LAMP5 - a Novel Target of MLL-Fusion Proteins Is Required for the Propagation of Leukemia

Abstract Acute leukemias with Mixed Lineage Leukemia gene (MLL, also called KMT2A) translocations are associated with poor outcomes. These leukemias are most frequently encountered in infants and as secondary malignancies, and can be either lymphoid or myeloid. In spite of aggressive treatments, including bone marrow transplantation, infants with MLL-leukemias face a grim prognosis, with predicted survival of only 20%. Novel, effective therapies are thus urgently needed. In search for molecular targets, we observed that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5, also known as C20orf103). This observation was consistent across several gene-expression-profiling studies and occurred in both ALL and AML. Moreover, data from the TCGA study on AML showed that patients with LAMP5 expression suffered worse prognosis compared to those lacking LAMP5. We first confirmed LAMP5 expression in human leukemia cell lines by immunoblot and RT-qPCR assays. We readily detected LAMP5 mRNA and protein in MLL-fusion leukemia cell lines (MV4;11, MOLM13, THP1, RS4;11, KOPN8), while no expression of LAMP5 was found in the non-MLL-cell lines (Kasumi, K562, REH). Published ChIP-seq studies on leukemia cell lines show the MLL-fusion protein directly bound at the promotor region of LAMP5. To validate that the MLL-fusion protein activates the expression of LAMP5, we transformed human CD34+ cord blood cells with an inducible (Tet-off) retrovirus carrying the MLL-AF9 fusion cDNA. In this system, addition of Doxycycline represses expression of the MLL-AF9 oncogene. We found that LAMP5 expression directly correlated with MLL-AF9 levels, with levels of both decreasing upon addition of Doxycycline. To investigate the role of LAMP5 in MLL-fusion leukemia, we studied the effect of shRNA-mediated knockdown. By screening several hairpin sequences, we identified one construct that efficiently inhibited LAMP5 expression in MLL-fusion leukemia cells but had no effect on LAMP5-negative cells, implying specificity. All MLL-fusion leukemia cell lines tested showed growth inhibition with LAMP5 knockdown. Specifically, growth of MV4;11, THP1 and MOLM13 cells was decreased by 69%, 73% and 80% respectively compared to controls (non-targeting shRNA). When cultured in semi-solid methylcellulose media for 10 days, LAMP-5-depleted MV4;11 cells formed significantly fewer colonies than control cells (64.3 ± 25.98 and 245.3 ± 27.42 colonies per 1000 cells respectively). To investigate the role of LAMP5 in leukemia-propagation in vivo, we transplanted control and LAMP5-depleted MV4;11 cells into Busulfan-conditioned immune-deficient (NRGS) mice (2x105cells/mouse). Preliminary results from bone marrow aspirates of transplanted mice at weeks 4 post-transplant showed abundant human leukemia cells in mice receiving control cells while the mice receiving LAMP5-knockdown cells showed near-absence of human leukemia cells. Collectively, these results demonstrate that LAMP5, a novel target of MLL-fusion proteins is required for the propagation of leukemia. In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it localizes to the ER-Golgi intermediate compartment (ERGIC). In leukemia cells, using immunofluorescent confocal microscopy we detected LAMP5 in the perinuclear zones where it co-localized with ERGIC-53, a marker of the ERGIC compartment. While little is known about the functions of LAMP5 in normal pDC, studies suggest that it functions as a co-chaperone with UNC93B1, a known Toll-like Receptor (TLR) chaperone, to shuttle the TLRs to their respective locations in the plasma membrane or endosomes. In ongoing experiments, we are determining the functions of LAMP5 in leukemia, including its association with UNC93B1 and with the TLR-NFKB signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we postulate that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias. Disclosures No relevant conflicts of interest to declare.

