murine leukemia virus.

PubMed Central

Kozak, C A; Hartley, J W; Morse, H C

1984-01-01

Mendelian segregation analysis was used to define genetic loci for the induction of infectious xenotropic murine leukemia virus in several laboratory and wild-derived mice. MA/My mice contain two loci for xenotropic virus inducibility, one of which, Bxv -1, is the only induction locus carried by five other inbred strains. The second, novel MA/My locus, designated Mxv -1, is unlinked to Bxv -1 and shows a lower efficiency of virus induction. The NZB mouse carries two induction loci; both are distinct from Bxv -1 since neither is linked to the Pep-3 locus on chromosome 1. Finally, one partially inbred strain derived from the wild Japanese mouse, Mus musculus molossinus, carries multiple (at least three) unlinked loci for induction of xenotropic virus. Although it is probable that inbred strains inherited xenotropic virus inducibility from Japanese mice, our data suggest that none of the induction loci carried by this particular M. m. molossinus strain are allelic with Bxv -1. PMID:6328046

Gene Therapy for Fracture Repair

DTIC Science & Technology

2005-12-01

therapeutic benefits. We have identified a murine leukemia virus (MLV) vector that provides robust transgene expression in fracture tissues, and applied it to...During the second year of funding, we used the surgical technique to apply the murine leukemia virus (MLV)-based vector to the fracture tissues and...trochanter. ii ) Fracture Injection The therapeutic gene chosen was the BMP-2/4 hybrid gene. To most accurately establish the expression of the

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

PubMed

Bedgood, R M; Stallcup, M R

1992-04-05

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.

Detection of Xenotropic Murine Leukemia Virus Related Virus (XMRV) in Gulf War Illness: Role in Pathogenesis or Biomarker?

DTIC Science & Technology

2013-10-01

Epstein   Barr   virus  (EBV)   □Yes   □No       Cytomegalovirus  (CMV)   □Yes   □No       Lyme...myalgia, arthralgia, depression, and memory loss; candidate etiologic agents include Epstein - Barr and other herpesviruses. • Syndrome thought to...Award Number: W81XWH-11-1-0766 TITLE: Detection of Xenotropic Murine Leukemia Virus Related Virus

Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors.

PubMed Central

Delviks, K A; Hu, W S; Pathak, V K

1997-01-01

We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events. PMID:9223521

Epigallocatechin gallate (EGCG), influences a murine WEHI-3 leukemia model in vivo through enhancing phagocytosis of macrophages and populations of T- and B-cells.

PubMed

Huang, An-Cheng; Cheng, Hsiu-Yueh; Lin, Tsu-Shun; Chen, Wen-Hsein; Lin, Ju-Hwa; Lin, Jen-Jyh; Lu, Chi-Cheng; Chiang, Jo-Hua; Hsu, Shu-Chun; Wu, Ping-Ping; Huang, Yi-Ping; Chung, Jing-Gung

2013-01-01

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea, and has been reported to have anticancer effects on many types of cancer cells. However, there is no report to show its effects on the immune response in a murine leukemia mouse model. Thus, in the present study, we investigated the effects of EGCG on the immune responses of murine WEHI-3 leukemia cells in vivo. WEHI-3 cells were intraperitoneally injected into normal BALB/c mice to establish leukemic BALB/c mice, which were then oral-treated with or without EGCG at 5, 20 and 40 mg/kg for two weeks. The results indicated that EGCG did not change the weight of the animals, nor the liver or spleen when compared to vehicle (olive oil) -treated groups. Furthermore, EGCG increased the percentage of cluster of differentiation 3 (CD3) (T-cell), cluster of differentiation 19 (CD19) (B-cell) and Macrophage-3 antigen (Mac-3) (macrophage) but reduced the percentage of CD11b (monocyte) cell surface markers in EGCG-treated groups as compared with the untreated leukemia group. EGCG promoted the phagocytosis of macrophages from 5 mg/kg treatment and promoted natural killer cell activity at 40 mg/kg, increased T-cell proliferation at 40 mg/kg but promoted B-cell proliferation at all three doses. Based on these observations, it appears that EGCG might exhibit an immune response in the murine WEHI-3 cell line-induced leukemia in vivo.

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed

Torrent, C; Gabus, C; Darlix, J L

1994-02-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Jing; Liu, Gexiu; Yan, Guoyao

By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway,more » β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.« less

An extract of Agaricus blazei Murill administered orally promotes immune responses in murine leukemia BALB/c mice in vivo.

PubMed

Lin, Jaung-Geng; Fan, Ming-Jen; Tang, Nou-Ying; Yang, Jai-Sing; Hsia, Te-Chun; Lin, Jen-Jyh; Lai, Kuang-Chi; Wu, Rick Sai-Chuen; Ma, Chia-Yu; Wood, W Gibson; Chung, Jing-Gung

2012-03-01

The edible mushroom (fungus) Agaricus blazei Murill (ABM) is a health food in many countries. Importantly, it has been shown to have antitumor and immune effects. There is no available information on ABM-affected immune responses in leukemia mice in vivo. Experimental Design. In this study, the authors investigated the immunopotentiating activities of boiled water-soluble extracts from desiccated ABM in WEHI-3 leukemia mice. The major characteristic of WEHI-3 leukemia mice are enlarged spleens and livers after intraperitoneal injection with murine leukemia WEHI-3 cells. Isolated T cells from spleens of ABM-treated mice resulted in increased T-cell proliferation compared with the untreated control with concanavalin A stimulation. ABM decreased the spleen and liver weights when compared with WEHI-3 leukemia mice and this effect was a dose-dependent response. ABM promoted natural killer cell activity and phagocytosis by macrophage/monocytes in leukemia mice in a dose-dependent manner. ABM also enhanced cytokines such as interleukin (IL)-1β, IL-6, and interferon-γ levels but reduced the level of IL-4 in WEHI-3 leukemia mice. Moreover, ABM increased the levels of CD3 and CD19 but decreased the levels of Mac-3 and CD11b in leukemia mice. The ABM extract is likely to stimulate immunocytes and regulate immune response in leukemia mice in vivo.

NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia

PubMed Central

LaRue, Rebecca S.; Nguyen, Hanh T.; Sachs, Karen; Noble, Klara E.; Mohd Hassan, Nurul Azyan; Diaz-Flores, Ernesto; Rathe, Susan K.; Sarver, Aaron L.; Bendall, Sean C.; Ha, Ngoc A.; Diers, Miechaleen D.; Nolan, Garry P.; Shannon, Kevin M.; Largaespada, David A.

2014-01-01

Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRASG12V). Using computational approaches to explore our gene-expression data sets, we found that NRASG12V enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9– and Myb-dependent leukemia self-renewal gene-expression program. NRASG12V was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRASG12V-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell–enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRASG12V withdrawal. NRASG12V maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell–specific therapies. Together, these experimental results define a RAS oncogene–driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction. PMID:25316678

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells.

PubMed

Cardona, Maria E; Simonson, Oscar E; Oprea, Iulian I; Moreno, Pedro M D; Silva-Lara, Maria F; Mohamed, Abdalla J; Christensson, Birger; Gahrton, Gösta; Dilber, M Sirac; Smith, C I Edvard; Arteaga, H Jose

2016-01-01

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

Diminished potential for B-lymphoid differentiation after murine leukemia virus infection in vivo and in EML hematopoietic progenitor cells.

PubMed

Finstad, Samantha L; Rosenberg, Naomi; Levy, Laura S

2007-07-01

Infection with a recombinant murine-feline gammaretrovirus, MoFe2, or with the parent virus, Moloney murine leukemia virus, caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective, in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state.

Gammaretroviral Vectors: Biology, Technology and Application

PubMed Central

Maetzig, Tobias; Galla, Melanie; Baum, Christopher; Schambach, Axel

2011-01-01

Retroviruses are evolutionary optimized gene carriers that have naturally adapted to their hosts to efficiently deliver their nucleic acids into the target cell chromatin, thereby overcoming natural cellular barriers. Here we will review—starting with a deeper look into retroviral biology—how Murine Leukemia Virus (MLV), a simple gammaretrovirus, can be converted into an efficient vehicle of genetic therapeutics. Furthermore, we will describe how more rational vector backbones can be designed and how these so-called self-inactivating vectors can be pseudotyped and produced. Finally, we will provide an overview on existing clinical trials and how biosafety can be improved. PMID:21994751

Evidence of B cell immune responses to acute lymphoblastic leukemia in murine allogeneic hematopoietic stem cell transplantation recipients treated with donor lymphocyte infusion and/or vaccination.

PubMed

Mullen, Craig A; Campbell, Andrew; Tkachenko, Olena; Jansson, Johan; Hsu, Yu-Chiao

2011-02-01

These experiments explored mechanisms of control of acute lymphoblastic leukemia (ALL) following allogeneic hematopoietic stem cell transplantation using a murine model of MHC-matched, minor histocompatibility antigen-mismatched transplantation. The central hypothesis examined was that addition of active vaccination against leukemia cells would substantially increase the effectiveness of allogeneic donor lymphocyte infusion (DLI) against ALL present in the host after transplantation. Although vaccination did increase the magnitude of type I T cell responses against leukemia cells associated with DLI, it did not lead to substantial improvement in long-term survival. Analysis of immunologic mechanisms of leukemia progression demonstrated that the failure of vaccination was not because of antigen loss in leukemia cells. However, analysis of survival provided surprising findings that, in addition to very modest type I T cell responses, a B cell response that produced antibodies that bind leukemia cells was found in long-term survivors. The risk of death from leukemia was significantly lower in recipients that had higher levels of such antibodies. These studies raise the hypothesis that stimulation of B cell responses after transplantation may provide a novel way to enhance allogeneic graft-versus-leukemia effects associated with transplantation. Copyright © 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Mouse chromosomal mapping of a murine leukemia virus integration region (Mis-1) first identified in rat thymic leukemia.

PubMed Central

Jolicoeur, P; Villeneuve, L; Rassart, E; Kozak, C

1985-01-01

We have previously identified a region of genomic DNA which constitutes the site of frequent provirus integration in rat thymomas induced by Moloney murine leukemia virus (Lemay and Jolicoeur, Proc. Natl. Acad. Sci. USA 81:38-42, 1984). This genetic locus is now designated Mis-1 (Moloney integration site). Cellular sequences homologous to Mis-1 are present in mouse DNA. Using a series of hamster-mouse somatic cell hybrids, we mapped the Mis-1 locus to mouse chromosome 15. Frequent chromosome 15 aberrations have been described in mouse thymomas. Mis-1 represents a putative new oncogene which might be involved in the initiation or maintenance or both of these neoplasms. Images PMID:4068142

Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines.

PubMed

Duisit, G; Salvetti, A; Moullier, P; Cosset, F L

1999-01-20

We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

PubMed Central

Torrent, C; Gabus, C; Darlix, J L

1994-01-01

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer. Images PMID:8289369

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

PubMed Central

Felsenstein, K M; Goff, S P

1992-01-01

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo. Images PMID:1404606

Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease.

PubMed

Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E; Dunn, Ben M; Wlodawer, Alexander

2011-11-01

Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K(i) , depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place. Journal compilation © 2011 FEBS. No claim to original US government works.

Role of CXCR4-mediated bone marrow colonization in CNS infiltration by T cell acute lymphoblastic leukemia.

PubMed

Jost, Tanja Rezzonico; Borga, Chiara; Radaelli, Enrico; Romagnani, Andrea; Perruzza, Lisa; Omodho, Lorna; Cazzaniga, Giovanni; Biondi, Andrea; Indraccolo, Stefano; Thelen, Marcus; Te Kronnie, Geertruy; Grassi, Fabio

2016-06-01

Infiltration of the central nervous system is a severe trait of T cell acute lymphoblastic leukemia. Inhibition of CXC chemokine receptor 4 significantly ameliorates T cell acute lymphoblastic leukemia in murine models of the disease; however, signaling by CXC chemokine receptor 4 is important in limiting the divagation of peripheral blood mononuclear cells out of the perivascular space into the central nervous system parenchyma. Therefore, Inhibition of CXC chemokine receptor 4 potentially may untangle T cell acute lymphoblastic leukemia cells from retention outside the brain. Here, we show that leukemic lymphoblasts massively infiltrate cranial bone marrow, with diffusion to the meninges without invasion of the brain parenchyma, in mice that underwent xenotransplantation with human T cell acute lymphoblastic leukemia cells or that developed leukemia from transformed hematopoietic progenitors. We tested the hypothesis that T cell acute lymphoblastic leukemia neuropathology results from meningeal infiltration through CXC chemokine receptor 4-mediated bone marrow colonization. Inhibition of leukemia engraftment in the bone marrow by pharmacologic CXC chemokine receptor 4 antagonism significantly ameliorated neuropathologic aspects of the disease. Genetic deletion of CXCR4 in murine hematopoietic progenitors abrogated leukemogenesis induced by constitutively active Notch1, whereas lack of CCR6 and CCR7, which have been shown to be involved in T cell and leukemia extravasation into the central nervous system, respectively, did not influence T cell acute lymphoblastic leukemia development. We hypothesize that lymphoblastic meningeal infiltration as a result of bone marrow colonization is responsible for the degenerative alterations of the neuroparenchyma as well as the alteration of cerebrospinal fluid drainage in T cell acute lymphoblastic leukemia xenografts. Therefore, CXC chemokine receptor 4 may constitute a pharmacologic target for T cell acute lymphoblastic leukemia neuropathology. © Society for Leukocyte Biology.

Sensitivity to. gamma. rays of avian sarcoma and murine leukemia viruses. [/sup 60/Co, uv

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toyoshima, K.; Niwa, O.; Yutsudo, M.

1980-09-01

The direct inactivation of avian and murine oncoviruses by ..gamma.. rays was examined using /sup 60/Co as a ..gamma..-ray source. The inactivation of murine leukemia virus (M-MuLV) followed single-hit kinetics while the subgroup D Schmidt-Ruppin strain of avian sarcoma virus (SR-RSV D) showed multihit inactivation kinetics with an extrapolation number of 5. The two viruses showed similar uv-inactivation kinetics. The genomic RNA of the SR-RSV D strain was degraded by ..gamma.. irradiation faster than its infectivity, but viral clones isolated from the foci formed after ..gamma.. irradiation had a complete genome. These results suggest that SR-RSV D has a strongmore » repair function, possibly connected with reverse transcriptase activity.« less

Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection

PubMed Central

Valle-Tenney, Roger; Opazo, Tatiana; Cancino, Jorge; Goff, Stephen P.

2016-01-01

ABSTRACT During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm—a crowded environment where diffusion is slow—is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex. PMID:27194765

A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing

PubMed Central

Déjardin, Jérôme; Bompard-Maréchal, Guillaume; Audit, Muriel; Hope, Thomas J.; Sitbon, Marc; Mougel, Marylène

2000-01-01

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD′) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD′ was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD′ without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells. PMID:10729146

RNA Related to That of a Murine Leukemia Virus in Burkitt's Tumors and Nasopharyngeal Carcinomas

PubMed Central

Kufe, D.; Hehlmann, R.; Spiegelman, S.

1973-01-01

RNA homologous to that of the Rauscher leukemia virus has been detected in Burkitt's lymphomas and nasopharyngeal carcinomas. Earlier excellent experimental evidence has linked these two human tumors with the Epstein-Barr virus, a DNA-containing agent. PMID:4346039

Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector.

PubMed

Dardalhon, V; Jaleco, S; Rebouissou, C; Ferrand, C; Skander, N; Swainson, L; Tiberghien, P; Spits, H; Noraz, N; Taylor, N

2000-08-01

Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA(+)) and memory (CD45RO(+)) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA(+) T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.

Selection of Optimal Polypurine Tract Region Sequences during Moloney Murine Leukemia Virus Replication

PubMed Central

Robson, Nicole D.; Telesnitsky, Alice

2000-01-01

Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT. PMID:11044073

Oncogenic Kras initiates leukemia in hematopoietic stem cells.

PubMed

Sabnis, Amit J; Cheung, Laurene S; Dail, Monique; Kang, Hio Chung; Santaguida, Marianne; Hermiston, Michelle L; Passegué, Emmanuelle; Shannon, Kevin; Braun, Benjamin S

2009-03-17

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras(G12D) from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras(G12D) expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras(G12D) HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras(G12D) expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras(G12D) HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras(G12D) mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.

Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kakoki, Katsura; Department of AIDS Research, Institute of Tropical Medicine, G-COE, Nagasaki University, Nagasaki 852-8523; Department of Urology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523

Highlights: • XMRV infection induces androgen-independent growth in LNCaP cells. • XMRV infection reduces expression of androgen receptor. • XMRV promotes appearance of androgen blocker-resistant prostate cancer cells. - Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage tomore » prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.« less

Commonly dysregulated genes in murine APL cells

PubMed Central

Yuan, Wenlin; Payton, Jacqueline E.; Holt, Matthew S.; Link, Daniel C.; Watson, Mark A.; DiPersio, John F.; Ley, Timothy J.

2007-01-01

To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (APL), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. Many genes were reproducibly expressed in restricted developmental “windows,” suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. We compared the normal myeloid developmental transcriptome with that of APL cells derived from mice expressing PML-RARα under control of the murine cathepsin G locus. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples, including Fos, Jun, Egr1, Tnf, and Vcam1. However, this set of commonly dysregulated genes was expressed normally in preleukemic, early myeloid cells from the same mouse model, suggesting that dysregulation occurs as a “downstream” event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis. PMID:17008535

Targeting of a Nuclease to Murine Leukemia Virus Capsids Inhibits Viral Multiplication

NASA Astrophysics Data System (ADS)

Natsoulis, Georges; Seshaiah, Partha; Federspiel, Mark J.; Rein, Alan; Hughes, Stephen H.; Boeke, Jef D.

1995-01-01

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

Leukemia cell-rhabdovirus vaccine: personalized immunotherapy for acute lymphoblastic leukemia.

PubMed

Conrad, David P; Tsang, Jovian; Maclean, Meaghan; Diallo, Jean-Simon; Le Boeuf, Fabrice; Lemay, Chantal G; Falls, Theresa J; Parato, Kelley A; Bell, John C; Atkins, Harold L

2013-07-15

Acute lymphoblastic leukemia (ALL) remains incurable in most adults. It has been difficult to provide effective immunotherapy to improve outcomes for the majority of patients. Rhabdoviruses induce strong antiviral immune responses. We hypothesized that mice administered ex vivo rhabdovirus-infected ALL cells [immunotherapy by leukemia-oncotropic virus (iLOV)] would develop robust antileukemic immune responses capable of controlling ALL. Viral protein production, replication, and cytopathy were measured in human and murine ALL cells exposed to attenuated rhabdovirus. Survival following injection of graded amounts of ALL cells was compared between cohorts of mice administered γ-irradiated rhabdovirus-infected ALL cells (iLOV) or multiple control vaccines to determine key immunotherapeutic components and characteristics. Host immune requirements were assessed in immunodeficient and bone marrow-transplanted mice or by adoptive splenocyte transfer from immunized donors. Antileukemic immune memory was ascertained by second leukemic challenge in long-term survivors. Human and murine ALL cells were infected and killed by rhabdovirus; this produced a potent antileukemia vaccine. iLOV protected mice from otherwise lethal ALL by developing durable leukemia-specific immune-mediated responses (P < 0.0001), which required an intact CTL compartment. Preexisting antiviral immunity augmented iLOV potency. Splenocytes from iLOV-vaccinated donors protected 60% of naïve recipients from ALL challenge (P = 0.0001). Injecting leukemia cells activated by, or concurrent with, multiple Toll-like receptor agonists could not reproduce the protective effect of iLOV. Similarly, injecting uninfected irradiated viable, apoptotic, or necrotic leukemia cells with/without concurrent rhabdovirus administration was ineffective. Rhabdovirus-infected leukemia cells can be used to produce a vaccine that induces robust specific immunity against aggressive leukemia.

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

PubMed Central

Chen, W; Qin, H; Chesebro, B; Cheever, M A

1996-01-01

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV. PMID:8892898

The xenotropic murine leukemia virus-related retrovirus debate continues at first international workshop.

PubMed

Stoye, Jonathan P; Silverman, Robert H; Boucher, Charles A; Le Grice, Stuart F J

2010-12-22

The 1st International Workshop on Xenotropic Murine Leukemia Virus-Related Retrovirus (XMRV), co-sponsored by the National Institutes of Health, The Department of Health and Human Services and Abbott Diagnostics, was convened on September 7/8, 2010 on the NIH campus, Bethesda, MD. Attracting an international audience of over 200 participants, the 2-day event combined a series of plenary talks with updates on different aspects of XMRV research, addressing basic gammaretrovirus biology, host response, association of XMRV with chronic fatigue syndrome and prostate cancer, assay development and epidemiology. The current status of XMRV research, concerns among the scientific community and suggestions for future actions are summarized in this meeting report.

Friend and Moloney murine leukemia viruses specifically recombine with different endogenous retroviral sequences to generate mink cell focus-forming viruses.

PubMed

Evans, L H; Cloyd, M W

1985-01-01

A group of mink cell focus-forming (MCF) viruses was derived by inoculation of NFS/N mice with Moloney murine leukemia virus (Mo-MuLV 1387) and was compared to a similarly derived group of MCF viruses from mice inoculated with Friend MuLV (Fr-MuLV 57). Antigenic analyses using monoclonal antibodies specific for MCF virus and xenotropic MuLV envelope proteins and genomic structural analyses by RNase T1-resistant oligonucleotide finger-printing indicated that the Moloney and Friend MCF viruses arose by recombination of the respective ecotropic MuLVs with different endogenous retrovirus sequences of NFS mice.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-02-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed Central

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation. Images PMID:2153242

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-I/sup n/ (NIH-3T3) and two Fv-I/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 genotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-1 cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses (host range restriction/Fv-1 gene/DNA transfection)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-1/sup n/ (NIH-3T3) and two Fv-1/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 geonotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-I cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Moloney leukemia virus immortalizes B lymphocytes in vitro.

PubMed Central

Runnels, J; Serunian, L; Thursby, M; Rosenberg, N

1991-01-01

An in vitro culture system in which Moloney murine leukemia virus induces immortalization of mature B lymphocytes has been developed. The cell lines derived in this way are nontumorigenic, and virus production is not required to sustain them. This system provides a new in vitro model with which to study the stepwise process of transformation by retroviruses lacking oncogenes. Images PMID:1895405

Modeling of Chronic Myeloid Leukemia: An Overview of In Vivo Murine and Human Xenograft Models

PubMed Central

Vellenga, Edo

2016-01-01

Over the past years, a wide variety of in vivo mouse models have been generated in order to unravel the molecular pathology of Chronic Myeloid Leukemia (CML) and to develop and improve therapeutic approaches. These models range from (conditional) transgenic models, knock-in models, and murine bone marrow retroviral transduction models followed by transplantation. With the advancement of immunodeficient xenograft models, it has become possible to use human stem/progenitor cells for in vivo studies as well as cells directly derived from CML patients. These models not only mimic CML but also have been instrumental in uncovering various fundamental mechanisms of CML disease progression and tyrosine kinase inhibitor (TKI) resistance. With the availability of iPSC technology, it has become feasible to derive, maintain, and expand CML subclones that are at least genetically identical to those in patients. The following review provides an overview of all murine as well as human xenograft models for CML established till date. PMID:27642303

Implication of a Central Cysteine Residue and the HHCC Domain of Moloney Murine Leukemia Virus Integrase Protein in Functional Multimerization

PubMed Central

Donzella, George A.; Leon, Oscar; Roth, Monica J.

1998-01-01

Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions important for catalysis of strand transfer and unimolecular and bimolecular disintegration reactions were investigated by using a panel of chemically modified M-MuLV IN proteins. Functional complementation of an HHCC-deleted protein (NΔ105) by an independent HHCC domain (CΔ232) was severely compromised by NEM modification of either subunit. Productive NΔ105 IN-DNA interactions with a disintegration substrate lacking a long terminal repeat 5′-single-stranded tail also required complementation by a functional HHCC domain. Virus encoding the C209A M-MuLV IN mutation exhibited delayed virion production and replication kinetics. PMID:9445080

Synthesis and evaluation of novel N-substituted-6-methoxynaphthalene-2-carboxamides as potential chemosensitizing agents for cancer.

PubMed

Lokhande, Tushar Narendra; Viswanathan, Chelakara Lakshmann; Juvekar, Aarti Shashikant

2008-07-01

A novel class of molecules with structure N-[3-(heteroaryl)propyl]-6-methoxynaphthalene-2-carboxamides 8-13 were synthesized by condensing 6-methoxy-2-naphthoyl chloride 1 with 3-(heteroaryl)propyl amines 2-7. Compounds 8-12 were evaluated in vitro, in P388 murine lymphocytic leukemia cell line (P388) using SRB assay for cytotoxicity and in adriamycin resistant P388 murine lymphocytic leukemia cell line (P388/ADR) using MTT assay for resistant reversal activity. Compounds 8-12 were non-toxic at lower dose of 20 microg/ml, and effectively reversed adriamycin resistance. However, at higher doses (40, 80 microg/ml) they showed significant cytotxicity and hence reversal potency was not determined at these concentrations.

Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo.

PubMed

Chan, Shih-Feng; Chen, Ya-Yin; Lin, Jen-Jyh; Liao, Ching-Lung; Ko, Yang-Ching; Tang, Nou-Ying; Kuo, Chao-Lin; Liu, Kuo-Ching; Chung, Jing-Gung

2017-02-01

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca 2+ release and mitochondrial membrane potential (ΔΨ m ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550-568, 2017. © 2016 Wiley Periodicals, Inc.

Replacement of Murine Leukemia Virus Readthrough Mechanism by Human Immunodeficiency Virus Frameshift Allows Synthesis of Viral Proteins and Virus Replication

PubMed Central

Brunelle, Marie-Noëlle; Brakier-Gingras, Léa; Lemay, Guy

2003-01-01

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model. PMID:12584361

Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia

PubMed Central

Kenderian, Saad S.; Shen, Feng; Ruella, Marco; Shestova, Olga; Kozlowski, Miroslaw; Li, Yong; Schrank-Hacker, April; Morrissette, Jennifer J. D.; Carroll, Martin; June, Carl H.; Grupp, Stephan A.; Gill, Saar

2017-01-01

We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA–electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti–CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML. PMID:28246194

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Genetic mapping of xenotropic murine leukemia virus-inducing loci in five mouse strains

PubMed Central

1980-01-01

A single mendelian gene was identified for induction of the endogenous xenotropic murine leukemia virus in five mouse strains (C57BL/10, C57L, C57BR, AKR, and BALB/c). This locus, designated Bxv-1, mapped to the same site on chromosome 1 in all strains: Id-1-Pep-3-[Bxv-1-Lp]. Thus, inducibility loci for xenotropic virus are more limited in number and chromosomal distribution than ecotropic inducibility loci. Virus expression in mice with Bxv-1 was induced by treatment of fibroblasts with 5-iododeoxyuridine or by exposure of spleen cells to a B cell mitogen, bacterial lipopolysaccharide. An analysis of the hamster X mouse somatic cell hybrids indicated that chromosome 1, alone, was sufficient for virus induction. PMID:6249881

Genetic mapping of xenotropic murine leukemia virus-inducing loci in five mouse strains.

PubMed

Kozak, C A; Rowe, W P

1980-07-01

A single mendelian gene was identified for induction of the endogenous xenotropic murine leukemia virus in five mouse strains (C57BL/10, C57L, C57BR, AKR, and BALB/c). This locus, designated Bxv-1, mapped to the same site on chromosome 1 in all strains: Id-1-Pep-3-[Bxv-1-Lp]. Thus, inducibility loci for xenotropic virus are more limited in number and chromosomal distribution than ecotropic inducibility loci. Virus expression in mice with Bxv-1 was induced by treatment of fibroblasts with 5-iododeoxyuridine or by exposure of spleen cells to a B cell mitogen, bacterial lipopolysaccharide. An analysis of the hamster X mouse somatic cell hybrids indicated that chromosome 1, alone, was sufficient for virus induction.

Differential Susceptibility of Spleen Focus-Forming Virus and Murine Leukemia Viruses to Ansamycin Antibiotics

PubMed Central

Horoszewicz, Julius S.; Leong, Susan S.; Carter, William A.

1977-01-01

The streptovaricin complex (SvCx) and rifamycin SV derivatives display potent antiviral activity against the polycythemic strain of Friend leukemia virus (FV-P), as measured by a reduction in the number of spleen foci produced in mice. Such reductions may be explained by inactivation of functions of (i) the spleen focus-forming virus (SFFV), (ii) its “helper” murine leukemia virus (MuLV), or (iii) both viruses normally present in FV-P. We noted that preincubation of FV-P with fractionation products of SvCx, or derivatives of rifamycin SV, at low concentrations (3 to 5 μg/ml) reduces the number of spleen foci 80 to 97%, whereas titers of MuLV (from the same inoculum) remain unaffected (MuLV titers were measured by XC, S+L−, and “helper activity” assays). Our findings indicate a remarkable biological selectivity of ansamycins, as well as nonansamycin components of SvCx, against the transforming and defective spleen focus-forming virus as compared to MuLV. Thus, the drugs might be useful in distinguishing other types of oncornaviruses. PMID:18986

In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

PubMed Central

Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

2013-01-01

SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

Antibody-mediated targeting of replication-competent retroviral vectors.

PubMed

Tai, Chien-Kuo; Logg, Christopher R; Park, Jinha M; Anderson, W French; Press, Michael F; Kasahara, Noriyuki

2003-05-20

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

Antagonism of SET using OP449 enhances the efficacy of tyrosine kinase inhibitors and overcome drug resistance in myeloid leukemia

PubMed Central

Agarwal, Anupriya; MacKenzie, Ryan J.; Pippa, Raffaella; Eide, Christopher A.; Oddo, Jessica; Tyner, Jeffrey W.; Sears, Rosalie; Vitek, Michael P.; Odero, María D.; Christensen, Dale; Druker, Brian J.

2014-01-01

Purpose The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A. Experimental Design In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis and colonogenic assays. Efficacy of target inhibition by OP449 is evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model. Results We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34+ CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1-mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells. Conclusions We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML. PMID:24436473

Synthesis of adriamycin-coupled polyglutaraldehyde microspheres and evaluation of their cytostatic activity

NASA Technical Reports Server (NTRS)

Tokes, Z. A.; Rogers, K. E.; Rembaum, A.

1982-01-01

Adriamycin was coupled to polyglutaraldehyde microspheres having an average diameter of 4500 A. The coupled microspheres remained stable during incubation with cells. Full cytostatic activity was observed when the coupled adriamycin was tested with murine or human leukemia and murine sarcoma cell lines. A 10-fold increase in sensitivity was obtained with drug-resistant human leukemia cell lines. Repeated use of the coupled microspheres in the cytostatic assays did not decrease their activity, indicating that these complexes can be recycled. The results suggest that coupled adriamycin sufficiently perturbs the plasma membrane to lead to cytostatic activity. It is proposed that this mode of drug delivery provides multiple and repetitious sites for drug-cell interactions. In addition, the drug-polymer complexes may overcome those forms of resistance that are the result of decreased drug binding at the cell surface.

Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling

PubMed Central

Bourquin, Jean-Pierre; Subramanian, Aravind; Langebrake, Claudia; Reinhardt, Dirk; Bernard, Olivier; Ballerini, Paola; Baruchel, André; Cavé, Hélène; Dastugue, Nicole; Hasle, Henrik; Kaspers, Gertjan L.; Lessard, Michel; Michaux, Lucienne; Vyas, Paresh; van Wering, Elisabeth; Zwaan, Christian M.; Golub, Todd R.; Orkin, Stuart H.

2006-01-01

Individuals with Down syndrome (DS) are predisposed to develop acute megakaryoblastic leukemia (AMKL), characterized by expression of truncated GATA1 transcription factor protein (GATA1s) due to somatic mutation. The treatment outcome for DS-AMKL is more favorable than for AMKL in non-DS patients. To gain insight into gene expression differences in AMKL, we compared 24 DS and 39 non-DS AMKL samples. We found that non-DS-AMKL samples cluster in two groups, characterized by differences in expression of HOX/TALE family members. Both of these groups are distinct from DS-AMKL, independent of chromosome 21 gene expression. To explore alterations of the GATA1 transcriptome, we used cross-species comparison with genes regulated by GATA1 expression in murine erythroid precursors. Genes repressed after GATA1 induction in the murine system, most notably GATA-2, MYC, and KIT, show increased expression in DS-AMKL, suggesting that GATA1s fail to repress this class of genes. Only a subset of genes that are up-regulated upon GATA1 induction in the murine system show increased expression in DS-AMKL, including GATA1 and BACH1, a probable negative regulator of megakaryocytic differentiation located on chromosome 21. Surprisingly, expression of the chromosome 21 gene RUNX1, a known regulator of megakaryopoiesis, was not elevated in DS-AMKL. Our results identify relevant signatures for distinct AMKL entities and provide insight into gene expression changes associated with these related leukemias. PMID:16492768

Ionizing Radiation–Inducible miR-27b Suppresses Leukemia Proliferation via Targeting Cyclin A2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Bo; Li, Dongping; Kovalchuk, Anna

2014-09-01

Purpose: Ionizing radiation is a common carcinogen that is important for the development of leukemia. However, the underlying epigenetic mechanisms remain largely unknown. The goal of the study was to explore microRNAome alterations induced by ionizing radiation (IR) in murine thymus, and to determine the role of IR-inducible microRNA (miRNA/miR) in the development of leukemia. Methods and Materials: We used the well-established C57BL/6 mouse model and miRNA microarray profiling to identify miRNAs that are differentially expressed in murine thymus in response to irradiation. TIB152 human leukemia cell line was used to determine the role of estrogen receptor–α (ERα) in miR-27bmore » transcription. The biological effects of ectopic miR-27b on leukemogenesis were measured by western immunoblotting, cell viability, apoptosis, and cell cycle analyses. Results: Here, we have shown that IR triggers the differential expression of miR-27b in murine thymus tissue in a dose-, time- and sex-dependent manner. miR-27b was significantly down-regulated in leukemia cell lines CCL119 and TIB152. Interestingly, ERα was overexpressed in those 2 cell lines, and it was inversely correlated with miR-27b expression. Therefore, we used TIB152 as a model system to determine the role of ERα in miR-27b expression and the contribution of miR-27b to leukemogenesis. β-Estradiol caused a rapid and transient reduction in miR-27b expression reversed by either ERα-neutralizing antibody or ERK1/2 inhibitor. Ectopic expression of miR-27b remarkably suppressed TIB152 cell proliferation, at least in part, by inducing S-phase arrest. In addition, it attenuated the expression of cyclin A2, although it had no effect on the levels of PCNA, PPARγ, CDK2, p21, p27, p-p53, and cleaved caspase-3. Conclusion: Our data reveal that β-estradiol/ERα signaling may contribute to the down-regulation of miR-27b in acute leukemia cell lines through the ERK1/2 pathway, and that miR-27b may function as a tumor suppressor that inhibits cell proliferation by targeting cyclin A2.« less

Progress and prospects: foamy virus vectors enter a new age.

PubMed

Erlwein, O; McClure, M O

2010-12-01

Foamy viruses, distantly related to the major subfamily of Retroviruses, Orthoretroviruses that include oncoviruses (for example, murine leukemia virus (MLV)) and lentiviruses (human immunodeficiency virus (HIV)), are endemic in mammalian species, but not in human populations. Humans infected by accidental or occupational exposure remain well. The virus is not transmitted to others, nor is it associated with any disease. These features added to its broad host range, efficient transduction of progenitor cells and an integration profile less likely to induce insertional mutagenesis, make these viruses attractive as vectors. Long-term reversal of disease phenotype in dogs with the genetic defect, leukocyte adhesion deficiency, by foamy virus vector therapy strengthens the case for their clinical exploitation.

A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo

PubMed Central

Kulcsár, Gyula; Gaál, Dezső; Kulcsár, Péter I; Schulcz, Ákos; Czömpöly, Tamás

2013-01-01

Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances (“active mixture”, AM: l-arginine, l-histidine, l-methionine, l-phenylalanine, l-tyrosine, l-tryptophan, l-ascorbate, d-biotin, pyridoxine, riboflavin, adenine, l(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance of our earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy. PMID:22858865

A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo.

PubMed

Kulcsár, Gyula; Gaál, Dezső; Kulcsár, Péter I; Schulcz, Ákos; Czömpöly, Tamás

2013-03-01

Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances ("active mixture", AM: L-arginine, L-histidine, L-methionine, L-phenylalanine, L-tyrosine, L-tryptophan, L-ascorbate, D-biotin, pyridoxine, riboflavin, adenine, L(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance of our earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy. Copyright © 2012 UICC.

Murine AIDS Protects Mice Against Experimental Cerebral Malaria: Down-Regulation by Interleukin 10 a T-Helper Type 1 CD4^+ Cell-Mediated Pathology

NASA Astrophysics Data System (ADS)

Eckwalanga, Michel; Marussig, Myriam; Dias Tavares, Marisa; Bouanga, Jean Claude; Hulier, Elisabeth; Henriette Pavlovitch, Jana; Minoprio, Paola; Portnoi, Denis; Renia, Laurent; Mazier, Dominique

1994-08-01

The retrovirus LP-BM5 murine leukemia virus induces murine AIDS in C57BL/6 mice that has many similarities with human AIDS; Plasmodium berghei ANKA causes experimental cerebral malaria in the same strain of mice. The outcome of malaria infection was studied in mice concurrently infected with the two pathogens. The retrovirus significantly reduced the gravity of the neurological manifestations associated with Plasmodium berghei ANKA infection. The protection against experimental cerebral malaria induced by murine AIDS increased with duration of viral infection and, hence, with the severity of the immunodeficiency. Interleukin 10, principally from splenic T cells, was shown to play a crucial role in this protection.

cDNA cloning, expression pattern, and chromosomal localization of Mlf1, murine homologue of a gene involved in myelodysplasia and acute myeloid leukemia.

PubMed

Hitzler, J K; Witte, D P; Jenkins, N A; Copeland, N G; Gilbert, D J; Naeve, C W; Look, A T; Morris, S W

1999-07-01

The NPM-MLF1 fusion protein is expressed in blasts from patients with myelodysplasia/acute myeloid leukemia (MDS/AML) containing the t(3;5) chromosomal rearrangement. Nucleophosmin (NPM), a previously characterized nucleolar phosphoprotein, contributes to two other fusion proteins found in lympho-hematopoietic malignancies, anaplastic large cell lymphoma (NPM-ALK) and acute promyelocytic leukemia (NPM-RARalpha). By contrast, the function of the carboxy-terminal fusion partner, myelodysplasia/myeloid leukemia factor 1 (MLF1), is unknown. To aid in understanding normal MLF1 function, we isolated the murine cDNA, determined the chromosomal localization of Mlf1, and defined its tissue expression by in situ hybridization. Mlf1 was highly similar to its human homologue (86% and 84% identical nucleotide and amino acid sequence, respectively) and mapped to the central region of chromosome 3, within a segment lacking known mouse mutations. Mlf1 tissue distribution was restricted during both development and postnatal life, with high levels present only in skeletal, cardiac, and selected smooth muscle, gonadal tissues, and rare epithelial tissues including the nasal mucosa and the ependyma/choroid plexus in the brain. Mlf1 transcripts were undetectable in the lympho-hematopoietic organs of both the embryonic and adult mouse, suggesting that NPM-MLF1 contributes to the genesis of MDS/AML in part by enforcing the ectopic overexpression of MLF1 within hematopoietic tissues.

Microenvironment Determines Lineage Fate in a Human Model of MLL-AF9 Leukemia

PubMed Central

Wei, Junping; Wunderlich, Mark; Fox, Catherine; Alvarez, Sara; Cigudosa, Juan C.; Wilhelm, Jamie S.; Zheng, Yi; Cancelas, Jose A.; Gu, Yi; Jansen, Michael; DiMartino, Jorge F.; Mulloy, James C.

2008-01-01

Summary Faithful modeling of mixed lineage leukemia in murine cells has been difficult to achieve. We show that expression of MLL-AF9 in human CD34+ cells induces acute myeloid, lymphoid or mixed lineage leukemia in immunodeficient mice. Some leukemia stem cells (LSC) were multipotent and could be lineage directed by altering either the growth factors or the recipient strain of mouse, highlighting the importance of microenviromental cues. Other LSC were strictly lineage committed, demonstrating the heterogeneity of the stem cell compartment in MLL disease. Targeting the Rac signaling pathway by pharmacologic or genetic means resulted in rapid and specific apoptosis of MLL-AF9 cells, suggesting that the Rac signaling pathway may be a valid therapeutic target in MLL-rearranged AML. PMID:18538732

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Gustchina, Alla; Matúz, Krisztina

Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K{sub i}, depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A weremore » solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.« less

Murine Leukemia Viruses: Objects and Organisms

PubMed Central

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes—protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera. PMID:22312342

Murine leukemia viruses: objects and organisms.

PubMed

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes-protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.

In Vivo Hypermutation of Xenotropic Murine Leukemia Virus-Related Virus DNA in Peripheral Blood Mononuclear Cells of Rhesus Macaque by APOBEC3 Proteins

PubMed Central

Zhang, Ao; Bogerd, Hal; Villinger, Francois; Gupta, Jaydip Das; Dong, Beihua; Klein, Eric A.; Hackett, John; Schochetman, Gerald; Cullen, Bryan R.; Silverman, Robert H.

2011-01-01

The gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), replicates to high titers in some human cell lines and is able to infect non-human primates. To determine whether APOBEC3 (A3) proteins restrict XMRV infections in a non-human primate model, we sequenced proviral DNA from peripheral blood mononuclear cells of XMRV-infected rhesus macaques. Hypermutation characteristic of A3DE, A3F and A3G activities was observed in the XMRV proviral sequences in vivo. Furthermore, expression of rhesus A3DE, A3F, or A3G in human cells inhibited XMRV infection and caused hypermutation of XMRV DNA. These studies show that some rhesus A3 isoforms are highly effective against XMRV in the blood of a non-human primate model of infection and in cultured human cells. PMID:21982221

Removal of xenotropic murine leukemia virus by nanocellulose based filter paper.

PubMed

Asper, M; Hanrieder, T; Quellmalz, A; Mihranyan, A

2015-11-01

The removal of xenotrpic murine leukemia virus (xMuLV) by size-exclusion filter paper composed of 100% naturally derived cellulose was validated. The filter paper was produced using cellulose nanofibers derived from Cladophora sp. algae. The filter paper was characterized using atomic force microscopy, scanning electron microscopy, helium pycnometry, and model tracer (100 nm latex beads and 50 nm gold nanoparticles) retention tests. Following the filtration of xMuLV spiked solutions, LRV ≥5.25 log10 TCID50 was observed, as limited by the virus titre in the feed solution and sensitivity of the tissue infectivity test. The results of the validation study suggest that the nanocellulose filter paper is useful for removal of endogenous rodent retroviruses and retrovirus-like particles during the production of recombinant proteins. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Podoscyphic acid, a new inhibitor of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptase from a Podoscypha species.

PubMed

Erkel, G; Anke, T; Velten, R; Steglich, W

1991-01-01

A novel enzyme inhibitor of RNA-directed DNA-polymerases of avian myeloblastosis and murine leukemia virus was isolated from fermentations of an tasmanian Podoscypha species. Its structure was elucidated by spectroscopic methods and oxidative degradation as (E)-4,5-dioxo-2-hexadecenoic acid (1). The enzyme inhibitor, which was named podoscyphic acid, did not inhibit DNA and RNA synthesis in permeabilized L 1210 cells nor did it affect RNA synthesis in isolated nuclei of L 1210 cells. 1 inhibits protein synthesis in whole L 1210 cells and rabbit reticulocyte lysate and shows very weak antimicrobial and cytotoxic properties. The testing of ethyl (E)-4,5-dioxo-2-hexadecenoate (2) and (E)-4-oxo-2-tetradecenoic acid (11) revealed the importance of the free gamma-oxoacrylic acid unit for the biological activities of 1.

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

PubMed Central

Sharma, Sanjai; Murai, Fukashi; Miyanohara, Atsushi; Friedmann, Theodore

1997-01-01

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 105 colony-forming units per ml. PMID:9380714

Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tress, E.; Pierotti, M.; DeLeo, A.B.

1982-02-01

To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulsechase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65/sup gag/, was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95/sup gag/, or its precursor, Pr75/sup gag/. No evidence was found for synthesis of gag-host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instancesmore » a monoclonal antibody, 35/56, which is specific for the NuLV G/sub IX/ antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same 35/56 phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.« less

Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant

PubMed Central

SONG, NINGXIA; GAO, LEI; QIU, HUIYING; HUANG, CHONGMEI; CHENG, HUI; ZHOU, HONG; LV, SHUQING; CHEN, LI; WANG, JIANMIN

2015-01-01

The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft-versus-host disease (aGVHD). However, the role of MSCs in graft-versus-leukemia remains to be determined. In the present study, we co-cultured C57BL/6 mouse bone marrow (BM)-derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit-8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)-10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies. PMID:25901937

Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant.

PubMed

Song, Ningxia; Gao, Lei; Qiu, Huiying; Huang, Chongmei; Cheng, Hui; Zhou, Hong; Lv, Shuqing; Chen, Li; Wang, Jianmin

2015-07-01

The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft‑versus‑host disease (aGVHD). However, the role of MSCs in graft‑versus‑leukemia remains to be determined. In the present study, we co‑cultured C57BL/6 mouse bone marrow (BM)‑derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit‑8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)‑10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies.

A Dominantly Acting Murine Allele of Mcm4 Causes Chromosomal Abnormalities and Promotes Tumorigenesis

PubMed Central

Bagley, Bruce N.; Keane, Thomas M.; Maklakova, Vilena I.; Marshall, Jonathon G.; Lester, Rachael A.; Cancel, Michelle M.; Paulsen, Alex R.; Bendzick, Laura E.; Been, Raha A.; Kogan, Scott C.; Cormier, Robert T.; Kendziorski, Christina; Adams, David J.; Collier, Lara S.

2012-01-01

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4D573H). MCM4 is part of the heterohexameric complex of MCM2–7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4D573H to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities. PMID:23133403

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

PubMed Central

Li, Fang; Ryu, Byoung Y.; Krueger, Robin L.; Heldt, Scott A.

2012-01-01

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 106 transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins. PMID:22013043

A dominantly acting murine allele of Mcm4 causes chromosomal abnormalities and promotes tumorigenesis.

PubMed

Bagley, Bruce N; Keane, Thomas M; Maklakova, Vilena I; Marshall, Jonathon G; Lester, Rachael A; Cancel, Michelle M; Paulsen, Alex R; Bendzick, Laura E; Been, Raha A; Kogan, Scott C; Cormier, Robert T; Kendziorski, Christina; Adams, David J; Collier, Lara S

2012-01-01

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4(D573H)). MCM4 is part of the heterohexameric complex of MCM2-7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4(D573H) to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities.

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

NASA Astrophysics Data System (ADS)

Dianhar, Hanhan; Syah, Yana Maolana; Mujahidin, Didin; Hakim, Euis Holisotan; Juliawaty, Lia Dewi

2014-03-01

Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though the extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR (1H-NMR and 13C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC50 value of 60.04 μg/mL and 5.40 μg/mL, respectively.

A flavone derivative from Sesbania sesban leaves and its cytotoxicity against murine leukemia P-388 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dianhar, Hanhan, E-mail: liadewi@chem.itb.ac.id; Syah, Yana Maolana, E-mail: liadewi@chem.itb.ac.id; Mujahidin, Didin, E-mail: liadewi@chem.itb.ac.id

2014-03-24

Sesbania sesban, locally named as Jayanti, is one of Indonesia plants belonging to Fabaceae family. This species is traditionally used by Indonesian people to cure digestive disorders, fever, or headache. Jayanti can grow well in tropical to subtropical region, such as in Asia and Africa. Based on literature, qualitative analysis of the methanol extract of leaves of S. sesban showed that it contained flavonoids, alkaloids, saponins and glycosides. In addition, the activity assay of extracts of different tissues of this species showed antitumor, antimalarial, and antidiabetic activityies (leaves and seed extracts), antioxidants (flower extract), and analgesic (wood extract). Though themore » extracts of S. sesban parts showed interesting activities, chemical study of those extracts have not been widely reported. Therefore, the objective of this research was to isolate the secondary metabolites from methanol extract of leaves of S. sesban and to determine their cytotoxicity against murine leukemia P-388 cells. One compound has been obtained and identified as 3-hydroxy-4',7-dimethoxyflavone (1), a new isolated compound from nature. This compound was obtained through separation of methanol extract using various chromatographic techniques, such as vacuum liquid chromatography and radial chromatography. The structure elucidation of isolated compound was based on 1D NMR ({sup 1}H-NMR and {sup 13}C-NMR) and 2D NMR (HMBC). The cytotoxicity of methanol extract and compound 1 against murine leukemia P-388 cells examined through MTT assay showed IC{sub 50} value of 60.04 μg/mL and 5.40 μg/mL, respectively.« less

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

PubMed Central

Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

2010-01-01

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector. PMID:20372106

cDNA Cloning, Expression Pattern, and Chromosomal Localization of Mlf1, Murine Homologue of a Gene Involved in Myelodysplasia and Acute Myeloid Leukemia

PubMed Central

Hitzler, Johann K.; Witte, David P.; Jenkins, Nancy A.; Copeland, Neal G.; Gilbert, Debra J.; Naeve, Clayton W.; Look, A. Thomas; Morris, Stephan W.

1999-01-01

The NPM-MLF1 fusion protein is expressed in blasts from patients with myelodysplasia/acute myeloid leukemia (MDS/AML) containing the t(3;5) chromosomal rearrangement. Nucleophosmin (NPM), a previously characterized nucleolar phosphoprotein, contributes to two other fusion proteins found in lympho-hematopoietic malignancies, anaplastic large cell lymphoma (NPM-ALK) and acute promyelocytic leukemia (NPM-RARα). By contrast, the function of the carboxy-terminal fusion partner, myelodysplasia/myeloid leukemia factor 1 (MLF1), is unknown. To aid in understanding normal MLF1 function, we isolated the murine cDNA, determined the chromosomal localization of Mlf1, and defined its tissue expression by in situ hybridization. Mlf1 was highly similar to its human homologue (86% and 84% identical nucleotide and amino acid sequence, respectively) and mapped to the central region of chromosome 3, within a segment lacking known mouse mutations. Mlf1 tissue distribution was restricted during both development and postnatal life, with high levels present only in skeletal, cardiac, and selected smooth muscle, gonadal tissues, and rare epithelial tissues including the nasal mucosa and the ependyma/choroid plexus in the brain. Mlf1 transcripts were undetectable in the lympho-hematopoietic organs of both the embryonic and adult mouse, suggesting that NPM-MLF1 contributes to the genesis of MDS/AML in part by enforcing the ectopic overexpression of MLF1 within hematopoietic tissues. PMID:10393836

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells.

PubMed

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine Kl; Berman, Jason N; Rupasinghe, Hp Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo . We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway.

