scholarly journals Establishment of a FDC-P1 murine cell line with human KIT N822K gene overexpression

Acta Naturae ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 51-55
Author(s):  
Elmira R. Vagapova ◽  
T. D. Lebedev ◽  
V. I. Popenko ◽  
O. G. Leonova ◽  
P. V. Spirin ◽  
...  
Keyword(s):  
Cell Line ◽  
Cellular Response ◽  
Ectopic Expression ◽  
Model Systems ◽  
Leukemia Cells ◽  
Gene Encoding ◽  
Murine Cell Line ◽  
Therapeutic Drugs ◽  
Kit Gene ◽  
Stromal Cell Line

The mechanism of resistance of leukemia cells to chemotherapeutic drugs remains poorly understood. New model systems for studying the processes of malignant transformation of hematopoietic cells are needed. Based on cytokine-dependent murine acute myeloid leukemia (AML) FDC-P1 cells, we generated a new cell line with ectopic expression of the KIT gene encoding mutant human receptor tyrosine kinase (N822K). We investigated the role played by overexpression of the mutant KIT in the survival of leukemia cells and their sensitivity to therapeutic drugs. We also generated a co-culture system consisting of FDC-P1 murine leukemia cells and a HS-5 human stromal cell line. Our data can be used for a further comprehensive analysis of the role of KIT N822K mutation in the cellular response to anti-leukemic drugs, growth factors, and cytokines. These data are of interest in the development of new effective therapeutic approaches to the treatment of acute leukemia.

scholarly journals In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Hong Kiat Lim ◽  
Pravin Periasamy ◽  
Helen C. O’Neill
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Model Systems ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Stromal Cell Line ◽  
Reticular Cells

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1026-1026
Author(s):  
Erica A. Lehotzky ◽  
Mark Y. Chiang
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Leukemia Cells ◽  
Inhibitory Effects ◽  
Notch Inhibition

Abstract Abstract 1026 Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

The Novel Immunoproteasome Inhibitor PR-924: Anti-Leukemic Activity and Mechanisms Of Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3841-3841
Author(s):  
Denise Niewerth ◽  
Johan van Meerloo ◽  
Yehuda Assaraf ◽  
Gerrit Jansen ◽  
Tessa C Hendrickx ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Line ◽  
Hematological Malignancies ◽  
Acquired Resistance ◽  
Proteasome Inhibition ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Gene Encoding ◽  
Exon 2 ◽  
Subunit Expression