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Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL

Blood ◽

10.1182/blood.v87.9.3837.bloodjournal8793837 ◽

1996 ◽

Vol 87 (9) ◽

pp. 3837-3843 ◽

Cited By ~ 96

Author(s):

A Benito ◽

M Silva ◽

D Grillot ◽

G Nunez ◽

JL Fernandez-Luna

Keyword(s):

Cell Death ◽

Cell Lines ◽

Leukemia Cell ◽

K562 Cells ◽

Erythroid Differentiation ◽

Leukemia Cells ◽

Human Leukemia ◽

Human Leukemia Cells ◽

Leukemia Cell Lines ◽

Wide Range

The induction of tumor cell differentiation represents an attractive strategy for the treatment of a wide range of malignancies. Differentiation of HL-60 promyelocytic leukemia cells towards neutrophils or monocytes has been shown to induce apoptotic cell death, which is inhibited by bcl-2 over-expression. However, the role of the bcl-2 gene family during erythroid differentiation of human leukemia cells remains unknown. We found that human erythroleukemia (HEL) and K562, two leukemia cell lines that undergo erythroid differentiation do not express Bcl-2, but express Bcl-XL, a related protein that functions as an inhibitor of apoptosis. Differentiation of HEL or K562 cells with inducers of erythroid differentiation (hemin, retinoic acid, or transforming growth factor-beta) was accompanied by progressive cell death and degradation of genomic DNA into oligonucleosomal fragments. The loss of cellular viability was associated with downregulation of bcl-xL mRNA and protein. In contrast, the levels of Bax, another Bcl-2 family member implicated in apoptosis remained unaltered. Constitutive expression of Bcl-XL by gene transfer inhibited apoptosis triggered by erythroid differentiation of HEL K562 cells. Yet, Bcl-XL did not alter the expression of epsilon-globin, which is induced during erythoid differentiation of HEL and K562 cells, arguing that apoptosis and differentiation can be uncoupled by Bcl-XL. These results indicate that Bcl-XL acts as an antiapoptosis protein in leukemia cells that undergo erythroid differentiation and that downregulation of bcl-x is a component of the apoptotic response that is coupled to differentiation in human leukemia cells.

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Imidazo[1,2-b]pyrazole-7-carboxamides Induce Apoptosis in Human Leukemia Cells at Nanomolar Concentrations

Molecules ◽

10.3390/molecules23112845 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2845 ◽

Cited By ~ 7

Author(s):

Gábor Szebeni ◽

József Balog ◽

András Demjén ◽

Róbert Alföldi ◽

Vanessza Végi ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Leukemia Cells ◽

Late Response ◽

Human Leukemia ◽

Altered Expression ◽

Human Leukemia Cells ◽

Leukemia Cell Lines ◽

Mitochondrial Membrane Depolarization ◽

Ic50 Values

Leukemia, the malignancy of the hematopoietic system accounts for 10% of cancer cases with poor overall survival rate in adults; therefore, there is a high unmet medical need for the development of novel therapeutics. Eight imidazo[1,2-b]pyrazole-7-carboxamides have been tested for cytotoxic activity against five leukemia cell lines: Acute promyelocytic leukemia (HL-60), acute monocytic leukemia (THP-1), acute T-lymphoblastic leukemia (MOLT-4), biphenotypic B myelomonocytic leukemia (MV-4-11), and erythroleukemia (K-562) cells in vitro. Imidazo[1,2-b]pyrazole-7-carboxamides hampered the viability of all five leukemia cell lines with different potential. Optimization through structure activity relationship resulted in the following IC50 values for the most effective lead compound DU385: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11 cells, respectively. Human primary fibroblasts were much less sensitive in the applied concentration range. Both monolayer or spheroid cultures of murine 4T1 and human MCF7 breast cancer cells were less sensitive to treatment with 1.5–10.8 μM IC50 values. Flow cytometry confirmed the absence of necrosis and revealed 60% late apoptotic population for MV-4-11, and 50% early apoptotic population for HL-60. MOLT-4 cells showed only about 30% of total apoptotic population. Toxicogenomic study of DU385 on the most sensitive MV-4-11 cells revealed altered expression of sixteen genes as early (6 h), midterm (12 h), and late response (24 h) genes upon treatment. Changes in ALOX5AP, TXN, and SOD1 expression suggested that DU385 causes oxidative stress, which was confirmed by depletion of cellular glutathione and mitochondrial membrane depolarization induction. Imidazo[1,2-b]pyrazole-7-carboxamides reported herein induced apoptosis in human leukemia cells at nanomolar concentrations.