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells

PubMed Central

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine KL; Berman, Jason N; Rupasinghe, HP Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo. We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway. PMID:29312799

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

PubMed Central

Laprevotte, I; Hampe, A; Sherr, C J; Galibert, F

1984-01-01

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing. PMID:6328019

Polyploidization without mitosis improves in vivo liver transduction with lentiviral vectors.

PubMed

Pichard, Virginie; Couton, Dominique; Desdouets, Chantal; Ferry, Nicolas

2013-02-01

Lentiviral vectors are efficient gene delivery vehicles for therapeutic and research applications. In contrast to oncoretroviral vectors, they are able to infect most nonproliferating cells. In the liver, induction of cell proliferation dramatically improved hepatocyte transduction using all types of retroviral vectors. However, the precise relationship between hepatocyte division and transduction efficiency has not been determined yet. Here we compared gene transfer efficiency in the liver after in vivo injection of recombinant lentiviral or Moloney murine leukemia viral (MoMuLV) vectors in hepatectomized rats treated or not with retrorsine, an alkaloid that blocks hepatocyte division and induces megalocytosis. Partial hepatectomy alone resulted in a similar increase in hepatocyte transduction using either vector. In retrorsine-treated and partially hepatectomized rats, transduction with MoMuLV vectors dropped dramatically. In contrast, we observed that retrorsine treatment combined with partial hepatectomy increased lentiviral transduction to higher levels than hepatectomy alone. Analysis of nuclear ploidy in single cells showed that a high level of transduction was associated with polyploidization. In conclusion, endoreplication could be exploited to improve the efficiency of liver-directed lentiviral gene therapy.

Polyploidization Without Mitosis Improves In Vivo Liver Transduction With Lentiviral Vectors

PubMed Central

Couton, Dominique; Desdouets, Chantal; Ferry, Nicolas

2013-01-01

Abstract Lentiviral vectors are efficient gene delivery vehicles for therapeutic and research applications. In contrast to oncoretroviral vectors, they are able to infect most nonproliferating cells. In the liver, induction of cell proliferation dramatically improved hepatocyte transduction using all types of retroviral vectors. However, the precise relationship between hepatocyte division and transduction efficiency has not been determined yet. Here we compared gene transfer efficiency in the liver after in vivo injection of recombinant lentiviral or Moloney murine leukemia viral (MoMuLV) vectors in hepatectomized rats treated or not with retrorsine, an alkaloid that blocks hepatocyte division and induces megalocytosis. Partial hepatectomy alone resulted in a similar increase in hepatocyte transduction using either vector. In retrorsine-treated and partially hepatectomized rats, transduction with MoMuLV vectors dropped dramatically. In contrast, we observed that retrorsine treatment combined with partial hepatectomy increased lentiviral transduction to higher levels than hepatectomy alone. Analysis of nuclear ploidy in single cells showed that a high level of transduction was associated with polyploidization. In conclusion, endoreplication could be exploited to improve the efficiency of liver-directed lentiviral gene therapy. PMID:23249390

Frequent Infection of Human Cancer Xenografts with Murine Endogenous Retroviruses in Vivo

PubMed Central

Naseer, Asif; Terry, Anne; Gilroy, Kathryn; Kilbey, Anna; Watts, Ciorsdaidh; Mackay, Nancy; Bell, Margaret; Mason, Susan; Blyth, Karen; Cameron, Ewan; Neil, James C.

2015-01-01

Infection of human cancer xenografts in mice with murine leukemia viruses (MLVs) is a long-standing observation, but the likelihood of infection in vivo and its biological consequences are poorly understood. We therefore conducted a prospective study in commonly used xenograft recipient strains. From BALB/c nude mice engrafted with MCF7 human mammary carcinoma cells, we isolated a virus that was virtually identical to Bxv1, a locus encoding replication-competent xenotropic MLV (XMLV). XMLV was detected in 9/17 (53%) independently isolated explants. XMLV was not found in primary leukemias or in THP1 leukemia cells grown in Bxv1-negative NSG (NOD/SCID/γCnull) mice, although MCF7 explants harbored replication-defective MLV proviruses. To assess the significance of infection for xenograft behavior in vivo, we examined changes in growth and global transcription in MCF7 and the highly susceptible Raji Burkitt lymphoma cell line chronically infected with XMLV. Raji cells showed a stronger transcriptional response that included up-regulation of chemokines and effectors of innate antiviral immunity. In conclusion, the risk of de novo XMLV infection of xenografts is high in Bxv1 positive mice, while infection can have positive or negative effects on xenograft growth potential with significant consequences for interpretation of many xenograft studies. PMID:25912714

Lentiviral Vector Induced Insertional Haploinsufficiency of Ebf1 Causes Murine Leukemia

PubMed Central

Heckl, Dirk; Schwarzer, Adrian; Haemmerle, Reinhard; Steinemann, Doris; Rudolph, Cornelia; Skawran, Britta; Knoess, Sabine; Krause, Johanna; Li, Zhixiong; Schlegelberger, Brigitte; Baum, Christopher; Modlich, Ute

2012-01-01

Integrating vectors developed on the basis of various retroviruses have demonstrated therapeutic potential following genetic modification of long-lived hematopoietic stem and progenitor cells. Lentiviral vectors (LV) are assumed to circumvent genotoxic events previously observed with γ-retroviral vectors, due to their integration bias to transcription units in comparison to the γ-retroviral preference for promoter regions and CpG islands. However, recently several studies have revealed the potential for gene activation by LV insertions. Here, we report a murine acute B-lymphoblastic leukemia (B-ALL) triggered by insertional gene inactivation. LV integration occurred into the 8th intron of Ebf1, a major regulator of B-lymphopoiesis. Various aberrant splice variants could be detected that involved splice donor and acceptor sites of the lentiviral construct, inducing downregulation of Ebf1 full-length message. The transcriptome signature was compatible with loss of this major determinant of B-cell differentiation, with partial acquisition of myeloid markers, including Csf1r (macrophage colony-stimulating factor (M-CSF) receptor). This was accompanied by receptor phosphorylation and STAT5 activation, both most likely contributing to leukemic progression. Our results highlight the risk of intragenic vector integration to initiate leukemia by inducing haploinsufficiency of a tumor suppressor gene. We propose to address this risk in future vector design. PMID:22472950

No Evidence for Xenotropic Murine Leukemia-Related Virus Infection in Sweden Using Internally Controlled Multiepitope Suspension Array Serology

PubMed Central

Blomberg, Fredrik; Sjösten, Anna; Sheikholvaezin, Ali; Bölin-Wiener, Agnes; Elfaitouri, Amal; Hessel, Sanna; Gottfries, Carl-Gerhard; Zachrisson, Olof; Öhrmalm, Christina; Jobs, Magnus; Pipkorn, Rüdiger

2012-01-01

Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control. PMID:22787191

Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors.

PubMed

López-Lastra, M; Gabus, C; Darlix, J L

1997-11-01

The murine leukemia virus (MLV)-related type C viruses constitute a major class of retroviruses that includes numerous endogenous and exogenous mammalian viruses and the related avian spleen necrosis virus (SNV). The MLV-related viruses possess a long and multifunctional 5' untranslated leader involved in key steps of the viral life cycle--splicing, translation, RNA dimerization, encapsidation, and reverse transcription. Recent studies have shown that the 5' leader of Friend murine leukemia virus and Moloney murine leukemia virus can direct cap independent translation of gag precursor proteins (Berlioz et al., 1995; Vagner et al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new internal ribosome entry segment (IRES) of retroviral origin. Here we describe an IRES element within the 5' leader of avian reticuloendotheliosis virus type A (REV-A) genomic RNA. Data show that the REV-A 5' IRES element maps downstream of the packaging/dimerization (E/DLS) sequence (Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES sequence appears to be within a 129 nt fragment (nucleotides 452-580) of the 5' leader, immediately upstream of the gag AUG codon. The REV-A IRES has been successfully utilized in the construction of novel high titer MLV-based retroviral vectors, containing one or more IRES elements of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene therapy, are also of interest as a model system of internal translation initiation and its possible regulation during development, cancer, or virus infection.

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

PubMed Central

Feddersen, R M; Van Ness, B G

1990-01-01

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed. Images PMID:2153918

Effects of murine leukemia virus env gene proteins on macrophage-mediated cytotoxicity in vitro

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

1991-01-01

F5b Tumor cells were incubated with concentrated culture supernatants taken from cells resistant (F5m) or sensitive (F5b) to contact-dependent macrophage cytotoxicity. Macrophage cell line B6MP102 and murine peritoneal macrophages killed targets incubated with supernatants taken from sensitive cells but poorly killed cells incubated in supernatants isolated from resistant cells. Membranes from cells resistant to macrophage killing, F5m, were fused into F5b cells. The fused F5b cells were killed significantly less than F5b cells fused with F5b cell membranes or untreated F5b cells. The decreased killing of F5b cells corresponded to increased concentrations of gp70(a) molecules on F5b cells. Affinity purified gp70(a) was added to cytotoxicity assays but failed to inhibit macrophage cytotoxicity. P15E molecules were detectable on both F5b and F5m cells. In addition, a synthetic peptide found to exhibit the inhibitory properties of p15E was added to cytotoxicity assays. P15E synthetic peptide also did not inhibit macrophage cytotoxicity. Therefore, env gene proteins of murine leukemia virus do not appear responsible for inducing tumor cell resistance to activated macrophage contact-dependent cytotoxicity.

Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin.

PubMed

Polonoff, E; Machida, C A; Kabat, D

1982-12-10

Addition of asparagine-linked oligosaccharides to nascent murine leukemia virus (MuLV)-encoded membrane glycoproteins was inhibited either completely by tunicamycin or specifically at Asn-X-Thr glycosylation sites by incorporation of the threonine analogue beta-hydroxynorvaline. In conditions of partial analogue substitution, a series of subglycosylated components is formed which are related by a constant apparent Mr difference when assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The total number of asparagine-linked oligosaccharides is then estimated by dividing the measured apparent Mr of one oligosaccharide into the total apparent Mr difference between the complete glycoprotein and the polypeptide chain that is synthesized in cells incubated with tunicamycin. Correct results were obtained using glycoproteins with known numbers of oligosaccharides. Our analyses indicate that the gp70 membrane envelope glycoproteins of certain ecotropic MuLVs contain seven oligosaccharides, whereas the GIX+ antigen-containing variant gp70 contains one fewer Asn-X-Thr-linked oligosaccharide. The membrane glycoprotein encoded by the gag gene of Friend MuLV contains only one asparagine-linked oligosaccharide. Similarly, the gp55 membrane glycoprotein encoded by Friend erythroleukemia virus contains four asparagine-linked oligosaccharides. Pulse-chase and cell surface iodination analyses indicate that MuLV membrane envelope glycoprotein processing by partial proteolysis and transport to the cell surface can be efficiently blocked by structural perturbations caused by incorporation of different amino acid analogues or by loss of oligosaccharides. Our data also suggest that loss of oligosaccharides may expose new antigenic sites in viral membrane glycoproteins and increase their susceptibility to intracellular proteolysis.

Induction of Protective CTL Responses in Newborn Mice by a Murine Retrovirus

NASA Astrophysics Data System (ADS)

Sarzotti, Marcella; Robbins, Deanna S.; Hoffman, Paul M.

1996-03-01

The susceptibility of neonates to virus-induced disease is thought to reflect, in part, the immaturity of their immune systems. However, inoculation of newborn mice with low doses of Cas-Br-M murine leukemia virus induced a protective cytotoxic T lymphocyte (CTL) response. The inability of neonates to develop a CTL response to high doses of virus was not the result of immunological immaturity but correlated with the induction of a nonprotective type 2 cytokine response. Thus, the initial viral dose is critical in the development of protective immunity in newborns.

Murine Leukemia Virus Reverse Transcriptase: Structural Comparison with HIV-1 Reverse Transcriptase

PubMed Central

Coté, Marie L.; Roth, Monica J.

2008-01-01

Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information. PMID:18294720

Infectivity and RNA patterns as functions of high- and low-dilution passage of murine sarcoma-leukemia virus: evidence for autointerference within an oncornavirus population.

PubMed

Bondurant, M C; Hackett, A J; Schaffer, F L

1973-05-01

Heterogeneity of buoyant density and RNA content of virions of Moloney murine leukemia-sarcoma complex [MSV (MLV)] was the result of passage at low dilution. Heterogeneous stocks revealed two major RNA components in the population, with the smaller component, apparent mol wt 4 x 10(6) to 5 x 10(6), becoming predominant upon serial passage at low dilution. Concomitantly, infectivity titers of both MLV and MSV decreased upon serial passage at low dilution. MSV (MLV) passaged at high dilution retained high titers and a rather homogeneous high-molecular-weight RNA population characteristic of high-buoyant-density virions. Interference of both MLV and MSV replication was demonstrated by employing mixed inocula containing both low- and high-dilution passage stocks of MSV (MLV). In contrast to results with MSV (MLV), MLV freed of MSV by limit dilution did not show heterogeneity of buoyant density or of RNA when propagated at low dilution.

Infectivity and RNA Patterns as Functions of High- and Low-Dilution Passage of Murine Sarcoma-Leukemia Virus: Evidence for Autointerference Within an Oncornavirus Population

PubMed Central

Bondurant, M. C.; Hackett, A. J.; Schaffer, F. L.

1973-01-01

Heterogeneity of buoyant density and RNA content of virions of Moloney murine leukemia-sarcoma complex [MSV (MLV)] was the result of passage at low dilution. Heterogeneous stocks revealed two major RNA components in the population, with the smaller component, apparent mol wt 4 × 106 to 5 × 106, becoming predominant upon serial passage at low dilution. Concomitantly, infectivity titers of both MLV and MSV decreased upon serial passage at low dilution. MSV (MLV) passaged at high dilution retained high titers and a rather homogeneous high-molecular-weight RNA population characteristic of high-buoyant-density virions. Interference of both MLV and MSV replication was demonstrated by employing mixed inocula containing both low- and high-dilution passage stocks of MSV (MLV). In contrast to results with MSV (MLV), MLV freed of MSV by limit dilution did not show heterogeneity of buoyant density or of RNA when propagated at low dilution. PMID:4350709

B lymphoma Moloney murine leukemia virus insertion region 1: An oncogenic mediator in prostate cancer.

PubMed

Liu, Qipeng; Li, Qiaqia; Zhu, Sen; Yi, Yang; Cao, Qi

2018-06-01

B lymphoma Moloney murine leukemia virus insertion region 1 (BMI1), a core member of polycomb repressive complex 1 (PRC1), has been intensely investigated in the field of cancer epigenetics for decades. Widely known as a critical regulator in cellular physiology, BMI1 is essential in self-renewal and differentiation in different lineages of stem cells. BMI1 also plays a significant role in cancer etiology for its involvement in pathological progress such as epithelial-mesenchymal transition (EMT) and cancer stem cell maintenance, propagation, and differentiation. Importantly, overexpression of BMI1 is predictive for drug resistance, tumor recurrence, and eventual therapy failure of various cancer subtypes, which renders the pharmacological targeting at BMI1 as a novel and promising therapeutic approach. The study on prostate cancer, a prevalent hormone-related cancer among men, has promoted enormous research advancements in cancer genetics and epigenetics. This review summarizes the role of BMI1 as an oncogenic and epigenetic regulator in tumor initiation, progression, and relapse of prostate cancer.

Murine and human leukemias.

PubMed

Burchenal, J H

1975-01-01

Essentially all the drugs which are active against human leukemias and lymphomas are active against one type or another of the rodent leukemias and lymphomas. Leukemia L1210 has been generally the most successful screening tool for clinically active compounds. Leukemia P388, however, seems to be better in detecting active antibiotics and natural products and P1534 is particularly sensitive to the Vinca alkaloids, while L5178Y, EARAD, and 6C3HED are useful in detecting the activities of various asparaginase containing fractions. Cell cultures of these leukemias can demonstrate mechanism of drug action and quantitate resistance. Spontaneous AKR leukemia is a model of the advanced human disease. In these leukemias vincristine and prednisone produce a 4 log cell kill. Cytoxan and arabinosyl cytosine (Ara-C) are also effective. On the other hand drugs such as mercaptopurine (6MP) and methotrexate which are highly active in the maintenance phase of acute lymphocytic leukemia (ALL) and in L1210 have little or no activity against the AKR spontaneous system. Mouse leukemias can also detect schedule dependence, synergistic combinations, cross resistance, oral activity, and the ability of drugs to pass the blood brain barrier. A case in point is the Ara-C analog 2,2'-anhydro-arabinofuranosyl-5-fluorocytosine (AAFC) which is not schedule dependent, is active orally, is potentiated by thioguanine, and is effective against intracerebrally inoculated mouse leukemia. AAFC and its analogs might thus be a considerable improvement over Ara-C which is at the present time the most important component of the combination treatment of acute myelogenous leukemia (AML).

A microfluidic method for synthesis of transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia

PubMed Central

Yang, Zhaogang; Yu, Bo; Zhu, Jing; Huang, Xiaomeng; Xie, Jing; Xu, Songlin; Yang, Xiaojuan; Wang, Xinmei; Yung, Bryant C.; Lee, L. James; Lee, Robert J.; Teng, Lesheng

2015-01-01

siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for targeted delivery of LOR-1284 siRNA to acute myeloid leukemia (AML) cells. In vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4–11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than free LOR-1284 in reducing the R2 mRNA and protein levels. Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific thereapeutic delivery of LOR-1284 to AML. PMID:25003978

Diet-induced obesity accelerates acute lymphoblastic leukemia progression in two murine models.

PubMed

Yun, Jason P; Behan, James W; Heisterkamp, Nora; Butturini, Anna; Klemm, Lars; Ji, Lingyun; Groffen, John; Müschen, Markus; Mittelman, Steven D

2010-10-01

Obesity is associated with an increased incidence of many cancers, including leukemia, although it is unknown whether leukemia incidence is increased directly by obesity or rather by associated genetic, lifestyle, health, or socioeconomic factors. We developed animal models of obesity and leukemia to test whether obesity could directly accelerate acute lymphoblastic leukemia (ALL) using BCR/ABL transgenic and AKR/J mice weaned onto a high-fat diet. Mice were observed until development of progressive ALL. Although obese and control BCR/ABL mice had similar median survival, older obese mice had accelerated ALL onset, implying a time-dependent effect of obesity on ALL. Obese AKR mice developed ALL significantly earlier than controls. The effect of obesity was not explained by WBC count, thymus/spleen weight, or ALL phenotype. However, obese AKR mice had higher leptin, insulin, and interleukin-6 levels than controls, and these obesity-related hormones all have potential roles in leukemia pathogenesis. In conclusion, obesity directly accelerates presentation of ALL, likely by increasing the risk of an early event in leukemogenesis. This is the first study to show that obesity can directly accelerate the progression of ALL. Thus, the observed associations between obesity and leukemia incidence are likely to be directly related to biological effects of obesity. ©2010 AACR.

Selenium suppresses leukemia through the action of endogenous eicosanoids

PubMed Central

Gandhi, Ujjawal H.; Kaushal, Naveen; Hegde, Shailaja; Finch, Emily R.; Kudva, Avinash K.; Kennett, Mary J.; Jordan, Craig T.; Paulson, Robert F.; Prabhu, K. Sandeep

2014-01-01

Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiological but non-toxic doses. In leukemia CSC, selenium treatment activated ATM-p53-dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR-ABL-expressing CSC revealed that the selenium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production of the cyclooxygenase-derived prostaglandins Δ12-PGJ2 and 15d-PGJ2. Accordingly, non-steroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia. PMID:24872387

Murine thymic lymphoma is associated with a species-specific hematopoietic progenitor cell subpopulation

NASA Technical Reports Server (NTRS)

Irons, R. D.; Colagiovanni, D. B.; Stillman, W. S.; Clarkson, T. W. (Principal Investigator)

1996-01-01

Many strains of laboratory mouse are uniquely susceptible to the development of T cell lymphoma/leukemia, either spontaneously or as a result of chemical or radiation exposure. In contrast, T cell leukemias or lymphomas which are relatively uncommon in human populations, are not easily induced by radiation, and are not generally associated with chemotherapy or chemical exposure. Evidence is presented to suggest that differences in the susceptibility to the development of these malignancies is related to subtle but important variations in the regulation of hematopoietic stem cell differentiation between these two species.

Toona sinensis Inhibits Murine Leukemia WEHI-3 Cells and Promotes Immune Response In Vivo.

PubMed

Yang, Hsin-Ling; Thiyagarajan, Varadharajan; Liao, Jiunn-Wang; Chu, Yu-Lin; Chang, Chia-Ting; Huang, Pei-Jane; Hsu, Chih-Jung; Hseu, You-Cheng

2017-09-01

Toona sinensis (TS) is one of the most popular vegetarian dishes in Taiwan. It has been shown to exhibit antioxidant, antiangiogenic, antiatherosclerotic, and anticancer properties. In this study, we demonstrated the ability of aqueous leaf extracts from TS to promote immune responses in BALB/c mice and to exhibit anti-leukemia activity in murine WEHI-3 cells. BALB/c mice were injected intravenously with WEHI-3 cells and then treated orally with TS (50 mg/kg). In vivo study showed that TS treatment reduced liver and spleen enlargement in WEHI-3 bearing mice compared with the untreated group. Furthermore, TS also decreased white blood cells (WBC), indicating inhibition of differentiation of the precursor of macrophages in WEHI-3 bearing mice. Treatment of WEHI-3 cells with TS (0-75 μg/mL for 24 hours) significantly reduced cell viability. Furthermore, TS treatment-induced late apoptosis was confirmed by Annexin-V/PI staining. Western blot analyses revealed that treatment of WEHI-3 cells with TS statistically increased the protein expression level of cytochrome c in the cytoplasm and activates caspase-3. Notably, TS treatment caused a dramatic reduction in Bcl-2 and increase in Bax protein levels. TS may disturb the Bcl-2 and Bax protein ratio and induce apoptosis. This reports confirms the antitumor activity of this nutritious vegetable potentially against leukemia.

Enhancers of Polycomb EPC1 and EPC2 sustain the oncogenic potential of MLL leukemia stem cells

PubMed Central

Huang, Xu; Spencer, Gary J; Lynch, James T; Ciceri, Filippo; Somerville, Tim D D; Somervaille, Tim C P

2013-01-01

Through a targeted knockdown (KD) screen of chromatin regulatory genes we identified the EP400 complex components EPC1 and EPC2 as critical oncogenic co-factors in acute myeloid leukemia (AML). EPC1 and EPC2 were required for the clonogenic potential of human AML cells of multiple molecular subtypes. Focusing on MLL-mutated AML as an exemplar, Epc1 or Epc2 KD induced apoptosis of murine MLL-AF9 AML cells and abolished leukemia stem cell potential. By contrast, normal hematopoietic stem and progenitor cells (HSPC) were spared. Similar selectivity was observed for human primary AML cells versus normal CD34+ HSPC. In keeping with these distinct functional consequences, Epc1 or Epc2 KD induced divergent transcriptional consequences in murine MLL-AF9 granulocyte-macrophage progenitor-like (GMP) cells versus normal GMP, with a signature of increased MYC activity in leukemic but not normal cells. This was caused by accumulation of MYC protein and was also observed following KD of other EP400 complex genes. Pharmacological inhibition of MYC:MAX dimerization, or concomitant MYC KD, reduced apoptosis following EPC1 KD, linking the accumulation of MYC to cell death. Therefore EPC1 and EPC2 are components of a complex which directly or indirectly serves to prevent MYC accumulation and AML cell apoptosis, thus sustaining oncogenic potential. PMID:24166297

Toona sinensis Inhibits Murine Leukemia WEHI-3 Cells and Promotes Immune Response In Vivo

PubMed Central

Yang, Hsin-Ling; Thiyagarajan, Varadharajan; Liao, Jiunn-Wang; Chu, Yu-Lin; Chang, Chia-Ting; Huang, Pei-Jane; Hsu, Chih-Jung; Hseu, You-Cheng

2016-01-01

Toona sinensis (TS) is one of the most popular vegetarian dishes in Taiwan. It has been shown to exhibit antioxidant, antiangiogenic, antiatherosclerotic, and anticancer properties. In this study, we demonstrated the ability of aqueous leaf extracts from TS to promote immune responses in BALB/c mice and to exhibit anti-leukemia activity in murine WEHI-3 cells. BALB/c mice were injected intravenously with WEHI-3 cells and then treated orally with TS (50 mg/kg). In vivo study showed that TS treatment reduced liver and spleen enlargement in WEHI-3 bearing mice compared with the untreated group. Furthermore, TS also decreased white blood cells (WBC), indicating inhibition of differentiation of the precursor of macrophages in WEHI-3 bearing mice. Treatment of WEHI-3 cells with TS (0-75 μg/mL for 24 hours) significantly reduced cell viability. Furthermore, TS treatment–induced late apoptosis was confirmed by Annexin-V/PI staining. Western blot analyses revealed that treatment of WEHI-3 cells with TS statistically increased the protein expression level of cytochrome c in the cytoplasm and activates caspase-3. Notably, TS treatment caused a dramatic reduction in Bcl-2 and increase in Bax protein levels. TS may disturb the Bcl-2 and Bax protein ratio and induce apoptosis. This reports confirms the antitumor activity of this nutritious vegetable potentially against leukemia. PMID:27151590

Amino- and carboxyl-terminal amino acid sequences of proteins coded by gag gene of murine leukemia virus

PubMed Central

Oroszlan, Stephen; Henderson, Louis E.; Stephenson, John R.; Copeland, Terry D.; Long, Cedric W.; Ihle, James N.; Gilden, Raymond V.

1978-01-01

The amino- and carboxyl-terminal amino acid sequences of proteins (p10, p12, p15, and p30) coded by the gag gene of Rauscher and AKR murine leukemia viruses were determined. Among these proteins, p15 from both viruses appears to have a blocked amino end. Proline was found to be the common NH2 terminus of both p30s and both p12s, and alanine of both p10s. The amino-terminal sequences of p30s are identical, as are those of p10s, while the p12 sequences are clearly distinctive but also show substantial homology. The carboxyl-terminal amino acids of both viral p30s and p12s are leucine and phenylalanine, respectively. Rauscher leukemia virus p15 has tyrosine as the carboxyl terminus while AKR virus p15 has phenylalanine in this position. The compositional and sequence data provide definite chemical criteria for the identification of analogous gag gene products and for the comparison of viral proteins isolated in different laboratories. On the basis of amino acid sequences and the previously proposed H-p15-p12-p30-p10-COOH peptide sequence in the precursor polyprotein, a model for cleavage sites involved in the post-translational processing of the precursor coded for by the gag gene is proposed. PMID:206897

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry.

PubMed

Viejo-Borbolla, A; Pizzato, M; Blair, E D; Schulz, T F

2005-03-01

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Chlorozotocin

PubMed Central

Panasci, Lawrence C.; Green, Dianna; Schein, Philip S.

1979-01-01

Chlorozotocin is a chloroethyl nitrosourea with a glucose carrier that has curative activity for the murine L1210 leukemia, but is nonmyelosuppressive in mice. To determine the mechanism for this unique property of reduced bone marrow toxicity, comparative studies were conducted with chlorozotocin and CCNU, a myelotoxic chloroethyl nitrosourea. Suspensions of L1210 leukemia and murine bone marrow cells were incubated for 2 h with 0.1 mM [14C]-chloroethyl chlorozotocin or CCNU. Chlorozotocin demonstrated a fourfold increased covalent binding of the chloroethyl group to L1210 nuclei when compared to equimolar CCNU. Chlorozotocin alkylation of L1210 cells resulted in the binding of 57 pmol of [14C]ethyl group/mg of DNA, which represented a 2.3-fold increased alkylation when compared to CCNU. In marked contrast, the binding of the chloroethyl group to bone marrow nuclei was equivalent for both drugs. In addition, chlorozotocin alkylation of murine bone marrow DNA, 45 pmol of [14C]ethyl group/mg of DNA, was equivalent to that of CCNU. The ratio of L1210:bone marrow DNA alkylation was 1.3 for chlorozotocin compared to 0.6 for CCNU. The intracellular carbamoylation of L1210 and bone marrow protein by CCNU was 400- to 600-fold greater than that produced by chlorozotocin. After a 2-h exposure to 0.1, 0.05, or 0.01 mM drug, both chlorozotocin and CCNU produced a reduction in the cloning efficiency of L1210 cells that was dose dependent. However, chlorozotocin was significantly more cytotoxic than CCNU at all three molar concentrations (P < 0.01). Chlorozotocin, 0.1 mM, reduced L1210 DNA synthesis to 1% of control by 48 h, in contrast to 16% with equimolar CCNU (P < 0.01). In mice bearing 105 L1210 cells, chlorozotocin produced its optimal antitumor activity (332% increased life span [ILS]) at doses of 48-64 μmol/kg, with >50% indefinite survivors. In contrast, CCNU at the same molar doses resulted in only a 191% ILS; a CCNU dose of 128 μmol/kg was required for comparable optimal L1210 antitumor activity, 413% ILS. On a molar basis, the dose of chlorozotocin that produced optimal in vivo L1210 antitumor activity was one-third to one-half that of CCNU. Chlorozotocin, unlike CCNU, produced no murine bone marrow toxicity at its optimal therapeutic dose. This unique combination of antitumor activity without myelosuppression can be correlated with the advantageous ratio of L1210:bone marrow in vitro DNA alkylation by chlorozotocin (1.3) as compared to equimolar CCNU (0.6). PMID:158033

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation

PubMed Central

Zou, Siying; Teixeira, Alexandra M.; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferruccio, Juliana; Zhang, Ping-xia; Hwa, John; Min, Wang; Krause, Diane S.

2018-01-01

Summary Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal hemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout, shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using 2 different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice. PMID:27345948

Synergy of sequential administration of a deglycosylated ricin A chain-containing combined anti-CD19 and anti-CD22 immunotoxin (Combotox) and cytarabine in a murine model of advanced acute lymphoblastic leukemia

PubMed Central

Barta, Stefan K.; Zou, Yiyu; Schindler, John; Shenoy, Niraj; Bhagat, Tushar D.; Steidl, Ulrich; Verma, Amit

2013-01-01

The outcome for patients with refractory or relapsed acute lymphoblastic leukemia (ALL) treated with conventional therapy is poor. Immunoconjugates present a novel approach and have recently been shown to have efficacy in this setting. Combotox is a mixture of two ricin-conjugated monoclonal antibodies (RFB4 and HD37) directed against CD19 and CD22, respectively, and has shown activity in pediatric and adult ALL. We created a murine xenograft model of advanced ALL using the NALM/6 cell line to explore whether the combination of Combotox with the cytotoxic agent cytarabine (Ara-C) results in better outcomes. In our model the combination of both low- and high-dose Combotox and Ara-C resulted in significantly longer median survival. Sequential administration of Ara-C and Combotox, however, was shown to be superior to concurrent administration. These findings have led to a phase I clinical trial exploring this combination in adults with relapsed or refractory B-lineage ALL (ClinicalTrials.gov identifier NCT01408160). PMID:22448921

Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Dongwen; Chung, Suhman; Miller, Maria

2012-06-19

The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site {alpha}-hydroxytropolone inhibitor. Enzymatic assays showed that the intactmore » RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with {beta}-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.« less

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

PubMed

Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

Effects of Iron Depletion on CALM-AF10 Leukemias

PubMed Central

Heath, Jessica L.; Weiss, Joshua M.

2014-01-01

Iron, an essential nutrient for cellular growth and proliferation, enters cells via clathrin-mediated endocytosis (CME). The clathrin assembly lymphoid myeloid (CALM) protein plays an essential role in the cellular import of iron by CME. CALM-AF10 leukemias harbor a single copy of the normal CALM gene, and may therefore be more sensitive to the growth inhibitory effect of iron restriction compared with normal hematopoietic cells. We found that Calm heterozygous (CalmHET) murine fibroblasts exhibit signs of iron deficiency, with increased surface transferrin receptor (sTfR) levels and reduced growth rates. CalmHET hematopoietic cells are more sensitive in vitro to iron chelators than their wild type counterparts. Iron chelation also displayed toxicity towards cultured CalmHET CALM-AF10 leukemia cells and this effect was additive to that of chemotherapy. In mice transplanted with CalmHET CALM-AF10 leukemia, we found that dietary iron restriction reduces tumor burden in the spleen. However, dietary iron restriction, used alone or in conjunction with chemotherapy, did not increase survival of mice with CalmHET CALM-AF10 leukemia. In summary, while Calm heterozygosity results in iron deficiency and increased sensitivity to iron chelation in vitro, our data in mice do not suggest that iron depletion strategies would be beneficial for the therapy of CALM-AF10 leukemia patients. PMID:25193880

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF.

PubMed

Meng, G L; Zur Nieden, N I; Liu, S Y; Cormier, J T; Kallos, M S; Rancourt, D E

2008-04-01

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders. Copyright 2007 Wiley-Liss, Inc.

AKT capture by feline leukemia virus.

PubMed

Kawamura, Maki; Umehara, Daigo; Odahara, Yuka; Miyake, Ariko; Ngo, Minh Ha; Ohsato, Yoshiharu; Hisasue, Masaharu; Nakaya, Masa-Aki; Watanabe, Shinya; Nishigaki, Kazuo

2017-04-01

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

Loss of p19Arf in a Rag1−/− B-cell precursor population initiates acute B-lymphoblastic leukemia

PubMed Central

Hauer, Julia; Mullighan, Charles; Morillon, Estelle; Wang, Gary; Bruneau, Julie; Brousse, Nicole; Lelorc'h, Marc; Romana, Serge; Boudil, Amine; Tiedau, Daniela; Kracker, Sven; Bushmann, Frederic D.; Borkhardt, Arndt; Fischer, Alain; Hacein-Bey-Abina, Salima

2011-01-01

In human B-acute lymphoblastic leukemia (B-ALL), RAG1-induced genomic alterations are important for disease progression. However, given that biallelic loss of the RAG1 locus is observed in a subset of cases, RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf−/−Rag1−/− mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model, we identified a new, Rag1-independent leukemia-initiating mechanism originating from a Sca1+CD19+ precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM, a similar CD34+CD19+ population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans. PMID:21622646

AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo.

PubMed

Yang, Jing; Ikezoe, Takayuki; Nishioka, Chie; Tasaka, Taizo; Taniguchi, Ayuko; Kuwayama, Yoshio; Komatsu, Naoki; Bandobashi, Kentaro; Togitani, Kazuto; Koeffler, H Phillip; Taguchi, Hirokuni; Yokoyama, Akihito

2007-09-15

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation.

Reciprocal Expression of Human ETS1 and ETS2 Genes during T-Cell Activation: Regulatory Role for the Protooncogene ETS1

DTIC Science & Technology

1990-05-01

viral v-ets oncogene of the E26 have studied the expression and regulation of ETS1 and ETS2 avian leukemia virus ( 1 . 2). The c-ets-i (3-5), c-ets-2...ets- 1 mRNA is detectable in different murine large granular lymphocytes, and CDll-bearing T cells. The (17-19) and human tissues (20. 21). c-ets- 1 mRNA...We have shown that: (i) the . (800 ng/ml) or a combination of PMA and ionomycin. An murine Ets-2 expression appears 1 day earlier than Ets-l optimal

A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia

NASA Astrophysics Data System (ADS)

Yang, Zhaogang; Yu, Bo; Zhu, Jing; Huang, Xiaomeng; Xie, Jing; Xu, Songlin; Yang, Xiaojuan; Wang, Xinmei; Yung, Bryant C.; Lee, L. James; Lee, Robert J.; Teng, Lesheng

2014-07-01

The siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for the targeted delivery of LOR-1284 siRNA into acute myeloid leukemia (AML) cells. The in vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4-11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than the free LOR-1284 in reducing the R2 mRNA and protein levels. The Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to the free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with the decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific therapeutic delivery of LOR-1284 into AML cells.

Ikaros gene expression and leukemia.

PubMed

Tonnelle, Cécile; Calmels, Boris; Maroc, Christine; Gabert, Jean; Chabannon, Christian

2002-01-01

The Ikaros (Ik) protein, or LyF1, was initially described as a protein binding to regulatory sequences of a number of genes expressed in murine lymphoid cells. Ikaros is a critical regulator of normal hematopoietic stem cell differentiation, as evidenced by dramatic defects in the lymphoid compartments, in homozygous animals with gene inactivation. Because differential splicing produces multiple isoforms with potentially different functions, Ikaros provides a unique model to study how post-transcriptional mechanisms may be involved in neoplastic processes. Indeed, several groups including ours have underlined evidences that expression of different Ikaros isoforms vary among different types of leukemias. The predominance of short isoforms in certain subsets is intriguing. Here, additional observations reinforced the hypothesis that Ikaros expression may be deregulated in human leukemias. Whether this is a cause or a consequence of the leukemic process remains speculative. Other human diseases however, provide examples of abnormal post-transcriptional regulations that have been further characterized.

Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard

2004-12-20

Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associatedmore » with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.« less

Synthesis, structure and cytotoxic activity of acetylenic derivatives of betulonic and betulinic acids

NASA Astrophysics Data System (ADS)

Bębenek, Ewa; Chrobak, Elwira; Wietrzyk, Joanna; Kadela, Monika; Chrobak, Artur; Kusz, Joachim; Książek, Maria; Jastrzębska, Maria; Boryczka, Stanisław

2016-02-01

A series of acetylenic derivatives of betulonic and betulinic acids has been synthesized and characterized by 1H and 13C NMR, IR and MS spectroscopy. The structure of propargyl betulonate 4 and propargyl betulinate-DMF solvate 8A was solved by X-ray diffraction. Thermal properties were examined using a DSC technique. The resulting alkynyl derivatives, as well as betulin 1 and betulinic acid 3, were evaluated in vitro for their cytotoxic activity against human T47D breast cancer, CCRF/CEM leukemia, SW707 colorectal, murine P388 leukemia and BALB3T3 normal fibroblasts cell lines. Several of the obtained compounds have a favorable cytotoxic profile than betulin 1. Propargyl betulinate 8 was the most active derivative, being up to 3-fold more potent than betulin 1 against the human leukemia (CCRF/CEM) cell line, with an IC50 value of 3.9 μg/mL.

Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

PubMed Central

Perkins, Archibald S.; Kirschmeier, Paul T.; Gattoni-Celli, Sebastiano; Weinstein, I. Bernard

1983-01-01

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique PstI site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt+ colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR-gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt+ phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells. Images PMID:6308426

Protein Transfer Into Human Cells by VSV-G-induced Nanovesicles

PubMed Central

Mangeot, Philippe-Emmanuel; Dollet, Sandra; Girard, Mathilde; Ciancia, Claire; Joly, Stéphane; Peschanski, Marc; Lotteau, Vincent

2011-01-01

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. PMID:21750535

Tumor Trp53 status and genotype affect the bone marrow microenvironment in acute myeloid leukemia

PubMed Central

Jacamo, Rodrigo; Davis, R. Eric; Ling, Xiaoyang; Sonnylal, Sonali; Wang, Zhiqiang; Ma, Wencai; Zhang, Min; Ruvolo, Peter; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Lowe, Scott; Zuber, Johannes; Kornblau, Steven M.; Konopleva, Marina; Andreeff, Michael

2017-01-01

The genetic heterogeneity of acute myeloid leukemia (AML) and the variable responses of individual patients to therapy suggest that different AML genotypes may influence the bone marrow (BM) microenvironment in different ways. We performed gene expression profiling of bone marrow mesenchymal stromal cells (BM-MSC) isolated from normal C57BL/6 mice or mice inoculated with syngeneic murine leukemia cells carrying different human AML genotypes, developed in mice with Trp53 wild-type or nullgenetic backgrounds. We identified a set of genes whose expression in BM-MSC was modulated by all four AML genotypes tested. In addition, there were sets of differentially-expressed genes in AML-exposed BM-MSC that were unique to the particular AML genotype or Trp53 status. Our findings support the hypothesis that leukemia cells alter the transcriptome of surrounding BM stromal cells, in both common and genotype-specific ways. These changes are likely to be advantageous to AML cells, affecting disease progression and response to chemotherapy, and suggest opportunities for stroma-targeting therapy, including those based on AML genotype. PMID:29137349

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses.

PubMed

O'Reilly, L; Roth, M J

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.

Second-Site Changes Affect Viability of Amphotropic/Ecotropic Chimeric Enveloped Murine Leukemia Viruses

PubMed Central

O'Reilly, Lucille; Roth, Monica J.

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells. PMID:10623753

Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models.

PubMed

Benito, Juliana; Ramirez, Marc S; Millward, Niki Zacharias; Velez, Juliana; Harutyunyan, Karine G; Lu, Hongbo; Shi, Yue-Xi; Matre, Polina; Jacamo, Rodrigo; Ma, Helen; Konoplev, Sergej; McQueen, Teresa; Volgin, Andrei; Protopopova, Marina; Mu, Hong; Lee, Jaehyuk; Bhattacharya, Pratip K; Marszalek, Joseph R; Davis, R Eric; Bankson, James A; Cortes, Jorge E; Hart, Charles P; Andreeff, Michael; Konopleva, Marina

2016-04-01

To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a "hypoxia index" compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins. ©2015 American Association for Cancer Research.

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome

PubMed Central

Alachkar, Houda; Santhanam, Ramasamy; Maharry, Kati; Metzeler, Klaus H.; Huang, Xiaomeng; Kohlschmidt, Jessica; Mendler, Jason H.; Benito, Juliana M.; Hickey, Christopher; Neviani, Paolo; Dorrance, Adrienne M.; Anghelina, Mirela; Khalife, Jihane; Tarighat, Somayeh S.; Volinia, Stefano; Whitman, Susan P.; Paschka, Peter; Hoellerbauer, Pia; Wu, Yue-Zhong; Han, Lina; Bolon, Brad N.; Blum, William; Mrózek, Krzysztof; Carroll, Andrew J.; Perrotti, Danilo; Andreeff, Michael; Caligiuri, Michael A.; Konopleva, Marina; Garzon, Ramiro; Bloomfield, Clara D.; Marcucci, Guido

2014-01-01

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML. PMID:24590286

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome.

PubMed

Alachkar, Houda; Santhanam, Ramasamy; Maharry, Kati; Metzeler, Klaus H; Huang, Xiaomeng; Kohlschmidt, Jessica; Mendler, Jason H; Benito, Juliana M; Hickey, Christopher; Neviani, Paolo; Dorrance, Adrienne M; Anghelina, Mirela; Khalife, Jihane; Tarighat, Somayeh S; Volinia, Stefano; Whitman, Susan P; Paschka, Peter; Hoellerbauer, Pia; Wu, Yue-Zhong; Han, Lina; Bolon, Brad N; Blum, William; Mrózek, Krzysztof; Carroll, Andrew J; Perrotti, Danilo; Andreeff, Michael; Caligiuri, Michael A; Konopleva, Marina; Garzon, Ramiro; Bloomfield, Clara D; Marcucci, Guido

2014-04-01

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

miR-150 exerts antileukemia activity in vitro and in vivo through regulating genes in multiple pathways

PubMed Central

Fang, Zhi Hong; Wang, Si Li; Zhao, Jin Tao; Lin, Zhi Juan; Chen, Lin Yan; Su, Rui; Xie, Si Ting; Carter, Bing Z; Xu, Bing

2016-01-01

MicroRNAs, a class of small noncoding RNAs, have been implicated to regulate gene expression in virtually all important biological processes. Although accumulating evidence demonstrates that miR-150, an important regulator in hematopoiesis, is deregulated in various types of hematopoietic malignancies, the precise mechanisms of miR-150 action are largely unknown. In this study, we found that miR-150 is downregulated in samples from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia, and normalized after patients achieved complete remission. Restoration of miR-150 markedly inhibited growth and induced apoptosis of leukemia cells, and reduced tumorigenicity in a xenograft leukemia murine model. Microarray analysis identified multiple novel targets of miR-150, which were validated by quantitative real-time PCR and luciferase reporter assay. Gene ontology and pathway analysis illustrated potential roles of these targets in small-molecule metabolism, transcriptional regulation, RNA metabolism, proteoglycan synthesis in cancer, mTOR signaling pathway, or Wnt signaling pathway. Interestingly, knockdown one of four miR-150 targets (EIF4B, FOXO4B, PRKCA, and TET3) showed an antileukemia activity similar to that of miR-150 restoration. Collectively, our study demonstrates that miR-150 functions as a tumor suppressor through multiple mechanisms in human leukemia and provides a rationale for utilizing miR-150 as a novel therapeutic agent for leukemia treatment. PMID:27899822

CD8+T cells expressing both PD-1 and TIGIT but not CD226 are dysfunctional in acute myeloid leukemia (AML) patients.

PubMed

Wang, Mengjie; Bu, Jin; Zhou, Maohua; Sido, Jessica; Lin, Yu; Liu, Guanfang; Lin, Qiwen; Xu, Xiuzhang; Leavenworth, Jianmei W; Shen, Erxia

2018-05-01

Acute myeloid leukemia (AML) is one of the most common types of leukemia among adults with an overall poor prognosis and very limited treatment management. Immune checkpoint blockade of PD-1 alone or combined with other immune checkpoint blockade has gained impressive results in murine AML models by improving anti-leukemia CD8 + T cell function, which has greatly promoted the strategy to utilize combined immune checkpoint inhibitors to treat AML patients. However, the expression profiles of these immune checkpoint receptors, such as co-inhibitory receptors PD-1 and TIGIT and co-stimulatory receptor CD226, in T cells from AML patients have not been clearly defined. Here we have defined subsets of CD8 + and CD4 + T cells in the peripheral blood (PB) from newly diagnosed AML patients and healthy controls (HCs). We have observed increased frequencies of PD-1- and TIGIT- expressing CD8 + T cells but decreased occurrence of CD226-expressing CD8 + T cells in AML patients. Further analysis of these CD8 + T cells revealed a unique CD8 + T cell subset that expressed PD-1 and TIGIT but displayed lower levels of CD226 was associated with failure to achieve remission after induction chemotherapy and FLT3-ITD mutations which predict poor clinical prognosis in AML patients. Importantly, these PD-1 + TIGIT + CD226 - CD8 + T cells are dysfunctional with lower expression of intracellular IFN-γ and TNF-α than their counterparts in HCs. Therefore, our studies revealed that an increased frequency of a unique CD8 + T cell subset, PD-1 + TIGIT + CD226 - CD8 + T cells, is associated with CD8 + T cell dysfunction and poor clinical prognosis of AML patients, which may reveal critical diagnostic or prognostic biomarkers and direct more efficient therapeutic strategies. Copyright © 2017. Published by Elsevier Inc.

A Replication-incompetent Rift Valley Fever Vaccine: Chimeric Virus-like Particles Protect Mice and Rats Against Lethal Challenge

PubMed Central

Mandell, Robert B.; Koukuntla, Ramesh; Mogler, Laura J. K.; Carzoli, Andrea K.; Freiberg, Alexander N.; Holbrook, Michael R.; Martin, Brian K.; Staplin, William R.; Vahanian, Nicholas N.; Link, Charles J.; Flick, Ramon

2009-01-01

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins GN and GC, nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate. PMID:19932911

Vital Role for CD8+ Cells in Controlling Retroviral Infections ▿

PubMed Central

Kane, Melissa; Case, Laure K.; Golovkina, Tatyana V.

2011-01-01

Antiviral adaptive immune defenses consist of humoral and cell-mediated responses, which together eliminate extracellular and intracellular virus. As most retrovirus-infected individuals do not raise efficient protective antivirus immune responses, the relative importance of humoral and cell-mediated responses in restraining retroviral infection is not well understood. We utilized retrovirus-resistant I/LnJ mice, which control infection with mouse mammary tumor virus (MMTV) and murine leukemia virus (MuLV) via an adaptive immune mechanism, to assess the contribution of cellular responses and virus-neutralizing antibodies (Abs) to the control of retroviral infection. We found that in retrovirus-infected CD8-deficient I/LnJ mice, viral titers exceed the neutralizing capability of antiviral Abs, resulting in augmented virus spread and disease induction. Thus, even in the presence of robust neutralizing Ab responses, CD8-mediated responses are essential for full protection against retroviral infection. PMID:21248041

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

PubMed Central

Millet, Jean Kaoru; Whittaker, Gary R.

2016-01-01

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay. PMID:28018942

Ludwik Gross, Sarah Stewart, and the 1950s discoveries of Gross murine leukemia virus and polyoma virus.

PubMed

Morgan, Gregory J

2014-12-01

The Polish-American scientist Ludwik Gross made two important discoveries in the early 1950s. He showed that two viruses - murine leukemia virus and parotid tumor virus - could cause cancer when they were injected into susceptible animals. At first, Gross's discoveries were greeted with skepticism: it seemed implausible that viruses could cause a disease as complex as cancer. Inspired by Gross's initial experiments, similar results were obtained by Sarah Stewart and Bernice Eddy who later renamed the parotid tumor virus SE polyoma virus after finding it could cause many different types of tumors in mice, hamsters, and rats. Eventually the "SE" was dropped and virologists adopted the name "polyoma virus." After Gross's work was published, additional viruses capable of causing solid tumors or blood-borne tumors in mice were described by Arnold Graffi, Charlotte Friend, John Moloney and others. By 1961, sufficient data had been accumulated for Gross to confidently publish an extensive monograph--Oncogenic Viruses--the first history of tumor virology, which became a standard reference work and marked the emergence of tumor virology as a distinct, legitimate field of study. Copyright © 2014 Elsevier Ltd. All rights reserved.

Early detection of a two-long-terminal-repeat junction molecule in the cytoplasm of recombinant murine leukemia virus-infected cells.

PubMed

Serhan, Fatima; Penaud, Magalie; Petit, Caroline; Leste-Lasserre, Thierry; Trajcevski, Stéphane; Klatzmann, David; Duisit, Ghislaine; Sonigo, Pierre; Moullier, Philippe

2004-06-01

We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19. Using cell fractionation combined with careful controls, we found that a two-long-terminal-repeat (two-LTR) junction molecule was detectable in the cytoplasm as early as 2 h post virus entry. Altogether, our data indicated that the neosynthesized retroviral DNA led to the early formation of structures including true two-LTR junctions in the cytoplasm of MLV-infected cells. Thus, the classical assumption that two-LTR circles are a mitosis-dependent dead-end product accumulating in the nucleus must be reconsidered. MLV-derived products containing a two-LTR junction can no longer be used as an exclusive surrogate for the preintegration complex nuclear translocation event.

The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

PubMed Central

Liu, Xiu-Huai; Xu, Wenqin; Russ, Jill; Eiden, Lee E.; Eiden, Maribeth V.

2011-01-01

Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease. PMID:21464894

Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

PubMed Central

Driessen, Emma M.C.; van Roon, Eddy H.J.; Spijkers-Hagelstein, Jill A.P.; Schneider, Pauline; de Lorenzo, Paola; Valsecchi, Maria Grazia; Pieters, Rob; Stam, Ronald W.

2013-01-01

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211–8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients. PMID:23403319

Leukemia cell infiltration causes defective erythropoiesis partially through MIP-1α/CCL3.