Abstract Proteasome inhibition with bortezomib (BTZ) is an effective treatment for hematological malignancies and an emerging treatment strategy for acute leukemia. However, the side-effects of BTZ and emergence of BTZ-resistance call for alternative therapeutic approaches. The immunoproteasome may serve as a candidate since its expression is higher than constitutive proteasome expression in cells of hematological malignancies, including acute lymphocytic leukemia (ALL). To this end, the new immunoproteasome inhibitor PR-924 may elicit anti-leukemic activity, since it bears greater specificity for the β5i subunit of the immunoproteasome compared to other proteasome inhibitors including BTZ and carfilzomib. The aim of the current study was to examine the proteasome inhibition capacity and cell growth inhibitory impact of PR-924 in acute leukemia cells and sublines with acquired resistance to BTZ. We further determined whether PR-924 itself would be prone to resistance development and if so, to explore the underlying molecular basis. To assess the anti-leukemic activity of PR-924, its cytotoxicity was determined in two human cell lines of hematological origin: the T-ALL cell line CCRF-CEM and the acute myeloid leukemia cell line THP1, and their 150-fold BTZ-resistant sublines CEM/BTZ200, and THP1/BTZ200, respectively. Parental CEM and THP1 cells displayed similar sensitivity to PR-924 (IC50 CEM: 1.8 µM ± 0.4, THP1: 1.5 µM ± 0.2), whereas their BTZ-resistant lines displayed a moderate 10-12 fold cross-resistance to PR-924 (IC50 CEM/BTZ200: 21.1 µM ± 0.5, THP1/BTZ200: 15.6 µM ± 1.6). Flow cytometric analysis revealed that PR-924-induced cell death was mediated by induction of apoptosis. Moreover, PR-924 exposure resulted in a (up to 50%) reduction of cell surface expression of HLA Class I. To determine whether PR-924 activity relies on specific inhibition of β5i, proteasome activity inhibition experiments were performed over a range of 10 nM – 10 µM PR-924. Notably, in parental cells, β5i activity was already inhibited by 20% at 10 nM PR-924 and > 90% inhibition was achieved at 100 nM PR-924. At higher PR-924 concentrations of 1-10 µM, inhibition of both β5 and β1i activities was observed, thus indicating that PR-924 blocks the β5i activity far below concentrations that exert anti-proliferative activity (1.5 µM), consistent with earlier studies of PR-924 (Parlati et al. Blood, 2009). Next, acquired resistance to PR-924 was provoked in CEM and THP1 cells by step-wise increasing PR-924 concentrations in cell culture, starting at the IC50 concentrations. Following this process, CEM cells resistant to 20 µM PR-924 (CEM/PR20) and THP1 cells resistant to 12 mM PR-924 (THP1/PR12) exhibited IC50 values of 22.1 µM PR-924 (resistance factor 13) and 14.3 µM PR-924 (resistance factor 10), respectively. In addition, these PR-924 resistant cells displayed 10-fold cross-resistance to BTZ. To explore mechanisms of PR-924 resistance, we first sequenced exon 2/3 of the PSMB8 gene (encoding β5i), a similar functioning coding region that was previously reported to harbor PSMB5 mutations in BTZ-resistant leukemia cells (Franke et al. Leukemia, 2012). However, no mutations were found in PSMB8 exon 2/3. Remarkably, we did identify mutations in exon 2 of the PSMB5 gene (encoding the S1 pocket of β5) in both CEM/PR20 (Met45Ile) and of THP1/PR12 (Ala49Thr); these amino acid substitutions are similar to those observed in BTZ-resistant sublines. Finally, we characterized proteasome subunit expression by ProCISE analysis in the PR-924 resistant cell lines. This method is a subunit-specific active-site ELISA assay, which utilizes a purified proteasome standard curve to calculate ng of subunit per µg total protein. Expression of constitutive proteasome subunits in CEM/PR20 and THP1/PR8 cells were upregulated up to 2.5-fold, compared to their parental counterparts, whereas immunoproteasome subunit expression was moderately decreased (up to 2-fold) in CEM/PR20 and increased in THP1/PR8 (up to 1.6-fold). In conclusion, PR-924 displayed significant anti-leukemic activity. Although there was cross-resistance to BTZ, PR-924 retained activity in BTZ-resistant leukemia cells. Despite PR-924 specificity to the β5i subunit of the proteasome, its anti-leukemic effect requires concentrations that block both β5 and β5i subunits. This notion is underscored by emergence of acquired mutations in PSMB5 rather than in PSMB8. Disclosures: Kirk: Onyx Pharmaceuticals: Employment, Equity Ownership. Anderl:Onyx: Employment.

scholarly journals Characteristics of live parameters of the HS-5 human bone marrow stromal cell line cocultured with the leukemia cells in hypoxia, for the studies of leukemia-stroma cross-talk

2018 ◽  
Vol 93 (9) ◽  
pp. 929-940
Author(s):  
Paulina Podszywalow-Bartnicka ◽  
Agata Kominek ◽  
Magdalena Wolczyk ◽  
Marta D. Kolba ◽  
Julian Swatler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cross Talk ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Leukemia Cells ◽  
Marrow Stromal Cell ◽  
Stromal Cell Line

A Novel B Cell Precursor ALL Cell Line (018Z) with Prominent Neurotropism and Isolated CNS Leukemia in a NOD/SCID/huALL Xenotransplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1630-1630 ◽  
Author(s):  
Eckhoff M. Eckhoff ◽  
Manon Queudeville ◽  
Klaus-Michael Debatin ◽  
Lueder H. Meyer
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Rapid Onset ◽  
Scid Mice ◽  
Model Systems ◽  
Leukemia Cells ◽  
Cell Precursor ◽  
Cns Disease ◽  
Two Samples ◽  
Xenotransplantation Model