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mTOR Inhibitor Rapamycin Interacts Synergistically with Farnesyltransferase Inhibitor FTI-277 To Induce Growth Inhibition in Human Leukemia Cells.

Blood ◽

10.1182/blood.v110.11.1821.1821 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1821-1821 ◽

Cited By ~ 1

Author(s):

June-Won Cheong ◽

Ju In Eom Eom ◽

Hye Won Lee ◽

In-Hae Park ◽

Yuri Kim ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Mtor Inhibitor ◽

Leukemia Cell ◽

Leukemia Cells ◽

Human Leukemia ◽

Farnesyltransferase Inhibitor ◽

Human Leukemia Cells ◽

Leukemia Cell Lines ◽

Primary Leukemia

Abstract Proteins regulating the mammalian target of rapamycin (mTOR), as well as some of the targets of the mTOR kinase, are overexpressed or mutated in cancer. Rapamycin inhibits the growth of cell lines derived from multiple tumor types in vitro, and tumor models in vivo. However, it has been suggested that substantial proportion of acute myeloid leukemia (AML) cells showed resistance to rapamycin-induced growth inhibition. Aim: We aim to investigate the effects of the farnesyltransferase inhibitor (FTI)-277 on the rapamycin-induced growth inhibition of human leukemia cells. Patients and methods: Flow cytometric evaluation and Western blot analysis for mTOR and Ras-like GTPase Rheb expression in the leukemia cell lines (HL60,NB4,THP1,KG1,U937) and primary leukemia cells obtained from AML patients were performed. We also observed the inhibition of cell growth and the changes in expression of mTOR and up- or down-streams of mTOR after mTOR inhibitor rapamycin treatment with or without FTI-277. Results: Both flow cytometric evaluation and Western blot analysis demonstrated that mTOR expression in the leukemia cell lines (HL60, NB4, THP1, KG1, U937) and primary leukemia cells obtained from AML patients were significantly higher compared to normal bone marrow mononuclear cells (p<0.001). Expression of Ras-like GTPase Rheb, a mTOR upstream, was also significantly increased in the leukemia cell lines and primary AML cells compared to normal bone marrow mononuclear (p<0.001 and p<0.005, respectively). We observed the inhibition rate of leukemia cell growth after treatment of cells with mTOR inhibitor rapamycin (100mM) in the absence or presence of farnesyltransferase inhibitor FTI-277 (10mM). Clonogenic cell growth in the leukemia cell lines was 69.3 ± 5.3% in the rapamycin group and 78.7 ± 4.4% in the FTI-277 group compared to that of the control group. Cotreatment of THP1 and HL-60 leukemia cells with rapamycin and FTI-277 exerted synergistic decrease in the clonogenic cell growth, as well as arrest at the G2/M phase of cell cycle, in a dose-and time-dependent manner (p<0.01). This was associated with marked attenuation of protein levels of Rheb, phospho-mTOR, and mTOR downstreams phospho-p70S6 kinase, phospho-4E-BP1. Interestingly decreased expression of mTOR upstreams Akt/PKB activity, Akt/PKB phosphorylation and PTEN phosphorylation was also observed in these leukemia cells after cotreatment with FTI-277 and rapamycin. These findings were also observed in the primary leukemia cells obtained from untreated patients with AML. Conclusions: Taken together, these findings indicate that farnesyltransferase inhibitor FTI-277 potentially enhance the growth-inhibitory property of rapamycin, with inducing multiple perturbations in PI3K - Akt/PKB - mTOR signaling pathway in human leukemia cells. Combined rapamycin and FTI blockade can exert powerful anti-leukemia effects and could be developed into a novel therapeutic strategy for AML.

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Evaluation of the Role of PlGF and the Therapeutic Potential of Anti-PlGF in BCR/ABL+ Leukemia.

Blood ◽

10.1182/blood.v112.11.1094.1094 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1094-1094

Author(s):

Sonja Loges ◽

Thomas Schmidt ◽

Maria Kleppe ◽

Kim De Keersmaecker ◽

Marc Tjwa ◽

...