PubMed

Wang, Y; Gao, A; Zhao, H; Lu, P; Cheng, H; Dong, F; Gong, Y; Ma, S; Zheng, Y; Zhang, H; Zhang, Y; Xu, J; Zhu, X; Yuan, W; Zhang, X; Hao, S; Cheng, T

2016-09-01

Leukemia often results in severe anemia, which may significantly contribute to patient mortality and morbidity. However, the mechanisms underlying defective erythropoiesis in leukemia have not been fully elucidated. In this study, we demonstrated that insufficient erythropoiesis in an immunocompetent acute myeloid leukemia (AML) murine model was due to reduced proliferation of megakaryocyte erythroid progenitors and increased apoptosis of erythroblasts. Colony-forming cell assays indicated that the leukemic bone marrow (BM) plasma inhibited erythroid colony formation, whereas they had no inhibitory effect on other types of colonies. Cytokine array analysis demonstrated that the chemokine CCL3 was elevated in the plasma of AML mice and patients. CCL3 inhibited erythroid differentiation of hematopoietic stem cells, common myeloid progenitors and especially megakaryocytic-erythroid progenitors. Administration of the CCR1 antagonist partially recovered the yield of erythroid colonies in the presence of CCL3 or leukemic BM plasma. Mechanistically, we observed an increase of p38 phosphorylation and subsequent downregulation of GATA1 after CCL3 treatment. Furthermore, knockdown of CCL3 attenuated leukemic progression and alleviated anemia. Therefore, our results demonstrate that elevated CCL3 in the leukemic environment suppresses erythropoiesis via CCR1-p38 activation, suggesting a novel mechanism for the erythroid defects observed in leukemia.

Suppression of graft-versus-host disease and retention of graft-versus-tumour reaction by murine genetically engineered dendritic cells following bone marrow transplantation.

PubMed

Huang, Yihong; Feng, Saran; Xu, Yujie; Chen, Wanru; Wang, Shuhua; Li, Depeng; Li, Zhenyu; Lu, Qunxian; Pan, Xiuying; Xu, Kailin

2015-05-01

The effect of infusion of lentiviral vector‑mediated, genetically engineered dendritic cells (DCs) following allogeneic bone marrow transplantation (allo‑BMT) on graft‑versus‑host disease (GVHD) and graft‑versus‑leukemia (GVL) was investigated in a mouse model. Lentivirus‑mediated expression of soluble tumor necrosis factor receptor 1 (sTNFR1) converted immature DCs (imDCs) from BABL/c mice into engineered DCs in vitro. An EL4 leukemia allo‑BMT model of BABL/c to C57BL/6 mice was established. Engineered DCs with donor bone marrow cells and splenocytes were subsequently transplanted into myeloablatively irradiated recipients. The average survival duration in the sTNFR1‑ and pXZ9‑imDC groups was significantly prolonged compared with that of the allo‑BMT group (P<0.05). Mild histological changes in GVHD or leukemia were observed in the recipients in the sTNFR1‑imDC group and clinical GVHD scores in this group were significantly decreased compared with those of the transplantation and pXZ9‑imDC groups. Serum interferon‑γ levels were decreased in the pXZ9‑imDC and sTNFR1‑imDC groups compared with those in the allo‑BMT group (P<0.05), with the reduction being more significant in the sTNFR1‑imDC group (P<0.05). Serum interleukin‑4 expression levels were decreased in the allo‑BMT group, but gradually increased in the pXZ9‑imDC and sTNFR1‑imDC groups (P<0.05). Co‑injection of donor genetically‑engineered imDCs was able to efficiently protect recipient mice from lethal GVHD while preserving GVL effects during allo‑BMT.

Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis

PubMed Central

Valerio, Daria G.; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E.; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J.; Huang, Chun-Hao; Lujambio, Amaia; Zheng, George; Zuber, Johannes; Pandita, Tej K.; Lowe, Scott W.; Armstrong, Scott A.

2017-01-01

Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore Mof inactivation suppressed leukemia development in a NUP98-HOXA9 driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. PMID:28202522

A novel cytotoxic flavonoid glycoside from Physalis angulata.

PubMed

Ismail, N; Alam, M

2001-08-01

A new flavonol glycoside, myricetin 3-O-neohesperidoside (1) was isolated from a cytotoxic MeOH extract of the leaves of Physalis angulata. Compound 1 showed remarkable cytotoxicity in vitro against murine leukemia cell line P-388, epidermoid carcinoma of the nasopharynx KB-16 cells, and lung adenocarcinoma A-549 with ED(50) values of 0.048, 0.50 and 0.55 microg ml(-1), respectively.

Detection of Xenotropic Murine Leukemia Virus-Related Virus (XMRV) in Gulf War Illness: Role in Pathogenesis or Biomarker?

DTIC Science & Technology

2012-10-01

Co-­‐Infections           Epstein   Barr   virus  (EBV)   □Yes...fever, lymphadenopathy, headache, myalgia, arthralgia, depression, and memory loss; candidate etiologic agents include Epstein - Barr and other... Virus -Related Virus (XMRV) in Gulf War Illness: Role in Pathogenesis or Biomarker? PRINCIPAL INVESTIGATOR: Vincent C Lombardi

Recombination Origin of Retrovirus XMRV | Center for Cancer Research

Cancer.gov

Xenotropic murine leukemia virus–related virus (XMRV) was first reported in samples from a human prostate tumor in 2006, and, at that time, claims were made that XMRV infection rates ranged from 6 to 27 percent of human prostate cancers.  Later research reported XMRV in the blood of 67 percent of people with chronic fatigue syndrome (CFS). When follow-up studies failed to

Expression and associations of TRAF1, BMI-1, ALDH1, and Lin28B in oral squamous cell carcinoma.

PubMed

Wu, Tian-Fu; Li, Yi-Cun; Ma, Si-Rui; Bing-Liu; Zhang, Wen-Feng; Sun, Zhi-Jun

2017-04-01

Tumor necrosis factor receptor-associated factor 1, an adaptor protein of tumor necrosis factor 2, is involved in classical nuclear factor (NF)-κB activation and lymphocyte recruitment. However, less is known about the expression and association of tumor necrosis factor receptor-associated factor 1 with cancer stem cell markers in oral squamous cell carcinoma. This study aimed to investigate the expression of tumor necrosis factor receptor-associated factor 1 and stem cell characteristic markers (lin28 homolog B, B cell-specific Moloney murine leukemia virus integration site 1, and aldehyde dehydrogenase 1) in oral squamous cell carcinoma and analyze their relations. Paraffin-embedded tissues of 78 oral squamous cell carcinomas, 39 normal oral mucosa, and 12 oral dysplasia tissues were employed in tissue microarrays, and the expression of tumor necrosis factor receptor-associated factor 1, B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B was measured by immunohistostaining and digital pathological analysis. The expression of tumor necrosis factor receptor-associated factor 1 was higher in the oral squamous cell carcinoma group as compared with the expression in the oral mucosa (p < 0.01) and oral dysplasia (p < 0.001) groups. In addition, the expression of tumor necrosis factor receptor-associated factor 1 was associated with those of B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B (p = 0.032, r 2  = 0.109; p < 0.0001, r 2  = 0.64; and p < 0.001, r 2  = 0.16) in oral squamous cell carcinoma. The patient survival rate was lower in the highly expressed tumor necrosis factor receptor-associated factor 1 group, although the difference was not significant. The clustering analysis showed that tumor necrosis factor receptor-associated factor 1 was most related to aldehyde dehydrogenase 1. These findings suggest that tumor necrosis factor receptor-associated factor 1 has potential direct/indirect regulations with the cancer stem cell markers in oral squamous cell carcinoma, which may help in further analysis of the cancer stem cell characteristics.

The transcription factor IRF8 counteracts BCR-ABL to rescue dendritic cell development in chronic myelogenous leukemia.

PubMed

Watanabe, Tomoya; Hotta, Chie; Koizumi, Shin-ichi; Miyashita, Kazuho; Nakabayashi, Jun; Kurotaki, Daisuke; Sato, Go R; Yamamoto, Michio; Nakazawa, Masatoshi; Fujita, Hiroyuki; Sakai, Rika; Fujisawa, Shin; Nishiyama, Akira; Ikezawa, Zenro; Aihara, Michiko; Ishigatsubo, Yoshiaki; Tamura, Tomohiko

2013-11-15

BCR-ABL tyrosine kinase inhibitors (TKI) have dramatically improved therapy for chronic myelogenous leukemia (CML). However, several problems leading to TKI resistance still impede a complete cure of this disease. IFN regulatory factor-8 (IRF8) is a transcription factor essential for the development and functions of immune cells, including dendritic cells. Irf8(-/-) mice develop a CML-like disease and IRF8 expression is downregulated in patients with CML, suggesting that IRF8 is involved in the pathogenesis of CML. In this study, by using a murine CML model, we show that BCR-ABL strongly inhibits a generation of dendritic cells from an early stage of their differentiation in vivo, concomitant with suppression of Irf8 expression. Forced expression of IRF8 overrode BCR-ABL (both wild-type and T315I-mutated) to rescue dendritic cell development in vitro, indicating that the suppression of Irf8 causes dendritic cell deficiency. Gene expression profiling revealed that IRF8 restored the expression of a significant portion of BCR-ABL-dysregulated genes and predicted that BCR-ABL has immune-stimulatory potential. Indeed, IRF8-rescued BCR-ABL-expressing dendritic cells were capable of inducing CTLs more efficiently than control dendritic cells. Altogether, our findings suggest that IRF8 is an attractive target in next-generation therapies for CML. ©2013 AACR

Pyrimethamine as a Potent and Selective Inhibitor of Acute Myeloid Leukemia Identified by High-throughput Drug Screening.

PubMed

Sharma, Amit; Jyotsana, Nidhi; Lai, Courteney K; Chaturvedi, Anuhar; Gabdoulline, Razif; Görlich, Kerstin; Murphy, Cecilia; Blanchard, Jan E; Ganser, Arnold; Brown, Eric; Hassell, John A; Humphries, R Keith; Morgan, Michael; Heuser, Michael

2016-01-01

Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.

Radiation-induced leukemia: Comparative studies in mouse and man

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haas, M.

1991-01-01

We now have a clear understanding of the mechanism by which radiation-induced (T-cell) leukemia occurs. In irradiated mice (radiation-induced thymic leukemia) and in man (acute lymphoblastic T-cell leukemia, T-ALL) the mechanism of leukemogenesis is surprisingly similar. Expressed in the most elementary terms, T-cell leukemia occurs when T-cell differentiation is inhibited by a mutation, and pre-T cells attempt but fail to differentiate in the thymus. Instead of leaving the thymus for the periphery as functional T-cells they continue to proliferate in the thymus. The proliferating pre- (pro-) T-cells constitute the (early) acute T-cell leukemia (A-TCL). This model for the mechanism ofmore » T-cell leukemogenesis accounts for all the properties of both murine and human A-TCL. Important support for the model has recently come from work by Ilan Kirsch and others, who have shown that mutations/deletions in the genes SCL (TAL), SIL, and LCK constitute primary events in the development of T-ALL, by inhibiting differentiation of thymic pre- (pro-) T-cells. This mechanism of T-cell leukemogenesis brings several specific questions into focus: How do early A-TCL cells progress to become potently tumorigenic and poorly treatable Is it feasible to genetically suppress early and/or progressed A-TCL cells What is the mechanism by which the differentiation-inhibited (leukemic) pre-T cells proliferate During the first grant year we have worked on aspects of all three questions.« less

Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.

PubMed

Greve, Tine; Clasen-Linde, Erik; Andersen, Morten T; Andersen, Mette K; Sørensen, Stine D; Rosendahl, Mikkel; Ralfkiaer, Elisabeth; Andersen, Claus Yding

2012-11-22

Some women suffering from leukemia require bone marrow transplantation to be cured. Bone marrow transplantation is associated with a high risk of sterility, and some patients are offered fertility preservation by cryopreservation of the ovarian cortex. Transplantation of the ovarian cortex to women cured of leukemia who became menopausal is currently not performed because of the risk of introducing the disease. In this study, individual pieces of ovarian cortex intended for reimplantation from 25 patients with leukemia were transplanted to each of 25 nude mice for 20 weeks. The ovarian cortex was examined before and after transplantation by histology and immunohistochemistry, and RT-quantitative PCR (in the 7 patients with a known marker). Seventeen patients had the ovarian cortex retrieved when they were in complete remission. Before transplantation, 4 of 7 pieces (2 from patients in complete remission) of ovarian cortex had a positive RT-quantitative PCR. After transplantation, none of the mice revealed any sign of disease, neither in the pieces of ovarian cortex transplanted nor in any of the murine organs evaluated. Thus, the ovaries from patients in complete remission do not appear to contain viable malignant cells contrasting ovarian tissue retrieved before treatment.

Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration

PubMed Central

Dias, Sergio; Hattori, Koichi; Zhu, Zhenping; Heissig, Beate; Choy, Margaret; Lane, William; Wu, Yan; Chadburn, Amy; Hyjek, Elizabeth; Gill, Muhammad; Hicklin, Daniel J.; Witte, Larry; Moore, M.A.S.; Rafii, Shahin

2000-01-01

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF165 induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF165 also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF165-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation. PMID:10953026

Myeloid leukemia factor 1 stabilizes tumor suppressor C/EBPα to prevent Trib1-driven acute myeloid leukemia.

PubMed

Nakamae, Ikuko; Kato, Jun-Ya; Yokoyama, Takashi; Ito, Hidenori; Yoneda-Kato, Noriko

2017-09-12

C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis.

Myeloid leukemia factor 1 stabilizes tumor suppressor C/EBPα to prevent Trib1-driven acute myeloid leukemia

PubMed Central

Nakamae, Ikuko; Kato, Jun-ya; Yokoyama, Takashi; Ito, Hidenori

2017-01-01

C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis. PMID:29296815

Becton-Dickson Model 420 Fluorescence-Activated Cell Sorter (FACS).

DTIC Science & Technology

1986-05-01

respectively) have been associated with certain autoimmune or immunodeficient diseases. The effects of UDMH on Lyt. antigens were previously evaluated...measured in cells from feline leukemia virus (FeLV)-infected cats and normal cat cells. The measurements are performed using the calcium-specific dye...ucavd as a stimulator, which allows for quantitation of " . phagocytosis activity of the cells. c) Quantitation of IL-2 receptor site on feline and murine

Insights into the nuclear export of murine leukemia virus intron-containing RNA.

PubMed

Pessel-Vivares, Lucie; Houzet, Laurent; Lainé, Sébastien; Mougel, Marylène

2015-01-01

The retroviral genome consists of an intron-containing transcript that has essential cytoplasmic functions in the infected cell. This viral transcript can escape splicing, circumvent the nuclear checkpoint mechanisms and be transported to the cytoplasm by hijacking the host machinery. Once in the cytoplasm, viral unspliced RNA acts as mRNA to be translated and as genomic RNA to be packaged into nascent viruses. The murine leukemia virus (MLV) is among the first retroviruses discovered and is classified as simple Retroviridae due to its minimal encoding capacity. The oncogenic and transduction abilities of MLV are extensively studied, whereas surprisingly the crucial step of its nuclear export has remained unsolved until 2014. Recent work has revealed the recruitment by MLV of the cellular NXF1/Tap-dependent pathway for export. Unconventionally, MLV uses of Tap to export both spliced and unspliced viral RNAs. Unlike other retroviruses, MLV does not harbor a unique RNA signal for export. Indeed, multiple sequences throughout the MLV genome appear to promote export of the unspliced MLV RNA. We review here the current understanding of the export mechanism and highlight the determinants that influence MLV export. As the molecular mechanism of MLV export is elucidated, we will gain insight into the contribution of the export pathway to the cytoplasmic fate of the viral RNA.

Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

PubMed

Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

2015-11-01

Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

Murine genetically engineered and human xenograft models of chronic lymphocytic leukemia.

PubMed

Chen, Shih-Shih; Chiorazzi, Nicholas

2014-07-01

Chronic lymphocytic leukemia (CLL) is a genetically complex disease, with multiple factors having an impact on onset, progression, and response to therapy. Genetic differences/abnormalities have been found in hematopoietic stem cells from patients, as well as in B lymphocytes of individuals with monoclonal B-cell lymphocytosis who may develop the disease. Furthermore, after the onset of CLL, additional genetic alterations occur over time, often causing disease worsening and altering patient outcomes. Therefore, being able to genetically engineer mouse models that mimic CLL or at least certain aspects of the disease will help us understand disease mechanisms and improve treatments. This notwithstanding, because neither the genetic aberrations responsible for leukemogenesis and progression nor the promoting factors that support these are likely identical in character or influences for all patients, genetically engineered mouse models will only completely mimic CLL when all of these factors are precisely defined. In addition, multiple genetically engineered models may be required because of the heterogeneity in susceptibility genes among patients that can have an effect on genetic and environmental characteristics influencing disease development and outcome. For these reasons, we review the major murine genetically engineered and human xenograft models in use at the present time, aiming to report the advantages and disadvantages of each. Copyright © 2014 Elsevier Inc. All rights reserved.

Loss of Function of TET2 Cooperates with Constitutively Active KIT in Murine and Human Models of Mastocytosis

PubMed Central

De Vita, Serena; Schneider, Rebekka K.; Garcia, Michael; Wood, Jenna; Gavillet, Mathilde; Ebert, Benjamin L.; Gerbaulet, Alexander; Roers, Axel; Levine, Ross L.; Mullally, Ann; Williams, David A.

2014-01-01

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM. PMID:24788138

MLV integration site selection is driven by strong enhancers and active promoters

PubMed Central

LaFave, Matthew C.; Varshney, Gaurav K.; Gildea, Derek E.; Wolfsberg, Tyra G.; Baxevanis, Andreas D.; Burgess, Shawn M.

2014-01-01

Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3 700 000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors. PMID:24464997

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.

PubMed

Kines, Kristine J; Mann, Victoria H; Morales, Maria E; Shelby, Bryan D; Kalinna, Bernd H; Gobert, Geoffrey N; Chirgwin, Sharon R; Brindley, Paul J

2006-04-01

Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.

Clearance of phenylalanine ammonia-lyase from normal and tumor-bearing mice.

PubMed

Shen, R S; Fritz, R R; Abell, C W

1977-04-01

Yeast phenylalanine ammonia-lyase was administered i.p. to normal and tumor-bearing mice, and its clearance from plasma was studied. Single and multiple weekly injections at dosages of 10,20,50 and 100 units/kg were administered to C57BL female, C57BL X DBA/2F1 male, and A/J female mice. L5178Y murine lymphoblastic leukemia, B16 melanoma, BW10232 adenocarcinoma, and 15091A anaplastic carcinoma were implanted 7 to 11 days prior to enzyme injection in the appropriate host. After a single injection, the average plasma half-lives of phenylalanine ammonia-lyase were 18 to 24 hr in all groups studied. While the other tumors had no effect on the plasma level of phenylalanine ammonia-lyase after a single injection, L5178Y murine lymphoblastic leukemia and 15091A anaplastic carcinoma significantly depressed the maximal level of phenylalanine ammonia-lyase attained in the plasma. After repeated injections of phenylalanine ammonia-lyase, the initial plasma enzyme level was significantly reduced when 20 units/kg were administered, and the clearance of the enzyme from the plasma was greatly accelerated regardless of the amount administered. Furthermore, in tumor-bearing mice, the rate of clearance was significantly more rapid than in the appropriate non-tumor-bearing control.

Coordinated disintegration reactions mediated by Moloney murine leukemia virus integrase.

PubMed Central

Donzella, G A; Jonsson, C B; Roth, M J

1996-01-01

The protein-DNA and protein-protein interactions important for function of the integrase (IN) protein of Moloney murine leukemia virus (M-MuLV) were investigated by using a coordinated-disintegration assay. A panel of M-MuLV IN mutants and substrate alterations highlighted distinctions between the intermolecular and intramolecular reactions of coordinated disintegration. Mispairing of the crossbone single-strand region and altered long terminal repeat (LTR) positioning affected the intermolecular, but not the intramolecular, reactions of coordinated disintegration. Partial components of the crossbone substrate were coordinated by M-MuLV IN, indicating a reliance on both LTR and target DNA determinants for substrate assembly. The intramolecular reaction was dependent on the presence of either the HHCC domain or a crossbone LTR 5' single-stranded tail. An M-MuLV IN mutant without the HHCC domain (Ndelta105) catalyzed reduced levels of double disintegration but not single disintegration. A separately purified HHCC domain protein (Cdelta232) stimulated double disintegration mediated by Ndelta105, suggesting a role of the N-terminal HHCC domain in stable IN-IN and IN-DNA interactions. Significantly, crossbone substrates lacking the LTR 5' tails were not recognized by the fingerless Ndelta105 protein. Collectively, these data suggest similar roles of the HHCC domain and 5' LTR tail in substrate recognition and modulation of IN activity. PMID:8648728

Tumor suppressors BTG1 and IKZF1 cooperate during mouse leukemia development and increase relapse risk in B-cell precursor acute lymphoblastic leukemia patients.

PubMed

Scheijen, Blanca; Boer, Judith M; Marke, René; Tijchon, Esther; van Ingen Schenau, Dorette; Waanders, Esmé; van Emst, Liesbeth; van der Meer, Laurens T; Pieters, Rob; Escherich, Gabriele; Horstmann, Martin A; Sonneveld, Edwin; Venn, Nicola; Sutton, Rosemary; Dalla-Pozza, Luciano; Kuiper, Roland P; Hoogerbrugge, Peter M; den Boer, Monique L; van Leeuwen, Frank N

2017-03-01

Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1 -deleted B-cell precursor acute lymphoblastic leukemia ( P =0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival ( P =0.0003) and a higher 5-year cumulative incidence of relapse ( P =0.005), when compared with IKZF1 -deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1 , did not affect the outcome of IKZF1 -deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1 -deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1 +/- mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1 +/- displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1 -deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function. Copyright© Ferrata Storti Foundation.

Chronic Lymphocytic Leukemia-Derived IL-10 Suppresses Antitumor Immunity.

PubMed

Alhakeem, Sara S; McKenna, Mary K; Oben, Karine Z; Noothi, Sunil K; Rivas, Jacqueline R; Hildebrandt, Gerhard C; Fleischman, Roger A; Rangnekar, Vivek M; Muthusamy, Natarajan; Bondada, Subbarao

2018-04-30

Chronic lymphocytic leukemia (CLL) patients progressively develop an immunosuppressive state. CLL patients have more plasma IL-10, an anti-inflammatory cytokine, than healthy controls. In vitro human CLL cells produce IL-10 in response to BCR cross-linking. We used the transgenic Eμ-T cell leukemia oncogene-1 ( TCL1 ) mouse CLL model to study the role of IL-10 in CLL associated immunosuppression. Eμ-TCL mice spontaneously develop CLL because of a B cell-specific expression of the oncogene, TCL1. Eμ- TCL1 mouse CLL cells constitutively produce IL-10, which is further enhanced by BCR cross-linking, CLL-derived IL-10 did not directly affect survival of murine or human CLL cells in vitro. We tested the hypothesis that the CLL-derived IL-10 has a critical role in CLL disease in part by suppressing the host immune response to the CLL cells. In IL-10R -/- mice, wherein the host immune cells are unresponsive to IL-10-mediated suppressive effects, there was a significant reduction in CLL cell growth compared with wild type mice. IL-10 reduced the generation of effector CD4 and CD8 T cells. We also found that activation of BCR signaling regulated the production of IL-10 by both murine and human CLL cells. We identified the transcription factor, Sp1, as a novel regulator of IL-10 production by CLL cells and that it is regulated by BCR signaling via the Syk/MAPK pathway. Our results suggest that incorporation of IL-10 blocking agents may enhance current therapeutic regimens for CLL by potentiating host antitumor immune response. Copyright © 2018 by The American Association of Immunologists, Inc.

Novel Quinazolinone MJ-29 Triggers Endoplasmic Reticulum Stress and Intrinsic Apoptosis in Murine Leukemia WEHI-3 Cells and Inhibits Leukemic Mice

PubMed Central

Lu, Chi-Cheng; Yang, Jai-Sing; Chiang, Jo-Hua; Hour, Mann-Jen; Lin, Kuei-Li; Lin, Jen-Jyh; Huang, Wen-Wen; Tsuzuki, Minoru

2012-01-01

The present study was to explore the biological responses of the newly compound, MJ-29 in murine myelomonocytic leukemia WEHI-3 cells in vitro and in vivo fates. We focused on the in vitro effects of MJ-29 on ER stress and mitochondria-dependent apoptotic death in WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice. Our results indicated that a concentration-dependent decrease of cell viability was shown in MJ-29-treated cells. DNA content was examined utilizing flow cytometry, whereas apoptotic populations were determined using annexin V/PI, DAPI staining and TUNEL assay. Increasing vital factors of mitochondrial dysfunction by MJ-29 were further investigated. Thus, MJ-29-provaked apoptosis of WEHI-3 cells is mediated through the intrinsic pathway. Importantly, intracellular Ca2+ release and ER stress-associated signaling also contributed to MJ-29-triggered cell apoptosis. We found that MJ-29 stimulated the protein levels of calpain 1, CHOP and p-eIF2α pathways in WEHI-3 cells. In in vivo experiments, intraperitoneal administration of MJ-29 significantly improved the total survival rate, enhanced body weight and attenuated enlarged spleen and liver tissues in leukemic mice. The infiltration of immature myeloblastic cells into splenic red pulp was reduced in MJ-29-treated leukemic mice. Moreover, MJ-29 increased the differentiations of T and B cells but decreased that of macrophages and monocytes. Additionally, MJ-29-stimulated immune responses might be involved in anti-leukemic activity in vivo. Based on these observations, MJ-29 suppresses WEHI-3 cells in vitro and in vivo, and it is proposed that this potent and selective agent could be a new chemotherapeutic candidate for anti-leukemia in the future. PMID:22662126

Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration

PubMed Central

De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferris, Andrea L.; Hughes, Stephen H.

2014-01-01

ABSTRACT Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors. PMID:24501411

Abrogation of graft-vs.-leukemia activity after depletion of CD3+ T cells in a murine model of MHC-matched peripheral blood progenitor cell transplantation (PBPCT).

PubMed

Uharek, L; Glass, B; Zeis, M; Dreger, P; Steinmann, J; Löffler, H; Schmitz, N

1998-02-01

Using a murine transplantation model, we simulated a clinical situation in which major histocompatibility complex (MHC)-identical allogeneic peripheral blood progenitor cells (PBPCs) are transplanted for the treatment of a malignant disease that is resistant to resting natural killer (NK) cells but sensitive to cytokine-activated NK cells and T cell-mediated antitumor activity. We determined the influence of selective T cell depletion of allogeneic PBPC grafts on graft-vs.-leukemia (GVL) activity and investigated the effectiveness of ex vivo treatment with NK cell-activating cytokines to compensate for the putative loss of T cell-derived factors stimulating natural cytotoxicity. After pretreatment of Balb/c (H-2d) recipients with 7.5 Gy of total body irradiation, 2x10(7) rhG-CSF-mobilized PBPCs of splenectomized syngeneic or MHC-identical DBA (H-2d) mice were transferred. Selective T cell depletion (TCD) was performed by immunomagnetic purging with a mononoclonal antibody directed against CD3. In some experimental groups, T cell-depleted PBPCs were incubated with 200 U/mL interleukin (IL)-2 and 100 U/mL IL-12 for 24 hours. To investigate antileukemic activity in vivo, recipient mice were inoculated with 1x10(5) A20 cells (a B-lymphoblastic leukemia of Balb/c origin) 2 days before PBPC transplantation (PBPCT). After transplantation of unmanipulated allogeneic cells, 25% of the animals died with signs of graft-vs.-host disease (GVHD) but 71% were free from relapse 100 days after PBPCT. After TCD of allogeneic grafts with anti-CD3, the incidence of GVH-related mortality was below 5% but the leukemia-free survival rate was significantly (p < 0.05) decreased to 25% and thus was similar to that observed after syngeneic PBPCT (17%). When CD3-depleted grafts were incubated with IL-2 and IL-12, 45% of the animals remained free from leukemia; however, the difference was not statistically significant. Our results suggest that ex vivo activation of residual NK cells with IL-2 and IL-12 does not fully compensate for the abrogation of GVL activity after depletion of CD3+ T cells from MHC-matched PBPCT.

Intracellular trafficking of hybrid gene delivery vectors.

PubMed

Keswani, Rahul K; Lazebnik, Mihael; Pack, Daniel W

2015-06-10

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles. Copyright © 2015 Elsevier B.V. All rights reserved.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

PubMed

Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

2015-01-01

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis.

PubMed

Valerio, Daria G; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J; Huang, Chun-Hao; Lujambio, Amaia; Zheng, YuJun George; Zuber, Johannes; Pandita, Tej K; Lowe, Scott W; Armstrong, Scott A

2017-04-01

Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9 -driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof -deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9 -driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL -rearranged leukemias. Cancer Res; 77(7); 1753-62. ©2017 AACR . ©2017 American Association for Cancer Research.

Structure of a gene encoding a murine thymus leukemia antigen, and organization of Tla genes in the BALB/c mouse

PubMed Central

1985-01-01

We have determined the DNA sequence of a gene encoding a thymus leukemia (TL) antigen in the BALB/c mouse, and have more definitively mapped the cloned BALB/c Tla-region class I gene clusters. Analysis of the sequence shows that the Tla gene is less closely related to the H-2 genes than H-2 genes are to one another or to a Qa-2,3-region genes. The Tla gene, 17.3A, contains an apparent gene conversion. Comparison of the BALB/c Tla genes with those from C57BL shows that BALB/c has more Tla-region class I genes, and that one of the genes absent in C57BL is gene 17.3A. PMID:3894562

Radiation-induced leukemia: Comparative studies in mouse and man. Annual performance report, June 1, 1991--October 31, 1991

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haas, M.

1991-12-31

We now have a clear understanding of the mechanism by which radiation-induced (T-cell) leukemia occurs. In irradiated mice (radiation-induced thymic leukemia) and in man (acute lymphoblastic T-cell leukemia, T-ALL) the mechanism of leukemogenesis is surprisingly similar. Expressed in the most elementary terms, T-cell leukemia occurs when T-cell differentiation is inhibited by a mutation, and pre-T cells attempt but fail to differentiate in the thymus. Instead of leaving the thymus for the periphery as functional T-cells they continue to proliferate in the thymus. The proliferating pre- (pro-) T-cells constitute the (early) acute T-cell leukemia (A-TCL). This model for the mechanism ofmore » T-cell leukemogenesis accounts for all the properties of both murine and human A-TCL. Important support for the model has recently come from work by Ilan Kirsch and others, who have shown that mutations/deletions in the genes SCL (TAL), SIL, and LCK constitute primary events in the development of T-ALL, by inhibiting differentiation of thymic pre- (pro-) T-cells. This mechanism of T-cell leukemogenesis brings several specific questions into focus: How do early A-TCL cells progress to become potently tumorigenic and poorly treatable? Is it feasible to genetically suppress early and/or progressed A-TCL cells? What is the mechanism by which the differentiation-inhibited (leukemic) pre-T cells proliferate? During the first grant year we have worked on aspects of all three questions.« less

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

PubMed

Hampton, T A; Conry, R M; Khazaeli, M B; Shaw, D R; Curiel, D T; LoBuglio, A F; Strong, T V

2000-03-01

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less

Search for infective mammalian type-C virus-related genes in the DNA of human sarcomas and leukemias.

PubMed

Nicolson, M O; Gilden, R V; Charman, H; Rice, N; Heberling, R; McAllister, R M

1978-06-15

DNA was extracted from two human sarcoma cell lines, TE-32 and TE-418, and the leukemic cells from five children with acute myelocytic leukemia, three children with acute lymphocytic leukemia and four adults with acute myelocytic leukemia. The DNAs, assayed for infectivity by transfection techniques, induced no measurable virus by methods which would detect known mammalian C-type antigens or RNA-directed DNA polymerase in TE-32, D-17 dog cells and other indicator cells, nor did they recombine with or rescue endogenous human or exogenous murine or baboon type-C virus. Model systems used as controls were human sarcoma cells, TE-32 and HT-1080, and human lymphoma cells TE-543, experimentally infected with KiMuLV, GaLV or baboon type-C virus, all of which released infectious virus and whose DNAs were infectious for TE-32 and D-17 dog cells. Other model systems included two baboon placentas and one embryonic cell strain spontaneously releasing infectious endogenous baboon virus and yielding DNAs infectious for D-17 dog cells but not for TE-32 cells. Four other baboon embryonic tissues and two embryonic cell strains, releasing either low levels of virus or no virus, did not yield infectious DNA.

RARα-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia

PubMed Central

Guidez, Fabien; Parks, Sarah; Wong, Henna; Jovanovic, Jelena V.; Mays, Ashley; Gilkes, Amanda F.; Mills, Kenneth I.; Guillemin, Marie-Claude; Hobbs, Robin M.; Pandolfi, Pier Paolo; de Thé, Hugues; Solomon, Ellen; Grimwade, David

2007-01-01

Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARα by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARα oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARα-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARα+RARα/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARα APL. RARα-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARα-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype. PMID:18000064

Rheb1 promotes tumor progression through mTORC1 in MLL-AF9-initiated murine acute myeloid leukemia.

PubMed

Gao, Yanan; Gao, Juan; Li, Minghao; Zheng, Yawei; Wang, Yajie; Zhang, Hongyan; Wang, Weili; Chu, Yajing; Wang, Xiaomin; Xu, Mingjiang; Cheng, Tao; Ju, Zhenyu; Yuan, Weiping

2016-04-12

The constitutive hyper-activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways has frequently been associated with acute myeloid leukemia (AML). While many inhibitors targeting these pathways have been developed, the anti-leukemic effect was not as robust as expected. As part of the molecular link between PI3K/Akt and mTOR kinase, the role of Rheb1 in AML remains unexplored. Our study aims to explore the role of Rheb1 in AML and estimate whether Rheb1 could be a potential target of AML treatment. The expressions of Rheb1 and other indicated genes were analyzed using real-time PCR. AML mouse model was established by retrovirus transduction. Leukemia cell properties and related signaling pathways were dissected by in vitro and in vivo studies. The transcriptional changes were analyzed via gene chip analysis. Molecular reagents including mTOR inhibitor and mTOR activator were used to evaluate the function of related signaling pathway in the mouse model. We observed that Rheb1 is overexpressed in AML patients and the change of Rheb1 level in AML patients is associated with their median survival. Using a Rheb1-deficient MLL-AF9 murine AML model, we revealed that Rheb1 deletion prolonged the survival of AML mice by weakening LSC function. In addition, Rheb1 deletion arrested cell cycle progression and enhanced apoptosis of AML cells. Furthermore, while Rheb1 deletion reduced mTORC1 activity in AML cells, additional rapamycin treatment further decreased mTORC1 activity and increased the apoptosis of Rheb1 (Δ/Δ) AML cells. The mTOR activator 3BDO partially rescued mTORC1 signaling and inhibited apoptosis in Rheb1 (Δ/Δ) AML cells. Our data suggest that Rheb1 promotes AML progression through mTORC1 signaling pathway and combinational drug treatments targeting Rheb1 and mTOR might have a better therapeutic effect on leukemia.

Anthocyanins from black rice (Oryza sativa) promote immune responses in leukemia through enhancing phagocytosis of macrophages in vivo.

PubMed

Fan, Ming-Jen; Yeh, Ping-Hsuan; Lin, Jing-Pin; Huang, An-Cheng; Lien, Jin-Cherng; Lin, Hui-Yi; Chung, Jing-Gung

2017-07-01

Rice is a staple food in numerous countries around the world. Anthocyanins found in black rice have been reported to reduce the risk of certain diseases, but the effects of crude extract of anthocyanins from Asia University-selected purple glutinous indica rice (AUPGA) on immune responses have not yet been demonstrated. The current study aimed to investigate whether AUPGA treatment could affect immune responses in murine leukemia cells in vivo . Murine acute myelomonocytic leukemia WEHI-3 cells were intraperitoneally injected into normal BALB/c mice to generate leukemia mice. A total of 50 mice were randomly divided into five groups (n=10 in each group) and were fed a diet supplemented with AUPGA at 0, 20, 50 or 100 mg/kg for three weeks. All mice were weighed and the blood, liver and spleen were collected for further experiments. The results indicated that AUPGA did not significantly affect animal body weight, but significantly increased spleen weight (P<0.05) and decreased liver weight (P<0.05) when compared with the control group. AUPGA significantly increased the T cell (CD3) population at treatments of 20 and 100 mg/kg (P<0.05). However, it only significantly increased the B cell (CD19) population at a treatment of 20 mg/kg (P<0.05). Furthermore, AUPGA at 50 and 100 mg/kg significantly increased the monocyte (CD11b) population and the level of macrophages (Mac-3; P<0.05 for both). AUPGA at 50 and 100 mg/kg significantly promoted macrophage phagocytosis in peripheral blood mononuclear cells (P<0.05), and all doses of AUPGA treatment significantly promoted macrophage phagocytotic activity in the peritoneum (P<0.05). AUPGA treatment significantly decreased natural killer cell activity from splenocytes (P<0.05). Finally, AUPGA treatment at 20 mg/kg treatment significantly promoted T cell proliferation (P<0.05), and treatment at 50 and 100 mg/kg significantly decreased B cell proliferation compared with the control group (P<0.05).

Anthocyanins from black rice (Oryza sativa) promote immune responses in leukemia through enhancing phagocytosis of macrophages in vivo

PubMed Central

Fan, Ming-Jen; Yeh, Ping-Hsuan; Lin, Jing-Pin; Huang, An-Cheng; Lien, Jin-Cherng; Lin, Hui-Yi; Chung, Jing-Gung

2017-01-01

Rice is a staple food in numerous countries around the world. Anthocyanins found in black rice have been reported to reduce the risk of certain diseases, but the effects of crude extract of anthocyanins from Asia University-selected purple glutinous indica rice (AUPGA) on immune responses have not yet been demonstrated. The current study aimed to investigate whether AUPGA treatment could affect immune responses in murine leukemia cells in vivo. Murine acute myelomonocytic leukemia WEHI-3 cells were intraperitoneally injected into normal BALB/c mice to generate leukemia mice. A total of 50 mice were randomly divided into five groups (n=10 in each group) and were fed a diet supplemented with AUPGA at 0, 20, 50 or 100 mg/kg for three weeks. All mice were weighed and the blood, liver and spleen were collected for further experiments. The results indicated that AUPGA did not significantly affect animal body weight, but significantly increased spleen weight (P<0.05) and decreased liver weight (P<0.05) when compared with the control group. AUPGA significantly increased the T cell (CD3) population at treatments of 20 and 100 mg/kg (P<0.05). However, it only significantly increased the B cell (CD19) population at a treatment of 20 mg/kg (P<0.05). Furthermore, AUPGA at 50 and 100 mg/kg significantly increased the monocyte (CD11b) population and the level of macrophages (Mac-3; P<0.05 for both). AUPGA at 50 and 100 mg/kg significantly promoted macrophage phagocytosis in peripheral blood mononuclear cells (P<0.05), and all doses of AUPGA treatment significantly promoted macrophage phagocytotic activity in the peritoneum (P<0.05). AUPGA treatment significantly decreased natural killer cell activity from splenocytes (P<0.05). Finally, AUPGA treatment at 20 mg/kg treatment significantly promoted T cell proliferation (P<0.05), and treatment at 50 and 100 mg/kg significantly decreased B cell proliferation compared with the control group (P<0.05). PMID:28672893

Prevalence of Abelson murine leukemia viral oncogene homolog-breakpoint cluster region fusions and correlation with peripheral blood parameters in chronic myelogenous leukemia patients in Lorestan Province, Iran.

PubMed

Kiani, Ali Asghar; Shahsavar, Farhad; Gorji, Mojtaba; Ahmadi, Kolsoum; Nazarabad, Vahideh Heydari; Bahmani, Banafsheh

2016-01-01

Chronic myelogenous leukemia (CML) is a chronic malignancy of myeloid linage associated with a significant increase in granulocytes in bone marrow and peripheral blood. CML diagnosis is based on detection of Philadelphia chromosome and "Abelson murine leukemia viral oncogene homolog" (ABL)-"breakpoint cluster region protein" fusions (ABL-BCR fusions). In this study, patients with CML morphology were studied according to ABL-BCR fusions and the relationship between the fusions and peripheral blood cell changes was examined. All patients suspected to chronic myeloproliferative disorders in Lorestan Province visiting subspecialist hematology clinics who were confirmed by oncologist were studied over a period of 5 years. After completing basic data questionnaire, blood samples were obtained with informed consent from the patients. Blood cell count and morphology were investigated and RNA was extracted from blood samples. cDNA was synthesized from RNA and ABL-BCR fusions including b3a2 and b2a2 (protein 210 kd or p210), e1a2 (protein 190 kdor p190), and e19a2 (protein 230 kdor p230) were studied by multiplex reverse transcription polymerase chain reaction method. Coexistence of e1a2 and b2a2 (p210/p190) fusions was also studied. The prevalence of mutations and their correlation with the blood parameters were statistically analyzed. Of 58 patients positive for ABL-BCR fusion, 18 (30.5%) had b2a2 fusion, 37 (62.71%) had b3a2 fusion and three (3.08%) had e1a2 fusion. Coexistence of e1a2 and b2a2 (p210/p190) was not observed. There was no significant correlation between ABL-BCR fusions and white blood cell count, platelet count, and hemoglobin concentration. The ABL-BCR fusions in Lorestan Province were similar to other studies in Iran, and b3a2 fusion had the highest prevalence in the studied patients studied.

A. cantoniensis inhibits the proliferation of murine leukemia WEHI-3 cells in vivo and promotes immunoresponses in vivo.

PubMed

Tan, Tzu-Wei; Lin, Yuh-Tzy; Yang, Jai-Sing; Lu, Chi-Cheng; Chiang, Jo-Hua; Wu, Chang-Lin; Lin, Jing-Pin; Tang, Nou-Ying; Yeh, Chin-Chung; Fan, Ming-Jen; Chung, Jing-Gung

2009-01-01

Ampelopsis cantoniensis (AC) has been used as a folk medicine for reducing pain in the Taiwanese population. Our previous studies have shown that the crude extract of AC induced apoptosis in human promyelocytic leukemia HL-60 cells. In this study, the in vivo effects of AC on leukemia WEHI-3 cells and immune responses such as phagocytosis and natural killer (NK) cell activity were investigated. The weights of the livers and spleens were decreased in the AC-treated groups compared to the control groups. The AC treatment increased the percentage of CD3 and CD19 marker cells in WEHI-3-injected mice, indicating that the precursors of T and B cells were inhibited. The AC treatment promoted the activity of macrophage phagocytosis in the peripheral blood mononuclear cells (PBMC) and peritoneal cells. It was found that the NK cells from mice after treatment with AC can kill the YAC-1 target cells. Therefore, the AC treatment increased NK cell activity. In conclusion, AC can affect WEHI-3 cells in vivo and promote macrophage and NK cell activities.

Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation

PubMed Central

Jo, Stephanie Y.; Granowicz, Eric M.; Maillard, Ivan; Thomas, Dafydd

2011-01-01

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis, and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, whereas cells with other leukemic oncogenes, such as Hoxa9/Meis1 and E2A-HLF, did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes. PMID:21398221

Therapy-induced PML/RARA proteolysis and acute promyelocytic leukemia cure.

PubMed

Nasr, Rihab; Lallemand-Breitenbach, Valérie; Zhu, Jun; Guillemin, Marie-Claude; de Thé, Hugues

2009-10-15

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that yields the PML/RARA fusion gene. Clinically, besides chemotherapy, two drugs induce clinical remissions: retinoic acid (RA) and arsenic trioxide (As). Both agents directly target PML/RARA-mediated transcriptional repression and protein stability, inducing to various extent promyelocyte differentiation and clinical remission of APL patients. RA targets the RARA moiety of the fusion, whereas arsenic targets its PML part. PML/RARA expression in the mouse is sufficient to initiate APL. The RA-As association, which synergizes for PML/RARA degradation but not for differentiation, rapidly clears leukemia initiating cells (LIC), resulting in APL eradication in murine APL models, but also in several APL clinical trials. Cyclic AMP triggered PML/RARA phosphorylation also enhances RA-induced APL regression, PML/RARA degradation, and LIC clearance, raising new options for therapy-resistant patients. Although differentiation has a major role in debulking of the tumor, PML/RARA degradation seems to be the primary basis for APL eradication by the RA-As association. Oncoprotein degradation could be a general therapeutic strategy that may be extended beyond APL.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed

Darlix, J L; Gabus, C; Allain, B

1992-12-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed Central

Darlix, J L; Gabus, C; Allain, B

1992-01-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein. Images PMID:1331519

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses.

PubMed Central

Khan, A S

1984-01-01

The sequence of 363 nucleotides near the 3' end of the pol gene and 564 nucleotides from the 5' terminus of the env gene in an endogenous murine leukemia viral (MuLV) DNA segment, cloned from AKR/J mouse DNA and designated as A-12, was obtained. For comparison, the nucleotide sequence in an analogous portion of AKR mink cell focus-forming (MCF) 247 MuLV provirus was also determined. Sequence features unique to MCF247 MuLV DNA in the 3' pol and 5' env regions were identified by comparison with nucleotide sequences in analogous regions of NFS -Th-1 xenotropic and AKR ecotropic MuLV proviruses. These included (i) an insertion of 12 base pairs encoding four amino acids located 60 base pairs from the 3' terminus of the pol gene and immediately preceding the env gene, (ii) the deletion of 12 base pairs (encoding four amino acids) and the insertion of 3 base pairs (encoding one amino acid) in the 5' portion of the env gene, and (iii) single base substitutions resulting in 2 MCF247 -specific amino acids in the 3' pol and 23 in the 5' env regions. Nucleotide sequence comparison involving the 3' pol and 5' env regions of AKR MCF247 , NFS xenotropic, and AKR ecotropic MuLV proviruses with the cloned endogenous MuLV DNA indicated that MCF247 proviral DNA sequences were conserved in the cloned endogenous MuLV proviral segment. In fact, total nucleotide sequence identity existed between the endogenous MuLV DNA and the MCF247 MuLV provirus in the 3' portion of the pol gene. In the 5' env region, only 4 of 564 nucleotides were different, resulting in three amino acid changes between AKR MCF247 MuLV DNA and the endogenous MuLV DNA present in clone A-12. In addition, nucleotide sequence comparison indicated that Moloney-and Friend-MCF MuLVs were also highly related in the 3' pol and 5' env regions to the cloned endogenous MuLV DNA. These results establish the role of endogenous MuLV DNA segments in generation of recombinant MCF viruses. PMID:6328017

Tissue responses to low protracted doses of high let radiations or photons - Early and late damage relevant to radio-protective countermeasures

NASA Technical Reports Server (NTRS)

Ainsworth, E. J.; Afzal, S. M. J.; Crouse, D. A.; Hanson, W. R.; Fry, R. J. M.

1989-01-01

Early and late murine tissue responses to single or fractionated low doses of heavy charged particles, fission-spectrum neutrons or gamma rays are considered. Damage to the hematopoietic system is emphasized, but results on acute lethality, host response to challenge with transplanted leukemia cells and life-shortening are presented. Recent studies on protection against early and late effects by aminothiols, prostaglandins, and other compounds are discussed.

Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag▿

PubMed Central

Datta, Siddhartha A. K.; Zuo, Xiaobing; Clark, Patrick K.; Campbell, Stephen J.; Wang, Yun-Xing; Rein, Alan

2011-01-01

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed. PMID:21917964

Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus.

PubMed Central

Manley, N R; O'Connell, M A; Sharp, P A; Hopkins, N

1989-01-01

Nondefective Friend murine leukemia virus (MuLV) causes erythroleukemia when injected into newborn NFS mice, while Moloney MuLV causes T-cell lymphoma. Exchange of the Friend virus enhancer region, a sequence of about 180 nucleotides including the direct repeat and a short 3'-adjacent segment, for the corresponding region in Moloney MuLV confers the ability to cause erythroid disease on Moloney MuLV. We have used the electrophoretic mobility shift assay and methylation interference analysis to identify cellular factors which bind to the Friend virus enhancer region and compared these with factors, previously identified, that bind to the Moloney virus direct repeat (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987). We identified five binding sites for sequence-specific DNA-binding proteins in the Friend virus enhancer region. While some binding sites are present in both the Moloney and Friend virus enhancers, both viruses contain unique sites not present in the other. Although none of the factors identified in this report which bind to these unique sites are present exclusively in T cells or erythroid cells, they bind to three regions of the enhancer shown by genetic analysis to encode disease specificity and thus are candidates to mediate the tissue-specific expression and distinct disease specificities encoded by these virus enhancer elements. Images PMID:2778872

Protein Kinase Inhibitor γ reciprocally regulates osteoblast and adipocyte differentiation by downregulating Leukemia Inhibitory Factor

PubMed Central

Chen, Xin; Hausman, Bryan S.; Luo, Guangbin; Zhou, Guang; Murakami, Shunichi; Rubin, Janet; Greenfield, Edward M.

2013-01-01

The Protein Kinase Inhibitor (Pki) gene family inactivates nuclear PKA and terminates PKA-induced gene expression. We previously showed that Pkig is the primary family member expressed in osteoblasts and that Pkig knockdown increases the effects of parathyroid hormone and isoproterenol on PKA activation, gene expression, and inhibition of apoptosis. Here, we determined whether endogenous levels of Pkig regulate osteoblast differentiation. Pkig is the primary family member in MEFs, murine marrow-derived mesenchymal stem cells, and human mesenchymal stem cells. Pkig deletion increased forskolin-dependent nuclear PKA activation and gene expression and Pkig deletion or knockdown increased osteoblast differentiation. PKA signaling is known to stimulate adipogenesis; however, adipogenesis and osteogenesis are often reciprocally regulated. We found that the reciprocal regulation predominates over the direct effects of PKA since adipogenesis was decreased by Pkig deletion or knockdown. Pkig deletion or knockdown simultaneously increased osteogenesis and decreased adipogenesis in mixed osteogenic/adipogenic medium. Pkig deletion increased PKA-induced expression of Leukemia Inhibitory Factor (Lif) mRNA and LIF protein. LIF neutralizing antibodies inhibited the effects on osteogenesis and adipogenesis of either Pkig deletion in MEFs or PKIγ knockdown in both murine and human mesenchymal stem cells. Collectively, our results show that endogenous levels of Pkig reciprocally regulate osteoblast and adipocyte differentiation and that this reciprocal regulation is mediated in part by LIF. PMID:23963683

Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

PubMed

De Rocquigny, H; Ficheux, D; Gabus, C; Allain, B; Fournie-Zaluski, M C; Darlix, J L; Roques, B P

1993-02-25

The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger.

Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites.