Abstract Abstract 1630 Poster Board I-656 Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Despite efforts achieved in treatment resulting in improved long term survival about 20% of the patients encounter relapse of their disease. Involvement of the central nervous system (CNS) is a common feature at diagnosis but also at relapse. About 10% to 20% of relapse patients present with isolated CNS leukemia as first event mainly occurring early within 24 months after diagnosis and associated with a survival of about 50%. However, achievement of effective CNS control is essential for successful treatment. Model systems to study the mechanisms of CNS leukemia but also new treatment strategies are required. Here we present and characterize a new pediatric B cell precursor (BCP) ALL cell line (018Z) which was established by passage in a NOD/SCID/huALL xenotransplantation model and exhibits a distinct CNS phenotype if re-transplanted onto NOD/SCID mice. Leukemia cells isolated from a male, adolescent patient at diagnosis were transplanted onto unconditioned NOD/SCID mice by intravenous injection. The patient had no CNS disease at diagnosis, however he died in remission after induction therapy. Upon onset of leukemia in the recipients the animals were killed and the engrafted leukemia cells were isolated and further transplanted onto secondary and tertiary recipients. Cells isolated from subsequent recipients were transferred into cell culture flasks and kept under standard culture conditions in RPMI medium supplemented with 20% fetal calf serum. Autonomous proliferation of the cells was observed for now more than three years. The cell line remained immunophenotypically stable as regularly assessed by flowcytometry (every 20 passages, from cell line passage 20 up to 160) showing the same BCP-ALL subtype. Infection of the cells with Epstein Barr Virus or Human Herpes Virus 8 was ruled out. Cytogenetic analysis of the cell line revealed presence of 47, XY, +8, del(9)(p13). The same karyotype was found in two samples isolated from previous xenograft passages (P4 and P6) and in leukemia cells obtained at diagnosis. The same karyotype was also detected if the 018Z cell line cells were re-transplanted and leukemia cells isolated from the recipients. Interestingly, at diagnosis two additional karyotypes were observed but could not be detected in the xenografts or cell line indicating selection and subsequent proliferation of one clone engrafted [47, XY, +8, del(9)(p13)]. Interestingly, chromosome arm 9p abnormalities have been reported to be associated with adverse events in pediatric BCP ALL, including onset of CNS leukemia (Heerema et al., Blood, 1996, 94(5): 1537-44). The cell line was further characterized and re-transplanted in the NOD/SCIDhuALL model. Unlike leukemia manifestation observed after transplantation of other samples, recipients transplanted with the 018Z cell line presented with a rapid onset of symptoms like seizures and palsies of the limbs pointing to CNS involvement. Infiltration of BCP ALL cells in the CNS of the recipients was detected by immunohistochemistry of the brain and by flowcytometry in cell suspensions prepared from the mice' meninges while only below 5% of human leukemia cells were detected in peripheral blood, spleen or bone marrow. Taken together, we established a novel BCP ALL cell line showing manifestation of CNS leukemia with rapid onset of about 6 weeks after re-transplantation onto NOD/SCID mice and providing a model system to study CNS disease in ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Molecular Cloning and Biological Characterization of a Novel Murine Lymphoid Growth Factor

2000 ◽  
Vol 192 (5) ◽  
pp. 671-680 ◽  
Author(s):  
John E. Sims ◽  
Douglas E. Williams ◽  
Philip J. Morrissey ◽  
Kirsten Garka ◽  
Diane Foxworthe ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cell ◽  
Biological Characterization ◽  
Gene Encoding ◽  
Interleukin 7 ◽  
Stromal Cell Line ◽  
Thymic Stromal Cell

Using a bioassay consisting of the proliferation of a murine B cell line, a cDNA of a gene whose product supports the growth of that cell line was isolated from a thymic stromal cell line. This factor, termed thymic stromal lymphopoietin (TSLP), is a protein of 140 amino acids. The gene encoding TSLP was mapped to murine chromosome 18. Purified recombinant TSLP supported the growth of pre-B cell colonies in vitro, but had no myelopoietic activity. TSLP had comitogenic activity for fetal thymocytes, but was not as potent as interleukin 7 in lobe submersion cultures. Injection of TSLP into neonatal mice induced the expansion of B220+BP-1+ pre-B cells.

Stromal Cell Line Enhances Survival of Myeloid Leukemia Cells by Regulating the Expression of bcl-2 Family Genes and Caspase-3.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4498-4498
Author(s):  
Zhenshu Xu ◽  
Yongjun Liu ◽  
Zhibo Han ◽  
Lulu Lv ◽  
Ting Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Caspase 3 ◽  
Serum Deprivation ◽  
Leukemia Cells ◽  
Late Relapse ◽  
Stromal Cell Line