Keyword(s):

Bone Marrow ◽

Solid Tumors ◽

Cell Lines ◽

Stromal Cells ◽

Therapeutic Potential ◽

Leukemia Cell ◽

Leukemia Cells ◽

Significant Prolongation ◽

Leukemia Cell Lines

Abstract Introduction of imatinib and second-generation BCR/ABL inhibitors has revolutionized treatment of patients with Philadelphia chromosome positive (Ph+) leukemia, but leukemia cells persist even in successfully treated patients, and some patients develop resistance and ultimately relapse. The reasons for these drawbacks are not entirely resolved, but besides BCR/ABL the host stroma potentially plays an important (independent) role. Placental Growth Factor (PlGF), a homologue of VEGF, was already proven to be abundantly secreted by stromal cells in solid tumors. Therefore, we investigated the role of PlGF, and the therapeutic potential of αPlGF, a monoclonal antibody against PlGF, which we recently reported to have a broad anti-tumoral potential in pre-clinical models of solid tumors (Fischer et al., Cell, 2007), in BCR/ABL+ lymphoid and myeloid leukemia. First, we studied expression of PlGF by 5 different human and murine BCR/ABL+ leukemia cell lines (Bv-173, BaF3, 32D, K562, KCL22) in vitro and found that neither of the cell lines secreted PlGF protein, but expressed its target receptor VEGFR-1. In contrast, primary murine adherent bone marrow stromal cells (BMDSC) expressed abundant amounts of PlGF protein (up to 105 pg/ml/105 cells), indicating a potential stroma-related function of PlGF. Second, we analyzed whether PlGF could induce proliferation and thereby revealed dose-dependent induction of proliferation by recombinant PlGF in all analyzed leukemia cell lines. This pro-proliferative effect of PlGF was nearly completely abrogated by both αPlGF and an extracellular anti-VEGFR-1 antibody, thus indicating that it is mediated primarily by VEGFR-1. Third, we studied potential paracrine interactions between BMDSCs and leukemia cells by performing co-culture experiments. Remarkably, coculture of BMDSC with leukemia cells significantly induced proliferation of both cell types. We hypothesized, that this induction of proliferation might be mediated by PlGF and indeed found its nearly complete abrogation upon addition of αPlGF to the co-cultures. Furthermore, BMDSCs significantly upregulated PlGF secretion (2.1 fold; N=3; P=0.005) when cultured in presence of leukemia cells. Thus, we conclude, that stromal derived PlGF promotes proliferation of leukemia cells in a paracrine fashion and at the same time acts as autocrine pro-proliferative signal for stromal cells. To test this hypothesis in vivo, we established 3 different murine models of BCR/ABL+ myeloid and lymphoid leukemia. Subsequently, we analyzed PlGF protein as present in blood and bone marrow of diseased mice in comparison to healthy mice, and detected no PlGF protein in the peripheral blood of healthy mice and low amounts of PlGF protein in their bone marrow. In contrast, leukemic mice showed PlGF protein (76.5 ± 18.4 pg / ml plasma; N=7) in their circulation at levels comparable to mice bearing solid tumors, and, interestingly more than 8.9 fold (N=7; P<0.0001) elevated PlGF levels in their bone marrow, compared to healthy mice, again indicating that PlGF represents a stroma derived, novel pathogenetic factor in Ph+ leukemia. In order to investigate the therapeutic potential of PlGF inhibition in murine BCR/ABL+ leukemia, we treated mice bearing leukemia induced by syngeneic BCR/ABL+ BaF3 cells with αPlGF, and found a significant prolongation of median survival by 18 days (N=9; P=0.015), compared to control antibodies. Encouraged by these positive results, we established models of imatinib-sensitive and imatinib-resistant (T315I mutant) CML by transducing primary bone marrow cells and subsequent transplantation into lethally irradiated recipient mice. Interestingly, also in these aggressive models, we found a significant prolongation of survival of diseased mice induced by blockade of PlGF compared to controls (median survival prolongation in wt BCR/ABL induced leukemia 5 days; N=11; P=0.002; in T315I mutant 4 days; N=12; P=0.039). Bone marrow histology and phenotypic analysis by FACS revealed decreased infiltration of spleen and bone marrow with leukemia cells (reduction in the bone marrow by 38% and in the spleen by 24%). In conclusion, these data indicate that PlGF represents a stromal derived factor promoting the progression of Ph+ leukemia, independent of BCR/ABL mutational status, and might represent a novel target produced by the leukemic stroma, potentially useful adjunct to BCR/ABL kinase inhibitors.