PubMed Central

Schultz, A M; Lockhart, S M; Rabin, E M; Oroszlan, S

1981-01-01

The structural relationships among the gag polyproteins Pr65gag, Pr75gag, and gPr80gag of Rauscher murine leukemia virus were studied by endoglycosidase H digestion and formic acid cleavage. Fragments were identified by precipitation with specific antisera to constituent virion structural proteins followed by one-dimensional mapping. Endoglycosidase H reduced the size of gPr80gag to that of Pr75gag. By comparing fragments of gPr80gag and the apoprotein Pr75gag, the former was shown to contain two mannose-rich oligosaccharide units. By comparing fragments of Pr65gag and Pr75gag, the latter was shown to differ from Pr65gag at the amino terminus by the presence of a leader peptide approximately 7,000 daltons in size. The internal and carboxyl-terminal peptides of the two unglycosylated polyproteins were not detectably different. The location of the two N-linked carbohydrate chains in gPr80gag has been specified. One occurs in the carboxyl-terminal half of the polyprotein at asparagine177 of the p30 sequence and the other is found in a 23,000-dalton fragment located in the amino-terminal region of gPr80gag and containing the additional amino acid sequences not found in Pr65gag plus a substantial portion of p15. Images PMID:7241663

Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies

PubMed Central

Zweidler-McKay, Patrick A.; He, Yiping; Xu, Lanwei; Rodriguez, Carlos G.; Karnell, Fredrick G.; Carpenter, Andrea C.; Aster, Jon C.; Allman, David; Pear, Warren S.

2005-01-01

Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)–translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies. PMID:16118316

Adoptively transferred TRAIL+ T cells suppress GVHD and augment antitumor activity

PubMed Central

Ghosh, Arnab; Dogan, Yildirim; Moroz, Maxim; Holland, Amanda M.; Yim, Nury L.; Rao, Uttam K.; Young, Lauren F.; Tannenbaum, Daniel; Masih, Durva; Velardi, Enrico; Tsai, Jennifer J.; Jenq, Robert R.; Penack, Olaf; Hanash, Alan M.; Smith, Odette M.; Piersanti, Kelly; Lezcano, Cecilia; Murphy, George F.; Liu, Chen; Palomba, M. Lia; Sauer, Martin G.; Sadelain, Michel; Ponomarev, Vladimir; van den Brink, Marcel R.M.

2013-01-01

Current strategies to suppress graft-versus-host disease (GVHD) also compromise graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to separate GVHD and GVT responses merely spare GVT function without actually enhancing it. We have previously shown that endogenously expressed TNF-related apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against certain malignancies in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we genetically engineered T cells to overexpress TRAIL and adoptively transferred donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We found that murine TRAIL+ T cells induced apoptosis of alloreactive T cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT response. Moreover, human TRAIL+ T cells mediated enhanced in vitro cytotoxicity against both human leukemia cell lines and against freshly isolated chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf, donor-unrestricted antitumor cellular therapy, in vitro–generated TRAIL+ precursor T cells from third-party donors also mediated enhanced GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing donor T cells could potentially enhance the curative potential of allo-HSCT by increasing GVT response and suppressing GVHD. PMID:23676461

Ferrate oxidation of murine leukemia virus reverse transcriptase: identification of the template-primer binding domain.

PubMed

Reddy, G; Nanduri, V B; Basu, A; Modak, M J

1991-08-20

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.

Cooperativity of CD44 and CD49d in leukemia cell homing, migration, and survival offers a means for therapeutic attack.

PubMed

Singh, Vibuthi; Erb, Ulrike; Zöller, Margot

2013-11-15

A CD44 blockade drives leukemic cells into differentiation and apoptosis by dislodging from the osteogenic niche. Because anti-CD49d also supports hematopoietic stem cell mobilization, we sought to determine the therapeutic efficacy of a joint CD49d/CD44 blockade. To unravel the underlying mechanism, the CD49d(-) EL4 lymphoma was transfected with CD49d or point-mutated CD49d, prohibiting phosphorylation and FAK binding; additionally, a CD44(-) Jurkat subline was transfected with murine CD44, CD44 with a point mutation in the ezrin binding site, or with cytoplasmic tail-truncated CD44. Parental and transfected EL4 and Jurkat cells were evaluated for adhesion, migration, and apoptosis susceptibility in vitro and in vivo. Ligand-binding and Ab-blocking studies revealed CD44-CD49d cooperation in vitro and in vivo in adhesion, migration, and apoptosis resistance. The cooperation depends on ligand-induced proximity such that both CD44 and CD49d get access to src, FAK, and paxillin and via lck to the MAPK pathway, with the latter also supporting antiapoptotic molecule liberation. Accordingly, synergisms were only seen in leukemia cells expressing wild-type CD44 and CD49d. Anti-CD44 together with anti-CD49d efficiently dislodged EL4-CD49d/Jurkat-CD44 in bone marrow and spleen. Dislodging was accompanied by increased apoptosis susceptibility that strengthened low-dose chemotherapy, the combined treatment most strongly interfering with metastatic settlement and being partly curative. Ab treatment also promoted NK and Ab-dependent cellular cytotoxicity activation, which affected leukemia cells independent of CD44/CD49d tail mutations. Thus, mostly owing to a blockade of joint signaling, anti-CD44 and anti-CD49d hamper leukemic cell settlement and break apoptosis resistance, which strongly supports low-dose chemotherapy.

Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fink, J.K.; Correll, P.H.; Perry, L.K.

1990-03-01

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, the authors developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py{sup +}/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hemaptopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeatmore » (Mo-MLV LTR) and levels of Py{sup +}/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py{sup +}/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity has been normalized.« less

miR-125b promotes MLL-AF9–driven murine acute myeloid leukemia involving a VEGFA-mediated non–cell-intrinsic mechanism

PubMed Central

Liu, Jun; Guo, Bo; Chen, Zhuo; Wang, Nayi; Iacovino, Michelina; Cheng, Jijun; Roden, Christine; Pan, Wen; Khan, Sajid; Chen, Suning; Kyba, Michael; Fan, Rong; Guo, Shangqin

2017-01-01

The hematopoietic stem cell–enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9. By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9–driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b. Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non–cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2. Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b. In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non–cell-intrinsic leukemia-promoting effects by an oncogenic miRNA. PMID:28053194

Retinoic acid-induced differentiation of retrovirus-infected HL-60 cells is associated with enhanced transcription from the viral long terminal repeat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, S.J.

1988-11-01

The author infected different human leukemic cell lines with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. In retrovirus-infected G418-resistant HL-60 cells, induction of granulocyte differentiation by retinoic acid was invariably accompanied by a marked increase (5- to 10-fold) in the transcriptional activity of the integrated retroviral long terminal repeat.

Detection of Xenotropic Murine Leukemia Virus-Related Virus (XMRV) in Gulf War Illness: Role in Pathogenesis or Biomarker?

DTIC Science & Technology

2014-12-01

Belz, C.M. Smith, R.J. Steptoe, W.R. Heath, K. Shortman, J.A. Villadangos, Most lymphoid organ dendritic cell types are phenotypically and...Nicolas, D. Kaiserlian, Innate CD4+CD25+ regulatory T cells are required for oral tolerance and inhibition of CD8+ T cells mediating skin inflammation...140] M. Guadalupe, E. Reay, S. Sankaran, T. Prindiville, J. Flamm, A. McNeil, S. Dandekar, Severe CD4+ T- cell depletion in gut lymphoid tissue during

[Synthesis and biological activity of 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone analogs. 1. Substitution at the S atom].

PubMed

Schulze, W; Gutsche, W; Wohlrabe, K; Fleck, W; Tresselt, D

1985-08-01

The synthesis of S-substituted derivatives of 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone is described. The obtained 1,4-benzoquinone-guanylhydrazone-S-alkyl (resp. aralkyl)-isothiosemicarbazones, in comparison with the unsubstituted standard compound, showed a significantly decreased biological activity against the murine leukemias L 1210 and P 388 as well as against the growth of several kinds of bacteria. Therefore the S-substitution seems not to be useful for reaching a maximum activity.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

8-CPT-cAMP/all-trans retinoic acid targets t(11;17) acute promyelocytic leukemia through enhanced cell differentiation and PLZF/RARα degradation

PubMed Central

Jiao, Bo; Ren, Zhi-Hong; Liu, Ping; Chen, Li-Juan; Shi, Jing-Yi; Dong, Ying; Ablain, Julien; Shi, Lin; Gao, Li; Hu, Jun-Pei; Ren, Rui-Bao; de Thé, Hugues; Chen, Zhu; Chen, Sai-Juan

2013-01-01

The refractoriness of acute promyelocytic leukemia (APL) with t(11;17)(q23;q21) to all-trans retinoic acid (ATRA)-based therapy concerns clinicians and intrigues basic researchers. By using a murine leukemic model carrying both promyelocytic leukemia zinc finger/retinoic acid receptor-α (PLZF/RARα) and RARα/PLZF fusion genes, we discovered that 8-chlorophenylthio adenosine-3′, 5′-cyclic monophosphate (8-CPT-cAMP) enhances cellular differentiation and improves gene trans-activation by ATRA in leukemic blasts. Mechanistically, in combination with ATRA, 8-CPT-cAMP activates PKA, causing phosphorylation of PLZF/RARα at Ser765 and resulting in increased dissociation of the silencing mediator for retinoic acid and thyroid hormone receptors/nuclear receptor corepressor from PLZF/RARα. This process results in changes of local chromatin and transcriptional reactivation of the retinoic acid pathway in leukemic cells. Meanwhile, 8-CPT-cAMP also potentiated ATRA-induced degradation of PLZF/RARα through its Ser765 phosphorylation. In vivo treatment of the t(11;17) APL mouse model demonstrated that 8-CPT-cAMP could significantly improve the therapeutic effect of ATRA by targeting a leukemia-initiating cell activity. This combined therapy, which induces enhanced differentiation and oncoprotein degradation, may benefit t(11;17) APL patients. PMID:23382200

K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors

PubMed Central

Cornejo, Melanie G.; Scholl, Claudia; Liu, Jianing; Leeman, Dena S.; Haydu, J. Erika; Fröhling, Stefan; Lee, Benjamin H.; Gilliland, D. Gary

2008-01-01

To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways. PMID:18663146

Preclinical evaluation of amiodarone for the treatment of murine leukemia P388. In vivo and in vitro investigation.

PubMed

Papageorgiou, A D; Dalezis, P; Mourelatos, C; Lioutas, K; Sahpazidou, D; Geromichalou, E; Geromichalos, G; Lialiaris, Th; Athanasiadou, P; Athanasiadis, P

2010-01-01

The purpose of the present study was the investigation of antileukemic effect of amiodarone in leukemia P388 BDF1 bearing mice and its genotoxic and cytostatic effect in cultured normal human lymphocytes. Leukemia P388 was used in this study. BDF1 mice were used for chemotherapy evaluation in vivo. The antitumor activity was assessed by the oncostatic parameter T/C, representing the increase of life span of drug-treated animals vs. controls. Lymphocyte cultures were used to study the genotoxic and cytostatic effect in vitro, expressed by enhanced sister chromatid exchange (SCE) and reduced proliferation rate indices (PRIS). Amiodarone was found to exert antileukemic potency against leukemia P388 bearing mice at all three different treatment schedules used, yielding T/C values of 155%, 163% with one cure and 230%. In the in vitro cytogenic experiments, significant increase of SCE rates by amiodarone was observed at 0.2 μM, while at the same concentration significant suppression of PRIS was achieved. According to the National Cancer Institute (NCI), a compound is characterized as potential chemotherapeutic deserving further evaluation if it produces T/C values≥125%. On the other hand the SCE assay has predictive value as a clinical assay for drugs exhibiting a strong correlation between cell killing and induction of SCEs. Further studies are warranted to clarify the structure-activity relationship of amiodarone.

Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors.

PubMed

Oyallon, Bruno; Brachet-Botineau, Marie; Logé, Cédric; Bonnet, Pascal; Souab, Mohamed; Robert, Thomas; Ruchaud, Sandrine; Bach, Stéphane; Berthelot, Pascal; Gouilleux, Fabrice; Viaud-Massuard, Marie-Claude; Denevault-Sabourin, Caroline

2018-05-11

We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1) kinase. Seventeen analogues were synthesized providing useful insight into structure-activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1 are consistent with an unclassical binding mode of these inhibitors. The lead compound 1 was able to block HsPim-1 enzymatic activity at nanomolar concentrations (IC 50 of 74 nM), with a good selectivity profile against a panel of mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia cell line KU812 showed an antitumor activity at micromolar concentrations. As a result, compound 1 represents a promising lead for the design of novel anticancer targeted therapies. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

PubMed Central

Zajac, Pawel; Islam, Saiful; Hochgerner, Hannah; Lönnerberg, Peter; Linnarsson, Sten

2013-01-01

Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3´ end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing. However, the base preference of the terminal transferase activity is not known in detail, which may lead to inefficiencies in template switching when starting from tiny amounts of mRNA. Here, we used fully degenerate oligos to determine the exact base preference at the template switching site up to a distance of ten nucleotides. We found a strong preference for guanosine at the first non-templated nucleotide, with a greatly reduced bias at progressively more distant positions. Based on this result, and a number of careful optimizations, we report conditions for efficient template switching for cDNA amplification from single cells. PMID:24392002

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

PubMed Central

Williams, J H; Daly, L N; Ingley, E; Beaumont, J G; Tilbrook, P A; Lalonde, J P; Stillitano, J P; Klinken, S P

1999-01-01

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells. PMID:10523300

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

PubMed

Williams, J H; Daly, L N; Ingley, E; Beaumont, J G; Tilbrook, P A; Lalonde, J P; Stillitano, J P; Klinken, S P

1999-10-15

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

Cafestol, a diterpene molecule found in coffee, induces leukemia cell death.

PubMed

Lima, Cauê S; Spindola, Daniel G; Bechara, Alexandre; Garcia, Daniel M; Palmeira-Dos-Santos, Caroline; Peixoto-da-Silva, Janaina; Erustes, Adolfo G; Michelin, Luis F G; Pereira, Gustavo J S; Smaili, Soraya S; Paredes-Gamero, Edgar; Calgarotto, Andrana K; Oliveira, Carlos R; Bincoletto, Claudia

2017-08-01

To evaluate the antitumor properties of Cafestol four leukemia cell lines were used (NB4, K562, HL60 and KG1). Cafestol exhibited the highest cytotoxicity against HL60 and KG1 cells, as evidenced by the accumulation of cells in the sub-G1 fraction, mitochondrial membrane potential reduction, accumulation of cleaved caspase-3 and phosphatidylserine externalization. An increase in CD11b and CD15 differentiation markers with attenuated ROS generation was also observed in Cafestol-treated HL60 cells. These results were similar to those obtained following exposure of the same cell line to cytarabine (Ara-C), an antileukemic drug. Cafestol and Ara-C reduced the clonogenic potential of HL60 cells by 100%, but Cafestol spared murine colony forming unit- granulocyte/macrophage (CFU-GM), which retained their clonogenicity. The co-treatment of Cafestol and Ara-C reduced HL60 cell viability compared with both drugs administered alone. In conclusion, despite the distinct molecular mechanisms involved in the activity of Cafestol and Ara-C, a similar cytotoxicity towards leukemia cells was observed, which suggests a need for prophylactic-therapeutic pre-clinical studies regarding the anticancer properties of Cafestol. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Eradication of acute promyelocytic leukemia-initiating cells by PML/RARA-targeting.

PubMed

Nasr, Rihab; de Thé, Hugues

2010-06-01

Acute promyelocytic leukemia (APL) is characterized by a t(15;17) translocation that yields a PML/RARA fusion protein. Expression of PML/RARA, a potent transcriptional repressor, induces APL in mice. Both retinoic acid (RA) and arsenic trioxide directly target PML/RARA-mediated transcriptional repression and protein stability, inducing rapid differentiation of the promyelocytes and clinical remission in most APL patients. RA also triggers growth arrest and progressive clearance of leukemia initiating cells (LIC), both ex vivo and in vivo. Suboptimal RA concentrations or expression of the PLZF/RARA variant allows complete RA-induced differentiation, but neither LIC clearance nor disease remission. Thus, RA-induced differentiation and LIC clearance may be uncoupled. The RA/arsenic trioxide association, which dramatically synergizes for PML/RARA degradation but not for differentiation, rapidly clears LIC in a proteasome-dependent manner, resulting in APL eradication in murine models and patients. Collectively, these results demonstrate that LIC clearance, which mirrors PML/RARA degradation, is the primary basis for APL cure by the RA/arsenic trioxide association, rather than differentiation. Oncogene degradation could be a generally applicable therapeutic strategy to clear LICs in several types of tumors.

Pretargeted Radioimmunotherapy Using Anti-CD45 Monoclonal Antibodies to Deliver Radiation to Murine Hematolymphoid Tissues and Human Myeloid Leukemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pagel, John M.; Matthews, Dana C.; Kenoyer, Aimee L.

2009-01-01

The efficacy of radioimmunotherapy (RIT) for treatment of patients with hematological malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by delivery of a biotinylated clearing agent and radiolabeled-DOTA-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the pretargeted RIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly-labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a largemore » amount of labeled antibody for a prolonged time. Therapy experiments demonstrated that 90Y-DOTA-biotin significantly prolonged survival of mice treated pretargeted with anti-hCD45 Ab-SA compared to mice treated with conventional RIT using 90Y-labeled anti-hCD45 Ab at the maximally tolerated dose (400 µCi). Since human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and pretargeted RIT using an anti-murine (m)CD45 Ab (A20 ) in a model where the target antigen is present on normal hematopoietic tissues. After 24 hours, 27.3 ± 2.8% of the injected dose of radionuclide was delivered per gram (% ID/g) of lymph node using 131I-A20-Ab compared with 40.0 ± 5.4% ID/g for pretargeted 111In-DOTA-biotin (p value). These data suggest that multi-step pretargeted methods for delivering RIT are superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.« less

Efficacy of Antiretroviral Agents against Murine Replication-Competent Retrovirus Infection in Human Cells

PubMed Central

Powell, Sharon K.; Artlip, Moria; Kaloss, Michele; Brazinski, Scott; Lyons, Russette; McGarrity, Gerard J.; Otto, Edward

1999-01-01

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone. PMID:10482636

A non-genetic approach to labelling acute myeloid leukemia and bone marrow cells with quantum dots.

PubMed

Zheng, Yanwen; Tan, Dongming; Chen, Zheng; Hu, Chenxi; Mao, Zhengwei J; Singleton, Timothy P; Zeng, Yan; Shao, Xuejun; Yin, Bin

2014-06-01

The difficulty in manipulation of leukemia cells has long hindered the dissection of leukemia pathogenesis. We have introduced a non-genetic approach of marking blood cells, using quantum dots. We compared quantum dots complexed with different vehicles, including a peptide Tat, cationic polymer Turbofect and liposome. Quantum dots-Tat showed the highest efficiency of marking hematopoietic cells among the three vehicles. Quantum dots-Tat could also label a panel of leukemia cell lines at varied efficiencies. More uniform intracellular distributions of quantum dots in mouse bone marrow and leukemia cells were obtained with quantum dots-Tat, compared with the granule-like formation obtained with quantum dots-liposome. Our results suggest that quantum dots have provided a photostable and non-genetic approach that labels normal and malignant hematopoietic cells, in a cell type-, vehicle-, and quantum dot concentration-dependent manner. We expect for potential applications of quantum dots as an easy and fast marking tool assisting investigations of various types of blood cells in the future.

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus.

PubMed

Doležal, Michal; Hadravová, Romana; Kožíšek, Milan; Bednárová, Lucie; Langerová, Hana; Ruml, Tomáš; Rumlová, Michaela

2016-09-23

The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Accelerated appearance of multiple B cell lymphoma types in NFS/N mice congenic for ecotropic murine leukemia viruses.

PubMed

Hartley, J W; Chattopadhyay, S K; Lander, M R; Taddesse-Heath, L; Naghashfar, Z; Morse, H C; Fredrickson, T N

2000-02-01

Spontaneous lymphomas occur at high frequency in NFS x V+ mice, strains congenic for ecotropic murine leukemia virus (MuLV) proviral genes and expressing virus at high titer. In the present study, a total of 703 NFS x V+ lymphomas were studied by histopathology, immunophenotypic analysis, immunoglobulin heavy chain or T cell receptor beta chain rearrangements, and somatic ecotropic MuLV integrations; 90% of the lymphomas tested were of B cell lineage. Low-grade tumors included small lymphocytic, follicular, and splenic marginal zone lymphomas, while high-grade tumors comprised diffuse large-cell (centroblastic and immunoblastic types), splenic marginal zone, and lymphoblastic lymphomas. Comparison of mice of similar genetic background except for presence (NFS x V+) or absence (NFS x V-) of functional ecotropic MuLV genomes showed that NFS x V-clonal lymphomas developed at about one-half the rate of those occurring in NFS x V+ mice, and most were low-grade B cell lymphomas with extended latent periods. In NFS x V+ mice, clonal outgrowth, defined by Ig gene rearrangements, was associated with acquisition of somatic ecotropic proviral integrations, suggesting that, although generation of B cell clones can be virus independent, ecotropic virus may act to increase the rate of generation of clones and speed their evolution to lymphoma. The mechanism remains undefined, because only rare rearrangements were detected in several cellular loci previously associated with MuLV insertional mutagenesis.

Activation of Membrane Fusion by Murine Leukemia Viruses Is Controlled in cis or in trans by Interactions between the Receptor-Binding Domain and a Conserved Disulfide Loop of the Carboxy Terminus of the Surface Glycoprotein

PubMed Central

Lavillette, Dimitri; Boson, Bertrand; Russell, Stephen J.; Cosset, François-Loïc

2001-01-01

Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive. Here we establish that a critical interaction is formed between the receptor-binding domain (RBD) and the major disulfide loop of the carboxy-terminal domain (C domain) of the murine leukemia virus SU. Receptor binding causes an alteration of this interaction and, in turn, promotes further events of Env fusion activation. We characterize mutations which, by lowering this interaction and reducing the compatibility between the RBD and C domains of Env glycoprotein chimeras, affect both Env fusogenicity and sensitivity to receptor interference. Additionally, we demonstrate that suboptimal interactions in such mutant Env proteins can be compensated in trans by soluble RBDs in a manner that depends on their compatibility with the C domain. Our results therefore indicate that RBD/C domain interactions may occur in cis, via the proper RBD of the viral Env itself, or in trans, via a distinct RBD expressed by virion-free Env glycoproteins expressed endogenously by the infected cells or provided by neighboring Env trimers. PMID:11264358

Phenethyl ester and amide of Ferulic Acids: Synthesis and bioactivity against P388 Leukemia Murine Cells

NASA Astrophysics Data System (ADS)

Firdaus; Soekamto, N. H.; Seniwati; Islam, M. F.; Sultan

2018-03-01

Bioactivity of a compound is closely related to the molecular structure of the compound concerned, its strength being the quantitative relation of the strength of the activity of the group it possesses. The combining of moieties of the active compounds will produce more active compounds. Most phenolic compounds as well as compounds containing moiety phenethyl groups have potential activity as anticancer. Combining phenolic groups and phenethyl groups in a compound will result in compounds having strong anticancer bioactivity. This study aims to combine the feruloyl and phenethyl groups to form esters and amides by synthesize of phenethyl trans-3-(4-hydroxy-3-methoxyphenyl)acrylate (5) and trans-3-(4- hydroxy-3-methoxyphenyl)-N-phenethylacrylamide (6) from ferulic acid with phenethyl alcohol and phenethylamine, and to study their bioactivity as anticancer. The synthesis of both compounds was conducted via indirect reaction, including acetylation, chlorination, esterfication/amidation, and deacetylation. Structures of products were characterized by FTIR and NMR data, and their bioactivity assay of the compounds against P388 Leukemia Murine Cells was conducted by an MTT method. Results showed that the compound 5 was obtained as a yellow gel with the IC50 of 10.79 μg/mL (36.21 μΜ), and the compound 6 was a yellowish solid with a melting point of 118-120°C and the IC50 of 29.14 μg/mL (97.79 μΜ). These compounds were more active than the analog compounds.

Neoplasia Driven by Mutant c-KIT Is Mediated by Intracellular, Not Plasma Membrane, Receptor Signaling▿

PubMed Central

Xiang, Zhifu; Kreisel, Frederike; Cain, Jennifer; Colson, AnnaLynn; Tomasson, Michael H.

2007-01-01

Activating mutations in c-KIT are associated with gastrointestinal stromal tumors, mastocytosis, and acute myeloid leukemia. In attempting to establish a murine model of human KITD816V (hKITD816V)-mediated leukemia, we uncovered an unexpected relationship between cellular transformation and intracellular trafficking. We found that transport of hKITD816V protein was blocked at the endoplasmic reticulum in a species-specific fashion. We exploited these species-specific trafficking differences and a set of localization domain-tagged KIT mutants to explore the relationship between subcellular localization of mutant KIT and cellular transformation. The protein products of fully transforming KIT mutants localized to the Golgi apparatus and to a lesser extent the plasma membrane. Domain-tagged KITD816V targeted to the Golgi apparatus remained constitutively active and transforming. Chemical inhibition of intracellular transport demonstrated that Golgi localization is sufficient, but plasma membrane localization is dispensable, for downstream signaling mediated by KIT mutation. When expressed in murine bone marrow, endoplasmic reticulum-localized hKITD816V failed to induce disease in mice, while expression of either Golgi-localized HyKITD816V or cytosol-localized, ectodomain-deleted KITD816V uniformly caused fatal myeloproliferative diseases. Taken together, these data demonstrate that intracellular, non-plasma membrane receptor signaling is sufficient to drive neoplasia caused by mutant c-KIT and provide the first animal model of myelomonocytic neoplasia initiated by human KITD816V. PMID:17060458

Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag

DOE Office of Scientific and Technical Information (OSTI.GOV)

Datta, Siddhartha A.K.; Zuo, Xiaobing; Clark, Patrick K.

2012-05-09

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV),more » a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of {approx}7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.« less

Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

PubMed Central

Kadan, M J; Sturm, S; Anderson, W F; Eglitis, M A

1992-01-01

Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments. PMID:1312632

Decreased Virus Population Diversity in p53-Null Mice Infected with Weakly Oncogenic Abelson Virus

PubMed Central

Marchlik, Erica; Kalman, Richard; Rosenberg, Naomi

2005-01-01

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53−/− tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV. PMID:16140739

Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

PubMed Central

De Rocquigny, H; Ficheux, D; Gabus, C; Allain, B; Fournie-Zaluski, M C; Darlix, J L; Roques, B P

1993-01-01

The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger. Images PMID:8451185

Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production

PubMed Central

Wang, Margaret Q; Kim, Wankee; Gao, Guangxia; Torrey, Ted A; Morse, Herbert C; De Camilli, Pietro; Goff, Stephen P

2004-01-01

Background The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. Results A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. Conclusions This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production. PMID:14659004

Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

PubMed Central

Döring, Jessica

2017-01-01

Abstract Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication. PMID:28160599

Assembly and composition of intracellular particles formed by Moloney murine leukemia virus.

PubMed Central

Hansen, M; Jelinek, L; Jones, R S; Stegeman-Olsen, J; Barklis, E

1993-01-01

Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections. Images PMID:8350394

Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration.

PubMed Central

Jonsson, C B; Roth, M J

1993-01-01

Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate. Images PMID:8350412

Leukemia diagnosis and testing of complement-fixing antibodies for bone marrow purging in acute lymphoid leukemia.

PubMed

Campana, D; Janossy, G

1986-12-01

In this paper a microplate method is described for diagnosing acute leukemia and for investigating the reactivity of monoclonal antibodies (MoAbs) against membrane antigens in combination with rabbit or murine antibodies to nuclear terminal transferase (TdT). The speed of this method facilitates the investigation of fresh leukemic cells from individual patients and assesses the cytolytic efficacy of the relevant MoAbs in the presence of complement (C'). Lymphoblasts (TdT+) are mixed in equal proportions with known numbers of "inert" cells, eg, RBC or nonleukemic bone marrow (BM). Following incubation with MoAbs and C' the ratio of residual TdT+ cells and inert cells is determined on cytospin preparations. Initially, percentages of TdT+ cells are counted in a unit volume of 5,000 inert cells, followed by the scanning of greater than 2 X 10(4) inert cells on entire slides. With this method more than 4 log cytoreduction of TdT + cells is detected. The method is also applicable for studying the cytolysis of malignant B cells by using mostly monoclonal lg expression rather than TdT for the identification of residual B cells. Ten representative patients selected from a group of greater than 100 are reported. In some cases cytoreduction of greater than 4 log with no identifiable residual TdT + cells is achieved by a single C'-fixing MoAb: anti-CD10 (RFAL3) in common acute lymphoid leukemia (ALL) and anti-CD7 (RFT2) in T cell ALL (T-ALL). Other cases require cocktails of anti-CD10, anti-CD19, and anti-CD24 in common ALL or anti-CD7 and anti-CD8 in T-ALL. In T-ALL a few TdT + cells remain that exhibit the features of normal TdT + BM cells (CD7-, HLA-DR+). This is particularly noticeable when patients are studied in partial remission or if nonleukemic BM is used as a source of inert cells. The methods described here contribute to establishing a range of MoAbs (ie, of IgM class) and techniques for efficient purging and to comparing the efficacy of "clean-up," in remission, of common ALL, T-ALL, and B cell malignancies.

Polyamine-iron chelator conjugate.

PubMed

Bergeron, Raymond J; McManis, James S; Franklin, April M; Yao, Hua; Weimar, William R

2003-12-04

The current study demonstrates unequivocally that polyamines can serve as vectors for the intracellular delivery of the bidentate chelator 1,2-dimethyl-3-hydroxypyridin-4-one (L1). The polyamine-hydroxypyridinone conjugate 1-(12-amino-4,9-diazadodecyl)-2-methyl-3-hydroxy-4(1H)-pyridinone is assembled from spermine and 3-O-benzylmaltol. The conjugate is shown to form a 3:1 complex with Fe(III) and to be taken up by the polyamine transporter 1900-fold against a concentration gradient. The K(i) of the conjugate is 3.7 microM vs spermidine for the polyamine transporter. The conjugate is also at least 230 times more active in suppressing the growth of L1210 murine leukemia cells than is the parent ligand, decreases the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase, and upregulates spermidine-spermine N (1)-acetyltransferase. However, the effect on native polyamine pools is a moderate one. These findings are in keeping with the idea that polyamines can also serve as efficient vectors for the intracellular delivery of other iron chelators.

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furukawa, Ayako; Okamura, Hideyasu; Morishita, Ryo

2012-08-24

Highlights: Black-Right-Pointing-Pointer Protease (PR) of XMR virus (XMRV) was successfully synthesized with cell-free system. Black-Right-Pointing-Pointer Interface of XMRV PR with an inhibitor, amprenavir (APV), was identified with NMR. Black-Right-Pointing-Pointer Structural heterogeneity is induced for two PR protomers in the APV:PR = 1:2 complex. Black-Right-Pointing-Pointer Structural heterogeneity is transmitted even to distant regions from the interface. Black-Right-Pointing-Pointer Long-range transmission of structural change may be utilized for drug discovery. -- Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nudemore » mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially {sup 15}N-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [{sup 1}H, {sup 15}N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) . A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR = 1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.« less

Inhibition of IRAK1/4 sensitizes T cell acute lymphoblastic leukemia to chemotherapies

PubMed Central

Li, Zhaoyang; Younger, Kenisha; Gartenhaus, Ronald; Joseph, Ann Mary; Hu, Fang; Baer, Maria R.; Brown, Patrick; Davila, Eduardo

2015-01-01

Signaling via the MyD88/IRAK pathway in T cells is indispensable for cell survival; however, it is not known whether this pathway functions in the progression of T acute lymphoblastic leukemia (T-ALL). Here, we determined that compared with thymic and peripheral T cells, T-ALL cells from patients have elevated levels of IRAK1 and IRAK4 mRNA as well as increased total and phosphorylated protein. Targeted inhibition of IRAK1 and IRAK4, either with shRNA or with a pharmacological IRAK1/4 inhibitor, dramatically impeded proliferation of T-ALL cells isolated from patients and T-ALL cells in a murine leukemia model; however, IRAK1/4 inhibition had little effect on cell death. We screened several hundred FDA-approved compounds and identified a set of drugs that had enhanced cytotoxic activity when combined with IRAK inhibition. Administration of an IRAK1/4 inhibitor or IRAK knockdown in combination with either ABT-737 or vincristine markedly reduced leukemia burden in mice and prolonged survival. IRAK1/4 signaling activated the E3 ubiquitin ligase TRAF6, increasing K63-linked ubiquitination and enhancing stability of the antiapoptotic protein MCL1; therefore, IRAK inhibition reduced MCL1 stability and sensitized T-ALL to combination therapy. These studies demonstrate that IRAK1/4 signaling promotes T-ALL progression through stabilization of MCL1 and suggest that impeding this pathway has potential as a therapeutic strategy to enhance chemotherapeutic efficacy. PMID:25642772

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

PubMed

Machado, Kátia da Conceição; Sousa, Lívia Queiroz de; Lima, Daisy Jereissati Barbosa; Soares, Bruno Marques; Cavalcanti, Bruno Coêlho; Maranhão, Sarah Sant'Anna; Noronha, Janaina da Costa de; Rodrigues, Domingos de Jesus; Militão, Gardenia Carmen Gadelha; Chaves, Mariana Helena; Vieira-Júnior, Gerardo Magela; Pessoa, Cláudia; Moraes, Manoel Odorico de; Sousa, João Marcelo de Castro E; Melo-Cavalcante, Ana Amélia de Carvalho; Ferreira, Paulo Michel Pinheiro

2018-03-15

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC 50 values ranging from 0.15 (leukemia) to 7.35 (larynx) μM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC 50: 10.88 μM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC 50 : 7.5 μM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Isometachromin, a new cytotoxic sesquiterpenoid from a deep water sponge of the family Spongiidae.

PubMed

McConnell, O J; Longley, R; Gunasekera, M

1992-09-15

Isometachromin (1), a new sesquiterpene-quinone that is related structurally to metachromin C (2), and the known compounds ilimaquinone (3) and 5-epi-ilimaquinone (4), were isolated from a deep water sponge in the family Spongiidae; the structure of isometachromin was elucidated by spectral methods. Isometachromin exhibits in vitro cytotoxicity against the human lung cancer cell line A549 (IC50 = 2.6 micrograms/ml), but not against P388 murine leukemia (IC 50 > or equal to 10 micrograms/ml) and also exhibits antimicrobial activity.

Recombination Origin of Retrovirus XMRV | Center for Cancer Research

Cancer.gov

Xenotropic murine leukemia virus–related virus (XMRV) was first reported in samples from a human prostate tumor in 2006, and, at that time, claims were made that XMRV infection rates ranged from 6 to 27 percent of human prostate cancers.  Later research reported XMRV in the blood of 67 percent of people with chronic fatigue syndrome (CFS). When follow-up studies failed to detect XMRV in multiple sets of specimens from people with prostate cancer or CFS and healthy controls, the original reports came under closer scrutiny.

Mutations in the Nucleolar Phosphoprotein, Nucleophosmin, Promote the Expression of the Oncogenic Transcription Factor MEF/ELF4 in Leukemia Cells and Potentiates Transformation*

PubMed Central

Ando, Koji; Tsushima, Hideki; Matsuo, Emi; Horio, Kensuke; Tominaga-Sato, Shinya; Imanishi, Daisuke; Imaizumi, Yoshitaka; Iwanaga, Masako; Itonaga, Hidehiro; Yoshida, Shinichiro; Hata, Tomoko; Moriuchi, Ryozo; Kiyoi, Hitoshi; Nimer, Stephen; Mano, Hiroyuki; Naoe, Tomoki; Tomonaga, Masao; Miyazaki, Yasushi

2013-01-01

Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis. PMID:23393136

Mutations in the nucleolar phosphoprotein, nucleophosmin, promote the expression of the oncogenic transcription factor MEF/ELF4 in leukemia cells and potentiates transformation.

PubMed

Ando, Koji; Tsushima, Hideki; Matsuo, Emi; Horio, Kensuke; Tominaga-Sato, Shinya; Imanishi, Daisuke; Imaizumi, Yoshitaka; Iwanaga, Masako; Itonaga, Hidehiro; Yoshida, Shinichiro; Hata, Tomoko; Moriuchi, Ryozo; Kiyoi, Hitoshi; Nimer, Stephen; Mano, Hiroyuki; Naoe, Tomoki; Tomonaga, Masao; Miyazaki, Yasushi

2013-03-29

Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis.

Dunaliella salina exhibits an antileukemic immunity in a mouse model of WEHI-3 leukemia cells.

PubMed

Chuang, Wen-Chen; Ho, Yung-Chyuan; Liao, Jiunn-Wang; Lu, Fung-Jou

2014-11-26

Dunaliella salina has been shown to have antioxidant property and induce apoptotic cell death of human cancer cells in vitro. However, there is no information available on D. salina showing an antileukemia effect or immunomodulatory activity in vivo. This study applied D. salina to syngeneic leukemia-implanted mice (BALB/c and WEHI-3) to investigate its immunological and antileukemia properties. Oral administration of D. salina (184.5, 369, and 922.5 mg/kg) inhibited spleen metastasis and prolonged the survival in BALB/c mice that had received an intravenous injection of WEHI-3 cells. The results revealed that D. salina had reduced spleen enlargement in murine leukemia. It had also increased the population and proliferation of T-cells (CD3) and B-cells (CD19) following Con A/LPS treatment on flow cytometry and MTT assay, respectively. Furthermore, D. salina increased the phagocytosis of macrophages and enhanced the cytotoxicity of natural killer cells on flow cytometry and LDH assay. Moreover, D. salina enhanced the levels of interferon-γ and interleukin 2 (IL-2) but reduced the levels of IL-4 and IL-10 in leukemic mice. In conclusion, these results demonstrated that the application of D. salina had beneficial effects on WEHI-3 leukemic mice by prolonging survival via modulating the immune responses.

Integrated computational biology analysis to evaluate target genes for chronic myelogenous leukemia.

PubMed

Zheng, Yu; Wang, Yu-Ping; Cao, Hongbao; Chen, Qiusheng; Zhang, Xi

2018-06-05

Although hundreds of genes have been linked to chronic myelogenous leukemia (CML), many of the results lack reproducibility. In the present study, data across multiple modalities were integrated to evaluate 579 CML candidate genes, including literature‑based CML‑gene relation data, Gene Expression Omnibus RNA expression data and pathway‑based gene‑gene interaction data. The expression data included samples from 76 patients with CML and 73 healthy controls. For each target gene, four metrics were proposed and tested with case/control classification. The effectiveness of the four metrics presented was demonstrated by the high classification accuracy (94.63%; P<2x10‑4). Cross metric analysis suggested nine top candidate genes for CML: Epidermal growth factor receptor, tumor protein p53, catenin β 1, janus kinase 2, tumor necrosis factor, abelson murine leukemia viral oncogene homolog 1, vascular endothelial growth factor A, B‑cell lymphoma 2 and proto‑oncogene tyrosine‑protein kinase. In addition, 145 CML candidate pathways enriched with 485 out of 579 genes were identified (P<8.2x10‑11; q=0.005). In conclusion, weighted genetic networks generated using computational biology may be complementary to biological experiments for the evaluation of known or novel CML target genes.

Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells

PubMed Central

Peng, Xing-Xiang; Tiwari, Amit K.; Wu, Hsiang-Chun; Chen, Zhe-Sheng

2012-01-01

Imatinib, a breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) tyrosine kinase inhibitor (TKI), has revolutionized the treatment of chronic myelogenous leukemia (CML). However, development of multidrug resistance (MDR) limits the use of imatinib. In the present study, we aimed to investigate the mechanisms of cellular resistance to imatinib in CML. Therefore, we established an imatinib-resistant human CML cell line (K562-imatinib) through a stepwise selection process. While characterizing the phenotype of these cells, we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells. In addition, these cells were cross-resistant to second- and third-generation BCR-ABL TKIs. Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein (P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells. In addition, accumulation of [14C]6-mercaptopurine (6-MP) was decreased, whereas the ATP-dependent efflux of [14C] 6-MP and [3H]methotrexate transport were increased in K562-imatinib cells. These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells. PMID:22098951

Host thiopurine methyltransferase status affects mercaptopurine antileukemic effectiveness in a murine model.

PubMed

Ramsey, Laura B; Janke, Laura J; Edick, Mathew J; Cheng, Cheng; Williams, Richard T; Sherr, Charles J; Evans, William E; Relling, Mary V

2014-05-01

Thiopurines are used for many cancers, including acute lymphoblastic leukemia (ALL). Patients with an inherited host defect in thiopurine methyltransferase (TPMT) are at high risk for life-threatening toxicity if treated with conventional dosages, but the impact on antileukemic efficacy is less clear. We treated thiopurine-sensitive BCR-ABL+Arf-null Tpmt+/+ ALL in Tpmt+/+, +/-, or -/- recipient mice to test the impact of the host polymorphism on antileukemic efficacy. Median survival was similar in untreated mice of different Tpmt genotypes (16-18 days). However, in mice treated with low-dose mercaptopurine (such as tolerated by TPMT-/- patients), the difference in 30-day leukemia-free survival by Tpmt genotype was profound: 5% (±9%) for Tpmt+/+ mice, 47% (±26%) for Tpmt+/- mice, and 85% (±14%) for Tpmt-/- mice (P=5×10), indicating a substantial impact of host Tpmt status on thiopurine effectiveness. Among Tpmt+/+ recipient mice, leukemia-free survival improved with higher doses of mercaptopurine (similar to doses tolerated by wild-type patients) compared with lower doses, and at higher doses was comparable (P=0.6) to the survival of Tpmt-/- mice treated with the lower dose. These findings support the notion that germline polymorphisms in Tpmt affect not only host tissue toxicity but also antitumor effectiveness.

Effective targeting of chronic myeloid leukemia initiating activity with the combination of arsenic trioxide and interferon alpha.

PubMed

El Eit, Rabab M; Iskandarani, Ahmad N; Saliba, Jessica L; Jabbour, Mark N; Mahfouz, Rami A; Bitar, Nizar M A; Ayoubi, Hanadi R El; Zaatari, Ghazi S; Mahon, Francois-Xavier; De Thé, Hugues B; Bazarbachi, Ali A; Nasr, Rihab R

2014-02-15

Imatinib is the standard of care in chronic meloid leukemia (CML) therapy. However, imatinib is not curative since most patients who discontinue therapy relapse indicating that leukemia initiating cells (LIC) are resistant. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, improved outcome was reported with the combination of interferon and imatinib. Arsenic trioxide was suggested to decrease CML LIC. We investigated the effects of arsenic and IFN on human CML cell lines or primary cells and the bone marrow retroviral transduction/transplantation murine CML model. In vitro, the combination of arsenic and IFN inhibited proliferation and activated apoptosis. Importantly, arsenic and IFN synergistically reduced the clonogenic activity of primary bone marrow cells derived from CML patients. Finally, in vivo, combined interferon and arsenic treatment, but not single agents, prolonged the survival of primary CML mice. Importantly, the combination severely impaired engraftment into untreated secondary recipients, with some recipients never developing the disease, demonstrating a dramatic decrease in CML LIC activity. Arsenic/IFN effect on CML LIC activity was significantly superior to that of imatinib. These results support further exploration of this combination, alone or with imatinib aiming at achieving CML eradication rather than long-term disease control. © 2013 UICC.

Dynamic changes in clonal cytogenetic architecture during progression of chronic lymphocytic leukemia in patients and patient-derived murine xenografts

PubMed Central

Davies, Nicholas J.; Kwok, Marwan; Gould, Clive; Oldreive, Ceri E.; Mao, Jingwen; Parry, Helen; Smith, Edward; Agathanggelou, Angelo; Pratt, Guy; Taylor, Alexander Malcolm R.; Moss, Paul; Griffiths, Mike; Stankovic, Tatjana

2017-01-01

Subclonal heterogeneity and clonal selection influences disease progression in chronic lymphocytic leukemia (CLL). It is therefore important that therapeutic decisions are made based on an understanding of the CLL clonal architecture and its dynamics in individual patients. Identification of cytogenetic abnormalities by FISH remains the cornerstone of contemporary clinical practice and provides a simple means for prognostic stratification. Here, we demonstrate that multiplexed-FISH can enhance recognition of CLL subclonal repertoire and its dynamics during disease progression, both in patients and CLL patient-derived xenografts (PDX). We applied a combination of patient-specific FISH probes to 24 CLL cases before treatment and at relapse, and determined putative ancestral relationships between subpopulations with different cytogenetic features. We subsequently established 7 CLL PDX models in NOD/Shi-SCID/IL-2Rγctm1sug/Jic (NOG) mice. Application of multiplexed-FISH to these models demonstrated that all of the identified cytogenetic subpopulations had leukemia propagating activity and that changes in their representation during disease progression could be spontaneous, accelerated by treatment or treatment-induced. We conclude that multiplexed-FISH in combination with PDX models have the potential to distinguish between spontaneous and treatment-induced clonal selection, and therefore provide a valuable tool for the pre-clinical evaluation of novel therapies. PMID:28496009

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

PubMed Central

Huang, M; Jolicoeur, P

1990-01-01

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus. Images PMID:2243376

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

PubMed

Rotter, V; Boss, M A; Baltimore, D

1981-04-01

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.

The prognostic value of polycomb group protein B-cell-specific moloney murine leukemia virus insertion site 1 in stage II colon cancer patients.

PubMed

Espersen, Maiken L M; Linnemann, Dorte; Christensen, Ib J; Alamili, Mahdi; Troelsen, Jesper T; Høgdall, Estrid

2016-07-01

The aim of this study was to investigate the prognostic value of B-cell-specific moloney murine leukemia virus insertion site 1 (BMI1) protein expression in primary tumors of stage II colon cancer patients. BMI1 protein expression was assessed by immunohistochemistry in a retrospective patient cohort consisting of 144 stage II colon cancer patients. BMI1 expression at the invasive front of the primary tumors correlated with mismatch repair status of the tumors. Furthermore, BMI1 expression at the luminal surface correlated with T-stage, tumor location, and the histological subtypes of the tumors. In a univariate Cox proportional hazard analysis, no statistical significant association between risk of relapse and BMI1 protein expression at the invasive front (HR: 1.12; 95% CI 0.78-1.60; p = 0.53) or at the luminal surface of the tumor (HR: 1.06; 95% CI 0.75-1.48; p = 0.70) was found. Likewise, there was no association between 5-year overall survival and BMI1 expression at the invasive front (HR: 1.12; 95% CI 0.80-1.56; p = 0.46) or at the luminal surface of the tumor (HR: 1.16; 95% CI 0.86-1.60; p = 0.33). In conclusion, BMI1 expression in primary tumors of stage II colon cancer patients could not predict relapse or overall survival of the patients, thus having a limited prognostic value in stage II colon cancer patients. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Poliomyelitis in MuLV-infected ICR-SCID mice after injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus.

PubMed

Carlson Scholz, Jodi A; Garg, Rohit; Compton, Susan R; Allore, Heather G; Zeiss, Caroline J; Uchio, Edward M

2011-10-01

The arterivirus lactate dehydrogenase-elevating virus (LDV) causes life-long viremia in mice. Although LDV infection generally does not cause disease, infected mice that are homozygous for the Fv1(n) allele are prone to develop poliomyelitis when immunosuppressed, a condition known as age-dependent poliomyelitis. The development of age-dependent poliomyelitis requires coinfection with endogenous murine leukemia virus. Even though LDV is a common contaminant of transplantable tumors, clinical signs of poliomyelitis after inadvertent exposure to LDV have not been described in recent literature. In addition, LDV-induced poliomyelitis has not been reported in SCID or ICR mice. Here we describe the occurrence of poliomyelitis in ICR-SCID mice resulting from injection of LDV-contaminated basement membrane matrix. After exposure to LDV, a subset of mice presented with clinical signs including paresis, which was associated with atrophy of the hindlimb musculature, and tachypnea; in addition, some mice died suddenly with or without premonitory signs. Mice presenting within the first 6 mo after infection had regions of spongiosis, neuronal necrosis and astrocytosis of the ventral spinal cord, and less commonly, brainstem. Axonal degeneration of ventral roots prevailed in more chronically infected mice. LDV was identified by RT-PCR in 12 of 15 mice with typical neuropathology; positive antiLDV immunolabeling was identified in all PCR-positive animals (n = 7) tested. Three of 8 mice with neuropathology but no clinical signs were LDV negative by RT-PCR. RT-PCR yielded murine leukemia virus in spinal cords of all mice tested, regardless of clinical presentation or neuropathology.

mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

PubMed Central

Hibbert, Catherine S.; Mirro, Jane; Rein, Alan

2004-01-01

Prior work by others has shown that insertion of ψ (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3′ untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Ψ+ mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops. PMID:15452213

Infection of XC Cells by MLVs and Ebola Virus Is Endosome-Dependent but Acidification-Independent

PubMed Central

Kamiyama, Haruka; Kakoki, Katsura; Yoshii, Hiroaki; Iwao, Masatomo; Igawa, Tsukasa; Sakai, Hideki; Hayashi, Hideki; Matsuyama, Toshifumi; Yamamoto, Naoki; Kubo, Yoshinao

2011-01-01

Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells. PMID:22022555

Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell migration inhibitors

PubMed Central

Salum, Lívia B.; Altei, Wanessa F.; Chiaradia, Louise D.; Cordeiro, Marlon N.S.; Canevarolo, Rafael R.; Melo, Carolina P.S.; Winter, Evelyn; Mattei, Bruno; Daghestani, Hikmat N.; Santos-Silva, Maria Cláudia; Creczynski-Pasa, Tânia B.; Yunes, Rosendo A.; Yunes, José A.; Andricopulo, Adriano D.; Day, Billy W.; Nunes, Ricardo J.; Vogt, Andreas

2013-01-01

Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays. PMID:23524161

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Helper-Dependent Properties of Friend Spleen Focus-Forming Virus: Effect of the Fv-1 Gene on the Late Stages in Virus Synthesis

PubMed Central

Eckner, Robert J.

1973-01-01

Co-infection of neonatal BALB/c mice with Friend virus (FV) complex (containing defective spleen focus-forming virus [SFFV] and endogenous N-tropic leukemia-inducing helper virus [LLV-F]) and B-tropic Tennant leukemia virus (TenLV) resulted in the inhibition of LLV-F by the Fv-1b gene and recovery of a TenLV pseudotype of SFFV, abbreviated SFFV(TenLV). The host range of this pseudotype was B-tropic, since SFFV(TenLV) was 10 to 100 times more infectious for B-type (Fv-1bb) than for N-type (Fv-1nn) mice. The similar patterns of neutralization of N-tropic and B-tropic SFFV by type-specific murine antisera suggested that the difference in infectivity between these two SFFV preparations did not reside in envelope determinants. Rather, helper control of SFFV's host range was only apparent and dependent upon the ability of associated virus to provide a helper function for late stages in SFFV synthesis. Early stages in SFFV's infectious cycle were shown to be helper independent. The Fv-1 gene did not act at the level of the cell membrane to effectively restrict SFFV infection, since SFFV-induced transformed cells could be detected in the absence of spleen focus formation and SFFV synthesis. Further, the generation of these transformed cells by SFFV followed a one-hit, dose-response pattern, suggesting that SFFV-induced cell transformation is helper independent. Finally, restriction of helper function by Fv-1 may be an intracellular event, because both SFFV and its associated LLV-F helper share common envelope determinants and presumably adsorb onto and penetrate target cells with equal efficiency. PMID:4127030

The Effects of Curcuma longa L., Purple Sweet Potato, and Mixtures of the Two on Immunomodulation in C57BL/6J Mice Infected with LP-BM5 Murine Leukemia Retrovirus.