Abstract Approximately 20–30% acute myeloid leukemia (AML) patients are not cured by traditional chemotherapy. The possible explanation is that residual leukemia cells are harbored in association with stromal cells in the bone marrow, which causes late relapse of leukemia. This study was designed to investigate interactions between marrow stromal microenvironment and leukemia cells. AML cells were cultured in different conditions with or without serum, Ara-c, the monolayers or the conditioned medium of a stromal cell line HESS-5. Apoptosis frequencies, cell viability, cell cycle distribution, DNA fragment, and bcl-2/bcl-xl gene as well as caspase-3 expression were determined by Typan blue exclusion, MTT assay, flow cytometry analysis, PCR and Western blot respectively. Direct contact of HL-60 cells with HESS-5 significantly inhibited serum-deprivation- or Ara-c-induced apoptosis of HL-60 cells, and resulted in a significant increase of short-term proliferation and viability. Soluble factors had negligible effect. The interaction of HL-60 with fibronectin did not confer a significant resistance to serum-deprivation and Ara-c agent. HESS-5 cells were also able to maintain cell cycle progression of HL-60 during chemotherapy exposure. HESS-5 cells-mediated apoptosis inhibition was associated with increased bcl-2/bcl-xl expression and down-regulation of caspase-3 in primary myeloid leukemia cells. These results indicate that stromal cells enhance the survival of myeloid leukemia cells by regulating bcl-2 family genes as well as caspase-3 protein and suggest that specific interactions between stromal cells and leukemia cells may enhance the resistance of leukemia cells to chemotherapy.

scholarly journals The Nuclear Remodeling Induced by Helicobacter Cytolethal Distending Toxin Involves MAFB Oncoprotein

Toxins ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 174
Author(s):  
Christelle Péré-Védrenne ◽  
Wencan He ◽  
Lamia Azzi-Martin ◽  
Valérie Prouzet-Mauléon ◽  
Alice Buissonnière ◽  
...  
Keyword(s):  
Cellular Response ◽  
Target Genes ◽  
Ectopic Expression ◽  
Cell Periphery ◽  
Cytolethal Distending Toxin ◽  
Dna Double Strand Breaks ◽  
Gene Encoding ◽  
Γh2ax Foci ◽  
Hepatic Diseases

Enterohepatic Helicobacters, such as Helicobacter hepaticus and Helicobacter pullorum, are associated with several intestinal and hepatic diseases. Their main virulence factor is the cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro in HT-29 intestinal cells while following the ectopic expression of the active CdtB subunit of H. hepaticus CDT. A CdtB-dependent upregulation of the V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene encoding the MAFB oncoprotein was found, as well as the CdtB-dependent regulation of several MAFB target genes. The transduction and coculture experiments confirmed MAFB mRNA and protein induction in response to CDT and its CdtB subunit in intestinal and hepatic cell lines. An analysis of MAFB protein subcellular localization revealed a strong nuclear and perinuclear localization in the CdtB-distended nuclei in intestinal and hepatic cells. MAFB was also detected at the cell periphery of the CdtB-induced lamellipodia in some cells. The silencing of MAFB changed the cellular response to CDT with the formation of narrower lamellipodia, a reduction of the increase in nucleus size, and the formation of less γH2AX foci, the biomarker for DNA double-strand breaks. Taken together, these data show that the CDT of enterohepatic Helicobacters modulates the expression of the MAFB oncoprotein, which is translocated in the nucleus and is associated with the remodeling of the nuclei and actin cytoskeleton.

Method for Obtaining Daedaleopsis confragosa K-1326 Extracts with Antitumor Activity

2019 ◽  
Vol 35 (1) ◽  
pp. 68-72
Author(s):  
T.V. Teplyakova ◽  
E.A. Vyazovaya ◽  
E.D. Danilenko
Keyword(s):  
Antitumor Activity ◽  
Cell Line ◽  
Carcinoma Cell ◽  
The State ◽  
Carcinoma Cell Line ◽  
Epidermoid Carcinoma ◽  
State Assignment ◽  
Therapeutic Drugs ◽  
Prevention And Treatment ◽  
Epidermoid Carcinoma Cell Line

A method for extraction and fractionation of proteins and polysaccharides from the biomass of the basidial fungus Daedaleopsis confragosa K-1326, having antitumor activity, shown on the epidermoid carcinoma cell line A431 and the cells of melanoma В16, has been developed. This method allows for reproducible production of series of preparations and can be used to obtain preparations from other basidiomycetes. The results obtained in this work show the promising prospects for the development and use of drugs from basidiomycetes for treatment of cancer. Basidiomycetes, Daedaleopsis confragosa, antitumor activity, fractionation, extraction The work was performed under the State Assignment «Development of preventive and therapeutic drugs based on basidiomycetes for the prevention and treatment of influenza with pandemic potential».

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