PubMed

Park, Soo-Jeung; Lee, Dasom; Lee, Minhee; Kwon, Han-Ol; Kim, Hyesook; Park, Jeongjin; Jeon, Woojin; Cha, Minseok; Jun, Suhwa; Park, Kwangjin; Lee, Jeongmin

2018-06-04

The immune response is stimulated to protect the body from external antigens and is controlled by several types of immune cells. In the present study, the immunomodulatory effects of Curcuma longa L., purple sweet potato, and mixtures of the two (CPM) were investigated in C57BL/6 mice infected with LP-BM5 murine leukemia virus (MuLV). Mice were divided into seven groups as follows: normal control, infected control (LP-BM5 MuLV infection), positive control (LP-BM5 MuLV infection+dietary supplement of red ginseng 300 mg/kg body weight), the original powder of C. longa L. (C; LP-BM5 MuLV infection+dietary supplement of C 189 mg/kg body weight), the original powder of purple sweet potato (P; LP-BM5 MuLV infection+dietary supplement of P 1811 mg/kg body weight), CPM Low (CPL; LP-BM5 MuLV infection+CPM 2 g/kg body weight), and CPM High (CPH; LP-BM5 MuLV infection+CPM 5 g/kg body weight). Dietary supplementation lasted for 12 weeks. Dietary supplementation of CPM inhibited LP-BM5 MuLV-induced lymphadenopathy and splenomegaly and inhibited reduction of messenger RNA (mRNA) expression of major histocompatibility complex (MHC) I and II. Moreover, CPM reduced the decrease in T- and B cell proliferation, reduced the population of CD4(+)/CD8(+) T cells, and remedied the unbalanced production of T helper-1 (Th1)/T helper-2 (Th2) cytokines in LP-BM5 MuLV-infected mice. In addition, CPM inhibited reduction of phagocytosis in peritoneal macrophages and decreased serum levels of immunoglobulin A (IgA), immunoglobulin E (IgE), and immunoglobulin G (IgG). These results suggest that CPM had a positive effect on immunomodulation in C57BL/6 mice induced by LP-BM5 leukemia retrovirus infection.

Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts.

PubMed

Kirschner, Gyöngyi; Balla, Bernadett; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Kósa, János Pál; Lakatos, Péter

2016-09-01

Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

A BCR-ABL Kinase Activity-Independent Signaling Pathway in Chronic Myelogenous Leukemia

DTIC Science & Technology

2008-02-01

myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood. 1998;92:3780-3792. 17. Zhang X, Ren R. Bcr-Abl efficiently induces a... myeloproliferative disease and production of excess interleukin-3 and granulocyte-macrophage colony-stimulating factor in mice: a novel model for chronic...Xu L, et al. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced

Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

PubMed Central

Kurtova, Antonina V.; Balakrishnan, Kumudha; Chen, Rong; Ding, Wei; Schnabl, Susanne; Quiroga, Maite P.; Sivina, Mariela; Wierda, William G.; Estrov, Zeev; Keating, Michael J.; Shehata, Medhat; Jäger, Ulrich; Gandhi, Varsha; Kay, Neil E.; Plunkett, William

2009-01-01

Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL–MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL–MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell–cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC–CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures. PMID:19762485

Chimeric Antigen Receptor T-Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia

PubMed Central

Tomuleasa, Ciprian; Fuji, Shigeo; Berce, Cristian; Onaciu, Anca; Chira, Sergiu; Petrushev, Bobe; Micu, Wilhelm-Thomas; Moisoiu, Vlad; Osan, Ciprian; Constantinescu, Catalin; Pasca, Sergiu; Jurj, Ancuta; Pop, Laura; Berindan-Neagoe, Ioana; Dima, Delia; Kitano, Shigehisa

2018-01-01

Chimeric antigen receptor (CAR) T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects. PMID:29515572

Low Dose Total Body Irradiation Combined With Recombinant CD19-Ligand × Soluble TRAIL Fusion Protein is Highly Effective Against Radiation-resistant B-precursor Acute Lymphoblastic Leukemia in Mice☆

PubMed Central

Uckun, Fatih M.; Myers, Dorothea E.; Ma, Hong; Rose, Rebecca; Qazi, Sanjive

2015-01-01

In high-risk remission B-precursor acute lymphoblastic leukemia (BPL) patients, relapse rates have remained high post-hematopoietic stem cell transplantation (HSCT) even after the use of very intensive total body irradiation (TBI)-based conditioning regimens, especially in patients with a high “minimal residual disease” (MRD) burden. New agents capable of killing radiation-resistant BPL cells and selectively augmenting their radiation sensitivity are therefore urgently needed. We report preclinical proof-of-principle that the potency of radiation therapy against BPL can be augmented by combining radiation with recombinant human CD19-Ligand × soluble TRAIL (“CD19L–sTRAIL”) fusion protein. CD19L–sTRAIL consistently killed radiation-resistant primary leukemia cells from BPL patients as well as BPL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. Low dose total body irradiation (TBI) combined with CD19L–sTRAIL was highly effective against (1) xenografted CD19+ radiochemotherapy-resistant human BPL in NOD/SCID (NS) mice challenged with an otherwise invariably fatal dose of xenograft cells derived from relapsed BPL patients as well as (2) radiation-resistant advanced stage CD19+ murine BPL with lymphomatous features in CD22ΔE12xBCR-ABL double transgenic mice. We hypothesize that the incorporation of CD19L–sTRAIL into the pre-transplant TBI regimens of patients with very high-risk BPL will improve their survival outcome after HSCT. PMID:26097891

Chimeric Antigen Receptor T-Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia.

PubMed

Tomuleasa, Ciprian; Fuji, Shigeo; Berce, Cristian; Onaciu, Anca; Chira, Sergiu; Petrushev, Bobe; Micu, Wilhelm-Thomas; Moisoiu, Vlad; Osan, Ciprian; Constantinescu, Catalin; Pasca, Sergiu; Jurj, Ancuta; Pop, Laura; Berindan-Neagoe, Ioana; Dima, Delia; Kitano, Shigehisa

2018-01-01

Chimeric antigen receptor (CAR) T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects.

Profound Amplification of Pathogenic Murine Polytropic Retrovirus Release from Coinfected Cells

PubMed Central

Rosenke, Kyle; Lavignon, Marc; Malik, Frank; Kolokithas, Angelo; Hendrick, Duncan; Virtaneva, Kimmo; Peterson, Karin

2012-01-01

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98–F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general. PMID:22514353

Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists

PubMed Central

Petersen, Christopher T.; Hassan, Mojibade; Morris, Anna B.; Jeffery, Jasmin; Lee, Kunhee; Jagirdar, Neera; Staton, Ashley D.; Raikar, Sunil S.; Spencer, Harold T.; Sulchek, Todd; Flowers, Christopher R.

2018-01-01

Adoptive therapy with ex vivo–expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27+CD28+ T cells vs 27.4% in control cultures; P < .05). This strategy results in a mean of 83.7% more T cells cultured from lymphoma patients in the presence of PI3Kδ and VIP antagonists, increased survival of human T cells from a lymphoma patient in a murine xenograft model, enhanced cytotoxic activity of antigen-specific human CAR T cells and murine T cells against lymphoma, and increased transduction and expansion of anti-CD5 human CAR T cells. PI3Kδ and VIP antagonist-expanded T cells from lymphoma patients show reduced terminal differentiation, enhanced polyfunctional cytokine expression, and preservation of costimulatory molecule expression. Taken together, synergistic blockade of these pathways is an attractive strategy to enhance the expansion and functional capacity of ex vivo–expanded cancer-specific T cells. PMID:29386194

In vitro priming of adoptively transferred T cells with a RORγ agonist confers durable memory and stemness in vivo.

PubMed

Hu, Xiao; Majchrzak, Kinga; Liu, Xikui; Wyatt, Megan M; Spooner, Chauncey; Moisan, Jacques; Zou, Weiping; Carter, Laura L; Paulos, Chrystal M

2018-05-16

Adoptive T cell transfer therapy is an FDA-approved treatment for leukemia that relies on the ex vivo expansion and re-infusion of a patient's immune cells, which can be engineered with a chimeric antigen receptor (CAR) for more efficient tumor recognition. Type 17 T cells, controlled transcriptionally by RORγ, have been reported to mediate potent anti-tumor effects superior to those observed with conventionally expanded T cells. Here we demonstrate that addition of a synthetic, small molecule RORγ agonist during ex vivo expansion potentiates the anti-tumor activity of human Th17 and Tc17 cells redirected with a CAR. Likewise, ex vivo use of this agonist bolstered the anti-tumor properties of murine tumor-specific CD4+ and CD8+ T cells. Expansion in the presence of the RORγ agonist enhanced IL-17A production without compromising IFN-γ secretion in vitro. In vivo, cytokine neutralization studies revealed that IFN-γ and IL-17A were required to regress murine melanoma tumors. The enhanced anti-tumor effect of RORγ agonist treatment was associated with recovery of more donor T cells in the tumor and spleen; these cells produced elevated levels of cytokines months after infusion and expressed markers of long-lived stem and central memory cells such as Tcf7 and CD62L. Conversely, untreated cells mainly exhibited effector phenotypes in the tumor. Cured mice previously treated with agonist-primed T cells were protected from tumor re-challenge. Collectively, our work reveals that in vitro treatment with a RORγ agonist generates potent anti-tumor Type 17 effector cells that persist as long-lived memory cells in vivo. Copyright ©2018, American Association for Cancer Research.

Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology.

PubMed

Ishikawa, Yuichi; Maeda, Manami; Pasham, Mithun; Aguet, Francois; Tacheva-Grigorova, Silvia K; Masuda, Takeshi; Yi, Hai; Lee, Sung-Uk; Xu, Jian; Teruya-Feldstein, Julie; Ericsson, Maria; Mullally, Ann; Heuser, John; Kirchhausen, Tom; Maeda, Takahiro

2015-04-01

Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera. Copyright© Ferrata Storti Foundation.

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins.

PubMed

Ahi, Yadvinder S; Zhang, Shu; Thappeta, Yashna; Denman, Audrey; Feizpour, Amin; Gummuluru, Suryaram; Reinhard, Bjoern; Muriaux, Delphine; Fivash, Matthew J; Rein, Alan

2016-11-22

Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called "glycogag" (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes. Many murine leukemia viruses (MLVs) encode a protein called "glycogag." The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5. Copyright © 2016 Ahi et al.

Biochemical and Biological Studies of Mouse APOBEC3

PubMed Central

Nair, Smita; Sanchez-Martinez, Silvia; Ji, Xinhua

2014-01-01

ABSTRACT Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (ΔVif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferase–mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3′ ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious ΔVif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5′ end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation. IMPORTANCE One way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells. PMID:24453360

Host Thiopurine Methyltransferase Status Affects Mercaptopurine Antileukemic Effectiveness in a Murine Model

PubMed Central

Ramsey, Laura B.; Janke, Laura J.; Edick, Mathew J.; Cheng, Cheng; Williams, Richard T.; Sherr, Charles J.; Evans, William E.; Relling, Mary V.

2014-01-01

Thiopurines are used for many cancers, including acute lymphoblastic leukemia (ALL). Patients with an inherited host defect in thiopurine methyltransferase (TPMT) are at high risk for life-threatening toxicity if treated with conventional dosages, but the impact on antileukemic efficacy is less clear. Herein, we treated thiopurine-sensitive BCR-ABL+ Arf-null Tpmt+/+ ALL in Tpmt+/+, +/−, or −/− recipient mice to test the impact of the host polymorphism on antileukemic efficacy. Median survival was similar in untreated mice of different Tpmt genotypes (16–18 days). However, in mice treated with low-dose mercaptopurine (such as tolerated by TPMT−/− patients), the difference in 30-day leukemia-free survival by Tpmt genotype was profound: 5% (± 9%) for Tpmt+/+ mice, 47% (± 26%) for Tpmt+/− mice, and 85% (± 14%) for Tpmt−/− mice (p = 5×10−8), indicating a substantial impact of host Tpmt status on thiopurine effectiveness. Among Tpmt+/+ recipient mice, leukemia-free survival improved with higher doses of mercaptopurine (similar to doses tolerated by wild-type patients) compared to lower doses, and at higher doses was comparable (p =0.6) to the survival of Tpmt−/− mice treated with the lower dose. These findings support the notion that germline polymorphisms in Tpmt affect not only host tissue toxicity, but also antitumor effectiveness. PMID:24710003

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies

PubMed Central

Lv, Kaosheng; Jiang, Jing; Donaghy, Ryan; Riling, Christopher R.; Cheng, Ying; Chandra, Vemika; Rozenova, Krasimira; An, Wei; Mohapatra, Bhopal C.; Goetz, Benjamin T.; Pillai, Vinodh; Han, Xu; Todd, Emily A.; Jeschke, Grace R.; Langdon, Wallace Y.; Kumar, Suresh; Hexner, Elizabeth O.

2017-01-01

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b. Importantly, primary human CBL mutated (CBLmut) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies. PMID:28611190

The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.

PubMed Central

Dreyling, M H; Martinez-Climent, J A; Zheng, M; Mao, J; Rowley, J D; Bohlander, S K

1996-01-01

The translocation t(10;11)(p13;q14) is a recurring chromosomal abnormality that has been observed in patients with acute lymphoblastic leukemia as well as acute myeloid leukemia. We have recently reported that the monocytic cell line U937 has a t(10;11)(p13;q14) translocation. Using a combination of positional cloning and candidate gene approach, we cloned the breakpoint and were able to show that AF10 is fused to a novel gene that we named CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) located at 11q14. AF10, a putative transcription factor, had recently been cloned as one of the fusion partners of MLL. CALM has a very high homology in its N-terminal third to the murine ap-3 gene which is one of the clathrin assembly proteins. The N-terminal region of ap-3 has been shown to bind to clathrin and to have a high-affinity binding site for phosphoinositols. The identification of the CALM/AF10 fusion gene in the widely used U937 cell line will contribute to our understanding of the malignant phenotype of this line. Images Fig. 1 Fig. 3 PMID:8643484

Calorie restriction suppresses subgenomic mink cytopathic focus-forming murine leukemia virus transcription and frequency of genomic expression while impairing lymphoma formation.

PubMed Central

Shields, B A; Engelman, R W; Fukaura, Y; Good, R A; Day, N K

1991-01-01

Calorie restriction suppresses mammary proviral mRNA expression and protooncogene activation in breast tumor-prone C3H/Ou mice while inhibiting tumor formation. To determine whether the beneficial effects of chronic energy-intake restriction (CEIR) can be extended to an organ site of retrovirus-induced tumorigenesis where the dynamics of growth and sexual maturity are not paramount as they are in breast tissue, calorie restriction of 40% was imposed on thymic lymphoma-prone AKR mice when 4 weeks old. Recombination between various murine leukemia virus (MuLV) mRNAs, resulting in the generation of an 8.4-kilobase genomic-length transcript with mink cytopathic focus-forming (MCF) characteristics, is considered the proximal retroviral event in AKR lymphomagenesis. Thymic expression of subgenomic MCF MuLV mRNA was uniformly suppressed among 6- and 8-week-old CEIR mice (P less than 0.02). This suppression of MuLV transcription preceded a 25% reduction in the appearance of genomic-length MCF transcripts among CEIR mice and a 28% reduction in cumulative lymphoma mortality. The latency to median tumor incidence was extended greater than 3 months by calorie restriction, and median lifespan was extended approximately 50%. Survival curves for the full-fed and CEIR dietary cohorts were found to be significantly different (P less than 0.0001), with full-fed mice experiencing a 3 times greater risk of lymphoma mortality. These findings extend the known range of pathologic states influenced by CEIR in inbred mice and show that retroviral mechanisms involved in generation of lymphoid malignancy can be significantly impaired by calorie restriction. Images PMID:1763029

Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.

PubMed

Tirumalai, R S; Modak, M J

1991-07-02

We have labeled the primer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5' end labeled d(T)8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d(T)8 for the cross-linking reaction, (ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent, (iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P]d(T)8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the bound primer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acid residues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking site appears to be at or near Pro-76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively.

Precise identification of endogenous proviruses of NFS/N mice participating in recombination with moloney ecotropic murine leukemia virus (MuLV) to generate polytropic MuLVs.

PubMed

Alamgir, A S M; Owens, Nick; Lavignon, Marc; Malik, Frank; Evans, Leonard H

2005-04-01

Polytropic murine leukemia viruses (MuLVs) are generated by recombination of ecotropic MuLVs with env genes of a family of endogenous proviruses in mice, resulting in viruses with an expanded host range and greater virulence. Inbred mouse strains contain numerous endogenous proviruses that are potential donors of the env gene sequences of polytropic MuLVs; however, the precise identification of those proviruses that participate in recombination has been elusive. Three different structural groups of proviruses in NFS/N mice have been described and different ecotropic MuLVs preferentially recombine with different groups of proviruses. In contrast to other ecotropic MuLVs such as Friend MuLV or Akv that recombine predominantly with a single group of proviruses, Moloney MuLV (M-MuLV) recombines with at least two distinct groups. In this study, we determined that only three endogenous proviruses, two of one group and one of another group, are major participants in recombination with M-MuLV. Furthermore, the distinction between the polytropic MuLVs generated by M-MuLV and other ecotropic MuLVs is the result of recombination with a single endogenous provirus. This provirus exhibits a frameshift mutation in the 3' region of the surface glycoprotein-encoding sequences that is excluded in recombinants with M-MuLV. The sites of recombination between the env genes of M-MuLV and endogenous proviruses were confined to a short region exhibiting maximum homology between the ecotropic and polytropic env sequences and maximum stability of predicted RNA secondary structure. These observations suggest a possible mechanism for the specificity of recombination observed for different ecotropic MuLVs.

Restricted Replication of Xenotropic Murine Leukemia Virus-Related Virus in Pigtailed Macaques

PubMed Central

Del Prete, Gregory Q.; Kearney, Mary F.; Spindler, Jon; Wiegand, Ann; Chertova, Elena; Roser, James D.; Estes, Jacob D.; Hao, Xing Pei; Trubey, Charles M.; Lara, Abigail; Lee, KyeongEun; Chaipan, Chawaree; Bess, Julian W.; Nagashima, Kunio; Keele, Brandon F.; Macallister, Rhonda; Smedley, Jeremy; Pathak, Vinay K.; KewalRamani, Vineet N.; Coffin, John M.

2012-01-01

Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >1010 RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread. PMID:22238316

Seroprevalence of xenotropic murine leukemia virus-related virus in normal and retrovirus-infected blood donors.

PubMed

Qiu, Xiaoxing; Swanson, Priscilla; Tang, Ning; Leckie, Gregor W; Devare, Sushil G; Schochetman, Gerald; Hackett, John

2012-02-01

Xenotropic murine leukemia virus-related virus (XMRV) has been reported in patients with prostate cancer and chronic fatigue syndrome. Although results have been conflicting, the potential of XMRV as an infectious human retrovirus has raised concerns about transfusion safety. To address this issue, normal and retrovirus-infected blood donors were screened for evidence of XMRV infection. Plasma from 1000 US, 100 human immunodeficiency virus Type 1-infected Cameroonian, and 642 human T-lymphotropic virus Type I (HTLV-I)-infected or uninfected Japanese blood donors as well as 311 sexually transmitted disease diagnostic specimens were screened for antibodies to XMRV gp70 and p15E using chemiluminescent immunoassays (CMIAs). CMIA-reactive samples were evaluated by p30 CMIA, Western blot, and real-time reverse transcriptase polymerase chain reaction. XMRV seroreactivity was low (0%-0.6%) with the exception of the HTLV-I-infected donors (4.9%). Antibody was detected against only a single XMRV protein (p15E or gp70); none of the seroreactive samples had detectable XMRV pol or env sequences. The elevated seroreactivity in HTLV-I-infected donors was due to an increased p15E seroreactive rate (4.1%). Inspection of XMRV and HTLV sequences revealed a high level of conservation within the immunodominant region (IDR) of the transmembrane protein. In some cases, HTLV IDR peptide competitively reduced the XMRV p15E signal. Based on the low prevalence of seroreactivity, detection of antibody to only a single XMRV protein and the absence of XMRV sequences, this study finds no compelling evidence of XMRV in normal or retrovirus-infected blood donors. The increased p15E seroreactivity observed in HTLV infection is likely due to cross-reactive antibodies. © 2012 American Association of Blood Banks.

Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Lingling; Noncoding RNA Center, Yangzhou University, Yangzhou 225001; Zhao, Yingmin

Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feedermore » layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.« less

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

PubMed Central

Ahi, Yadvinder S.; Zhang, Shu; Thappeta, Yashna; Denman, Audrey; Feizpour, Amin; Reinhard, Bjoern; Muriaux, Delphine; Fivash, Matthew J.

2016-01-01

ABSTRACT Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes. PMID:27879338

Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial.

PubMed

Wayne, Alan S; Kreitman, Robert J; Findley, Harry W; Lew, Glen; Delbrook, Cynthia; Steinberg, Seth M; Stetler-Stevenson, Maryalice; Fitzgerald, David J; Pastan, Ira

2010-03-15

Although most children with B-lineage acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma are cured, new agents are needed to overcome drug resistance and reduce toxicities of chemotherapy. We hypothesized that the novel anti-CD22 immunotoxin, RFB4(dsFv)-PE38 (BL22, CAT-3888), would be active and have limited nonspecific side effects in children with CD22-expressing hematologic malignancies. We conducted the first preclinical and phase I clinical studies of BL22 in that setting. Lymphoblasts from children with B-lineage ALL were assessed for CD22 expression by flow cytometry and for BL22 sensitivity by in vitro cytotoxicity assay. BL22 was evaluated in a human ALL murine xenograft model. A phase I clinical trial was conducted for pediatric subjects with CD22+ ALL and non-Hodgkin lymphoma. All samples screened were CD22+. BL22 was cytotoxic to blasts in vitro (median IC(50), 9.8 ng/mL) and prolonged the leukemia-free survival of murine xenografts. Phase I trial cohorts were treated at escalating doses and schedules ranging from 10 to 40 microg/kg every other day for three or six doses repeated every 21 or 28 days. Treatment was associated with an acceptable safety profile, adverse events were rapidly reversible, and no maximum tolerated dose was defined. Pharmacokinetics were influenced by disease burden consistent with rapid drug binding by CD22+ blasts. Although no responses were observed, transient clinical activity was seen in most subjects. CD22 represents an excellent target and anti-CD22 immunotoxins offer therapeutic promise in B-lineage hematologic malignancies of childhood.

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

PubMed Central

Julias, J G; Kim, T; Arnold, G; Pathak, V K

1997-01-01

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis. PMID:9151812

Selective Packaging of Host tRNA's by Murine Leukemia Virus Particles Does Not Require Genomic RNA

PubMed Central

Levin, Judith G.; Seidman, J. G.

1979-01-01

The 4S RNA contained in RNA tumor virus particles consists of a selected population of host tRNA's. However, the mechanism by which virions select host tRNA's has not been elucidated. We have considered a model which specifies that 35S genomic RNA determines which tRNA's are to be encapsidated as well as the relative amounts of these tRNA's within the virion. The model was tested by comparing the free 4S RNA composition of normal murine leukemia virus (MuLV) particles and noninfectious virions from actinomycin D (ActD)-treated cells, which are deficient in genomic RNA (ActD virions). Viral 4S RNA was analyzed by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the patterns obtained for control and ActD 4S RNA were identical to each other and were clearly distinct from the cell 4S RNA pattern. The viral patterns had three prominent areas of radioactivity. One of the spots was identified on the basis of its oligonucleotide fingerprint as tRNA Pro, the primer for MuLV RNA-directed DNA synthesis. These results were obtained with two different MuLV strains, AKR and Moloney, each grown in SC-1 cells. The demonstration that ActD virions contain primer tRNA and in general exhibit the characteristic MuLV tRNA pattern rather than the complete representation of cell 4S RNA leads to the conclusion that genomic RNA is not the major determinant in selective packaging of host tRNA's. A possible role for one or more viral proteins, including reverse transcriptase, is suggested. Images PMID:219227

NK Cell–Mediated Antitumor Effects of a Folate-Conjugated Immunoglobulin are Enhanced by Cytokines

PubMed Central

Kondadasula, SriVidya; Skinner, Cassandra C.; Mundy-Bosse, Bethany L.; Luedke, Eric; Jones, Natalie B.; Mani, Aruna; Roda, Julie; Karpa, Volodymyr; Li, Hong; Li, Jilong; Elavazhagan, Saranya; La Perle, Krista M.; Schmitt, Alessandra C.; Lu, Yanhui; Zhang, Xiaoli; Pan, Xueliang; Mao, Hsaioyin; Davis, Melanie; Jarjoura, David; Butchar, Jonathan P.; Poi, Ming; Phelps, Mitch; Tridandapani, Susheela; Byrd, John C.; Caligiuri, Michael A.; Lee, Robert J.; Carson, William E.

2016-01-01

Optimally effective antitumor therapies would not only activate immune effector cells, but engage them at the tumor. Folate-conjugated to immunoglobulin (F-IgG) could direct innate immune cells with Fc receptors to folate receptor–expressing cancer cells. F-IgG bound to human KB and HeLa cells, as well as murine L1210JF, a folate receptor (FR) overexpressing cancer cell line, as determined by flow cytometry. Recognition of F-IgG by NK cell Fc receptors led to phosphorylation of the ERK transcription factor and increased NK cell expression of CD69. Lysis of KB tumor cells by NK cells increased about 5-fold after treatment with F-IgG, an effect synergistically enhanced by treatment with IL2, IL12, IL15, or IL21 (P < 0.001). F-IgG also enhanced the lysis of chronic lymphocytic leukemia cells by autologous NK cells. NK cells significantly increased production of IFNγ, MIP-1α, and RANTES in response to F-IgG–coated KB target cells in the presence of the NK cell–activating cytokine IL12, and these coculture supernatants induced significant T cell chemotaxis P < 0.001). F-IgG–coated targets also stimulated FcR-mediated monocyte effector functions. Studies in a murine leukemia model confirmed the intratumoral localization and antitumor activity of F-IgG, as well as enhancement of its effects by IL12 (P = 0.05). The antitumor effect of this combination was dependent on NK cells and led to decreased tumor cell proliferation in vivo. Thus, F-IgG can induce an immune response against FR-positive tumor cells that is mediated by NK cells and can be augmented by cytokine therapy. PMID:26865456

The possible role of liver kinase B1 in hydroquinone-induced toxicity of murine fetal liver and bone marrow hematopoietic stem cells.

PubMed

Li, Zhen; Wang, Chunhong; Zhu, Jie; Bai, YuE; Wang, Wei; Zhou, Yanfeng; Zhang, Shaozun; Liu, Xiangxiang; Zhou, Sheng; Huang, Wenting; Bi, Yongyi; Wang, Hong

2016-07-01

Epidemiological studies suggest that the increasing incidence of childhood leukemia may be due to maternal exposure to benzene, which is a known human carcinogen; however, the mechanisms involved remain unknown. Liver Kinase B1 (LKB1) acts as a regulator of cellular energy metabolism and functions to regulate hematopoietic stem cell (HSC) homeostasis. We hypothesize that LKB1 contributes to the deregulation of fetal or bone hematopoiesis caused by the benzene metabolite hydroquinone (HQ). To evaluate this hypothesis, we compared the effects of HQ on murine fetal liver hematopoietic stem cells (FL-HSCs) and bone marrow hematopoietic stem cells (BM-HSCs). FL-HSCs and BM-HSCs were isolated and enriched by a magnetic cell sorting system and exposed to various concentrations of HQ (0, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 h. We found that the inhibition of differentiation and growth, as well as the apoptosis rate of FL-HSCs, induced by HQ were consistent with the changes in BM-HSCs. Furthermore, G1 cell cycle arrest was observed in BM-HSCs and FL-HSCs in response to HQ. Importantly, FL-HSCs were more sensitive than BM-HSCs after exposure to HQ. The highest induction of LKB1 and adenosine monophosphate-activated protein kinase (AMPK) was observed with a much lower concentration of HQ in FL-HSCs than in BM-HSCs. LKB1 may play a critical role in apoptosis and cell cycle arrest of HQ-treated HSCs. This research has developed innovative ideas concerning benzene-induced hematopoietic toxicity or embryotoxicity, which can provide a new experimental evidence for preventing childhood leukemia. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 830-841, 2016. © 2014 Wiley Periodicals, Inc.

Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells.

PubMed

Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Kobayashi, Shouhei; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

2016-08-01

Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery. © 2016 Federation of European Biochemical Societies.

Acute myeloid leukemia stem cell markers in prognosis and targeted therapy: potential impact of BMI-1, TIM-3 and CLL-1.

PubMed

Darwish, Noureldien H E; Sudha, Thangirala; Godugu, Kavitha; Elbaz, Osama; Abdelghaffar, Hasan A; Hassan, Emad E A; Mousa, Shaker A

2016-09-06

Acute myeloid leukemia (AML) patients show high relapse rates and some develop conventional chemotherapy resistance. Leukemia Stem Cells (LSCs) are the main player for AML relapses and drug resistance. LSCs might rely on the B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) in promoting cellular proliferation and survival. Growth of LSCs in microenvironments that are deprived of nutrients leads to up-regulation of the signaling pathways during the progression of the disease, which may illustrate the sensitivity of LSCs to inhibitors of those signaling pathways as compared to normal cells. We analyzed the expression of LSC markers (CD34, CLL-1, TIM-3 and BMI-1) using quantitative RT-PCR in bone marrow samples of 40 AML patients of different FAB types (M1, M2, M3, M4, M5, and M7). We also studied the expression of these markers in 2 AML cell lines (Kasumi-1 and KG-1a) using flow cytometry and quantitative RT-PCR. The overexpression of TIM-3, CLL-1, and BMI-1 was markedly correlated with poor prognosis in these patients. Our in vitro findings demonstrate that targeting BMI-1, which markedly increased in the leukemic cells, was associated with marked decrease in leukemic burden. This study also presents results for blocking LSCs' surface markers CD44, CLL-1, and TIM-3. These markers may play an important role in elimination of AML. Our study indicates a correlation between the expression of markers TIM-3, CLL-1, and especially of BMI-1 and the aggressiveness of AML and thus the potential impact of prognosis and therapies that target LSCs on improving the cure rates.

Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.

PubMed

Hollyman, Daniel; Stefanski, Jolanta; Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Taylor, Clare; Yeh, Raymond; Capacio, Vanessa; Olszewska, Malgorzata; Hosey, James; Sadelain, Michel; Brentjens, Renier J; Rivière, Isabelle

2009-01-01

On the basis of promising preclinical data demonstrating the eradication of systemic B-cell malignancies by CD19-targeted T lymphocytes in vivo in severe combined immunodeficient-beige mouse models, we are launching phase I clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia. We present here the validation of the bioprocess which we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semiclosed culture system using the Wave Bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in severe combined immunodeficient-beige mice bearing disseminated tumors. The validation requirements in terms of T-cell expansion, T-cell transduction with the 1928z CAR, biologic activity, quality control testing, and release criteria were met for all 4 validation runs using apheresis products from patients with CLL. Additionally, after expansion of the T cells, the diversity of the skewed Vbeta T-cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemorefractory CLL and in patients with relapsed acute lymphoblastic leukemia. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any CAR or T-cell receptor.

In-depth characterization of the microRNA transcriptome in a leukemia progression model

PubMed Central

Kuchenbauer, Florian; Morin, Ryan D.; Argiropoulos, Bob; Petriv, Oleh I.; Griffith, Malachi; Heuser, Michael; Yung, Eric; Piper, Jessica; Delaney, Allen; Prabhu, Anna-Liisa; Zhao, Yongjun; McDonald, Helen; Zeng, Thomas; Hirst, Martin; Hansen, Carl L.; Marra, Marco A.; Humphries, R. Keith

2008-01-01

MicroRNAs (miRNAs) have been shown to play important roles in physiological as well as multiple malignant processes, including acute myeloid leukemia (AML). In an effort to gain further insight into the role of miRNAs in AML, we have applied the Illumina massively parallel sequencing platform to carry out an in-depth analysis of the miRNA transcriptome in a murine leukemia progression model. This model simulates the stepwise conversion of a myeloid progenitor cell by an engineered overexpression of the nucleoporin 98 (NUP98)–homeobox HOXD13 fusion gene (ND13), to aggressive AML inducing cells upon transduction with the oncogenic collaborator Meis1. From this data set, we identified 307 miRNA/miRNA* species in the ND13 cells and 306 miRNA/miRNA* species in ND13+Meis1 cells, corresponding to 223 and 219 miRNA genes. Sequence counts varied between two and 136,558, indicating a remarkable expression range between the detected miRNA species. The large number of miRNAs expressed and the nature of differential expression suggest that leukemic progression as modeled here is dictated by the repertoire of shared, but differentially expressed miRNAs. Our finding of extensive sequence variations (isomiRs) for almost all miRNA and miRNA* species adds additional complexity to the miRNA transcriptome. A stringent target prediction analysis coupled with in vitro target validation revealed the potential for miRNA-mediated release of oncogenes that facilitates leukemic progression from the preleukemic to leukemia inducing state. Finally, 55 novel miRNAs species were identified in our data set, adding further complexity to the emerging world of small RNAs. PMID:18849523

The Pim kinases: new targets for drug development.

PubMed

Swords, Ronan; Kelly, Kevin; Carew, Jennifer; Nawrocki, Stefan; Mahalingam, Devalingam; Sarantopoulos, John; Bearss, David; Giles, Francis

2011-12-01

The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation.

Studies on the inhibition of Moloney murine leukemia virus reverse transcriptase by N-tritylamino acids and N-tritylamino acid-nucleotide compounds.

PubMed

Hawtrey, Arthur; Pieterse, Anton; van Zyl, Johann; Van der Bijl, Pieter; Van der Merwe, Marichen; Nel, William; Ariatti, Mario

2008-09-01

N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5 '-phosphate were prepared and studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-aminohexyl) amino-5'-AMP inhibited the enzyme partially using poly (rA).oligo d(pT)(16-18) as template-primer with [(3)H]dTTP. In order to increase hydrophobicity in the acyl component tethered to the 8-(6-aminohexyl) amino group on the adenine nucleotide, N-trityl-L-phenylalanine and the N-trityl derivatives of the o, m, and p-fluoro-DL-phenylalanine were initially examined for inhibition of the enzyme using the above template-primer system. The compounds all inhibited the reverse transcriptase with IC(50) values of approximately 60-80 microM. However, when N-trityl-m-fluoro-DL-phenylalanine was coupled to the nucleotide 8-(6-aminohexyl) amino-adenosine-5'-phosphate, the inhibitory activity of this compound increased significantly (IC(50) = 5 microM).

β-Arrestin2 mediates progression of murine primary myelofibrosis.

PubMed

Rein, Lindsay Am; Wisler, James W; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Minyong; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia Kl; Premont, Richard T; Lefkowitz, Robert J

2017-12-21

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2-/- cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.

PubMed Central

Skoda, R C; Seldin, D C; Chiang, M K; Peichel, C L; Vogt, T F; Leder, P

1993-01-01

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells. Images PMID:8334987

The mechano-chemistry of a monomeric reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei

2017-01-01

Abstract Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex. PMID:29165701

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

PubMed

Kessler, J H; Bres-Vloemans, S A; van Veelen, P A; de Ru, A; Huijbers, I J G; Camps, M; Mulder, A; Offringa, R; Drijfhout, J W; Leeksma, O C; Ossendorp, F; Melief, C J M

2006-10-01

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

PubMed

Zou, Siying; Teixeira, Alexandra M; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferrucio, Juliana; Zhang, Ping-Xia; Hwa, John; Min, Wang; Krause, Diane S

2016-08-30

Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.

Evaluation of viral removal by nanofiltration using real-time quantitative polymerase chain reaction.

PubMed

Zhao, Xiaowen; Bailey, Mark R; Emery, Warren R; Lambooy, Peter K; Chen, Dayue

2007-06-01

Nanofiltration is commonly introduced into purification processes of biologics produced in mammalian cells to serve as a designated step for removal of potential exogenous viral contaminants and endogenous retrovirus-like particles. The LRV (log reduction value) achieved by nanofiltration is often determined by cell-based infectivity assay, which is time-consuming and labour-intensive. We have explored the possibility of employing QPCR (quantitative PCR) to evaluate LRV achieved by nanofiltration in scaled-down studies using two model viruses, namely xenotropic murine leukemia virus and murine minute virus. We report here the successful development of a QPCR-based method suitable for quantification of virus removal by nanofiltration. The method includes a nuclease treatment step to remove free viral nucleic acids, while viral genome associated with intact virus particles is shielded from the nuclease. In addition, HIV Armored RNA was included as an internal control to ensure the accuracy and reliability of the method. The QPCRbased method described here provides several advantages such as better sensitivity, faster turnaround time, reduced cost and higher throughput over the traditional cell-based infectivity assays.

Micro-computed tomography in murine models of cerebral cavernous malformations as a paradigm for brain disease.

PubMed

Girard, Romuald; Zeineddine, Hussein A; Orsbon, Courtney; Tan, Huan; Moore, Thomas; Hobson, Nick; Shenkar, Robert; Lightle, Rhonda; Shi, Changbin; Fam, Maged D; Cao, Ying; Shen, Le; Neander, April I; Rorrer, Autumn; Gallione, Carol; Tang, Alan T; Kahn, Mark L; Marchuk, Douglas A; Luo, Zhe-Xi; Awad, Issam A

2016-09-15

Cerebral cavernous malformations (CCMs) are hemorrhagic brain lesions, where murine models allow major mechanistic discoveries, ushering genetic manipulations and preclinical assessment of therapies. Histology for lesion counting and morphometry is essential yet tedious and time consuming. We herein describe the application and validations of X-ray micro-computed tomography (micro-CT), a non-destructive technique allowing three-dimensional CCM lesion count and volumetric measurements, in transgenic murine brains. We hereby describe a new contrast soaking technique not previously applied to murine models of CCM disease. Volumetric segmentation and image processing paradigm allowed for histologic correlations and quantitative validations not previously reported with the micro-CT technique in brain vascular disease. Twenty-two hyper-dense areas on micro-CT images, identified as CCM lesions, were matched by histology. The inter-rater reliability analysis showed strong consistency in the CCM lesion identification and staging (K=0.89, p<0.0001) between the two techniques. Micro-CT revealed a 29% greater CCM lesion detection efficiency, and 80% improved time efficiency. Serial integrated lesional area by histology showed a strong positive correlation with micro-CT estimated volume (r(2)=0.84, p<0.0001). Micro-CT allows high throughput assessment of lesion count and volume in pre-clinical murine models of CCM. This approach complements histology with improved accuracy and efficiency, and can be applied for lesion burden assessment in other brain diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Adoptive T-cell therapy for Leukemia.

PubMed

Garber, Haven R; Mirza, Asma; Mittendorf, Elizabeth A; Alatrash, Gheath

2014-01-01

Allogeneic stem cell transplantation (alloSCT) is the most robust form of adoptive cellular therapy (ACT) and has been tremendously effective in the treatment of leukemia. It is one of the original forms of cancer immunotherapy and illustrates that lymphocytes can specifically recognize and eliminate aberrant, malignant cells. However, because of the high morbidity and mortality that is associated with alloSCT including graft-versus-host disease (GvHD), refining the anti-leukemia immunity of alloSCT to target distinct antigens that mediate the graft-versus-leukemia (GvL) effect could transform our approach to treating leukemia, and possibly other hematologic malignancies. Over the past few decades, many leukemia antigens have been discovered that can separate malignant cells from normal host cells and render them vulnerable targets. In concert, the field of T-cell engineering has matured to enable transfer of ectopic high-affinity antigen receptors into host or donor cells with greater efficiency and potency. Many preclinical studies have demonstrated that engineered and conventional T-cells can mediate lysis and eradication of leukemia via one or more leukemia antigen targets. This evidence now serves as a foundation for clinical trials that aim to cure leukemia using T-cells. The recent clinical success of anti-CD19 chimeric antigen receptor (CAR) cells for treating patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia displays the potential of this new therapeutic modality. In this review, we discuss some of the most promising leukemia antigens and the novel strategies that have been implemented for adoptive cellular immunotherapy of lymphoid and myeloid leukemias. It is important to summarize the data for ACT of leukemia for physicians in-training and in practice and for investigators who work in this and related fields as there are recent discoveries already being translated to the patient setting and numerous accruing clinical trials. We primarily focus on ACT that has been used in the clinical setting or that is currently undergoing preclinical testing with a foreseeable clinical endpoint.

Dependence of erythroid differentiation on cell replication in dimethyl sulfoxide-treated friend leukemia-virus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiens, A.W.; McClintock, P.R.; Papaconstantinou, J.

1976-01-01

The dimethyl sulfoxide (Me/sub 2/SO)-mediated induction of hemoglobin synthesis in Friend leukemia cells (a murine erythroblastoid cell line) is coupled with the number of cell replications occurring in the presence of inducer. Varying concentrations of proflavine increase the generation time of these cells from 24 hours to over 50 hours, and in each case the induction of hemoglobin synthesis follows the completion of two cell doublings. Once the induction is initiated, the rate of hemoglobin accumulation is not affected by proflavine. These data indicate that proflavine does not affect the transcription or translation of globin mRNA and that the delaymore » in induction of hemoglobin synthesis is due to its effect on the rate of cellular replication. In experiments using high concentrations of thymidine to block replication, hemoglobin accumulation is prevented only if the cells are blocked prior to 36 hours after Me/sub 2/SO addition. If the cells have completed two generations in the presence of Me/sub 2/SO, there is no effect upon their ability to synthesize hemoglobin even though their growth is arrested. Thus, the inhibition of hemoglobin synthesis by proflavine is not merely the result of a toxic effect on newly subcultured cells but is due to its effect on cellular replication. These experiments confirm that, after addition of Me/sub 2/SO, Friend leukemia cells require more than one complete cell cycle in order to synthesize hemoglobin.« less

Cannabinoids induce incomplete maturation of cultured human leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murison, G.; Chubb, C.B.H.; Maeda, S.

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. Themore » THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.« less

Lineage Switching in Acute Leukemias: A Consequence of Stem Cell Plasticity?

PubMed Central

Dorantes-Acosta, Elisa; Pelayo, Rosana

2012-01-01

Acute leukemias are the most common cancer in childhood and characterized by the uncontrolled production of hematopoietic precursor cells of the lymphoid or myeloid series within the bone marrow. Even when a relatively high efficiency of therapeutic agents has increased the overall survival rates in the last years, factors such as cell lineage switching and the rise of mixed lineages at relapses often change the prognosis of the illness. During lineage switching, conversions from lymphoblastic leukemia to myeloid leukemia, or vice versa, are recorded. The central mechanisms involved in these phenomena remain undefined, but recent studies suggest that lineage commitment of plastic hematopoietic progenitors may be multidirectional and reversible upon specific signals provided by both intrinsic and environmental cues. In this paper, we focus on the current knowledge about cell heterogeneity and the lineage switch resulting from leukemic cells plasticity. A number of hypothetical mechanisms that may inspire changes in cell fate decisions are highlighted. Understanding the plasticity of leukemia initiating cells might be fundamental to unravel the pathogenesis of lineage switch in acute leukemias and will illuminate the importance of a flexible hematopoietic development. PMID:22852088

Dendritic Cells Pulsed with Leukemia Cell-Derived Exosomes More Efficiently Induce Antileukemic Immunities

PubMed Central

Wei, Wei; Shen, Chang; Deng, Xiaohui; Chen, Linjun; Ma, Liyuan; Hao, Siguo

2014-01-01

Dendritic cells (DCs) and tumor cell-derived exosomes have been used to develop antitumor vaccines. However, the biological properties and antileukemic effects of leukemia cell-derived exosomes (LEXs) are not well described. In this study, the biological properties and induction of antileukemic immunity of LEXs were investigated using transmission electron microscopy, western blot analysis, cytotoxicity assays, and animal studies. Similar to other tumor cells, leukemia cells release exosomes. Exosomes derived from K562 leukemia cells (LEXK562) are membrane-bound vesicles with diameters of approximately 50–100 μm and harbor adhesion molecules (e.g., intercellular adhesion molecule-1) and immunologically associated molecules (e.g., heat shock protein 70). In cytotoxicity assays and animal studies, LEXs-pulsed DCs induced an antileukemic cytotoxic T-lymphocyte immune response and antileukemic immunity more effectively than did LEXs and non-pulsed DCs (P<0.05). Therefore, LEXs may harbor antigens and immunological molecules associated with leukemia cells. As such, LEX-based vaccines may be a promising strategy for prolonging disease-free survival in patients with leukemia after chemotherapy or hematopoietic stem cell transplantation. PMID:24622345

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

PubMed Central

Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

2016-01-01

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples.

PubMed

Nekouei, Omid; Durocher, Jean; Keefe, Greg

2016-07-01

This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples

PubMed Central

Nekouei, Omid; Durocher, Jean; Keefe, Greg

2016-01-01

This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs. PMID:27429469

No Evidence for XMRV Nucleic Acids, Infectious Virus or Anti-XMRV Antibodies in Canadian Patients with Chronic Fatigue Syndrome

PubMed Central

Steffen, Imke; Tyrrell, D. Lorne; Stein, Eleanor; Montalvo, Leilani; Lee, Tzong-Hae; Zhou, Yanchen; Lu, Kai; Switzer, William M.; Tang, Shaohua; Jia, Hongwei; Hockman, Darren; Santer, Deanna M.; Logan, Michael; Landi, Amir; Law, John; Houghton, Michael; Simmons, Graham

2011-01-01

The gammaretroviruses xenotropic murine leukemia virus (MLV)-related virus (XMRV) and MLV have been reported to be more prevalent in plasma and peripheral blood mononuclear cells of chronic fatigue syndrome (CFS) patients than in healthy controls. Here, we report the complex analysis of whole blood and plasma samples from 58 CFS patients and 57 controls from Canada for the presence of XMRV/MLV nucleic acids, infectious virus, and XMRV/MLV-specific antibodies. Multiple techniques were employed, including nested and qRT-PCR, cell culture, and immunoblotting. We found no evidence of XMRV or MLV in humans and conclude that CFS is not associated with these gammaretroviruses. PMID:22114717

Curative chemotherapy for acute myeloid leukemia: the development of high-dose ara-C from the laboratory to bedside.

PubMed

Capizzi, R L

1996-01-01

In the bench to bedside development of drugs to treat patients with cancer, the common guide to dose and schedule selection is toxicity to normal organs patterned after the preclinical profile of the drug. An understanding of the cellular pharmacology of the drug and specifically the cellular targets linked to the drug's effect is of substantial value in assisting the clinical investigator in selecting the proper dose and schedule of drug administration. The clinical development of ara-C for the treatment of acute myeloid leukemia (AML) provides a useful paradigm for the study of this process. An understanding of the cellular pharmacology, cytokinetics and pharmacokinetics of ara-C in leukemic mice showed substantial schedule-dependency. Exposure to high doses for a short duration (C x t) resulted in a palliative therapeutic outcome. In marked contrast, exposure to lower doses for a protracted period (c x T) was curative. Clinical use of ara-C in patients with AML patterned after the murine experience, c x T approach, has been of limited benefit in terms of long-term disease-free survival. Studies with human leukemia blasts from patients have shown that for the majority of patients, the initial rate-limiting step is membrane transport, the characteristics of which are substantially affected by extracellular drug concentration (dose). This pharmacologic impediment is eliminated with the blood levels attained during the infusion of gram doses (1-3 gm/m2) of the drug (high-dose ara-C, HiDaC) for shorter periods of time, a C x t approach. Clinical confirmation of these pharmacologic observations is evident in the therapeutic efficacy of HiDaC in patients with relapsed or SDaC-refractory acute leukemia. This is further emphasized by the significantly improved leukemia-free survival of patients with AML treated with HiDaC intensification during remission compared to those patients treated with milligram doses typical of SDaC protocols. Thus, the identification and monitoring of important parameters of drug action in tumors during the course of a clinical trial can be of substantial assistance in optimizing drug dose and schedule so as to attain the best therapeutic index.

β-Arrestin2 mediates progression of murine primary myelofibrosis

PubMed Central

Rein, Lindsay A.M.; Wisler, James W.; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia K.L.; Premont, Richard T.; Lefkowitz, Robert J.

2017-01-01

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2–/–) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2–/– cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2–/– cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease. PMID:29263312

ATL response to arsenic/interferon therapy is triggered by SUMO/PML/RNF4-dependent Tax degradation.

PubMed

Dassouki, Zeina; Sahin, Umut; El Hajj, Hiba; Jollivet, Florence; Kfoury, Youmna; Lallemand-Breitenbach, Valérie; Hermine, Olivier; de Thé, Hugues; Bazarbachi, Ali

2015-01-15

The human T-cell lymphotropic virus type I (HTLV-1) Tax transactivator initiates transformation in adult T-cell leukemia/lymphoma (ATL), a highly aggressive chemotherapy-resistant malignancy. The arsenic/interferon combination, which triggers degradation of the Tax oncoprotein, selectively induces apoptosis of ATL cell lines and has significant clinical activity in Tax-driven murine ATL or human patients. However, the role of Tax loss in ATL response is disputed, and the molecular mechanisms driving degradation remain elusive. Here we demonstrate that ATL-derived or HTLV-1-transformed cells are dependent on continuous Tax expression, suggesting that Tax degradation underlies clinical responses to the arsenic/interferon combination. The latter enforces promyelocytic leukemia protein (PML) nuclear body (NB) formation and partner protein recruitment. In arsenic/interferon-treated HTLV-1 transformed or ATL cells, Tax is recruited onto NBs and undergoes PML-dependent hyper-sumoylation by small ubiquitin-like modifier (SUMO)2/3 but not SUMO1, ubiquitination by RNF4, and proteasome-dependent degradation. Thus, the arsenic/interferon combination clears ATL through degradation of its Tax driver, and this regimen could have broader therapeutic value by promoting degradation of other pathogenic sumoylated proteins. © 2015 by The American Society of Hematology.

Developmentally induced Mll1 loss reveals defects in postnatal haematopoiesis.

PubMed

Gan, T; Jude, C D; Zaffuto, K; Ernst, P

2010-10-01

The mixed lineage leukemia (MLL) gene is disrupted by chromosomal translocations in acute leukemia, producing a fusion oncogene with altered properties relative to the wild-type gene. Murine loss-of-function studies have shown an essential role for Mll in developing the haematopoietic system, yet studies using different conditional knockout models have yielded conflicting results regarding the requirement for Mll during adult steady-state haematopoiesis. In this study, we used a loxP-flanked Mll allele (Mll(F)) and a developmentally regulated, haematopoietic-specific VavCre transgene to reassess the consequences of Mll loss in the haematopoietic lineage, without the need for inducers of Cre recombinase. We show that VavCre;Mll mutants exhibit phenotypically normal fetal haematopoiesis, but rarely survive past 3 weeks of age. Surviving animals are anemic, thrombocytopenic and exhibit a significant reduction in bone marrow haematopoietic stem/progenitor populations, consistent with our previous findings using the inducible Mx1Cre transgene. Furthermore, the analysis of VavCre mutants revealed additional defects in B-lymphopoiesis that could not be assessed using Mx1Cre-mediated Mll deletion. Collectively, these data support the conclusion that Mll has an essential role in sustaining postnatal haematopoiesis.

Light scattering application for quantitative estimation of apoptosis

NASA Astrophysics Data System (ADS)

Bilyy, Rostyslav O.; Stoika, Rostyslav S.; Getman, Vasyl B.; Bilyi, Olexander I.

2004-05-01

Estimation of cell proliferation and apoptosis are in focus of instrumental methods used in modern biomedical sciences. Present study concerns monitoring of functional state of cells, specifically the development of their programmed death or apoptosis. The available methods for such purpose are either very expensive, or require time-consuming operations. Their specificity and sensitivity are frequently not sufficient for making conclusions which could be used in diagnostics or treatment monitoring. We propose a novel method for apoptosis measurement based on quantitative determination of cellular functional state taking into account their physical characteristics. This method uses the patented device -- laser microparticle analyser PRM-6 -- for analyzing light scattering by the microparticles, including cells. The method gives an opportunity for quick, quantitative, simple (without complicated preliminary cell processing) and relatively cheap measurement of apoptosis in cellular population. The elaborated method was used for studying apoptosis expression in murine leukemia cells of L1210 line and human lymphoblastic leukemia cells of K562 line. The results obtained by the proposed method permitted measuring cell number in tested sample, detecting and quantitative characterization of functional state of cells, particularly measuring the ratio of the apoptotic cells in suspension.

cis-Acting elements important for retroviral RNA packaging specificity.

PubMed

Beasley, Benjamin E; Hu, Wei-Shau

2002-05-01

Spleen necrosis virus (SNV) proteins can package RNA from distantly related murine leukemia virus (MLV), whereas MLV proteins cannot package SNV RNA efficiently. We used this nonreciprocal recognition to investigate regions of packaging signals that influence viral RNA encapsidation specificity. Although the MLV and SNV packaging signals (Psi and E, respectively) do not contain significant sequence homology, they both contain a pair of hairpins. This hairpin pair was previously proposed to be the core element in MLV Psi. In the present study, MLV-based vectors were generated to contain chimeric SNV/MLV packaging signals in which the hairpins were replaced with the heterologous counterpart. The interactions between these chimeras and MLV or SNV proteins were examined by virus replication and RNA analyses. SNV proteins recognized all of the chimeras, indicating that these chimeras were functional. We found that replacing the hairpin pair did not drastically alter the ability of MLV proteins to package these chimeras. These results indicate that, despite the important role of the hairpin pair in RNA packaging, it is not the major motif responsible for the ability of MLV proteins to discriminate between the MLV and SNV packaging signals. To determine the role of sequences flanking the hairpins in RNA packaging specificity, vectors with swapped flanking regions were generated and evaluated. SNV proteins packaged all of these chimeras efficiently. In contrast, MLV proteins strongly favored chimeras with the MLV 5'-flanking regions. These data indicated that MLV Gag recognizes multiple elements in the viral packaging signal, including the hairpin structure and flanking regions.

Baculovirus GP64-mediated entry into mammalian cells.

PubMed

Kataoka, Chikako; Kaname, Yuuki; Taguwa, Shuhei; Abe, Takayuki; Fukuhara, Takasuke; Tani, Hideki; Moriishi, Kohji; Matsuura, Yoshiharu

2012-03-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

The Gag Cleavage Product, p12, is a Functional Constituent of the Murine Leukemia Virus Pre-Integration Complex

PubMed Central

Laham-Karam, Nihay; Selig, Sara; Ehrlich, Marcelo; Bacharach, Eran

2010-01-01

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration. PMID:21085616

Genetic interactions in the spontaneous production of endogenous murine leukemia virus in low leukemic mouse strains

PubMed Central

1982-01-01

The spontaneous expression of ecotropic murine leukemia virus (MuLV) in spleen cells of BALB/c, C57BL/6 (B6), and derivative mice was examined as a function of age. The patterns of spontaneous virus induction in vivo correlate with the patterns of virus induction in vitro, which result from the action of two loci, Inc-l and Inb-l (7). Whereas mice carrying Inc-l or Inb-l have similar phenotypes in vitro, they have significantly different phenotypes in vivo. Mice of the Inb-l+/+ genotype, e.g., B6, rarely expressed MuLV, and the titer of MuLV recovered from rare MuLV-positive mice of this genotype was usually low. Mice of the Inc-l+/+ genotype, e.g., BALB/c, expressed low amounts of MuLV early in life, however, from 6-12 mo of age approximately one- half of the Inc-l+/+ mice expressed virus, frequently of high titer. Equal numbers of N-tropic and B-tropic MuLV were recovered from Inb-l+ mice, but predominantly N-tropic MuLV was recovered from Inc-l+ mice. Strains that carry dominant (+) alleles at both Inc-l and Inb-l show higher titers of MuLV earlier in life than strains that carry only Inc- l or Inb-l. The presence of dominant alleles at both loci results in the appearance of predominantly N-tropic virus early in life. These results demonstrate that the principal determinants of spontaneous virus expression in these low leukemic strains of mice are the In loci or genes linked to them. A further inference that can be drawn from these studies is that the appearance of B-tropic virus is by no means a random process but rather results from predictable patterns of MuLV expression and alteration. PMID:6284854

Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikuta, K.; Luftig, R.B.

1988-01-01

The authors detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using /sup 32/P/sub i/-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the /sup 32/P label added to infected cells. When immunoprecipitates from M-MuLV lysates labeled with /sup 32/P/sub i/ were compared with those labeled with (/sup 35/S)methionine, it was calculated that the degree of phosphorylation at themore » p30 domain of Pr65/sup gag/ was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the /sup 32/P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the (/sup 35/S) methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with > 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with (/sup 14/C)sesrine, (/sup 35/S)methionine, and /sup 32/P/sub i/, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH/sub 2/-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.« less

Down-regulation of Jab1, HIF-1alpha, and VEGF by Moloney murine leukemia virus-ts1 infection: a possible cause of neurodegeneration.

PubMed

Lungu, Gina F; Stoica, George; Wong, Paul K Y

2008-05-01

Moloney murine leukemia virus-temperature sensitive (MoMuLV-ts1)-mediated neuronal death is a result of both loss of glial support and release of cytokines and neurotoxins from ts1-infected glial cells. Here the authors propose vascular endothelial growth factor (VEGF) down-regulation as another contributory factor in neuronal degeneration induced by ts1 infection. To determine how ts1 affects VEGF expression in ts1-infected brain, the authors examined the expression of several proteins that are important in regulating the expression of VEGF. The authors found significant decreases in Jun-activating domain-binding protein 1 (Jab1), hypoxia-inducible factor (HIF)-1alpha, and VEGF levels and increases in p53 protein levels in ts1-infected brains compared to noninfected control brains. The authors suggest that a decrease Jab1 expression in ts1 infection leads to accumulation of p53, which binds to HIF-1alpha to accelerate its degradation. A rapid degradation of HIF-1alpha leads to decreased VEGF production and secretion. Considering that endothelial cells are the most conspicuous in virus replication and production in ts1 infection, but are not killed by the infection, the authors examined the expression of these proteins using infected and noninfected mouse cerebrovascular endothelial (CVE) cells. The ts1- infected CVE cells showed decreased Jab1, HIF-1alpha, and VEGF mRNA and protein levels and increased p53 protein levels compared with noninfected cells, consistent with the results found in vivo. These results confirm that ts1 infection results in insufficient secretion of VEGF from endothelial cells and may result in decreased neuroprotection. This study suggested that ts1-mediated neuropathology in mice may result from changes in expression and activity of Jab1, p53, and HIF-1alpha, with a final target on VEGF expression and neuronal degeneration.

A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients.

PubMed

Vezzalini, Marzia; Mafficini, Andrea; Tomasello, Luisa; Lorenzetto, Erika; Moratti, Elisabetta; Fiorini, Zeno; Holyoake, Tessa L; Pellicano, Francesca; Krampera, Mauro; Tecchio, Cristina; Yassin, Mohamed; Al-Dewik, Nader; Ismail, Mohamed A; Al Sayab, Ali; Monne, Maria; Sorio, Claudio

2017-06-21

Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34 + /CD38 bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions.

U3 long terminal repeat-mediated induction of intracellular immunity by a murine retrovirus: a novel model of latency for retroviruses.

PubMed Central

Gorska-Flipot, I; Huang, M; Cantin, M; Rassart, E; Massé, G; Jolicoeur, P

1992-01-01

BL/VL3 radiation leukemia virus (RadLV) is a thymotropic, highly leukemogenic murine leukemia virus (MuLV) which is unable to replicate in vitro in mouse fibroblasts. We have previously reported that the U3 long terminal repeat region of its genome is responsible for this block (E. Rassart, Y. Paquette, and P. Jolicoeur, J. Virol. 62:3840-3848, 1988). By using hybrids of permissive and resistant cells infected with BL/VL3 RadLV or fibrotropic MuLV, we found that the resistant phenotype was dominant. Investigation to determine at which step of the virus cycle the block operates revealed that integration, transcription, and translation of the BL/VL3 viral genome occurred at normal levels in nonpermissive cells. The BL/VL3 RadLV Pr65gag proteins made in nonpermissive cells were also myristylated and located at the membrane, and the levels of their cleaved products were similar to those of fibrotropic MuLV. However, processing of BL/VL3 RadLV Pr85env was impaired in nonpermissive cells. Virions were not released into the culture medium of nonpermissive cells, as measured by reverse transcriptase activity and by content in p30 or gp70 protein and as documented by lower levels of budding particles seen by electron microscopy. These results indicate that BL/VL3 RadLV replication is blocked at a late stage of the virus cycle, i.e., at virion assembly. Interestingly, these BL/VL3 RadLV-infected nonpermissive fibroblasts were resistant to superinfection by fibrotropic Moloney MuLV, and this resistance also occurred at a late step of the Moloney virus cycle. Since this block is dominant, it appears that the U3 long terminal repeat region of the BL/VL3 viral genome has the ability to induce a cellular suppressor factor(s), thus bringing intracellular immunity against itself and against other ecotropic MuLVs. Images PMID:1433513

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Control of proliferating potential of myeloid leukemia cells during long-term treatment with vitamin D3 analogues and other differentiation inducers in combination with antileukemic drugs: in vitro and in vivo studies.

PubMed

Kasukabe, T; Honma, Y; Hozumi, M; Suda, T; Nishii, Y

1987-01-15

Growth inhibition of murine and human myeloid leukemia cells by differentiation inducers during long-term culture was examined to improve the strategy for therapy of myeloid leukemia by differentiation inducers. When the effect of 1 alpha,25-dihydroxyvitamin D3, a typical differentiation inducer, on proliferation of mouse myeloid leukemia M1 cells was examined at a constant product of time and concentration (480 nM in 20 days), the continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3 was the most effective for inhibition of cell proliferation. After 20 days, the cumulative cell number was reduced about 3 X 10(5) times by continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. Similar results were obtained when M1 cells were treated continuously with dexamethasone. M1 cells resistant to 1 alpha,25-dihydroxyvitamin D3 appeared about 25 days after the start of continuous treatment with 24 nM 1 alpha,25-dihydroxyvitamin D3. On the other hand, when M1 cells were treated continuously with 1 alpha,25-dihydroxyvitamin D3 and noncytotoxic doses of antileukemic drugs such as 1-beta-D-arabinofuranosylcytosine and daunomycin, resistant cells did not appear for at least 35 days. A similar effect of 1 alpha,25-dihydroxyvitamin D3 and antileukemic drugs on cell proliferation was observed with the human monoblast-like cell line U937. The survival of syngeneic SL mice inoculated with M1 cells was prolonged more by treatment with both 1 alpha-hydroxyvitamin D3 and daunomycin than by treatment with either drug alone. These results suggest that continuous treatment with both differentiation inducers and certain antileukemic drugs may be more effective therapeutically than treatment with a differentiation inducer alone.

Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia.

PubMed

Wang, Lan; Zhou, Guang-Biao; Liu, Ping; Song, Jun-Hong; Liang, Yang; Yan, Xiao-Jing; Xu, Fang; Wang, Bing-Shun; Mao, Jian-Hua; Shen, Zhi-Xiang; Chen, Sai-Juan; Chen, Zhu

2008-03-25

To enhance therapeutic efficacy and reduce adverse effects, practitioners of traditional Chinese medicine (TCM) prescribe a combination of plant species/minerals, called formulae, based on clinical experience. Nearly 100,000 formulae have been recorded, but the working mechanisms of most remain unknown. In trying to address the possible beneficial effects of formulae with current biomedical approaches, we use Realgar-Indigo naturalis formula (RIF), which has been proven to be very effective in treating human acute promyelocytic leukemia (APL) as a model. The main components of RIF are realgar, Indigo naturalis, and Salvia miltiorrhiza, with tetraarsenic tetrasulfide (A), indirubin (I), and tanshinone IIA (T) as major active ingredients, respectively. Here, we report that the ATI combination yields synergy in the treatment of a murine APL model in vivo and in the induction of APL cell differentiation in vitro. ATI causes intensified ubiquitination/degradation of promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) oncoprotein, stronger reprogramming of myeloid differentiation regulators, and enhanced G(1)/G(0) arrest in APL cells through hitting multiple targets compared with the effects of mono- or biagents. Furthermore, ATI intensifies the expression of Aquaglyceroporin 9 and facilitates the transportation of A into APL cells, which in turn enhances A-mediated PML-RARalpha degradation and therapeutic efficacy. Our data also indicate A as the principal component of the formula, whereas T and I serve as adjuvant ingredients. We therefore suggest that dissecting the mode of action of clinically effective formulae at the molecular, cellular, and organism levels may be a good strategy in exploring the value of traditional medicine.

Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2–dependent adult T-cell leukemia

PubMed Central

Zhang, Meili; Mathews Griner, Lesley A.; Ju, Wei; Duveau, Damien Y.; Guha, Rajarshi; Petrus, Michael N.; Wen, Bernard; Maeda, Michiyuki; Shinn, Paul; Ferrer, Marc; Conlon, Kevin D.; Bamford, Richard N.; O’Shea, John J.; Thomas, Craig J.; Waldmann, Thomas A.

2015-01-01

Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1–encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine-dependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients’ PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL. PMID:26396258

The mouse Pol I terminator is more efficient than the hepatitis delta virus ribozyme in generating influenza-virus-like RNAs with precise 3' ends in a plasmid-only-based virus rescue system.

PubMed

Feng, Liqiang; Li, Feng; Zheng, Xuehua; Pan, Weiqi; Zhou, Kai; Liu, Yichu; He, Hongxuan; Chen, Ling

2009-01-01

Reverse genetics systems for generating recombinant influenza viruses are based on two different mechanisms for obtaining the 3' end of the viral RNA: one uses the self-cleaving hepatitis delta virus ribozyme (HDVR), and the other uses the murine RNA polymerase I (Pol I) terminator. In this study, we employed EGFP and Renilla luciferase reporter constructs to compare the efficiency of both methods. Our results indicate that the murine Pol I terminator was more efficient than the HDVR, which will be helpful in choosing an influenza virus rescue system, as well as in establishing other RNA virus rescue systems.

The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model

PubMed Central

Mercher, Thomas; Raffel, Glen D.; Moore, Sandra A.; Cornejo, Melanie G.; Baudry-Bluteau, Dominique; Cagnard, Nicolas; Jesneck, Jonathan L.; Pikman, Yana; Cullen, Dana; Williams, Ifor R.; Akashi, Koichi; Shigematsu, Hirokazu; Bourquin, Jean-Pierre; Giovannini, Marco; Vainchenker, William; Levine, Ross L.; Lee, Benjamin H.; Bernard, Olivier A.; Gilliland, D. Gary

2009-01-01

Acute megakaryoblastic leukemia (AMKL) is a form of acute myeloid leukemia (AML) associated with a poor prognosis. The genetics and pathophysiology of AMKL are not well understood. We generated a knockin mouse model of the one twenty-two–megakaryocytic acute leukemia (OTT-MAL) fusion oncogene that results from the t(1;22)(p13;q13) translocation specifically associated with a subtype of pediatric AMKL. We report here that OTT-MAL expression deregulated transcriptional activity of the canonical Notch signaling pathway transcription factor recombination signal binding protein for immunoglobulin κ J region (RBPJ) and caused abnormal fetal megakaryopoiesis. Furthermore, cooperation between OTT-MAL and an activating mutation of the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPL) efficiently induced a short-latency AMKL that recapitulated all the features of human AMKL, including megakaryoblast hyperproliferation and maturation block, thrombocytopenia, organomegaly, and extensive fibrosis. Our results establish that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage and suggest that specific targeting of these pathways could be of therapeutic value for human AMKL. PMID:19287095

Characterization of a protein that binds multiple sequences in mammalian type C retrovirus enhancers.

PubMed Central

Sun, W; O'Connell, M; Speck, N A

1993-01-01

Mammalian type C retrovirus enhancer factor 1 (MCREF-1) is a nuclear protein that binds several directly repeated sequences (CNGGN6CNGG) in the Moloney and Friend murine leukemia virus (MLV) enhancers (N. R. Manley, M. O'Connell, W. Sun, N. A. Speck, and N. Hopkins, J. Virol. 67:1967-1975, 1993). In this paper, we describe the partial purification of MCREF-1 from calf thymus nuclei and further characterize the binding properties of MCREF-1. MCREF-1 binds four sites in the Moloney MLV enhancer and three sites in the Friend MLV enhancer. Ethylation interference analysis suggests that the MCREF-1 binding site spans two adjacent minor grooves of DNA. Images PMID:8445719

Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

PubMed Central

Sudeep, H.V.; Gouthamchandra, K.; Venkatesh, B. J.; Prasad, K. Shyam

2017-01-01

Background: Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. Objective: In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. Materials and Methods: The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. Results: The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 μg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 μg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. Conclusion: We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. SUMMARY The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 μg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance. PMID:29491636

Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells.

PubMed

Sudeep, H V; Gouthamchandra, K; Venkatesh, B J; Prasad, K Shyam

2018-01-01

Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 μg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 μg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 μg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance.

Growth and differentiation of a murine interleukin-3-producing myelomonocytic leukemia cell line in a protein-free chemically defined medium.

PubMed

Kajigaya, Y; Ikuta, K; Sasaki, H; Matsuyama, S

1990-10-01

We established the continuous growth of WEHI-3B D+ cells in protein-free chemically defined F-12 medium by stepwise decreases in the concentration of fetal calf serum. This cell line, designated as WEHI-3B-Y1, has now been propagated in protein-free F-12 medium for 3 years. The population-doubling time of the cells in culture is about 24 hr. WEHI-3B-Y1 cells are immature undifferentiated cells which show positive staining for naphthol ASD chloroacetate esterase and alpha-naphthyl butyrate esterase and spontaneously exhibit a low level of differentiation to mature granulocytes and macrophages. Medium conditioned by WEHI-3B-Y1 cells stimulated the proliferation of an interleukin-3 (IL-3)-dependent FDCP-2 cell line. This conditioned medium was shown to have erythroid burst-promoting activity when assayed using normal murine bone marrow. The colony formation of WEHI-3B-Y1 cells in semi-solid agar culture was not stimulated by purified recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, in the presence of human transferrin, rhG-CSF enhanced the number of colonies of WEHI-3B-Y1 cells but did not induce their differentiation. These results suggest that WEHI-3B-Y1 cells cultured in protein-free medium produced murine IL-3. In addition, human G-CSF enhanced the clonal growth but did not induce the differentiation of WEHI-3B-Y1 cells cultured in serum-free medium.

ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

PubMed Central

Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

2017-01-01

The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

Human and Murine Innate Immune Cell Populations Display Common and Distinct Response Patterns during Their In Vitro Interaction with the Pathogenic Mold Aspergillus fumigatus.

PubMed

Hellmann, Anna-Maria; Lother, Jasmin; Wurster, Sebastian; Lutz, Manfred B; Schmitt, Anna Lena; Morton, Charles Oliver; Eyrich, Matthias; Czakai, Kristin; Einsele, Hermann; Loeffler, Juergen

2017-01-01

Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus . However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus . For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus . Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host-pathogen interaction studies.

Human and Murine Innate Immune Cell Populations Display Common and Distinct Response Patterns during Their In Vitro Interaction with the Pathogenic Mold Aspergillus fumigatus

PubMed Central

Hellmann, Anna-Maria; Lother, Jasmin; Wurster, Sebastian; Lutz, Manfred B.; Schmitt, Anna Lena; Morton, Charles Oliver; Eyrich, Matthias; Czakai, Kristin; Einsele, Hermann; Loeffler, Juergen

2017-01-01

Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus. However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus. For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus. Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host–pathogen interaction studies. PMID:29270175

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

PubMed Central

Lim, Kwang-il; Klimczak, Ryan; Yu, Julie H.; Schaffer, David V.

2010-01-01

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS. PMID:20616052

Thrombopoietin/MPL signaling confers growth and survival capacity to CD41-positive cells in a mouse model of Evi1 leukemia.

PubMed

Nishikawa, Satoshi; Arai, Shunya; Masamoto, Yosuke; Kagoya, Yuki; Toya, Takashi; Watanabe-Okochi, Naoko; Kurokawa, Mineo

2014-12-04

Ecotropic viral integration site 1 (Evi1) is a transcription factor that is highly expressed in hematopoietic stem cells and is crucial for their self-renewal capacity. Aberrant expression of Evi1 is observed in 5% to 10% of de novo acute myeloid leukemia (AML) patients and predicts poor prognosis, reflecting multiple leukemogenic properties of Evi1. Here, we show that thrombopoietin (THPO) signaling is implicated in growth and survival of Evi1-expressing cells using a mouse model of Evi1 leukemia. We first identified that the expression of megakaryocytic surface molecules such as ITGA2B (CD41) and the THPO receptor, MPL, positively correlates with EVI1 expression in AML patients. In agreement with this finding, a subpopulation of bone marrow and spleen cells derived from Evi1 leukemia mice expressed both CD41 and Mpl. CD41(+) Evi1 leukemia cells induced secondary leukemia more efficiently than CD41(-) cells in a serial bone marrow transplantation assay. Importantly, the CD41(+) cells predominantly expressing Mpl effectively proliferated and survived on OP9 stromal cells in the presence of THPO via upregulating BCL-xL expression, suggesting an essential role of the THPO/MPL/BCL-xL cascade in enhancing the progression of Evi1 leukemia. These observations provide a novel aspect of the diverse functions of Evi1 in leukemogenesis. © 2014 by The American Society of Hematology.

Temporal Variation in the Association between Benzene and Leukemia Mortality

PubMed Central

Richardson, David B.

2008-01-01

Background Benzene is a human carcinogen. Exposure to benzene occurs in occupational and environmental settings. Objective I evaluated variation in benzene-related leukemia with age at exposure and time since exposure. Methods I evaluated data from a cohort of 1,845 rubber hydrochloride workers. Benzene exposure–leukemia mortality trends were estimated by applying proportional hazards regression methods. Temporal variation in the impact of benzene on leukemia rates was assessed via exposure time windows and fitting of a multistage cancer model. Results The association between leukemia mortality and benzene exposures was of greatest magnitude in the 10 years immediately after exposure [relative rate (RR) at 10 ppm-years = 1.19; 95% confidence interval (CI), 1.10–1.29]; the association was of smaller magnitude in the period 10 to < 20 years after exposure (RR at 10 ppm-years = 1.05; 95% CI, 0.97–1.13); and there was no evidence of association ≥ 20 years after exposure. Leukemia was more strongly associated with benzene exposures accrued at ≥ 45 years of age (RR at 10 ppm-years = 1.11; 95% CI, 1.04–1.17) than with exposures accrued at younger ages (RR at 10 ppm-years = 1.01; 95% CI, 0.92–1.09). Jointly, these temporal effects can be efficiently modeled as a multistage process in which benzene exposure affects the penultimate stage in disease induction. Conclusions Further attention should be given to evaluating the susceptibility of older workers to benzene-induced leukemia. PMID:18335105

Synergistic Antileukemic Activity of Carnosic Acid-Rich Rosemary Extract and the 19-nor Gemini Vitamin D Analogue in a Mouse Model of Systemic Acute Myeloid Leukemia

PubMed Central

Shabtay, Ayelet; Sharabani, Hagar; Barvish, Zeev; Kafka, Michael; Amichay, Doron; Levy, Joseph; Sharoni, Yoav; Uskokovic, Milan R.; Studzinski, George P.; Danilenko, Michael

2008-01-01

Objective Differentiation therapy with the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D3), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D3 induces hypercalcemia at pharmacologically active doses. We investigated the in vitro and in vivoantileukemic efficacy of combined treatment with non-toxic doses of a low-calcemic 1,25D3 analogue, 1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol (19-nor-Gemini; Ro27-5646), and rosemary plant agents in a mouse model of AML. Methods Proliferation and differentiation of WEHI-3B D– (WEHI) murine myelomonocytic leukemia cellsin vitro were determined by standard assays. Reactive oxygen species, glutathione and protein expression levels were measured by flow cytometry, enzymatic assay and Western blotting, respectively. Systemic AML was developed by intravenous injection of WEHI cells in syngeneic Balb/c mice. Results 19-nor-Gemini had a higher potency than its parent compounds, Gemini (Ro27-2310) and 1,25D3, in the induction of differentiation (EC50 = 0.059 ± 0.011, 0.275 ± 0.093 and 0.652 ± 0.085 nM, respectively) and growth arrest (IC50 = 0.072 ± 0.018, 0.165 ± 0.061 and 0.895 ± 0.144 nM, respectively) in WEHI cells in vitro, and lower in vivo toxicity. Combined treatment of leukemia-bearing mice with 19-nor-Gemini (injected intraperitoneally) and standardized rosemary extract (mixed with food) resulted in a synergistic increase in survival (from 42.2 ± 2.5 days in untreated mice to 66.5 ± 4.2 days, n = 3) and normalization of white blood cell and differential counts. This was consistent with strong cooperative antiproliferative and differentiation effects of low concentrations of 19-nor-Gemini or 1,25D3 combined with rosemary extract or its major polyphenolic component, carnosic acid, as well as with the antioxidant action of rosemary agents and vitamin D derivatives in WEHI cell cultures. Conclusion Combined effectiveness of 1,25D3 analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia. PMID:18852491

The homeobox gene CDX2 is aberrantly expressed in most cases of acute myeloid leukemia and promotes leukemogenesis

PubMed Central

Scholl, Claudia; Bansal, Dimple; Döhner, Konstanze; Eiwen, Karina; Huntly, Brian J.P.; Lee, Benjamin H.; Rücker, Frank G.; Schlenk, Richard F.; Bullinger, Lars; Döhner, Hartmut; Gilliland, D. Gary; Fröhling, Stefan

2007-01-01

The homeobox transcription factor CDX2 plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. We have found that CDX2 is expressed in leukemic cells of 90% of patients with acute myeloid leukemia (AML) but not in hematopoietic stem and progenitor cells derived from normal individuals. Stable knockdown of CDX2 expression by RNA interference inhibited the proliferation of various human AML cell lines and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity, were able to be continuously propagated in liquid culture, generated fully penetrant and transplantable AML in BM transplant recipients, and displayed dysregulated expression of Hox family members in vitro and in vivo. These results demonstrate that aberrant expression of the developmental regulatory gene CDX2 in the adult hematopoietic compartment is a frequent event in the pathogenesis of AML; suggest a role for CDX2 as part of a common effector pathway that promotes the proliferative capacity and self-renewal potential of myeloid progenitor cells; and support the hypothesis that CDX2 is responsible, in part, for the altered HOX gene expression that is observed in most cases of AML. PMID:17347684

Combined Targeting of BCL-2 and BCR-ABL Tyrosine Kinase Eradicates Chronic Myeloid Leukemia Stem Cells

PubMed Central

Mak, Po Yee; Mu, Hong; Zhou, Hongsheng; Mak, Duncan H.; Schober, Wendy; Leverson, Joel D.; Zhang, Bin; Bhatia, Ravi; Huang, Xuelin; Cortes, Jorge; Kantarjian, Hagop; Konopleva, Marina

2016-01-01

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin−Sca-1+cKit+ cells of inducible CML in mice as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin−Sca-1+cKit+ cell numbers and long-term stem cell frequency, and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34+CD38−, CD34+CD38+, and quiescent stem/progenitor CD34+ cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic phase and BC CML. PMID:27605552

AML1/ETO induces self-renewal in hematopoietic progenitor cells via the Groucho-related amino-terminal AES protein.

PubMed

Steffen, Björn; Knop, Markus; Bergholz, Ulla; Vakhrusheva, Olesya; Rode, Miriam; Köhler, Gabriele; Henrichs, Marcel-Philipp; Bulk, Etmar; Hehn, Sina; Stehling, Martin; Dugas, Martin; Bäumer, Nicole; Tschanter, Petra; Brandts, Christian; Koschmieder, Steffen; Berdel, Wolfgang E; Serve, Hubert; Stocking, Carol; Müller-Tidow, Carsten

2011-04-21

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.

The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation

PubMed Central

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P.; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S.

2013-01-01

The adaptor molecule signaling lymphocytic activation molecule–associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase–mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)–induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell–mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA–mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS. PMID:23430111

Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3.

PubMed

Yoneda-Kato, Noriko; Tomoda, Kiichiro; Umehara, Mari; Arata, Yukinobu; Kato, Jun-ya

2005-05-04

Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3-COP1 pathway.

The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

PubMed

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

2013-04-25

The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells.

PubMed

Babu, Geetha; Waterfield, Michael; Chang, Mikyoung; Wu, Xuefeng; Sun, Shao-Cong

2006-05-19

Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-kappaB1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-kappaB by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.

Endogenous oncogenic Nras mutation promotes aberrant GM-CSF signaling in granulocytic/monocytic precursors in a murine model of chronic myelomonocytic leukemia.

PubMed

Wang, Jinyong; Liu, Yangang; Li, Zeyang; Du, Juan; Ryu, Myung-Jeom; Taylor, Philip R; Fleming, Mark D; Young, Ken H; Pitot, Henry; Zhang, Jing

2010-12-23

Oncogenic NRAS mutations are frequently identified in myeloid diseases involving monocyte lineage. However, its role in the genesis of these diseases remains elusive. We report a mouse bone marrow transplantation model harboring an oncogenic G12D mutation in the Nras locus. Approximately 95% of recipient mice develop a myeloproliferative disease resembling the myeloproliferative variant of chronic myelomonocytic leukemia (CMML), with a prolonged latency and acquisition of multiple genetic alterations, including uniparental disomy of oncogenic Nras allele. Based on single-cell profiling of phospho-proteins, a novel population of CMML cells is identified to display aberrant granulocyte-macrophage colony stimulating factor (GM-CSF) signaling in both the extracellular signal-regulated kinase (ERK) 1/2 and signal transducer and activator of transcription 5 (Stat5) pathways. This abnormal signaling is acquired during CMML development. Further study suggests that aberrant Ras/ERK signaling leads to expansion of granulocytic/monocytic precursors, which are highly responsive to GM-CSF. Hyperactivation of Stat5 in CMML cells is mainly through expansion of these precursors rather than up-regulation of surface expression of GM-CSF receptors. Our results provide insights into the aberrant cytokine signaling in oncogenic NRAS-associated myeloid diseases.

Subcutaneous Rituximab for the Treatment of B-Cell Hematologic Malignancies: A Review of the Scientific Rationale and Clinical Development.

PubMed

Davies, Andrew; Berge, Claude; Boehnke, Axel; Dadabhoy, Anjum; Lugtenburg, Pieternella; Rule, Simon; Rummel, Mathias; McIntyre, Christine; Smith, Rodney; Badoux, Xavier

2017-10-01

Rituximab (MabThera ® /Rituxan ® ), a chimeric murine/human monoclonal antibody that binds specifically to the transmembrane antigen CD20, was the first therapeutic antibody to enter clinical practice for the treatment of cancer. As monotherapy and in combination with chemotherapy, rituximab has been shown to prolong progression-free survival and, in some indications overall survival, in patients with various B-cell malignancies, while having a well-established and manageable safety profile and a wide therapeutic window. As a result, rituximab is considered to have revolutionized treatment practices for patients with B-cell malignancies. A subcutaneous (SC) formulation of rituximab has been developed, comprising the same monoclonal antibody as the originally marketed formulation [rituximab concentrate for solution for intravenous (IV) infusion], and has undergone a detailed, sequential clinical development program. This program demonstrated that, at fixed doses, rituximab SC achieves non-inferior serum trough concentrations in patients with non-Hodgkin lymphoma and chronic lymphocytic leukemia, with comparable efficacy and safety relative to the IV formulation. The added benefit of rituximab SC was demonstrated in dedicated studies showing that rituximab SC allows for simplified and shortened drug preparation and administration times resulting in a reduced treatment burden for patients as well as improved resource utilization (efficiency) at the treatment facility. The improved efficiency of delivering rituximab's benefit to patients may broaden patient access to rituximab therapy in areas with low levels of healthcare resources, including IV-chair capacity constraints. This article is a companion paper to G. Salles, et al., which is also published in this issue. F. Hoffmann-La Roche Ltd.

Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication

PubMed Central

Dang, Que; Hu, Wei-Shau

2001-01-01

Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3′ R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer. PMID:11134294

Single-use technology for solvent/detergent virus inactivation of industrial plasma products.

PubMed

Hsieh, Yao-Ting; Mullin, Lori; Greenhalgh, Patricia; Cunningham, Michael; Goodrich, Elizabeth; Shea, Jessica; Youssef, Eric; Burnouf, Thierry

2016-06-01

Virus inactivation of plasma products is conducted using stainless-steel vessels. Single-use technology can offer significant benefits over stainless such as operational flexibility, reduced capital infrastructure costs, and increased efficiency by minimizing the time and validation requirements associated with hardware cleaning. This study qualifies a single-use bag system for solvent/detergent (S/D) virus inactivation. Human plasma and immunoglobulin test materials were S/D-treated in Mobius single-use bags using 1% tri-n-butyl phosphate (TnBP) with 1% Triton X-100 or 1% Tween 80 at 31°C for 4 to 6 hours to evaluate the impact on protein quality. Volatile and nonvolatile organic leachables from low-density polyethylene film (Pureflex film) used in 1-L-scale studies after exposure to S/D in phosphate-buffered saline were identified compared to controls in glass containers. Virus inactivation studies were performed with xenotropic murine leukemia virus (XMuLV) and bovine viral diarrhea virus (BVDV) to determine the kinetics of virus inactivation, measured using infectivity assays. S/D treatment in Mobius bags did not impact the protein content and profile of plasma and immunoglobulin, including proteolytic enzymes and thrombin generation. Cumulative leachable levels after exposure to S/D were 1.5 and 1.85 ppm when using 0.3% TnBP combined with 1% Tween 80 or 1% Triton X-100, respectively. Efficient inactivation of both XMuLV and BVDV was observed, with differences in the rate of inactivation dependent on both virus and S/D mixture. Effective S/D virus inactivation in single-use container technology is achievable. It does not alter plasma proteins and induces minimal release of leachables. © 2016 The Authors. Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Upregulation of the proto-oncogene Bmi-1 predicts a poor prognosis in pediatric acute lymphoblastic leukemia.

PubMed

Peng, Hong-Xia; Liu, Xiao-Dan; Luo, Zi-Yan; Zhang, Xiao-Hong; Luo, Xue-Qun; Chen, Xiao; Jiang, Hua; Xu, Ling

2017-01-25

Bmi-1, the B cell-specific moloney murine leukemia virus insertion site 1, is a member of the Polycomb-group (PcG) family and acts as an oncogene in various tumors; however, its expression related to the prognosis of pediatric patients with acute lymphoblastic leukemia (ALL) has not been well studied. The Bmi-1 expression levels in the bone marrow of 104 pediatric ALL patients and 18 normal control subjects were determined by using qRT-PCR. The association between the Bmi-1 expression and the clinicopathological characteristics of pediatric ALL patients was analyzed, and the correlation between Bmi-1 and the prognosis of pediatric ALL was calculated according to the Kaplan-Meier method. Furthermore, the association between Bmi-1 expression and its transcriptional regulator Sall4 was investigated. Compared to normal control subjects, patients with primary pediatric ALL exhibited upregulated levels of Bmi-1. However, these levels were sharply decreased in patients who achieved complete remission. A significant positive association between elevated Bmi-1 levels and a poor response to prednisone as well as an increased clinical risk was observed. Patients who overexpressed Bmi-1 at the time of diagnosis had a lower relapse-free survival (RFS) rate (75.8%), whereas patients with lower Bmi-1 expression had an RFS of 94.1%. Furthermore, in ALL patients, the mRNA expression of Bmi-1 was positively correlated to the mRNA expression of Sall4a. Taken together, these data suggest that Bmi-1 could serve as a novel prognostic biomarker in pediatric primary ALL and may be partially regulated by Sall4a. Our study also showed that Bmi-1 could serve as a new therapeutic target for the treatment of pediatric ALL.

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia-lymphoma cells.

PubMed

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-12-01

Adult T-cell leukemia-lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia

PubMed Central

Wang, Lan; Zhou, Guang-Biao; Liu, Ping; Song, Jun-Hong; Liang, Yang; Yan, Xiao-Jing; Xu, Fang; Wang, Bing-Shun; Mao, Jian-Hua; Shen, Zhi-Xiang; Chen, Sai-Juan; Chen, Zhu

2008-01-01

To enhance therapeutic efficacy and reduce adverse effects, practitioners of traditional Chinese medicine (TCM) prescribe a combination of plant species/minerals, called formulae, based on clinical experience. Nearly 100,000 formulae have been recorded, but the working mechanisms of most remain unknown. In trying to address the possible beneficial effects of formulae with current biomedical approaches, we use Realgar-Indigo naturalis formula (RIF), which has been proven to be very effective in treating human acute promyelocytic leukemia (APL) as a model. The main components of RIF are realgar, Indigo naturalis, and Salvia miltiorrhiza, with tetraarsenic tetrasulfide (A), indirubin (I), and tanshinone IIA (T) as major active ingredients, respectively. Here, we report that the ATI combination yields synergy in the treatment of a murine APL model in vivo and in the induction of APL cell differentiation in vitro. ATI causes intensified ubiquitination/degradation of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) oncoprotein, stronger reprogramming of myeloid differentiation regulators, and enhanced G1/G0 arrest in APL cells through hitting multiple targets compared with the effects of mono- or biagents. Furthermore, ATI intensifies the expression of Aquaglyceroporin 9 and facilitates the transportation of A into APL cells, which in turn enhances A-mediated PML-RARα degradation and therapeutic efficacy. Our data also indicate A as the principal component of the formula, whereas T and I serve as adjuvant ingredients. We therefore suggest that dissecting the mode of action of clinically effective formulae at the molecular, cellular, and organism levels may be a good strategy in exploring the value of traditional medicine. PMID:18344322

Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy

PubMed Central

Hollyman, Daniel; Stefanski, Jolanta; Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Taylor, Clare; Yeh, Raymond; Capacio, Vanessa; Olszewska, Malgorzata; Hosey, James; Sadelain, Michel; Brentjens, Renier J.; Rivière, Isabelle

2009-01-01

Summary Based on promising pre-clinical data demonstrating the eradication of systemic B cell malignancies by CD19-targeted T lymphocytes in vivo in SCID beige mouse models, we are launching Phase 1 clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We present here the validation of the bioprocess we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads® CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semi-closed culture system using the Wave bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in SCID beige mice bearing disseminated tumors. The validation requirements in terms of T cell expansion, T cell transduction with the 1928z CAR, biological activity, quality control testing and release criteria were met for all four validation runs using apheresis products from patients with CLL. Additionally, following expansion of the T cells, the diversity of the skewed Vβ T cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemo-refractory CLL and in patients with relapsed ALL. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any chimeric antigen receptor or T cell receptor. PMID:19238016

CCR7 guides migration of mesenchymal stem cell to secondary lymphoid organs: a novel approach to separate GvHD from GvL effect.

PubMed

Li, Hong; Jiang, YanMing; Jiang, XiaoXia; Guo, XiMin; Ning, HongMei; Li, YuHang; Liao, Li; Yao, HuiYu; Wang, XiaoYan; Liu, YuanLin; Zhang, Yi; Chen, Hu; Mao, Ning

2014-07-01

Inefficient homing of systemically infused mesenchymal stem cells (MSCs) limits the efficacy of existing MSC-based clinical graft-versus-host disease (GvHD) therapies. Secondary lymphoid organs (SLOs) are the major niches for generating immune responses or tolerance. MSCs home to a wide range of organs, but rarely to SLOs after intravenous infusion. Thus, we hypothesized that targeted migration of MSCs into SLOs may significantly improve their immunomodulatory effect. Here, chemokine receptor 7 (CCR7) gene, encoding a receptor that specifically guides migration of immune cells into SLOs, was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (MSCs/CCR7). We found that infusion of MSCs/CCR7 potently prolonged the survival of GvHD mouse model. The infused MSCs/CCR7 migrate to SLOs, relocate in proximity with T lymphocytes, therefore, potently inhibited their proliferation, activation, and cytotoxicity. Natural killer (NK) cells contribute to the early control of leukemia relapse. Although MSCs/CCR7 inhibited NK cell activity in vitro coculture, they did not impact on the proportion and cytotoxic capacities of NK cells in the peripheral blood of GvHD mice. In an EL4 leukemia cell loaded GvHD model, MSCs/CCR7 infusion preserved the graft-versus-leukemia (GvL) effect. In conclusion, this study demonstrates that CCR7 guides migration of MSCs to SLOs and thus highly intensify their in vivo immunomodulatory effect while preserving the GvL activity. This exciting therapeutic strategy may improve the clinical efficacy of MSC based therapy for immune diseases. © 2014 AlphaMed Press.

Synthesis of Nanodiamond-Daunorubicin Conjugates to Overcome Multidrug Chemoresistance in Leukemia

PubMed Central

Man, Han B.; Kim, Hansung; Kim, Ho-Joong; Robinson, Erik; Liu, Wing Kam; Chow, Edward Kai-Hua; Ho, Dean

2013-01-01

Nanodiamonds (NDs) are promising candidates in nanomedicine, demonstrating significant potential as gene/drug delivery platforms for cancer therapy. We have synthesized ND vectors capable of chemotherapeutic loading and delivery with applications towards chemoresistant leukemia. The loading of Daunorubicin (DNR) onto NDs was optimized by adjusting reaction parameters such as acidity and concentration. The resulting conjugate, a novel therapeutic payload for NDs, was characterized extensively for size, surface charge, and loading efficiency. A K562 human myelogenous leukemia cell line, with multidrug resistance conferred by incremental DNR exposure, was used to demonstrate the efficacy enhancement resulting from ND-based delivery. While resistant K562 cells were able to overcome treatment from DNR alone, as compared with non-resistant K562 cells, NDs were able to improve DNR delivery into resistant K562 cells. By overcoming efflux mechanisms present in this resistant leukemia line, ND-enabled therapeutics have demonstrated the potential to improve cancer treatment efficacy, especially towards resistant strains. PMID:23916889

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics.

PubMed

Tung, Yi-Chung; Torisawa, Yu-suke; Futai, Nobuyuki; Takayama, Shuichi

2007-11-01

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.

Multiplexing clonality: combining RGB marking and genetic barcoding

PubMed Central

Cornils, Kerstin; Thielecke, Lars; Hüser, Svenja; Forgber, Michael; Thomaschewski, Michael; Kleist, Nadja; Hussein, Kais; Riecken, Kristoffer; Volz, Tassilo; Gerdes, Sebastian; Glauche, Ingmar; Dahl, Andreas; Dandri, Maura; Roeder, Ingo; Fehse, Boris

2014-01-01

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies. PMID:24476916

Long-term maintenance combination chemotherapy with OPEC/MPEC (vincristine or methotrexate, prednisolone, etoposide and cyclophosphamide) or with daily oral etoposide and prednisolone can improve survival and quality of life in adult T-cell leukemia/lymphoma.

PubMed

Matsushita, K; Matsumoto, T; Ohtsubo, H; Fujiwara, H; Imamura, N; Hidaka, S; Kukita, T; Tei, C; Matsumoto, M; Arima, N

1999-12-01

Acute leukemia and lymphoma varieties of adult T-cell leukemia/lymphoma (ATL) usually carry a poor prognosis. While etoposide is generally useful for treating ATL, especially as a daily oral maintenance regimen, etoposide has not proven effective in severe types of ATL efficient in some patients. Of 87 ATL patients whom we have treated, 51 had acute leukemia, 22 lymphoma and 14 progressive chronic leukemia. Seventy-nine patients were treated with a long term maintenance combination protocol, OPEC/MPEC (weekly doses of vincristine, 0.7 mg/m2 or methotrexate, 14 mg/m2; prednisolone, 20 mg/m2; etoposide, 70 mg/m2 and cyclophosphamide, 200 mg/m2). The other 8 patients, 3 with acute leukemia, 2 with lymphoma and 3 with progressive chronic leukemia, were treated with daily oral administration of 25 mg of etoposide and 10 mg of prednisolone (DOEP). The dose administered was modified in individual cases to maintain the granulocyte count and reduce the number of ATL cells. Considering both protocols, a complete response and a partial response were achieved in 31.0% and 58.6% patients, respectively. Median survival times (MST) of all patients and, acute leukemia, lymphoma and progressive chronic leukemia types were 7.5, 6.7, 9.6 and 12.4 months, respectively. Respective MST of patients treated with OPEC/MPEC or DOEP protocols were 7.1 and 18.0 months. Relatively normal WBC counts, lower lactate dehydrogenase concentration and normal calcium concentration, limited numbers of anatomic sites involved, good performance status and good response to chemotherapy were significantly associated with long survival time. Drug toxicity was not apparent, and about half of patients were treated in an outpatient setting.

ΔNp73 overexpression promotes resistance to apoptosis but does not cooperate with PML/RARA in the induction of an APL-leukemic phenotype

PubMed Central

Lucena-Araujo, Antonio R.; Coelho-Silva, Juan L.; Pereira-Martins, Diego A.; Thomé, Carolina; Scheucher, Priscila S.; Lange, Ana P.; Paiva, Helder H.; Hemmelgarn, Benjamin T.; Morais-Sobral, Mariana C.; Azevedo, Elisa A.; Franca-Neto, Pedro L.; Franca, Rafael F.; Silva, Cleide L.; Krause, Alexandre; Rego, Eduardo M.

2017-01-01

Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis. PMID:28035072

EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

PubMed Central

Gu, Chao-Jiang; Kelschenbach, Jennifer; Kim, Boe-Hyun; Arancio, Ottavio; Suh, Jin; Polsky, Bruce; Edagwa, Benson; Gendelman, Howard E.

2018-01-01

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication. PMID:29879225

EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment.

PubMed

Gu, Chao-Jiang; Borjabad, Alejandra; Hadas, Eran; Kelschenbach, Jennifer; Kim, Boe-Hyun; Chao, Wei; Arancio, Ottavio; Suh, Jin; Polsky, Bruce; McMillan, JoEllyn; Edagwa, Benson; Gendelman, Howard E; Potash, Mary Jane; Volsky, David J

2018-06-01

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication.

Absence of suppressor of cytokine signalling 3 reduces self-renewal and promotes differentiation in murine embryonic stem cells.

PubMed

Forrai, Ariel; Boyle, Kristy; Hart, Adam H; Hartley, Lynne; Rakar, Steven; Willson, Tracy A; Simpson, Ken M; Roberts, Andrew W; Alexander, Warren S; Voss, Anne K; Robb, Lorraine

2006-03-01

Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-beta and gp130 and signals via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3-null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3-null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK-STAT and extracellular signal-related kinase 1/2 (ERK-1/2)-mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain-containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3-null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK-STAT and SHP-2-ERK-1/2-MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.

Modular MLV-VLPs co-displaying ovalbumin peptides and GM-CSF effectively induce expansion of CD11b+ APC and antigen-specific T cell responses in vitro.

PubMed

Gogesch, Patricia; Schülke, Stefan; Scheurer, Stephan; Mühlebach, Michael D; Waibler, Zoe

2018-05-28

The development of novel vaccination strategies is a persistent challenge to provide effective prophylactic treatments to encounter viral infections. In general, the physical conjugation of selected vaccine components, e.g. antigen and adjuvant, has been shown to enhance the immunogenicity and hence, can increase effectiveness of the vaccine. In our proof-of-concept study, we generated non-infectious, replication deficient Murine Leukemia Virus (MLV)-derived virus-like particles (VLPs) that physically link antigen and adjuvant in a modular fashion by co-displaying them on their surface. For this purpose, we selected the immunodominant peptides of the model antigen ovalbumin (OVA) and the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) as non-classical adjuvant. Our results show that murine GM-CSF displayed on MLV-VLPs mediates expansion and proliferation of CD11b + cells within murine bone marrow and total spleen cells. Moreover, we show increased immunogenicity of modular VLPs co-displaying OVA peptides and GM-CSF by their elevated capacity to induce OVA-specific T cell-activation and -proliferation within OT-I and OT-II splenocyte cultures. These enhanced effects were not achieved by using an equimolar mixture of VLPs displaying either OVA or GM-CSF. Taken together, OVA and GM-CSF co-displaying MLV-VLPs are able to target and expand antigen presenting cells which in turn results in enhanced antigen-specific T cell activation and proliferation in vitro. These data suggest MLV-VLPs to be an attractive platform to flexibly combine antigen and adjuvant for novel modular vaccination approaches. Copyright © 2018 Elsevier Ltd. All rights reserved.

A systems biology approach for elucidating the interaction of curcumin with Fanconi anemia FANC G protein and the key disease targets of leukemia.

PubMed

Mahato, David; Samanta, Dipayan; Mukhopadhyay, Sudit S; Krishnaraj, R Navanietha

2017-06-01

Fanconi anemia (FA) is an autosomal recessive disorder with a high risk of malignancies including acute myeloid leukemia and squamous cell carcinoma. There is a constant search out of new potential therapeutic molecule to combat this disorder. In most cases, patients with FA develop haematological malignancies with acute myeloid leukemia and acute lymphoblastic leukemia. Identifying drugs which can efficiently block the pathways of both these disorders can be an ideal and novel strategy to treat FA. The curcumin, a natural compound obtained from turmeric is an interesting therapeutic molecule as it has been reported in the literature to combat both FA as well as leukemia. However, its complete mechanism is not elucidated. Herein, a systems biology approach for elucidating the therapeutic potential of curcumin against FA and leukemia is investigated by analyzing the computational molecular interactions of curcumin ligand with FANC G of FA and seven other key disease targets of leukemia. The proteins namely DOT1L, farnesyl transferase (FDPS), histone decetylase (EP3000), Polo-like kinase (PLK-2), aurora-like kinase (AUKRB), tyrosine kinase (ABL1), and retinoic acid receptor alpha (RARA) were chosen as disease targets for leukemia and modeled structure of FANC G protein as the disease target for FA. The docking investigations showed that curcumin had a very high binding affinity of -8.1 kcal/mol with FANC G protein. The key disease targets of leukemia namely tyrosine kinase (ABL1), aurora-like kinase (AUKRB), and polo-like kinase (PLK-2) showed that they had the comparable binding affinities of -9.7 k cal/mol, -8.7 k cal/mol, and -8.6 k cal/mol, respectively with curcumin. Further, the percentage similarity scores obtained from PAM50 using EMBOSS MATCHER was shown to provide a clue to understand the structural relationships to an extent and to predict the binding affinity. This investigation shows that curcumin effectively interacts with the disease targets of both FA and leukemia.

GRB2 Interaction with the Ecotropic Murine Leukemia Virus Receptor, mCAT-1, Controls Virus Entry and Is Stimulated by Virus Binding

PubMed Central

Chen, Zeming; Kolokoltsov, Andrey A.; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A.

2012-01-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking. PMID:22090132

GRB2 interaction with the ecotropic murine leukemia virus receptor, mCAT-1, controls virus entry and is stimulated by virus binding.

PubMed

Chen, Zeming; Kolokoltsov, Andrey A; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A; Davey, Robert A

2012-02-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376

L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells.

PubMed

Dewannieux, Marie; Heidmann, Thierry

2005-06-03

SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

PubMed

Han, Zongchao; Zhong, Li; Maina, Njeri; Hu, Zhongbo; Li, Xiaomiao; Chouthai, Nitin S; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

2008-03-01

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain.

PubMed

Vanmierlo, Tim; Weingärtner, Oliver; van der Pol, Susanne; Husche, Constanze; Kerksiek, Anja; Friedrichs, Silvia; Sijbrands, Eric; Steinbusch, Harry; Grimm, Marcus; Hartmann, Tobias; Laufs, Ulrich; Böhm, Michael; de Vries, Helga E; Mulder, Monique; Lütjohann, Dieter

2012-04-01

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.

An Fc-engineered CD19 antibody eradicates MRD in patient-derived MLL-rearranged acute lymphoblastic leukemia xenografts.

PubMed

Schewe, Denis M; Alsadeq, Ameera; Sattler, Cornelia; Lenk, Lennart; Vogiatzi, Fotini; Cario, Gunnar; Vieth, Simon; Valerius, Thomas; Rosskopf, Sophia; Meyersieck, Fabian; Alten, Julia; Schrappe, Martin; Gramatzki, Martin; Peipp, Matthias; Kellner, Christian

2017-09-28

Antibody therapy constitutes a major advance in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To evaluate the efficacy and the mechanisms of action of CD19 monoclonal antibody therapy in pediatric BCP-ALL, we tested an Fc-engineered CD19 antibody carrying the S239D/I332E mutation for improved effector cell recruitment (CD19-DE). Patient-derived xenografts (PDX) of pediatric mixed-lineage leukemia gene ( MLL )-rearranged ALL were established in NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. Antibody CD19-DE was efficient in prolonging the survival of NSG mice in a minimal residual disease (MRD) model. The majority of surviving mice remained polymerase chain reaction (PCR)-MRD negative after treatment. When antibody therapy was initiated in overt leukemia, antibody CD19-DE was still efficient in prolonging survival of xenografted mice in comparison with nontreated control animals, but the effects were less pronounced than in the MRD setting. Importantly, the combination of antibody CD19-DE and cytoreduction by chemotherapy (dexamethasone, vincristine, PEG-asparaginase) resulted in significantly improved survival rates in xenografted mice. Antibody CD19-DE treatment was also efficient in a randomized phase 2-like PDX trial using 13 MLL -rearranged BCP-ALL samples. Macrophage depletion by liposomal clodronate resulted in a reversal of the beneficial effects of CD19-DE, suggesting an important role for macrophages as effector cells. In support of this finding, CD19-DE was found to enhance phagocytosis of patient-derived ALL blasts by human macrophages in vitro. Thus, Fc-engineered CD19 antibodies may represent a promising treatment option for infants and children with MLL -rearranged BCP-ALL who have a poor outcome when treated with chemotherapy only. © 2017 by The American Society of Hematology.

Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights.

PubMed

Harrison, Nicholas R; Laroche, Fabrice J F; Gutierrez, Alejandro; Feng, Hui

2016-01-01

Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients.

Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights

PubMed Central

Harrison, Nicholas R.; Laroche, Fabrice J.F.; Gutierrez, Alejandro

2016-01-01

Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients. PMID:27165361

Viral/Nonviral Chimeric Nanoparticles to Synergistically Suppress Leukemia Proliferation via Simultaneous Gene Transduction and Silencing

PubMed Central

Hong, Cheol Am; Cho, Soo Kyung; Edson, Julius A.; Kim, Jane; Ingato, Dominique; Pham, Bryan; Chuang, Anthony; Fruman, David; Kwon, Young Jik

2017-01-01

Single modal cancer therapy that targets one pathological pathway often turns out to be inefficient. For example, relapse of Chronic Myelogenous Leukemia (CML) after inhibiting BCR-ABL fusion protein using tyrosine kinase inhibitors (TKI) (e.g., Imatinib) is of significant clinical concern. This study developed a dual modal gene therapy that simultaneously tackles two key BCR-ABL-linked pathways using viral/nonviral chimeric nanoparticles (ChNPs). Consisting of an adeno-associated virus (AAV) core and an acid-degradable polymeric shell, the ChNPs were designed to simultaneously induce pro-apoptotic BIM expression by the AAV core and silence pro-survival MCL-1 by the small interfering RNA (siRNA) encapsulated in the shell. The resulting BIM/MCL-1 ChNPs were able to efficiently suppress the proliferation of BCR-ABL+ K562 and FL5.12/p190 cells in vitro and in vivo via simultaneously expressing BIM and silencing MCL-1. Interestingly, the synergistic anti-leukemic effects generated by BIM/MCL-1 ChNPs were specific to BCR-ABL+ cells and independent of a proliferative cytokine, IL-3. The AAV core of ChNPs was efficiently shielded from inactivation by anti-AAV serum and avoided the generation of anti-AAV serum, without acute toxicity. This study demonstrates the development of a synergistically efficient, specific, and safe therapy for leukemia using gene carriers that simultaneously manipulate multiple and inter-linked pathological pathways. PMID:27472284

Inhibition of West Nile Virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells.

PubMed

Yang, Yongbo; Wu, Chengxiang; Wu, Jianguo; Nerurkar, Vivek R; Yanagihara, Richard; Lu, Yuanan

2008-05-01

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.

Rates of spontaneous mutation among RNA viruses.

PubMed Central

Drake, J W

1993-01-01

Simple methods are presented to estimate rates of spontaneous mutation from mutant frequencies and population parameters in RNA viruses. Published mutant frequencies yield a wide range of mutation rates per genome per replication, mainly because mutational targets have usually been small and, thus, poor samples of the mutability of the average base. Nevertheless, there is a clear central tendency for lytic RNA viruses (bacteriophage Q beta, poliomyelitis, vesicular stomatitis, and influenza A) to display rates of spontaneous mutation of approximately 1 per genome per replication. This rate is some 300-fold higher than previously reported for DNA-based microbes. Lytic RNA viruses thus mutate at a rate close to the maximum value compatible with viability. Retroviruses (spleen necrosis, murine leukemia, Rous sarcoma), however, mutate at an average rate about an order of magnitude lower than lytic RNA viruses. PMID:8387212

XMRV Discovery and Prostate Cancer-Related Research.

PubMed

Kang, David E; Lee, Michael C; Das Gupta, Jaydip; Klein, Eric A; Silverman, Robert H

2011-01-01

Xenotropic murine leukemia virus-related virus (XMRV) was first reported in 2006 in a study of human prostate cancer patients with genetic variants of the antiviral enzyme, RNase L. Subsequent investigations in North America, Europe, Asia, and Africa have either observed or failed to detect XMRV in patients (prostate cancer, chronic fatigue syndrome-myalgic encephalomyelitis (CFS-ME), and immunosuppressed with respiratory tract infections) or normal, healthy, control individuals. The principal confounding factors are the near ubiquitous presence of mouse-derived reagents, antibodies and cells, and often XMRV itself, in laboratories. XMRV infects and replicates well in many human cell lines, but especially in certain prostate cancer cell lines. XMRV also traffics to prostate in a nonhuman primate model of infection. Here, we will review the discovery of XMRV and then focus on prostate cancer-related research involving this intriguing virus.

Negative regulation of retrovirus expression in embryonal carcinoma cells mediated by an intragenic domain.

PubMed

Loh, T P; Sievert, L L; Scott, R W

1988-11-01

An intragenic region spanning the tRNA primer binding site of a Moloney murine leukemia virus recombinant retrovirus was found to restrict expression specifically in embryonal carcinoma (EC) cells. When the inhibitory domain was present, the levels of steady-state RNA synthesized from integrated recombinant templates in stable cotransformation assays were reduced 20-fold in EC cells but not in C2 myoblast cells. Transient-cotransfection assays showed that repression of a template containing the EC-specific inhibitory component was relieved by an excess of specific competitor DNA. In addition, repression mediated by the inhibitory component was orientation independent. This evidence demonstrates the presence of a saturable, trans-acting negative regulatory factor(s) in EC cells and suggests that the interaction of the factor(s) with the intragenic inhibitory component occurs at the DNA level.

Reduced Differentiation Efficiency of Murine Embryonic Stem Cells in Stirred Suspension Bioreactors

PubMed Central

Taiani, Jaymi T.; Krawetz, Roman J.; zur Nieden, Nicole I.; Wu, Yiru Elizabeth; Kallos, Michael S.; Matyas, John R.

2010-01-01

The use of embryonic stem cells (ESCs) for regenerative medicine has generated increased attention due to the favorable attributes of these cells; namely, they are pluripotent and possess long-term self-renewal capacity. The initial aims of the present study were: (i) to use stirred suspension bioreactors to expand and differentiate ESCs into osteogenic and chondrogenic cell types and (ii) to explore if these ESC-derived cells influenced skeletal healing in an in vivo fracture model. We show that differentiation protocols used in static culture are insufficient when applied directly to suspension culture bioreactors. Moreover, when bioreactor-differentiated cells are transplanted into a burr-hole defect in bone, severe disruption of the bone architecture was noted at the fracture site, as determined by microcomputed tomography (microCT) imaging and histopathology. Further characterization of the bioreactor-differentiated cultures revealed that a subpopulation of cells in the resulting aggregates expressed the pluripotency marker Oct-4 in the nucleus. Nuclear Oct-4 expression persisted even after 30 days of culture in the absence of leukemia inhibitory factor (LIF). Remarkably, and unlike ESCs differentiated into skeletal cell types in static cultures, bioreactor-differentiated aggregates implanted subcutaneously into SCID mice formed teratomas. The development of effective ESC differentiation protocols for suspension bioreactors will require a more complete understanding of the environmental conditions within these culture systems and the influence that these conditions have on the regulation of pluripotency and differentiation in ESCs. PMID:19775198

Leukemia inhibitory factor: a novel bone-active cytokine.

PubMed

Reid, L R; Lowe, C; Cornish, J; Skinner, S J; Hilton, D J; Willson, T A; Gearing, D P; Martin, T J

1990-03-01

A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.

Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

PubMed Central

Chong, Seon-Ha; Kim, Kyunhoo; Choi, Dong Kyu; Thi Vu, Thu Trang; Nguyen, Minh Tan; Jeong, Boram; Ryu, Han-Bong; Kim, Injune; Jang, Yeon Jin; Robinson, Robert Charles; Choe, Han

2013-01-01

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research. PMID:24358310

The RNA-binding protein tristetraprolin schedules apoptosis of pathogen-engaged neutrophils during bacterial infection

PubMed Central

Ebner, Florian; Ivin, Masa; Kratochvill, Franz; Gratz, Nina; Villunger, Andreas; Sixt, Michael

2017-01-01

Protective responses against pathogens require a rapid mobilization of resting neutrophils and the timely removal of activated ones. Neutrophils are exceptionally short-lived leukocytes, yet it remains unclear whether the lifespan of pathogen-engaged neutrophils is regulated differently from that in the circulating steady-state pool. Here, we have found that under homeostatic conditions, the mRNA-destabilizing protein tristetraprolin (TTP) regulates apoptosis and the numbers of activated infiltrating murine neutrophils but not neutrophil cellularity. Activated TTP-deficient neutrophils exhibited decreased apoptosis and enhanced accumulation at the infection site. In the context of myeloid-specific deletion of Ttp, the potentiation of neutrophil deployment protected mice against lethal soft tissue infection with Streptococcus pyogenes and prevented bacterial dissemination. Neutrophil transcriptome analysis revealed that decreased apoptosis of TTP-deficient neutrophils was specifically associated with elevated expression of myeloid cell leukemia 1 (Mcl1) but not other antiapoptotic B cell leukemia/lymphoma 2 (Bcl2) family members. Higher Mcl1 expression resulted from stabilization of Mcl1 mRNA in the absence of TTP. The low apoptosis rate of infiltrating TTP-deficient neutrophils was comparable to that of transgenic Mcl1-overexpressing neutrophils. Our study demonstrates that posttranscriptional gene regulation by TTP schedules the termination of the antimicrobial engagement of neutrophils. The balancing role of TTP comes at the cost of an increased risk of bacterial infections. PMID:28504646

Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3

PubMed Central

Yoneda-Kato, Noriko; Tomoda, Kiichiro; Umehara, Mari; Arata, Yukinobu; Kato, Jun-ya

2005-01-01

Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3–COP1 pathway. PMID:15861129

Cytotoxic chalcones from some Indonesian Cryptocarya

NASA Astrophysics Data System (ADS)

Kurniadewi, F.; Syah, Y. M.; Juliawaty, L. D.; Hakim, E. H.; Koyama, K.; Kinoshita, K.

2017-07-01

Malignant tumors are one of the main causes of death in the world. Until now the search for cytotoxic (antitumor) compounds from nature, particularly from plants, is being a continuation activities. One group of plants that produce potential cytotoxic compounds is the Cryptocarya, one of the large genera of the Lauraceae family. As a part of our chemical and cytotoxic evaluation of the Cryptocarya species, we examined three species of Indonesian Cryptocarya. The sample of the wood of C. konishii hayata was collected from Cibodas Botanical Garden, West Java while the stem bark of C. phoebeopsis and C. cagayanensis were obtained from Sorong, Papua. Our investigation of flavonoid constituents on these species afforded three chalcone compounds i.e. desmethylinfectocaryone (1), infectocaryone (2) and cryptocaryone (3). The molecular structures of the isolated compounds were determined based on spectroscopic data, including UV, IR, 1D and 2D NMR. Cytotoxic effects of the compounds were evaluated using MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. Compound 1, 2 and 3 displayed strong cytotoxic properties (IC50 < 2 μg/mL) against Murine Leukemia P388 and HL 60 (blood premyelocytic leukemia) cells whereas 2 and 3 exhibited strong cytotoxicity properties against HCT116 (colon cancer). Cryptocaryone (3) also showed moderate cytotoxic properties (IC50 < 10 μg/mL) towards A549 (human lung adenocarcinoma epithelial) cells.

Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells.

PubMed

Wang, Zhujun; Chen, Yuanyuan; Li, Sisi; Cheng, Yuping; Zhao, Haizhao; Jia, Ming; Luo, Zebin; Tang, Yongmin

2014-02-01

CD45RA has been found highly expressed on leukemia cells and may be a potential target of the disease. In this study, an anti-CD45RA single-chain antibody fragment (scFv3A4) was genetically linked to the N terminus of the enhanced green fluorescent protein (EGFP) to generate a scFv3A4-EGFP fusion protein. The scFv3A4-EGFP with a molecular weight of 57kDa was stably expressed and secreted from the transfected CHO cells through the ER/Golgi-dependent pathway. The fusion protein was soluble in the culture supernatant and the yield was 1350μg/L. Flow cytometry analysis showed that the scFv3A4-EGFP had the same binding site and a very similar reactivity pattern with its parental murine monoclonal antibody (mAb) 3A4. Furthermore, comparing to conventional labeled 3A4-FITC antibody, the scFv3A4-EGFP was more resistant to illumination and more suitable for immunofluorescence histology (IFH) detection. Therefore, the scFv3A4-EGFP fusion protein can be a powerful tool to investigate the targeting of CD45RA on leukemia cells, biological activity of the target and possibly for the genetic manipulation of the antibody. Copyright © 2013 Elsevier Inc. All rights reserved.

Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

PubMed

Vercauteren, Koen; Hoffman, Brad E; Zolotukhin, Irene; Keeler, Geoffrey D; Xiao, Jing W; Basner-Tschakarjan, Etiena; High, Katherine A; Ertl, Hildegund Cj; Rice, Charles M; Srivastava, Arun; de Jong, Ype P; Herzog, Roland W

2016-06-01

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo.

PubMed

Dani, Sergio U; Espindola, Rachel

2002-06-30

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer.

Prevention of EBV lymphoma development by oncolytic myxoma virus in a murine xenograft model of post-transplant lymphoproliferative disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Manbok, E-mail: manbok66@dankook.ac.kr; Rahman, Masmudur M.; Cogle, Christopher R.

Epstein–Barr virus (EBV) has been associated with a variety of epithelial and hematologic malignancies, including B-, T- and NK cell-lymphomas, Hodgkin's disease (HD), post-transplant lymphoproliferative diseases (LPDs), nasopharyngeal and gastric carcinomas, smooth muscle tumors, and HIV-associated lymphomas. Currently, treatment options for EBV-associated malignancies are limited. We have previously shown that myxoma virus specifically targets various human solid tumors and leukemia cells in a variety of animal models, while sparing normal human or murine tissues. Since transplant recipients of bone marrow or solid organs often develop EBV-associated post-transplant LPDs and lymphoma, myxoma virus may be of utility to prevent EBV-associated malignanciesmore » in immunocompromised transplant patients where treatment options are frequently limited. In this report, we demonstrate the safety and efficacy of myxoma virus purging as a prophylactic strategy for preventing post-transplant EBV-transformed human lymphomas, using a highly immunosuppressed mouse xenotransplantation model. This provides support for developing myxoma virus as a potential oncolytic therapy for preventing EBV-associated LPDs following transplantation of bone marrow or solid organ allografts. - Highlights: • Myxoma virus effectively infects and purges EBV lymphoma cells in vivo. • Oncolytic myxoma virus effectively eradicates oncogenic EBV tumorigenesis. • Ex vivo pre-treatment of myxoma virus can be effective as a preventive treatment modality for post-transplant lymphoproliferative diseases.« less

Growth-inhibitory effects of the red alga Gelidium amansii on cultured cells.

PubMed

Chen, Yue-Hwa; Tu, Ching-Jung; Wu, Hsiao-Ting

2004-02-01

The objective of this study was to investigate the effects of Gelidium amansii, an edible red agar cultivated off the northeast coast of Taiwan, on the growth of two lines of cancer cells, murine hepatoma (Hepa-1) and human leukemia (HL-60) cells, as well as a normal cell line, murine embryo fibroblast cells (NIH-3T3). The potential role of G. amansii on the induction of apoptosis was also examined. The results indicated that all extracts from G. amansii, including phosphate-buffered saline (PBS) and methanol extracts from dried algae as well as the dimethyl sulfoxide (DMSO) extract from freeze-dried G. amansii agar, inhibited the growth of Hepa-1 and NIH-3T3 cells, but not the growth of HL-60 cells. Annexin V-positive cells were observed in methanol and DMSO extract-treated, but not PBS extract-treated Hepa-1 and NIH-3T3 cells, suggesting that the lipid-soluble extracts of G. amansii induced apoptosis. In summary, extracts of G. amansii from various preparations exhibited antiproliferative effects on Hepa-1 and NIH-3T3 cells, and apoptosis may play a role in the methanol and DMSO extract-induced inhibitory effects. However, the antiproliferative effects of PBS extracts was not through apoptosis. Moreover, the growth-inhibitory effects of G. amansii were not specific to cancer cells.

The pan-B cell marker CD22 is expressed on gastrointestinal eosinophils and negatively regulates tissue eosinophilia¶

PubMed Central

Wen, Ting; Mingler, Melissa K.; Blanchard, Carine; Wahl, Benjamin; Pabst, Oliver; Rothenberg, Marc E.

2011-01-01

CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 monoclonal antibody having been used against B cell leukemia. Herein, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. In order to confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 days after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild-type control mice, while the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the “B cell-specific” inhibitory molecule CD22 on GI eosinophils. PMID:22190185

The pan-B cell marker CD22 is expressed on gastrointestinal eosinophils and negatively regulates tissue eosinophilia.

PubMed

Wen, Ting; Mingler, Melissa K; Blanchard, Carine; Wahl, Benjamin; Pabst, Oliver; Rothenberg, Marc E

2012-02-01

CD22 is currently recognized as a B cell-specific Siglec and has been exploited therapeutically with humanized anti-CD22 mAb having been used against B cell leukemia. In this study, tissue-specific eosinophil mRNA microarray analysis identified that CD22 transcript levels of murine gastrointestinal (GI) eosinophils are 10-fold higher than those of lung eosinophils. To confirm the mRNA data at the protein level, we developed a FACS-based protocol designed to phenotype live GI eosinophils isolated from the murine lamina propria. Indeed, we found that jejunum eosinophils expressed remarkably high levels of surface CD22, similar to levels found in B cells across multiple mouse strains. In contrast, CD22 was undetectable on eosinophils from the colon, blood, thymus, spleen, uterus, peritoneal cavity, and allergen-challenged lung. Eosinophils isolated from newborn mice did not express CD22 but subsequently upregulated CD22 expression to adult levels within the first 10 d after birth. The GI lamina propria from CD22 gene-targeted mice harbored more eosinophils than wild type control mice, whereas the GI eosinophil turnover rate was unaltered in the absence of CD22. Our findings identify a novel expression pattern and tissue eosinophilia-regulating function for the "B cell-specific" inhibitory molecule CD22 on GI eosinophils.

Models for mature T-cell lymphomas--a critical appraisal of experimental systems and their contribution to current T-cell tumorigenic concepts.

PubMed

Warner, Kathrin; Crispatzu, Giuliano; Al-Ghaili, Nabil; Weit, Nicole; Florou, Vaia; You, M James; Newrzela, Sebastian; Herling, Marco

2013-12-01

Mature T-cell lymphomas/leukemias (MTCL) have been understudied lymphoid neoplasms that currently receive growing attention. Our historically rudimentary molecular understanding and dissatisfactory interventional success in this complex and for the most part poor-prognostic group of tumors is only slightly improving. A major limiting aspect in further progress in these rare neoplasms is the lack of suitable model systems that would substantially facilitate pathogenic studies and pre-clinical drug evaluations. Such representations of MTCL have thus far not been systematically appraised. We therefore provide an overview on existing models and point out their particular advantages and limitations in the context of the specific scientific questions. After addressing issues of species-specific differences and classifications, we summarize data on MTCL cell lines of human as well as murine origin, on murine strain predispositions to MTCL, on available models of genetically engineered mice, and on transplant systems. From an in-silico meta-analysis of available primary data of gene expression profiles on human MTCL we cross-reference genes reported to transform T-cells in mice and reflect on their general vs entity-restricted relevance and on target-promoter influences. Overall, we identify the urgent need for new models of higher fidelity to human MTCL with respect to their increasingly recognized diversity and to predictions of drug response. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Essential oils from Schinus terebinthifolius leaves - chemical composition and in vitro cytotoxicity evaluation.

PubMed

Santana, Jeferson S; Sartorelli, Patricia; Guadagnin, Rafael C; Matsuo, Alisson L; Figueiredo, Carlos R; Soares, Marisi G; da Silva, Adalberto M; Lago, João Henrique G

2012-10-01

In folk medicine, Schinus terebinthifolius Raddi (Anacardiaceae), has been used as a remedy for ulcers, respiratory problems, wounds, rheumatism, gout, diarrhea, skin ailments and arthritis, as well as to treat tumors and leprosy. To investigate the chemical composition and cytotoxicity of essential oil from leaves of S. terebinthifolius as well as the identification of active compounds from this oil. Essential oil from S. terebinthifolius leaves, obtained by hydrodistillation using a Clevenger-type apparatus, was characterized in terms of its chemical composition. Also, the crude oil was subjected to chromatographic separation procedures to afford an active fraction composed of α- and β-pinenes. These compounds, including hydrogenation (pinane) and epoxydation (α-pinene oxide) derivatives from α-pinene, were tested in vitro against murine melanoma cell line (B16F10-Nex2) and human melanoma (A2058), breast adenocarcinoma (MCF7), leukemia (human leukemia (HL-60) and cervical carcinoma (HeLa) cell lines. Forty-nine constituents were identified in the oil (97.9% of the total), with germacrene D (23.7%), bicyclogermacrene (15.0%), β-pinene (9.1%) and β-longipinene (8.1%) as the main compounds. The crude essential oil showed cytotoxic effects in several cell lines, mainly on leukemia and human cervical carcinoma. Fractions composed mainly of α- and β-pinenes as well as those composed of individually pinenes showed effective activities against all tested cell lines. Aiming to determinate preliminary structure/activity relationships, α-pinene was subjected to epoxydation and hydrogenation procedures whose obtained α-pinene oxide showed an expressive depression in its cytotoxicity effect, similar as observed to pinane derivative. The obtained results indicated that the monoterpenes α- and β-pinenes could be responsible to the cytotoxic activity detected in the crude oil from leaves of S. terebinthifolius. In addition, it was possibly inferred that the presence of double bond in their structures, mainly at endocyclic position, is crucial to cytotoxic potential detected in these derivatives.

Murine cell glycolipids customization by modular expression of glycosyltransferases.

PubMed

Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

2013-01-01

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

PubMed

Shi, Junwei; Wang, Eric; Milazzo, Joseph P; Wang, Zihua; Kinney, Justin B; Vakoc, Christopher R

2015-06-01

CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We also show that the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

ENDOGENOUS RETROVIRUSES MOBILIZED DURING FRIEND MURINE LEUKEMIA VIRUS INFECTION

PubMed Central

Hansen, Ethan; Hendrick, Duncan; Malik, Frank; Evans, Leonard H.

2016-01-01

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses. PMID:27657834

Structural and biochemical characterization of the inhibitor complexes of XMRV protease

PubMed Central

Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander

2012-01-01

Summary Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus (XMRV) and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of Ki, depending on exact experimental conditions. TL-3, a universal inhibitor of retroviral proteases, as well as some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of XMRV PR with TL-3, amprenavir, and pepstatin A were solved at high resolution and compared to the structures of these inhibitors complexed with other retropepsins. Whereas TL-3 and amprenavir bind in a predictable manner spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bind simultaneously in an unprecedented manner, leaving the catalytic water molecule in place. PMID:21951660

Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors

PubMed Central

Mikkelsen, Jacob Giehm; Lund, Anders H.; Dybkær, Karen; Duch, Mogens; Pedersen, Finn Skou

1998-01-01

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed. PMID:9499117

Platinum Nanoparticles Induce Apoptosis on Raw 264.7 Macrophage Cells.

PubMed

Loan, Ta Thi; Do, Le Thanh; Yoo, Hoon

2018-02-01

The cellular effects of platinum nanoparticles (PNP05, average size of 5 nm, and PNP30, average size of 30 nm) were investigated on murine leukemia Raw 264.7 cells. Cells treated with various concentrations of PNPs showed size-dependent cytotoxicity in an MTT assay with PNP5 of smaller nanoparticles higher toxicity than PNP30. Investigations on cell morphology, Annexin V assay, DNA fragmentation and the activity of caspase-3/-7 showed that PNPs induced apoptosis on Raw 264.7 cells by changing cell morphology and density, increasing cell population in apoptosis and causing nucleus fragmentation. Further study on caspase activity by Western blotting revealed that the apoptosis was induced by the activation of caspase-3 and -7. In addition, PNPs inactivated DNA repair system, generating dose-dependent DNA ladder bands on agarose gel electrophoresis. Taken together, PNPs triggered cytotoxicity on Raw 264.7 cells by suppressing cell growth/survival and inducing apoptosis.

In vitro and in vivo effects of CpG-Oligodeoxynucleotides (CpG-ODN) on murine transitional cell carcinoma and on the native murine urinary bladder wall.

PubMed

Olbert, Peter Jochen; Schrader, Andres Jan; Simon, Corinna; Dalpke, Alexander; Barth, Peter; Hofmann, Rainer; Hegele, Axel

2009-06-01

Intravesical BCG instillation is established and efficient in the prophylaxis of recurrent transitional cell carcinoma. A Th-1 biased immune response is postulated. Recent work has proven the efficacy of synthetic CpG-Oligodeoxynucleotides (ODN) as inducers and adjuvants for a strong Th1-response and there is evidence for a direct and/or adjuvant anti-neoplastic effect. The purpose of this study was to examine the local effects of CpG-ODN on the murine bladder wall after intravesical instillation and the effects on cytokine expression in an orthotopic murine bladder cancer model. Histopathology, immunohistochemistry and fluorescence microscopy were performed after different instillation schedules of stimulatory, non-stimulatory biotinylized and FITC-labelled CpG-ODN into the murine bladder. MB-49 murine bladder cancer cells were tested for TLR-9 expression to exclude a potential direct responsiveness to CpG-ODN. Furthermore induction of apoptosis was tested by annexin V staining and FACS analysis of CpG-ODN stimulated tumor cells. In an orthotopic C57/Bl6 murine bladder cancer model, the expressions of IL-12, IFNgamma, IL-10 and TGF-beta were evaluated after repeated CpG-ODN treatment. Single and repeated instillation of CpG-ODN induced subepithelial and urothelial lymphocytic infiltrations with consecutive apoptoses. PBS and non-stimulative ODN induced no visible reaction. Bladder submucosa stained positive for biotin. Controls showed no endogenic biotin staining. FITC-labelled ODN adhered to the bladder mucosa and penetration of the mucosal barrier was not detected. MB-49 TCC cells did not express TLR-9 and CpG-ODN did not induce apoptosis in these cells. Repeated intravesical instillations of CpG-ODN in orthotopic murine tumor bearing urinary bladders resulted in significant up-regulation of both Th-1 and Th-2 cytokines. CpG-ODNs have promising anti-neoplastic potential. They exert a pronounced immunological response both in the native murine urinary bladder and in murine TCC. The mechanisms of action appear to be mediated immunologically, There was no direct effect of CpG-ODN on the tumor cells in this model.

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons.

PubMed

Matikainen, S; Ronni, T; Lehtonen, A; Sareneva, T; Melén, K; Nordling, S; Levy, D E; Julkunen, I

1997-06-01

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

Expression of human factor IX gene in murine plasma through lentiviral vector-infected haematopoietic stem cells.

PubMed

Chen, Haoming; Yao, Hengmei; Huang, Lu; Shen, Qi; Jia, William; Xue, Jinglun

2006-12-01

1. Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX (hFIX) gene, respectively, to infect HSC. 2. High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate-mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro. Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence-activated cell sorting, whereas the expression of hFIX was detected by ELISA. 3. The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)-3, IL-6 and stem cell factor. 4. Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [(60)Co]-irradiated non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 +/- 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice. 5. The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.

Ultrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice.

PubMed

Suzuki, Jun-ichi; Ogawa, Masahito; Takayama, Kiyoshi; Taniyama, Yoshiaki; Morishita, Ryuichi; Hirata, Yasunobu; Nagai, Ryozo; Isobe, Mitsuaki

2010-03-02

The purpose of this study was to investigate the efficiency of small interfering ribonucleic acid (siRNA) in murine arteries. We transfected it using a nonviral ultrasound-microbubble-mediated in vivo gene delivery system. siRNA is an effective methodology to suppress gene function. The siRNA can be synthesized easily; however, a major obstacle in the use of siRNA as therapeutics is the difficulty involved in effective in vivo delivery. To investigate the efficiency of nonviral ultrasound-microbubble-mediated in vivo siRNA delivery, we used a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA in murine arteries. Murine femoral arteries were injured using flexible wires to establish arterial injury. The fluorescein-labeled siRNA and GFP siRNA showed that this nonviral approach could deliver siRNA into target arteries effectively without any tissue damage and systemic adverse effects. ICAM-1 siRNA transfection into murine injured arteries significantly suppressed the development of neointimal formation in comparison to those in the control group. Immunohistochemistry revealed that accumulation of T cells and adhesion molecule positive cells was observed in nontreated injured arteries, whereas siRNA suppressed accumulation. The nonviral ultrasound-microbubble delivery of siRNA ensures effective transfection into target arteries. ICAM-1 siRNA has the potential to suppress arterial neointimal formation. Transfection of siRNA can be beneficial for the clinical treatment of cardiovascular and other inflammatory diseases. Copyright 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

CD56bright NK cells exhibit potent antitumor responses following IL-15 priming

PubMed Central

Wagner, Julia A.; Berrien-Elliott, Melissa M.; Schneider, Stephanie E.; Leong, Jeffrey W.; Sullivan, Ryan P.; Jewell, Brea A.; Becker-Hapak, Michelle; Abdel-Latif, Sara; Ireland, Aaron R.; Jaishankar, Devika; King, Justin A.; Vij, Ravi; Clement, Dennis; Goodridge, Jodie; Malmberg, Karl-Johan; Wong, Hing C.; Fehniger, Todd A.

2017-01-01

NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56dim NK cell subset is thought to mediate antitumor responses, whereas the CD56bright subset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56bright NK cells. Priming improved multiple CD56bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56bright cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56bright cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56bright NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15–based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1–, CD2-, and NKG2D-dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56bright compared with CD56dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy. PMID:28972539

Gammaretrovirus-Specific Antibodies in Free-Ranging and Captive Namibian Cheetahs

PubMed Central

Krengel, Annika; Cattori, Valentino; Meli, Marina L.; Wachter, Bettina; Böni, Jürg; Bisset, Leslie R.; Thalwitzer, Susanne; Melzheimer, Jörg; Jago, Mark; Hofmann-Lehmann, Regina; Hofer, Heribert

2015-01-01

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers. PMID:25809630

Exposure of the Bone Marrow Microenvironment to Simulated Solar and Galactic Cosmic Radiation Induces Biological Bystander Effects on Human Hematopoiesis

DOE PAGES

Almeida-Porada, Graca; Rodman, Christopher; Kuhlman, Bradford; ...

2018-04-26

The stem cell compartment of the hematopoietic system constitutes one of the most radiosensitive tissues of the body and leukemias represent one of the most frequent radiogenic cancers with short latency periods. As such, leukemias may pose a particular threat to astronauts during prolonged space missions. Control of hematopoiesis is tightly governed by a specialized bone marrow (BM) microenvironment/niche. As such, any environmental insult that damages cells of this niche would be expected to produce pronounced effects on the types and functionality of hematopoietic/immune cells generated. We recently reported that direct exposure of human HSC to simulated SEP and GCRmore » radiation dramatically altered the differentiative potential of these cells, and that simulated GCR exposures can directly induce DNA damage and mutations within human HSC, which led to leukemic transformation when these cells repopulated murine recipients. In the present study, we performed the first in depth examination to define changes that occur in mesenchymal stem cells (MSC) present in the human BM niche following exposure to accelerated protons and iron ions, and assess the impact these changes have upon human hematopoiesis. Here, our data thus provides compelling evidence that simulated SEP/GCR exposures can also contribute to defective hematopoiesis/immunity through so-called “biological bystander effects” by damaging the stromal cells that comprise the human marrow microenvironment, thereby altering their ability to support normal hematopoiesis.« less

Antiplasmodial, cytotoxic activities and characterization of a new naturally occurring quinone methide pentacyclic triterpenoid derivative isolated from Salacia leptoclada Tul. (Celastraceae) originated from Madagascar

PubMed Central

Ruphin, Fatiany Pierre; Baholy, Robijaona; Emmanue, Andrianarivo; Amelie, Raharisololalao; Martin, Marie-Therese; Koto-te-Nyiwa, Ngbolua

2013-01-01

Objective To validate scientifically the traditional use of Salacia leptoclada Tul. (Celastraceae) (S. leptoclada) and to isolate and elucidate the structure of the biologically active compound. Methods Bioassay-guided fractionation of the acetonic extract of the stem barks of S. leptoclada was carried out by a combination of chromatography technique and biological experiments in viro using Plasmodium falciparum and P388 leukemia cell lines as models. The structure of the biologically active pure compound was elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. Results Biological screening of S. leptoclada extracts resulted in the isolation of a pentacyclic triterpenic quinone methide. The pure compound exhibited both in vitro a cytotoxic effect on murine P388 leukemia cells with IC50 value of (0.041±0.020) µg/mL and an antiplasmodial activity against the chloroquine-resistant strain FC29 of Plasmodium falciparum with an IC50 value of (0.052±0.030) µg/mL. Despite this interesting anti-malarial property of the lead compound, the therapeutic index was weak (0.788). In the best of our knowledge, the quinone methide pentacyclic triterpenoid derivative compound is reported for the first time in S. leptoclada. Conclusions The results suggest that furthers studies involving antineoplastic activity is needed for the development of this lead compound as anticancer drug. PMID:24075342

Antiplasmodial, cytotoxic activities and characterization of a new naturally occurring quinone methide pentacyclic triterpenoid derivative isolated from Salacia leptoclada Tul. (Celastraceae) originated from Madagascar.

PubMed

Ruphin, Fatiany Pierre; Baholy, Robijaona; Emmanue, Andrianarivo; Amelie, Raharisololalao; Martin, Marie-Therese; Koto-te-Nyiwa, Ngbolua

2013-10-01

To validate scientifically the traditional use of Salacia leptoclada Tul. (Celastraceae) (S. leptoclada) and to isolate and elucidate the structure of the biologically active compound. Bioassay-guided fractionation of the acetonic extract of the stem barks of S. leptoclada was carried out by a combination of chromatography technique and biological experiments in viro using Plasmodium falciparum and P388 leukemia cell lines as models. The structure of the biologically active pure compound was elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. Biological screening of S. leptoclada extracts resulted in the isolation of a pentacyclic triterpenic quinone methide. The pure compound exhibited both in vitro a cytotoxic effect on murine P388 leukemia cells with IC50 value of (0.041±0.020) μg/mL and an antiplasmodial activity against the chloroquine-resistant strain FC29 of Plasmodium falciparum with an IC50 value of (0.052±0.030) μg/mL. Despite this interesting anti-malarial property of the lead compound, the therapeutic index was weak (0.788). In the best of our knowledge, the quinone methide pentacyclic triterpenoid derivative compound is reported for the first time in S. leptoclada. The results suggest that furthers studies involving antineoplastic activity is needed for the development of this lead compound as anticancer drug. Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.

E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

PubMed Central

Hatchi, Elodie; Rodier, Genevieve; Lacroix, Matthieu; Caramel, Julie; Kirsh, Olivier; Jacquet, Chantal; Schrepfer, Emilie; Lagarrigue, Sylviane; Linares, Laetitia Karine; Lledo, Gwendaline; Tondeur, Sylvie; Dubus, Pierre

2011-01-01

The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA–mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest. PMID:21708927

Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice

PubMed Central

Walker, Emma C.; McGregor, Narelle E.; Poulton, Ingrid J.; Solano, Melissa; Pompolo, Sueli; Fernandes, Tania J.; Constable, Matthew J.; Nicholson, Geoff C.; Zhang, Jian-Guo; Nicola, Nicos A.; Gillespie, Matthew T.; Martin, T. John; Sims, Natalie A.

2010-01-01

Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr–/– osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer. PMID:20051625

Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML

PubMed Central

Carter, Bing Z.; Mak, Duncan H.; Schober, Wendy D.; Koller, Erich; Pinilla, Clemencia; Vassilev, Lyubomir T.; Reed, John C.

2010-01-01

Activation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a–induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic. PMID:19897582

Exposure of the Bone Marrow Microenvironment to Simulated Solar and Galactic Cosmic Radiation Induces Biological Bystander Effects on Human Hematopoiesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Almeida-Porada, Graca; Rodman, Christopher; Kuhlman, Bradford

The stem cell compartment of the hematopoietic system constitutes one of the most radiosensitive tissues of the body and leukemias represent one of the most frequent radiogenic cancers with short latency periods. As such, leukemias may pose a particular threat to astronauts during prolonged space missions. Control of hematopoiesis is tightly governed by a specialized bone marrow (BM) microenvironment/niche. As such, any environmental insult that damages cells of this niche would be expected to produce pronounced effects on the types and functionality of hematopoietic/immune cells generated. We recently reported that direct exposure of human HSC to simulated SEP and GCRmore » radiation dramatically altered the differentiative potential of these cells, and that simulated GCR exposures can directly induce DNA damage and mutations within human HSC, which led to leukemic transformation when these cells repopulated murine recipients. In the present study, we performed the first in depth examination to define changes that occur in mesenchymal stem cells (MSC) present in the human BM niche following exposure to accelerated protons and iron ions, and assess the impact these changes have upon human hematopoiesis. Here, our data thus provides compelling evidence that simulated SEP/GCR exposures can also contribute to defective hematopoiesis/immunity through so-called “biological bystander effects” by damaging the stromal cells that comprise the human marrow microenvironment, thereby altering their ability to support normal hematopoiesis.« less

Leukemia inhibitory factor protects against experimental lethal Escherichia coli septic shock in mice.

PubMed Central

Waring, P M; Waring, L J; Billington, T; Metcalf, D

1995-01-01

Leukemia inhibitory factor (LIF) has recently been associated with septic shock in humans. In this study we sought to determine, in mice, the role of LIF in septic shock. During sublethal endotoxemia, serum LIF levels, as determined by radio-receptor competition assay, peaked at 2 h and were low (3 ng/ml), whereas in lethal Escherichia coli septic shock serum LIF levels rose progressively (> 30 ng/ml) in the premorbid phase coincident with the development of tissue injury. Single i.v. injections of high doses (up to 50 micrograms per mouse) of recombinant murine LIF had no obvious acute detrimental effects, whereas continued i.p. administration (30 micrograms per mouse per day) for 3-4 days induced a fatal catabolic state without evidence of preceding hemodynamic collapse or shock. Simultaneous or subsequent administration of high doses of LIF had no effect on mortality from sublethal and lethal E. coli septic shock, whereas prior administration conferred significant protection against lethality (P << 0.001 by log-rank test), an effect that was dose and interval dependent. This protective effect resembled endotoxin tolerance and was characterized by suppression of E. coli-induced serum tumor necrosis factor concentration (P < 0.05), reduction in the number of viable bacteria (P < 0.05), and prevention of sepsis-induced tissue injury. These observations suggest that systemic LIF production is part of the host response to both endotoxin and sepsis-induced tissue injury. Images Fig. 2 Fig. 5 PMID:7877978

UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

PubMed Central

Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T. C.; Imren, Suzan; Lam, Vivian; Poon, Grace F. T.; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William

2016-01-01

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

Induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway delays the initiation but fails to eradicate established murine acute myeloid leukemia.

PubMed

Jaako, P; Ugale, A; Wahlestedt, M; Velasco-Hernandez, T; Cammenga, J; Lindström, M S; Bryder, D

2017-01-01

Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

Exposure of the Bone Marrow Microenvironment to Simulated Solar and Galactic Cosmic Radiation Induces Biological Bystander Effects on Human Hematopoiesis.

PubMed

Almeida-Porada, Graça; Rodman, Christopher; Kuhlman, Bradford; Brudvik, Egil; Moon, John; George, Sunil; Guida, Peter; Sajuthi, Satria P; Langefeld, Carl D; Walker, Stephen J; Wilson, Paul F; Porada, Christopher D

2018-04-26

The stem cell compartment of the hematopoietic system constitutes one of the most radiosensitive tissues of the body and leukemias represent one of the most frequent radiogenic cancers with short latency periods. As such, leukemias may pose a particular threat to astronauts during prolonged space missions. Control of hematopoiesis is tightly governed by a specialized bone marrow (BM) microenvironment/niche. As such, any environmental insult that damages cells of this niche would be expected to produce pronounced effects on the types and functionality of hematopoietic/immune cells generated. We recently reported that direct exposure of human hematopoietic stem cells (HSC) to simulated solar energetic particle (SEP) and galactic cosmic ray (GCR) radiation dramatically altered the differentiative potential of these cells, and that simulated GCR exposures can directly induce DNA damage and mutations within human HSC, which led to leukemic transformation when these cells repopulated murine recipients. In this study, we performed the first in-depth examination to define changes that occur in mesenchymal stem cells present in the human BM niche following exposure to accelerated protons and iron ions and assess the impact these changes have upon human hematopoiesis. Our data provide compelling evidence that simulated SEP/GCR exposures can also contribute to defective hematopoiesis/immunity through so-called "biological bystander effects" by damaging the stromal cells that comprise the human marrow microenvironment, thereby altering their ability to support normal hematopoiesis.

BCR-ABL1 promotes leukemia by converting p27 into a cytoplasmic oncoprotein

PubMed Central

Mackenzie, Ryan J.; Besson, Arnaud; Jeng, Sophia; Carey, Alyssa; LaTocha, Dorian H.; Fleischman, Angela G.; Duquesnes, Nicolas; Eide, Christopher A.; Vasudevan, Kavin B.; Loriaux, Marc M.; Firpo, Eduardo; Cortes, Jorge E.; McWeeney, Shannon; O’Hare, Thomas; Roberts, James M.; Druker, Brian J.; Deininger, Michael W.

2014-01-01

Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27CK−) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27T187A) or nuclear retention (p27S10A) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization. PMID:25293778

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

PubMed Central

Henderson, L E; Sowder, R; Copeland, T D; Smythers, G; Oroszlan, S

1984-01-01

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus. Images PMID:6333515

Lipid based noninvasive vesicular formulation of cytarabine: Nanodeformable liposomes.

PubMed

Raj, Rakesh; Raj, Pooja Mongia; Ram, Alpana

2016-06-10

Leukemia is the common cause of death and worldwide incidence of this disease is increasing. Chemotherapy is the first choice for leukemia treatment, but the major limitations of standard therapy are its side effects and poor patient compliances. Therefore it is imperative to look for a therapeutic system with lesser side effects urgently to address the underlying causes of poor treatment outcomes. In such a scenario transdermal route for delivery of chemotherapeutic drugs could be a better alternative to provide sustained drug level, enhanced activity, self administration and better patient compliances. The present work is focus on the design of nanolipid based transdermal carrier, deformable liposomes bearing cytarabine as a model drug for effective delivery of drug with enhanced transdermal flux. Developed nanocarriers were characterized for their size, morphology, entrapment efficiency, skin penetration and irritation. It could be concluded that nanodeformable liposomes accentuated transdermal flux of cytarabine and could provide a new strategy for leukemia. Copyright © 2016 Elsevier B.V. All rights reserved.

Gelatin-coated Gold Nanoparticles as Carriers of FLT3 Inhibitors for Acute Myeloid Leukemia Treatment.

PubMed

Suarasan, Sorina; Simon, Timea; Boca, Sanda; Tomuleasa, Ciprian; Astilean, Simion

2016-06-01

This study presents the design of a gold nanoparticle (AuNPs)-drug system with improved efficiency for the treatment of acute myeloid leukemia. The system is based on four different FLT3 inhibitors, namely midostaurin, sorafenib, lestaurtinib, and quizartinib, which were independently loaded onto gelatin-coated gold nanoparticles. Detailed investigation of the physicochemical properties of the formed complexes lead to the selection of quizartinib-loaded AuNPs for the in vitro evaluation of the biological effects of the formed complex against OCI-AML3 acute myeloid leukemia cells. Viability tests by MTT demonstrated that the proposed drug complex has improved efficacy when compared with the drug alone. The obtained results constitute a premise for further in vivo investigation of such drug vehicles based on AuNPs. To the best of our knowledge, this is the first study that investigates the delivery of the above-mentioned FLT3 inhibitors via gelatin-coated gold nanoparticles. © 2016 John Wiley & Sons A/S.

Azacytidine in combination with tyrosine kinase inhibitors induced durable responses in patients with advanced phase chronic myelogenous leukemia.

PubMed

Ruggiu, Mathilde; Oberkampf, Florence; Ghez, David; Cony-Makhoul, Pascale; Beckeriche, Florence; Cano, Isabelle; Taksin, Anne L; Benbrahim, Omar; Ghez, Stéphanie; Farhat, Hassan; Rigaudeau, Sophie; de Gunzburg, Noémie; Lara, Diane; Terre, Christine; Raggueneau, Victoria; Garcia, Isabel; Spentchian, Marc; De Botton, Stéphane; Rousselot, Philippe

2017-11-28

Although the tyrosine kinase inhibitor (TKI) era has brought great improvement in outcome in chronic myelogenous leukemia (CML), prognosis of accelerated phase or myeloid blast crisis patients or of de novo Philadelphia chromosome-positive acute myeloid leukemia remains poor. We conducted a retrospective study on patients with advanced phase disease treated with a TKI and azacytidine. Sixteen patients were eligible. Median age was 64.9 years, the median number of previous therapies was 2.5 lines, and median follow-up was 23.1 months. Hematologic response (HR) rate was 81.3%. Median overall survival (OS), event free survival and relapse-free survival (RFS) were 31.5, 23.3, and 32.2 months, respectively. All except one patient were treated as out-patients after the first cycle. Five patients were bridged to allogenic hematopoietic stem cells transplant. The combination of a TKI and azacytidine is a safe and efficient regiment for patients with CML patients in advanced phases.

Genetic control of T cell responsiveness to the Friend murine leukemia virus envelope antigen. Identification of class II loci of the H-2 as immune response genes

PubMed Central

1988-01-01

T cells primed specifically for the envelope glycoprotein of Friend murine leukemia helper virus (F-MuLV) were prepared by immunizing mice with a recombinant vaccinia virus that expressed the entire env gene of F-MuLV. Significant proliferative responses of F-MuLV envelope- specific, H-2a/b T cells were observed when the T cells were stimulated with antigen-pulsed peritoneal exudate cells (PEC) having the b allele at the K, A beta, A alpha, and E beta loci of the H-2. On the other hand, PEC having only the kappa allele at these loci did not induce the envelope-specific T cell proliferation, even when the PEC had the b allele at the E alpha, S, or D loci. F-MuLV envelope-specific proliferation of H-2a/b T cells under the stimulation of antigen- pulsed, H-2a/b PEC was specifically blocked with anti-I-Ab and anti-I- Ek mAbs but not with anti-Kb, anti-Kk, or anti-I-Ak mAbs. Moreover, (B10.MBR x A/WySn)F1 mice that have the b allele only at the K locus but not in I-A subregion were nonresponders to the envelope glycoprotein, and the bm12 mutation at the A beta locus completely abolished the T cell responsiveness to this antigen. These results indicate that proliferative T cells recognize a limited number of epitopes on F-MuLV envelope protein in the context of I-Ab, hybrid I- Ak/b, and/or hybrid I-Ek/b class II MHC molecules but fail to recognize the same envelope protein in the context of I-Ak or I-Ek molecules. This influence of the H-2I region on T cell recognition of the envelope glycoprotein appeared to control in vivo induction of protective immunity against Friend virus complex after immunization with the vaccinia-F-MuLV env vaccine. Thus, these results provide, for the first time, direct evidence for Ir gene-controlled responder/nonresponder phenotypes influencing the immune response to a pathogenic virus of mice. PMID:3141552

HZE Radiation Leukemogenesis in Mice

NASA Astrophysics Data System (ADS)

Peng, Yuanlin

Radiation exposure is a risk factor for acute myeloid leukemia (AML). The Leukemogenesis NSCOR was developed to compare this risk for low LET vs HZE radiations as a means to better assess the leukemia risk to astronauts posed by space radiation. Individual projects within the NSCOR explore HZE radiation leukemogenesis in murine model systems and extend the findings to AML in humans. AML sensitive CBA/CaJ mice have been irradiated with 1 GeV 56 Fe particles at NSRL and with 137 Cs gamma-rays at Colorado State University and followed to 800 days of age for the development of AML. Molecular and cytogenetic analyses of HZE- and gamma-induced AML, including assays for chromosomal aberrations, PU.1 deletion, gene expression, array CGH and microsatellite instability are ongoing. Preliminary data indicate that 56 Fe particles are no more effective in inducing AML or shortening lifespan than gamma-rays. Studies designed to address the individual molecular steps in leukemogenesis and determine the effects of radiation and genetic background on each step have been initiated using knockout mice. Deletion of the PU.1 gene on mouse chromosome 2 is a critical step in this murine model of radiation leukemogenesis. Two of the three HZE-induced AMLs that could be assayed and thirteen of fourteen γ-induced AMLs had PU.1 loss as determined by Fluorescence in Situ Hybridization (FISH). We have found that AML sensitive CBA/CaJ mice have a higher incidence of Chr. 2 deletion in bone marrow cells following 56 Fe irradiation than AML resistant C57BL/6 mice. This study is being extended to proton irradiated mice. Our preliminary results indicate that microsatellite instability may be common in HZE irradiated progenitor cells. To determine if these cytogenetic changes can be induced in human myeloid progenitor cells by gamma, proton or HZE irradiation we are generating NOD/SCID mice that have been "humanized" by being transplanted with human hematopoietic stem cells. We are currently irradiating the humanized NOD/SCID mice with gamma-rays and then harvesting human cells from their bone marrow. These cells will be assayed for specific cytogenetic and molecular changes consistent with AML. In addition to screening the cells for chromosomal aberrations and specific deletions and translocations, we will also screen them for microsatellite instability by small pool PCR.(Funded by NASA Grant NAG9 1569)

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

PubMed

D'Souza, V; Melamed, J; Habib, D; Pullen, K; Wallace, K; Summers, M F

2001-11-23

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds to a pocket or surface that only exists in the presence of all three stem loops. Copyright 2001 Academic Press.

DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection.

PubMed

Stavrou, Spyridon; Aguilera, Alexya N; Blouch, Kristin; Ross, Susan R

2018-06-05

Host recognition of viral nucleic acids generated during infection leads to the activation of innate immune responses essential for early control of virus. Retrovirus reverse transcription creates numerous potential ligands for cytosolic host sensors that recognize foreign nucleic acids, including single-stranded RNA (ssRNA), RNA/DNA hybrids, and double-stranded DNA (dsDNA). We and others recently showed that the sensors cyclic GMP-AMP synthase (cGAS), DEAD-box helicase 41 (DDX41), and members of the Aim2-like receptor (ALR) family participate in the recognition of retroviral reverse transcripts. However, why multiple sensors might be required and their relative importance in in vivo control of retroviral infection are not known. Here, we show that DDX41 primarily senses the DNA/RNA hybrid generated at the first step of reverse transcription, while cGAS recognizes dsDNA generated at the next step. We also show that both DDX41 and cGAS are needed for the antiretroviral innate immune response to murine leukemia virus (MLV) and HIV in primary mouse macrophages and dendritic cells (DCs). Using mice with cell type-specific knockout of the Ddx41 gene, we show that DDX41 sensing in DCs but not macrophages was critical for controlling in vivo MLV infection. This suggests that DCs are essential in vivo targets for infection, as well as for initiating the antiviral response. Our work demonstrates that the innate immune response to retrovirus infection depends on multiple host nucleic acid sensors that recognize different reverse transcription intermediates. IMPORTANCE Viruses are detected by many different host sensors of nucleic acid, which in turn trigger innate immune responses, such as type I interferon (IFN) production, required to control infection. We show here that at least two sensors are needed to initiate a highly effective innate immune response to retroviruses-DDX41, which preferentially senses the RNA/DNA hybrid generated at the first step of retrovirus replication, and cGAS, which recognizes double-stranded DNA generated at the second step. Importantly, we demonstrate using mice lacking DDX41 or cGAS that both sensors are needed for the full antiviral response needed to control in vivo MLV infection. These findings underscore the need for multiple host factors to counteract retroviral infection. Copyright © 2018 Stavrou et al.

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

PubMed

Finch, Leanne K; Ling, Roger; Napthine, Sawsan; Olspert, Allan; Michiels, Thomas; Lardinois, Cécile; Bell, Susanne; Loughran, Gary; Brierley, Ian; Firth, Andew E

2015-08-01

Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed-1 ribosomal frameshift (1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that-1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3= RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient-1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.

Sunitinib in Treating Patients With Idiopathic Myelofibrosis

ClinicalTrials.gov

2014-05-12

Accelerated Phase Chronic Myelogenous Leukemia; Acute Undifferentiated Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Blastic Phase Chronic Myelogenous Leukemia; Chronic Myelomonocytic Leukemia; Chronic Phase Chronic Myelogenous Leukemia; Mast Cell Leukemia; Meningeal Chronic Myelogenous Leukemia; Primary Myelofibrosis; Progressive Hairy Cell Leukemia, Initial Treatment; Prolymphocytic Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Refractory Chronic Lymphocytic Leukemia; Refractory Hairy Cell Leukemia; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; Stage I Chronic Lymphocytic Leukemia; Stage II Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage IV Chronic Lymphocytic Leukemia; T-cell Large Granular Lymphocyte Leukemia; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Adult Acute Myeloid Leukemia; Untreated Hairy Cell Leukemia

Direct Interaction of Jak1 and v-Abl Is Required for v-Abl-Induced Activation of STATs and Proliferation

PubMed Central

Danial, Nika N.; Losman, Julie A.; Lu, Tianhong; Yip, Natalie; Krishnan, Kartik; Krolewski, John; Goff, Stephen P.; Wang, Jean Y. J.; Rothman, Paul B.

1998-01-01

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl–Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene. PMID:9774693

Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA.

PubMed

Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

2016-07-01

The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of replacing the aspartic acid/glutamic acid residues of bullfrog sialic acid binding lectin with asparagine/glutamine and arginine on the inhibition of cell proliferation in murine leukemia P388 cells.

PubMed

Ogawa, Yuko; Iwama, Masanori; Ohgi, Kazuko; Tsuji, Tsutomu; Irie, Masachika; Itagaki, Tadashi; Kobayashi, Hiroko; Inokuchi, Norio

2002-06-01

The sialic acid binding lectin from bullfrog oocytes (cSBL) is known to have anti-tumor activity. In a previous report, to elucidate the relationship between the net charge and anti-tumor activity of cSBL, we examined the effect of chemical modifications of cSBL with a water-soluble carbodiimide in the presence of various nucleophiles. The results suggested that the anti-tumor activity and internalization into tumor cells increased with an increase in the net charge of cSBL. However, in the chemically modified cSBL, a modification site was observed on average in two of the carboxyl groups of cSBL. To confirm these previous results and to determine which modified carboxyl group contributes to the increase in anti-tumor activity, we prepared mutants with substitutions of Asn/Gln and Arg at three acidic amino acid residues of cSBL and studied their anti-tumor activity and internalization efficiency. The results showed the enhancing effect of charge on anti-tumor activity and internalization, and suggested that the replacement of D24 and E88 of cSBL with arginine is more effective than that of E97. The double mutant D24RE88R showed comparable anti-tumor activity to the ethylenediamine-modified cSBL reported previously. The mutant was well-characterized as a pure cSBL derivative suitable for studying the mechanism of the anti-tumor action of cSBL.

Synthetic studies of the zoanthamine alkaloids: the total syntheses of norzoanthamine and zoanthamine.

PubMed

Yoshimura, Fumihiko; Sasaki, Minoru; Hattori, Izumi; Komatsu, Kei; Sakai, Mio; Tanino, Keiji; Miyashita, Masaaki

2009-07-06

The zoanthamine alkaloids, a type of heptacyclic marine alkaloid isolated from colonial zoanthids of the genus Zoanthus sp., have distinctive biological and pharmacological properties in addition to their unique chemical structures with stereochemical complexity. Namely, norzoanthamine (1) can suppress the loss of bone weight and strength in ovariectomized mice and has been expected as a promising candidate for a new type of antiosteoporotic drug, while zoanthamine (2) has exhibited potent inhibitory activity toward phorbol myristate-induced inflammation in addition to powerful analgesic effects. Recently, norzoanthamine derivatives were demonstrated to inhibit strongly the growth of P-388 murine leukemia cell lines, in addition to their potent antiplatelet activities on human platelet aggregation. Their distinctive biological properties, combined with novel chemical structures, make this family of alkaloids extremely attractive targets for chemical synthesis. However, the chemical synthesis of the zoanthamine alkaloids has been impeded owing to their densely functionalized complex stereostructures. In this paper, we report the first and highly efficient total syntheses of norzoanthamine (1) and zoanthamine (2) in full detail, which involve stereoselective synthesis of the requisite triene (18) for an intramolecular Diels-Alder reaction via the sequential three-component coupling reactions, the key intramolecular Diels-Alder reaction, and subsequent crucial bis-aminoacetalization as the key steps. Ultimately, we achieved the total synthesis of norzoanthamine (1) in 41 steps with an overall yield of 3.5 % (an average of 92 % yield each step) and that of zoanthamine (2) in 43 steps with an overall yield of 2.2 % (an average of 91 % yield each step) starting from (R)-5-methylcyclohexenone (3), respectively.

SOCS1 cooperates with FLT3-ITD in the development of myeloproliferative disease by promoting the escape from external cytokine control.

PubMed

Reddy, Pavankumar N G; Sargin, Bülent; Choudhary, Chunaram; Stein, Stefan; Grez, Manuel; Müller-Tidow, Carsten; Berdel, Wolfgang E; Serve, Hubert; Brandts, Christian H

2012-08-23

Activating mutations in the receptor tyrosine kinase FLT3 are frequently found in acute myelogenous leukemia patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control that regulates normal hematopoiesis. We have recently demonstrated that FLT3-internal tandem duplication (ITD), when localized to the biosynthetic compartment, aberrantly activates STAT5. Here, we show that one of the target genes induced by STAT5 is suppressor of cytokine signaling (SOCS)1-a surprising finding for a known tumor suppressor. Although SOCS1 expression in murine bone marrow severely impaired cytokine-induced colony growth, it failed to inhibit FLT3-ITD-supported colony growth, indicating resistance of FLT3-ITD to SOCS1. In addition, SOCS1 coexpression did not affect FLT3-ITD-mediated signaling or proliferation. Importantly, SOCS1 coexpression inhibited interferon-α and interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-mediated growth inhibitory effects. In a murine bone marrow transplantation model, the coexpression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared with FLT3-ITD alone (P < .01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting leukemogenesis. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

Combined BTK and PI3Kδ Inhibition with Acalabrutinib and ACP-319 Improves Survival and Tumor Control in CLL Mouse Model.

PubMed

Niemann, Carsten U; Mora-Jensen, Helena I; Dadashian, Eman L; Krantz, Fanny; Covey, Todd; Chen, Shih-Shih; Chiorazzi, Nicholas; Izumi, Raquel; Ulrich, Roger; Lannutti, Brian J; Wiestner, Adrian; Herman, Sarah E M

2017-10-01

Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3Kδ is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3Kδ and BTK inhibitors. Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3Kδ inhibitor ACP-319 in vivo We compared single-agent with combination therapy in TCL1-192 cell-injected mice, a model of aggressive CLL. Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-κB signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy. Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814-23. ©2017 AACR . ©2017 American Association for Cancer Research.

NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Guohua; Shi Lingfang; Qiu Daoming

2005-05-01

NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mousemore » NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.« less

Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: studies in infant ADA-deficient mice and rhesus monkeys.

PubMed

Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

2014-10-01

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

Parameters determining the efficacy of adoptive CD8 T-cell therapy of cytomegalovirus infection.

PubMed

Ebert, Stefan; Podlech, Jürgen; Gillert-Marien, Dorothea; Gergely, Kerstin M; Büttner, Julia K; Fink, Annette; Freitag, Kirsten; Thomas, Doris; Reddehase, Matthias J; Holtappels, Rafaela

2012-11-01

Reactivation of latent cytomegalovirus (CMV) in the transient state of immunodeficiency after hematopoietic cell transplantation (HCT) is the most frequent and severe viral complication endangering leukemia therapy success. By infecting the bone marrow (BM) stroma of the transplantation recipient, CMV can directly interfere with BM repopulation by the transplanted donor-derived hematopoietic cells and thus delay immune reconstitution of the recipient. Cytopathogenic virus spread in tissues can result in CMV disease with multiple organ manifestations of which interstitial pneumonia is the most feared. There exists a 'window of risk' between hematoablative treatment and reconstitution of antiviral immunity after HCT, whereby timely reconstitution of antiviral CD8 T cells is a recognized positive prognostic parameter for the control of reactivated CMV infection and prevention of CMV disease. Supplementation of endogenous reconstitution by adoptive cell transfer of 'ready-to-go' effector and/or memory virus epitope-specific CD8 T cells is a therapeutic option to bridge the 'window of risk.' Preclinical research in murine models of CMV disease has been pivotal by providing 'proof of concept' for a benefit from CD8 T-cell therapy of HCT-associated CMV disease (reviewed in Holtappels et al. Med Microbiol Immunol 197:125-134, 2008). Here, we give an update of our previous review with focus on parameters that determine the efficacy of adoptive immunotherapy of CMV infection by antiviral CD8 T cells in the murine model.

EVI2B is a C/EBPα target gene required for granulocytic differentiation and functionality of hematopoietic progenitors.

PubMed

Zjablovskaja, Polina; Kardosova, Miroslava; Danek, Petr; Angelisova, Pavla; Benoukraf, Touati; Wurm, Alexander A; Kalina, Tomas; Sian, Stephanie; Balastik, Martin; Delwel, Ruud; Brdicka, Tomas; Tenen, Daniel G; Behre, Gerhard; Fiore, Fréderic; Malissen, Bernard; Horejsi, Vaclav; Alberich-Jorda, Meritxell

2017-04-01

Development of hematopoietic populations through the process of differentiation is critical for proper hematopoiesis. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is a master regulator of myeloid differentiation, and the identification of C/EBPα target genes is key to understand this process. Here we identified the Ecotropic Viral Integration Site 2B (EVI2B) gene as a direct target of C/EBPα. We showed that the product of the gene, the transmembrane glycoprotein EVI2B (CD361), is abundantly expressed on the surface of primary hematopoietic cells, the highest levels of expression being reached in mature granulocytes. Using shRNA-mediated downregulation of EVI2B in human and murine cell lines and in primary hematopoietic stem and progenitor cells, we demonstrated impaired myeloid lineage development and altered progenitor functions in EVI2B-silenced cells. We showed that the compromised progenitor functionality in Evi2b-depleted cells can be in part explained by deregulation of cell proliferation and apoptosis. In addition, we generated an Evi2b knockout murine model and demonstrated altered properties of hematopoietic progenitors, as well as impaired G-CSF dependent myeloid colony formation in the knockout cells. Remarkably, we found that EVI2B is significantly downregulated in human acute myeloid leukemia samples characterized by defects in CEBPA. Altogether, our data demonstrate that EVI2B is a downstream target of C/EBPα, which regulates myeloid differentiation and functionality of hematopoietic progenitors.

Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines.

PubMed

Hirabayashi, Masumi; Goto, Teppei; Tamura, Chihiro; Sanbo, Makoto; Hara, Hiromasa; Hochi, Shinichi

2014-03-07

This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines.

Efficient collection of peripheral blood stem cells using the Fresenius AS104 in chronic myelocytic leukemia patients with very high numbers of platelets.

PubMed

Komatsu, F; Ishida, Y

1997-04-01

For chronic myelocytic leukemia patients with very high numbers of platelets, we describe an efficient method for the collection of peripheral blood stem cells (PBSC) using the Fresenius AS104 cell separator. In these patients, it is difficult to collect a sufficient number of PBSC, due to the platelet band interfering with the machine's red cell interface sensor. We, therefore, tried a manual adjustment of the device. The collection phase was set automatically. When the whole blood began to separate into the red cell layer and plasma (plus mononuclear cell) layer, the red cell interface setting of "7:1" was changed to "OFF," and the plasma pump flow rate was controlled manually in order to locate the interface position 1 cm from the outside wall of the centrifuge chamber. After the collection phase, the procedure was returned to the automatic setting. By repeating this procedure, we were able to collect large numbers of PBSC.

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor.

PubMed

Do, Hyun-Jin; Song, Hyuk; Yang, Heung-Mo; Kim, Dong-Ku; Kim, Nam-Hyung; Kim, Jin-Hoi; Cha, Kwang-Yul; Chung, Hyung-Min; Kim, Jae-Hwan

2006-03-20

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236-252 and 249-267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

PubMed

Rozov, F N; Grinenko, T S; Levit, G L; Krasnov, V P; Belyavsky, A V

2010-09-15

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders. 2010 Elsevier Inc. All rights reserved.

Effects of Methylphenidate on Attention Deficits in Childhood Cancer Survivors

ClinicalTrials.gov

2015-03-16

ALL, Childhood; Leukemia, Lymphoblastic; Leukemia, Lymphoblastic, Acute; Leukemia, Lymphoblastic, Acute, L1; Leukemia, Lymphoblastic, Acute, L2; Leukemia, Lymphoblastic, Acute, Philadelphia-Positive; Leukemia, Lymphocytic, Acute; Leukemia, Lymphocytic, Acute, L1; Leukemia, Lymphocytic, Acute, L2; Lymphoblastic Leukemia; Lymphoblastic Leukemia, Acute; Lymphoblastic Leukemia, Acute, Childhood; Lymphoblastic Leukemia, Acute, L1; Lymphoblastic Leukemia, Acute, L2; Lymphoblastic Lymphoma; Lymphocytic Leukemia, Acute; Lymphocytic Leukemia, L1; Lymphocytic Leukemia, L2; Brain Tumors; Cancer of the Brain; Cancer of Brain; Malignant Primary Brain Tumors; Brain Neoplasms, Malignant

Tanespimycin and Cytarabine in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, Chronic Myelogenous Leukemia, Chronic Myelomonocytic Leukemia, or Myelodysplastic Syndromes

ClinicalTrials.gov

2013-09-27

Accelerated Phase Chronic Myelogenous Leukemia; Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Blastic Phase Chronic Myelogenous Leukemia; Chronic Myelomonocytic Leukemia; de Novo Myelodysplastic Syndromes; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Refractory Anemia With Excess Blasts in Transformation; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes

7-Hydroxystaurosporine and Perifosine in Treating Patients With Relapsed or Refractory Acute Leukemia, Chronic Myelogenous Leukemia or High Risk Myelodysplastic Syndromes

ClinicalTrials.gov

2013-09-27

Accelerated Phase Chronic Myelogenous Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Acute Promyelocytic Leukemia (M3); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Blastic Phase Chronic Myelogenous Leukemia; Myelodysplastic/Myeloproliferative Neoplasms; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; T-cell Adult Acute Lymphoblastic Leukemia; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Adult Acute Myeloid Leukemia

Nanoparticle targeted therapy against childhood acute lymphoblastic leukemia

NASA Astrophysics Data System (ADS)

Satake, Noriko; Lee, Joyce; Xiao, Kai; Luo, Juntao; Sarangi, Susmita; Chang, Astra; McLaughlin, Bridget; Zhou, Ping; Kenney, Elaina; Kraynov, Liliya; Arnott, Sarah; McGee, Jeannine; Nolta, Jan; Lam, Kit

2011-06-01

The goal of our project is to develop a unique ligand-conjugated nanoparticle (NP) therapy against childhood acute lymphoblastic leukemia (ALL). LLP2A, discovered by Dr. Kit Lam, is a high-affinity and high-specificity peptidomimetic ligand against an activated α4β1 integrin. Our study using 11 fresh primary ALL samples (10 precursor B ALL and 1 T ALL) showed that childhood ALL cells expressed activated α4β1 integrin and bound to LLP2A. Normal hematopoietic cells such as activated lymphocytes and monocytes expressed activated α4β1 integrin; however, normal hematopoietic stem cells showed low expression of α4β1 integrin. Therefore, we believe that LLP2A can be used as a targeted therapy for childhood ALL. The Lam lab has developed novel telodendrimer-based nanoparticles (NPs) which can carry drugs efficiently. We have also developed a human leukemia mouse model using immunodeficient NOD/SCID/IL2Rγ null mice engrafted with primary childhood ALL cells from our patients. LLP2A-conjugated NPs will be evaluated both in vitro and in vivo using primary leukemia cells and this mouse model. NPs will be loaded first with DiD near infra-red dye, and then with the chemotherapeutic agents daunorubicin or vincristine. Both drugs are mainstays of current chemotherapy for childhood ALL. Targeting properties of LLP2A-conjugated NPs will be evaluated by fluorescent microscopy, flow cytometry, MTS assay, and mouse survival after treatment. We expect that LLP2A-conjugated NPs will be preferentially delivered and endocytosed to leukemia cells as an effective targeted therapy.

Flavopiridol in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, or Chronic Myelogenous Leukemia

ClinicalTrials.gov

2013-06-03

Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Blastic Phase Chronic Myelogenous Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Relapsing Chronic Myelogenous Leukemia

Vorinostat, Cytarabine, and Etoposide in Treating Patients With Relapsed and/or Refractory Acute Leukemia or Myelodysplastic Syndromes or Myeloproliferative Disorders

ClinicalTrials.gov

2013-05-01

Accelerated Phase Chronic Myelogenous Leukemia; Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Acute Promyelocytic Leukemia (M3); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Blastic Phase Chronic Myelogenous Leukemia; Chronic Eosinophilic Leukemia; Chronic Myelomonocytic Leukemia; Chronic Neutrophilic Leukemia; de Novo Myelodysplastic Syndromes; Essential Thrombocythemia; Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Polycythemia Vera; Previously Treated Myelodysplastic Syndromes; Primary Myelofibrosis; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes

Treosulfan, Fludarabine Phosphate, and Total-Body Irradiation Before Donor Stem Cell Transplant in Treating Patients With High-Risk Acute Myeloid Leukemia, Myelodysplastic Syndrome, Acute Lymphoblastic Leukemia

ClinicalTrials.gov

2017-04-05

Accelerated Phase Chronic Myelogenous Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Blastic Phase Chronic Myelogenous Leukemia; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Myeloid Leukemia in Remission; Childhood Chronic Myelogenous Leukemia; Childhood Myelodysplastic Syndromes; Chronic Myelomonocytic Leukemia; de Novo Myelodysplastic Syndromes; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Childhood Acute Lymphoblastic Leukemia

Bortezomib and Combination Chemotherapy in Treating Younger Patients With Recurrent, Refractory, or Secondary Acute Myeloid Leukemia

ClinicalTrials.gov

2018-05-21

Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myelomonocytic Leukemia (M4); Childhood Acute Basophilic Leukemia; Childhood Acute Eosinophilic Leukemia; Childhood Acute Erythroleukemia (M6); Childhood Acute Megakaryocytic Leukemia (M7); Childhood Acute Minimally Differentiated Myeloid Leukemia (M0); Childhood Acute Monoblastic Leukemia (M5a); Childhood Acute Monocytic Leukemia (M5b); Childhood Acute Myeloblastic Leukemia With Maturation (M2); Childhood Acute Myeloblastic Leukemia Without Maturation (M1); Childhood Acute Myelomonocytic Leukemia (M4); Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia

Caspofungin Acetate or Fluconazole in Preventing Invasive Fungal Infections in Patients With Acute Myeloid Leukemia Who Are Undergoing Chemotherapy

ClinicalTrials.gov

2017-11-13

Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Childhood Acute Erythroleukemia (M6); Childhood Acute Megakaryocytic Leukemia (M7); Childhood Acute Minimally Differentiated Myeloid Leukemia (M0); Childhood Acute Monoblastic Leukemia (M5a); Childhood Acute Monocytic Leukemia (M5b); Childhood Acute Myeloblastic Leukemia With Maturation (M2); Childhood Acute Myeloblastic Leukemia Without Maturation (M1); Childhood Acute Myeloid Leukemia in Remission; Childhood Acute Myelomonocytic Leukemia (M4); Fungal Infection; Neutropenia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia; Untreated Childhood Acute Myeloid Leukemia and Other Myeloid Malignancies

Alemtuzumab and Combination Chemotherapy in Treating Patients With Untreated Acute Lymphoblastic Leukemia

ClinicalTrials.gov

2014-03-20

Acute Undifferentiated Leukemia; B-cell Adult Acute Lymphoblastic Leukemia; B-cell Childhood Acute Lymphoblastic Leukemia; L1 Adult Acute Lymphoblastic Leukemia; L1 Childhood Acute Lymphoblastic Leukemia; L2 Adult Acute Lymphoblastic Leukemia; L2 Childhood Acute Lymphoblastic Leukemia; Philadelphia Chromosome Negative Adult Precursor Acute Lymphoblastic Leukemia; Philadelphia Chromosome Positive Adult Precursor Acute Lymphoblastic Leukemia; Philadelphia Chromosome Positive Childhood Precursor Acute Lymphoblastic Leukemia; T-cell Adult Acute Lymphoblastic Leukemia; T-cell Childhood Acute Lymphoblastic Leukemia; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Childhood Acute Lymphoblastic Leukemia

SB-715992 in Treating Patients With Acute Leukemia, Chronic Myelogenous Leukemia, or Advanced Myelodysplastic Syndromes

ClinicalTrials.gov

2013-01-10

Acute Undifferentiated Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Acute Promyelocytic Leukemia (M3); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Blastic Phase Chronic Myelogenous Leukemia; de Novo Myelodysplastic Syndromes; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Refractory Anemia With Excess Blasts; Refractory Anemia With Excess Blasts in Transformation; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes; Untreated Adult Acute Myeloid Leukemia

Tipifarnib in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia

ClinicalTrials.gov

2013-02-01

Acute Myeloid Leukemia With Multilineage Dysplasia Following Myelodysplastic Syndrome; Adult Acute Erythroid Leukemia (M6); Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monoblastic Leukemia and Acute Monocytic Leukemia (M5); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Recurrent Adult Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Nilotinib and Imatinib Mesylate After Donor Stem Cell Transplant in Treating Patients With ALL or CML

ClinicalTrials.gov

2017-07-11

Accelerated Phase Chronic Myelogenous Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Blastic Phase Chronic Myelogenous Leukemia; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Chronic Myelogenous Leukemia; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Chronic Phase Chronic Myelogenous Leukemia; Philadelphia Positive Adult Acute Lymphoblastic Leukemia; Philadelphia Positive Childhood Acute Lymphoblastic Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Relapsing Chronic Myelogenous Leukemia; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Childhood Acute Lymphoblastic Leukemia

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging.

PubMed

Gidáli, J; Szamosvölgyi, S; Fehér, I; Kovács, P

1990-01-01

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.

Lenalidomide in Treating Older Patients With Acute Myeloid Leukemia

ClinicalTrials.gov

2014-07-25

Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Biological Therapy in Treating Patients With Advanced Myelodysplastic Syndrome, Acute or Chronic Myeloid Leukemia, or Acute Lymphoblastic Leukemia Who Are Undergoing Stem Cell Transplantation

ClinicalTrials.gov

2017-03-27

Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); B-cell Adult Acute Lymphoblastic Leukemia; B-cell Childhood Acute Lymphoblastic Leukemia; Childhood Chronic Myelogenous Leukemia; Childhood Myelodysplastic Syndromes; Chronic Myelomonocytic Leukemia; Essential Thrombocythemia; Polycythemia Vera; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Refractory Anemia With Excess Blasts; Refractory Anemia With Excess Blasts in Transformation; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; T-cell Adult Acute Lymphoblastic Leukemia; T-cell Childhood Acute Lymphoblastic Leukemia

Selective Depletion of CD45RA+ T Cells From Allogeneic Peripheral Blood Stem Cell Grafts in Preventing GVHD in Children

ClinicalTrials.gov

2018-04-23

Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Acute Biphenotypic Leukemia; Acute Leukemia of Ambiguous Lineage; Acute Undifferentiated Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Blast Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Myeloid Leukemia in Remission; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Myelodysplastic Syndrome With Excess Blasts-1; Myelodysplastic Syndrome With Excess Blasts-2; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Refractory Adult Acute Lymphoblastic Leukemia; Refractory Childhood Acute Lymphoblastic Leukemia

Collecting and Storing Malignant, Borderline Malignant Neoplasms, and Related Samples From Young Patients With Cancer

ClinicalTrials.gov

2017-12-11

Acute Undifferentiated Leukemia; Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Childhood Acute Lymphoblastic Leukemia; Childhood Acute Myeloid Leukemia/Other Myeloid Malignancies; Childhood Chronic Myelogenous Leukemia; Chronic Lymphocytic Leukemia; Hairy Cell Leukemia; Juvenile Myelomonocytic Leukemia; Mast Cell Leukemia; Neoplasm of Uncertain Malignant Potential; Prolymphocytic Leukemia; Secondary Acute Myeloid Leukemia; T-cell Large Granular Lymphocyte Leukemia; Unspecified Childhood Solid Tumor, Protocol Specific

Decitabine, Cytarabine, and Daunorubicin Hydrochloride in Treating Patients With Acute Myeloid Leukemia

ClinicalTrials.gov

2018-05-24

Acute Myeloid Leukemia; Adult Acute Basophilic Leukemia; Adult Acute Monoblastic Leukemia; Adult Acute Monocytic Leukemia; Adult Acute Myeloid Leukemia With Maturation; Adult Acute Myeloid Leukemia With t(9;11)(p22.3;q23.3); MLLT3-KMT2A; Adult Acute Myeloid Leukemia Without Maturation; Adult Acute Myelomonocytic Leukemia; Alkylating Agent-Related Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Azacitidine, Cytarabine, and Mitoxantrone Hydrochloride in Treating Patients With High-Risk Acute Myeloid Leukemia

ClinicalTrials.gov

2018-01-02

Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Eltrombopag Olamine in Treating Patients With Relapsed/Refractory Acute Myeloid Leukemia

ClinicalTrials.gov

2016-04-04

Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Recurrent Adult Acute Myeloid Leukemia

MS-275 and GM-CSF in Treating Patients With Myelodysplastic Syndrome and/or Relapsed or Refractory Acute Myeloid Leukemia or Acute Lymphocytic Leukemia

ClinicalTrials.gov

2017-06-16

Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Chronic Myelomonocytic Leukemia; de Novo Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Refractory Anemia; Refractory Anemia With Excess Blasts; Refractory Anemia With Ringed Sideroblasts; Refractory Cytopenia With Multilineage Dysplasia; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Adult Acute Myeloid Leukemia

Obatoclax, Fludarabine, and Rituximab in Treating Patients With Previously Treated Chronic Lymphocytic Leukemia

ClinicalTrials.gov

2013-09-27

B-cell Chronic Lymphocytic Leukemia; Leukemia; Prolymphocytic Leukemia; Refractory Chronic Lymphocytic Leukemia; Stage I Chronic Lymphocytic Leukemia; Stage II Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage IV Chronic Lymphocytic Leukemia

Tipifarnib in Treating Older Patients With Acute Myeloid Leukemia

ClinicalTrials.gov

2015-03-19

Adult Acute Megakaryoblastic Leukemia; Adult Acute Monoblastic Leukemia; Adult Acute Monocytic Leukemia; Adult Acute Myeloid Leukemia With Inv(16)(p13.1q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With Maturation; Adult Acute Myeloid Leukemia With Minimal Differentiation; Adult Acute Myeloid Leukemia With t(16;16)(p13.1;q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); RUNX1-RUNX1T1; Adult Acute Myeloid Leukemia With t(9;11)(p22;q23); MLLT3-MLL; Adult Acute Myeloid Leukemia Without Maturation; Adult Acute Myelomonocytic Leukemia; Adult Erythroleukemia; Adult Pure Erythroid Leukemia; Alkylating Agent-Related Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Idarubicin, Cytarabine, and Tipifarnib in Treating Patients With Newly Diagnosed Myelodysplastic Syndromes or Acute Myeloid Leukemia

ClinicalTrials.gov

2014-05-09

Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Childhood Myelodysplastic Syndromes; Chronic Myelomonocytic Leukemia; de Novo Myelodysplastic Syndromes; Refractory Anemia With Excess Blasts; Refractory Anemia With Excess Blasts in Transformation; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes; Untreated Adult Acute Myeloid Leukemia

Clofarabine, Cytarabine, and Filgrastim Followed by Infusion of Non-HLA Matched Ex Vivo Expanded Cord Blood Progenitors in Treating Patients With Acute Myeloid Leukemia

ClinicalTrials.gov

2014-08-13

Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Acute Promyelocytic Leukemia (M3); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Recurrent Adult Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Veliparib and Temozolomide in Treating Patients With Acute Leukemia

ClinicalTrials.gov

2018-04-20

Accelerated Phase of Disease; Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Adult Acute Myeloid Leukemia With Inv(16)(p13.1q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With t(16;16)(p13.1;q22); CBFB-MYH11; Adult Acute Myeloid Leukemia With t(8;21); (q22; q22.1); RUNX1-RUNX1T1; Adult Acute Myeloid Leukemia With t(9;11)(p22.3;q23.3); MLLT3-KMT2A; Adult Acute Promyelocytic Leukemia With PML-RARA; Adult B Acute Lymphoblastic Leukemia; Adult B Acute Lymphoblastic Leukemia With t(9;22)(q34.1;q11.2); BCR-ABL1; Adult T Acute Lymphoblastic Leukemia; Alkylating Agent-Related Acute Myeloid Leukemia; Blastic Phase; Chronic Myelomonocytic Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Disease; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Adult Acute Myeloid Leukemia

Omacetaxine Mepesuccinate, Cytarabine, and Decitabine in Treating Older Patients With Newly Diagnosed Acute Myeloid Leukemia

ClinicalTrials.gov

2016-04-05

Acute Myeloid Leukemia With Multilineage Dysplasia Following Myelodysplastic Syndrome; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Choline Magnesium Trisalicylate and Combination Chemotherapy in Treating Patients With Acute Myeloid Leukemia

ClinicalTrials.gov

2017-02-01

Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Recurrent Adult Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

MK2206 in Treating Younger Patients With Recurrent or Refractory Solid Tumors or Leukemia

ClinicalTrials.gov

2014-04-28

Accelerated Phase Chronic Myelogenous Leukemia; Acute Leukemias of Ambiguous Lineage; Acute Myeloid Leukemia/Transient Myeloproliferative Disorder; Acute Undifferentiated Leukemia; Aggressive NK-cell Leukemia; Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Blastic Phase Chronic Myelogenous Leukemia; Blastic Plasmacytoid Dendritic Cell Neoplasm; Childhood Burkitt Lymphoma; Childhood Chronic Myelogenous Leukemia; Childhood Diffuse Large Cell Lymphoma; Childhood Grade III Lymphomatoid Granulomatosis; Childhood Immunoblastic Large Cell Lymphoma; Childhood Nasal Type Extranodal NK/T-cell Lymphoma; Chronic Eosinophilic Leukemia; Chronic Myelomonocytic Leukemia; Chronic Neutrophilic Leukemia; Chronic Phase Chronic Myelogenous Leukemia; Intraocular Lymphoma; Juvenile Myelomonocytic Leukemia; Mast Cell Leukemia; Myeloid/NK-cell Acute Leukemia; Noncutaneous Extranodal Lymphoma; Post-transplant Lymphoproliferative Disorder; Primary Central Nervous System Hodgkin Lymphoma; Primary Central Nervous System Non-Hodgkin Lymphoma; Progressive Hairy Cell Leukemia, Initial Treatment; Prolymphocytic Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Recurrent Childhood Anaplastic Large Cell Lymphoma; Recurrent Childhood Grade III Lymphomatoid Granulomatosis; Recurrent Childhood Large Cell Lymphoma; Recurrent Childhood Lymphoblastic Lymphoma; Recurrent Childhood Small Noncleaved Cell Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Recurrent/Refractory Childhood Hodgkin Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Hairy Cell Leukemia; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; Small Intestine Lymphoma; Splenic Marginal Zone Lymphoma; Unspecified Childhood Solid Tumor, Protocol Specific; Waldenström Macroglobulinemia

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

PubMed

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-05-06

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification

PubMed Central

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-01-01

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines. PMID:27150638

Novel insights into the antiproliferative effects and synergism of quercetin and menadione in human leukemia Jurkat T cells.

PubMed

Baran, Irina; Ionescu, Diana; Filippi, Alexandru; Mocanu, Maria Magdalena; Iftime, Adrian; Babes, Ramona; Tofolean, Ioana Teodora; Irimia, Ruxandra; Goicea, Alexandru; Popescu, Valentin; Dimancea, Alexandru; Neagu, Andrei; Ganea, Constanta

2014-07-01

The flavonoid quercetin and menadione (vitamin K3) are known as potent apoptogens in human leukemia Jurkat T cells. We explored some underlying mechanisms and the potential relevance of the combination quercetin-menadione for clinical applications. In acute treatments, quercetin manifested a strong antioxidant character, but induced a transient loss of Δψm, likely mediated by opening of the mitochondrial permeability transition pore. After removal of quercetin, persistent mitochondrial hyperpolarization was generated via stimulation of respiratory Complex I. In contrast, menadione-induced Δψm dissipation was only partially and transiently reversed after menadione removal. Results indicate that Ca(2+) release is a necessary event in quercetin-induced cell death and that the survival response to quercetin is delineated within 1h from exposure. Depending on dose, the two agents exhibited either antagonistic or synergistic effects in reducing clonogenicity of Jurkat cells. 24-h combinatorial regimens at equimolar concentrations of 10-15 μM, which are compatible with a clinically achievable (and safe) scheme, reduced cell viability at efficient rates. Altogether, these findings support the idea that the combination quercetin-menadione could improve the outcome of conventional leukemia therapies, and warrant the utility of additional studies to investigate the therapeutic effects of this combination in different cellular or animal models for leukemia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Efficiency of use endobronchial laser doppler-flowmetry in patients with chronic leukemia

NASA Astrophysics Data System (ADS)

Vanina, E. A.; Voitsekhovskiy, V. V.; Landyshev, Y. S.; Tkacheva, S. I.

2016-11-01

In this work indicatorsendobronchial microcirculation were investigated in patients with chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), polycythemia vera (PV), idiopathic myelofibrosis (IMF). A diagnostic bronchoscopy was performed using fibreoptic «Olympus» (Japan).Endobronchial laser Doppler flowmetry was carried out on the laser analyzer capillary blood LAK-02 (Russia). Laser Doppler flowmetry indicators such as parameter of microcirculation, the oscillation amplitude in the endothelial, neurogenic, myogenic, cardiac and respiratory ranges were calculated by continuous the Wavelet transforms. Reduced cardiac and respiratory amplitudes in CML and CLL are primarily due to the development leukostasis. If PV is the case, this is due to sludge syndrome. And when MM occurs, it is caused by protein stasis in the vessels of the bronchial tubes. Increased endothelial oscillation amplitudes in the range in CML, PV, IMF and their reduction in MM indicate the presence of endothelial dysfunction in these patients. Increasing the amplitude of oscillations in the range of neurogenic indicates the development of arteriolar vasodilation as a compensatory response to the violation of blood flow. Increasing the amplitude of oscillations of myogenic tone indicating decrease precapillaries as a compensatory reaction to improve blood flow. It is concluded that endobronchial laser Doppler flowmetry is an important method allowing diagnosing the pathology of the microvasculature of the bronchi in chronic leukemia.

Donor Umbilical Cord Blood Stem Cell Transplant in Treating Patients With Hematologic Malignancies

ClinicalTrials.gov

2015-12-18

Acute Myeloid Leukemia With Multilineage Dysplasia Following Myelodysplastic Syndrome; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Erythroleukemia (M6a); Adult Nasal Type Extranodal NK/T-cell Lymphoma; Adult Pure Erythroid Leukemia (M6b); B-cell Adult Acute Lymphoblastic Leukemia; B-cell Childhood Acute Lymphoblastic Leukemia; Blastic Phase Chronic Myelogenous Leukemia; Burkitt Lymphoma; Childhood Acute Erythroleukemia (M6); Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Megakaryocytic Leukemia (M7); Childhood Acute Minimally Differentiated Myeloid Leukemia (M0); Childhood Acute Monoblastic Leukemia (M5a); Childhood Acute Monocytic Leukemia (M5b); Childhood Acute Myeloid Leukemia in Remission; Childhood Chronic Myelogenous Leukemia; Childhood Diffuse Large Cell Lymphoma; Childhood Immunoblastic Large Cell Lymphoma; Childhood Myelodysplastic Syndromes; Childhood Nasal Type Extranodal NK/T-cell Lymphoma; Chronic Myelomonocytic Leukemia; Chronic Phase Chronic Myelogenous Leukemia; Cutaneous B-cell Non-Hodgkin Lymphoma; de Novo Myelodysplastic Syndromes; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Juvenile Myelomonocytic Leukemia; Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Nodal Marginal Zone B-cell Lymphoma; Previously Treated Myelodysplastic Syndromes; Prolymphocytic Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Recurrent Childhood Anaplastic Large Cell Lymphoma; Recurrent Childhood Grade III Lymphomatoid Granulomatosis; Recurrent Childhood Large Cell Lymphoma; Recurrent Childhood Lymphoblastic Lymphoma; Recurrent Childhood Small Noncleaved Cell Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes; Secondary Myelofibrosis; Splenic Marginal Zone Lymphoma; Stage I Chronic Lymphocytic Leukemia; Stage II Chronic Lymphocytic Leukemia; Stage III Chronic Lymphocytic Leukemia; Stage IV Chronic Lymphocytic Leukemia; T-cell Adult Acute Lymphoblastic Leukemia; T-cell Childhood Acute Lymphoblastic Leukemia; T-cell Large Granular Lymphocyte Leukemia; Waldenstrom Macroglobulinemia

Trebananib With or Without Low-Dose Cytarabine in Treating Patients With Acute Myeloid Leukemia

ClinicalTrials.gov

2017-02-14

Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Recurrent Adult Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Metformin+Cytarabine for the Treatment of Relapsed/Refractory AML

ClinicalTrials.gov

2017-09-12

Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Blastic Phase Chronic Myelogenous Leukemia; Recurrent Adult Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Selective Depletion of CD45RA+ T Cells From Allogeneic Peripheral Blood Stem Cell Grafts From HLA-Matched Related and Unrelated Donors in Preventing GVHD

ClinicalTrials.gov

2017-10-25

Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Acute Biphenotypic Leukemia; Acute Leukemia of Ambiguous Lineage; Acute Undifferentiated Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Blast Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Blastic Plasmacytoid Dendritic Cell Neoplasm; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Myeloid Leukemia in Remission; Lymphoblastic Lymphoma; Myelodysplastic Syndrome With Excess Blasts; Myelodysplastic Syndrome With Excess Blasts-1; Myelodysplastic Syndrome With Excess Blasts-2; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Recurrent Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Refractory Acute Lymphoblastic Leukemia; Refractory Acute Myeloid Leukemia

A polysaccharide from the fruiting bodies of Agaricus blazei Murill induces caspase-dependent apoptosis in human leukemia HL-60 cells.

PubMed

Li, Xiaohui; Zhao, Xin; Wang, Hongmin; Han, Junqing; Liu, Li

2014-09-01

Polysaccharides are the major active ingredients of fungus Agaricus blazei for treating and preventing cancer. However, there are no reports showing anti-tumor activity of A. blazei polysaccharides (ABP) on human leukemia (HL)-60 cells in vitro and in vivo. In this study, we demonstrated that ABP efficiently inhibited proliferation of cultured HL-60 cells, which was associated with the induction of apoptosis. The increase in ABP-induced apoptosis was accompanied by loss of mitochondria membrane potential (∆Ψm), cytochrome c release from the mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase (PARP), and the elevated ratio of Bcl-2-associated X (Bax)/B-cell lymphoma 2 (Bcl-2). Moreover, z-DEVD-fmk, a caspase-3 inhibitor, reversed the cytotoxic effects and apoptotic characteristics induced by ABP in HL-60 cells. Furthermore, we confirmed that ABP could obviously inhibit the solid cancer growth of leukemia HL-60 in tumor xenograft model. These data demonstrated that ABP effectively induced the apoptosis of HL-60 cells via a signaling cascade of mitochondrial caspase-3-dependent pathway.

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

PubMed

Guitart, Amelie V; Panagopoulou, Theano I; Villacreces, Arnaud; Vukovic, Milica; Sepulveda, Catarina; Allen, Lewis; Carter, Roderick N; van de Lagemaat, Louie N; Morgan, Marcos; Giles, Peter; Sas, Zuzanna; Gonzalez, Marta Vila; Lawson, Hannah; Paris, Jasmin; Edwards-Hicks, Joy; Schaak, Katrin; Subramani, Chithra; Gezer, Deniz; Armesilla-Diaz, Alejandro; Wills, Jimi; Easterbrook, Aaron; Coman, David; So, Chi Wai Eric; O'Carroll, Donal; Vernimmen, Douglas; Rodrigues, Neil P; Pollard, Patrick J; Morton, Nicholas M; Finch, Andrew; Kranc, Kamil R

2017-03-06

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1 / Hoxa9 -driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation. © 2017 Guitart et al.

Quantification of Wilms' tumor 1 mRNA by digital polymerase chain reaction.

PubMed

Koizumi, Yuki; Furuya, Daisuke; Endo, Teruo; Asanuma, Kouichi; Yanagihara, Nozomi; Takahashi, Satoshi

2018-02-01

Wilms' tumor 1 (WT1) is overexpressed in various hematopoietic tumors and widely used as a marker of minimal residual disease. WT1 mRNA has been analyzed using quantitative real-time polymerase chain reaction (real-time PCR). In the present study, we analyzed 40 peripheral blood and bone marrow samples obtained from cases of acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome at Sapporo Medical University Hospital from April 2012 to January 2015. We performed quantification of WT1 was performed using QuantStudio 3D Digital PCR System (Thermo Fisher Scientific‎) and compared the results between digital PCR and real-time PCR technology. The correlation between digital PCR and real-time PCR was very strong (R = 0.99), and the detection limits of the two methods were equivalent. Digital PCR was able to accurately detect lower WT levels compared with real-time PCR. Digital PCR technology can thus be utilized to predict WT1/ABL1 expression level accurately and should thus be useful for diagnosis or the evaluation of drug efficiency in patients with leukemia.

Preventive and Therapeutic Strategies for Bovine Leukemia Virus: Lessons for HTLV

PubMed Central

Rodríguez, Sabrina M.; Florins, Arnaud; Gillet, Nicolas; de Brogniez, Alix; Sánchez-Alcaraz, María Teresa; Boxus, Mathieu; Boulanger, Fanny; Gutiérrez, Gerónimo; Trono, Karina; Alvarez, Irene; Vagnoni, Lucas; Willems, Luc

2011-01-01

Bovine leukemia virus (BLV) is a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1). BLV is a major animal health problem worldwide causing important economic losses. A series of attempts were developed to reduce prevalence, chiefly by eradication of infected cattle, segregation of BLV-free animals and vaccination. Although having been instrumental in regions such as the EU, these strategies were unsuccessful elsewhere mainly due to economic costs, management restrictions and lack of an efficient vaccine. This review, which summarizes the different attempts previously developed to decrease seroprevalence of BLV, may be informative for management of HTLV-1 infection. We also propose a new approach based on competitive infection with virus deletants aiming at reducing proviral loads. PMID:21994777

Gammaretrovirus-specific antibodies in free-ranging and captive Namibian cheetahs.

PubMed

Krengel, Annika; Cattori, Valentino; Meli, Marina L; Wachter, Bettina; Böni, Jürg; Bisset, Leslie R; Thalwitzer, Susanne; Melzheimer, Jörg; Jago, Mark; Hofmann-Lehmann, Regina; Hofer, Heribert; Lutz, Hans

2015-06-01

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

131I-Anti-CD45 Antibody Plus Busulfan and Cyclophosphamide before Allogeneic Hematophoietic Cell Transplantation for Treatment of Acute Myeloid Leukemia in First Remission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pagel, John M.; Appelbaum, Frederick R.; Eary, Janet F.

2006-03-01

In an attempt to improve outcomes for patients with acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (HCT), we conducted a Phase I/II study in which targeted irradiation delivered by 131I-anti-CD45 antibody was combined with targeted busulfan (BU; area-under-curve, 600-900 ng/ml) and cyclophosphamide (CY; 120 mg/kg). Fifty-two of 59 patients (88%) receiving a trace 131I-labeled dose of 0.5 mg/kg anti-CD45 murine antibody had higher estimated absorbed radiation in bone marrow and spleen than in any other organ. Forty-six patients were treated with 102-298 mCi 131I delivering an estimated 5.3-19 (mean 11.3) Gy to marrow, 17-72 (mean 29.7) Gy tomore » spleen, and 3.5 Gy (n=4) to 5.25 Gy (n=42) to the liver. The estimated 3-year non-relapse mortality and disease-free survival (DFS) were 21% and 61%, respectively. These results were compared to those from 509 similar International Bone Marrow Transplant Registry patients transplanted using BU/CY alone. After adjusting for differences in age and cytogenetics-risk, the hazard of mortality among all antibody-treated patients was 0.65 times that of the Registry patients (95% CI 0.39-1.08; p=.09). The addition of targeted hematopoietic irradiation to conventional BU/CY is feasible and well tolerated, and Phase II results are sufficiently encouraging to warrant further study.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Yu-Kyoung; Lee, Tae-Yoon; Choi, Jong-Soon

Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-α, PPAR-γ, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of thesemore » meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.« less

Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow

PubMed Central

Zhu, Jie; Wang, Hong; Yang, Shuo; Guo, Liqiao; Li, Zhen; Wang, Wei; Wang, Suhan; Huang, Wenting; Wang, Liping; Yang, Tan; Ma, Qiang; Bi, Yongyi

2013-01-01

Benzene is an occupational toxicant and an environmental pollutant that potentially causes hematotoxicity and leukemia in exposed populations. Epidemiological studies suggest an association between an increased incidence of childhood leukemia and benzene exposure during the early stages of pregnancy. However, experimental evidence supporting the association is lacking at the present time. It is believed that benzene and its metabolites target hematopoietic stem cells (HSCs) to cause toxicity and cancer in the hematopoietic system. In the current study, we compared the effects of hydroquinone (HQ), a major metabolite of benzene in humans and animals, on mouse embryonic yolk sac hematopoietic stem cells (YS-HSCs) and adult bone marrow hematopoietic stem cells (BM-HSCs). YS-HSCs and BM-HSCs were isolated and enriched, and were exposed to HQ at increasing concentrations. HQ reduced the proliferation and the differentiation and colony formation, but increased the apoptosis of both YS-HSCs and BM-HSCs. However, the cytotoxic and apoptotic effects of HQ were more apparent and reduction of colony formation by HQ was more severe in YS-HSCs than in BM-HSCs. Differences in gene expression profiles were observed in HQ-treated YS-HSCs and BM-HSCs. Cyp4f18 was induced by HQ both in YS-HSCs and BM-HSCs, whereas DNA-PKcs was induced in BM-HSCs only. The results revealed differential effects of benzene metabolites on embryonic and adult HSCs. The study established an experimental system for comparison of the hematopoietic toxicity and leukemogenicity of benzene and metabolites during mouse embryonic development and adulthood. PMID:23940708