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Autocrine loop through cholecystokinin-B/gastrin receptors involved in growth of human leukemia cells

Blood ◽

10.1182/blood.v88.7.2683.bloodjournal8872683 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2683-2689 ◽

Cited By ~ 24

Author(s):

N Iwata ◽

T Murayama ◽

Y Matsumori ◽

M Ito ◽

A Nagata ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Receptor Expression ◽

Leukemia Cells ◽

Human Leukemia ◽

Trophic Effect ◽

Gastrin Receptor ◽

Receptor Mrna ◽

Human Leukemia Cells ◽

Leukemia Cell Lines

The cholecystokinin (CCK)-B/gastrin receptor binds two brain-gut hormones, CCK and gastrin, with high affinities. These peptides have a trophic effect on gastrointestinal cells expressing the receptor in vivo as well as in vitro. Recently, this receptor mRNA was reported to be expressed in immunocytes localized in the lamina propria of normal rat stomach mucosa. Here, we studied the receptor expression in human hematopoietic cells in order to determine whether they play a role in cell growth. The CCK-B/gastrin receptor mRNA was detectable in the polymorphonuclear (PMN) cells but not in the mononuclear cells of normal peripheral white blood cells by reverse transcription-polymerase chain reaction. The receptor transcript was, however, expressed in human leukemia cell lines (14 of 18 cell lines tested) derived from not only myeloid, but also T- and B-lymphoid lineages. The CCK-B/gastrin receptors on several leukemia cell lines were shown to be biologically active by demonstrating ligand-dependent cell proliferation in serum-deprived medium. Interestingly, a human CCK-B/gastrin receptor specific antagonist, YM022, but not its stereotype isoform, selectively inhibited the DNA synthesis of THP-1, MOLT-16, MOLT-14, and CCRF-CEM in the absence of exogenous peptide ligands. Further investigation revealed that these leukemia cell lines and normal PMN cells also expressed gastrin mRNA. These results suggest that growth of human leukemia cells is promoted by an autocrine mechanism through the CCK-B/gastrin receptors.

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Targeting the Mnk Pathway Enhances Chemotherapy-Induced Antileukemic Responses.

Blood ◽

10.1182/blood.v112.11.1617.1617 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1617-1617

Author(s):

Jessica K Altman ◽

Heather Glaser ◽

Amanda Redig ◽

Antonella Sassano ◽

Martin S Tallman ◽

...

Keyword(s):

Bone Marrow ◽

Map Kinase ◽

Cell Lines ◽

Negative Feedback ◽

Peripheral Blood ◽

Leukemia Cell ◽

P38 Map Kinase ◽

Human Leukemia ◽

Inhibitory Effects ◽

Leukemia Cell Lines

Abstract Mnk kinases (mitogen-activated protein kinase [MAPK]-interacting kinases) are downstream effectors of Map kinase pathways, including the MEK/Erk and the p38 Map kinase signaling cascades. We have previously shown that Mnk kinases and the p38 Map kinase pathway are activated in a negative feedback regulatory manner during treatment of cells with arsenic trioxide, and that molecular or pharmacological inhibition of their activation enhances arsenic trioxide-dependent apoptosis and antileukemic responses (J Biol Chem. 283:12034–42, 2008, and Cancer Res. 66:6763–71, 2006). We examined the activation status of Mnk kinases in response to treatment of AML cells with chemotherapy and the function of these kinases in the generation of antileukemic responses. The human AML cell lines U937, K562, or MM6 were treated with cytarabine, in the presence or absence of a commercially available pharmacological Mnk-1 inhibitor (Calbiochem); and the phosphorylation of Mnk and its downstream effector, eIF4E, were assessed. Treatment with cytarabine increased phosphorylation of Mnk and eIF4E. The cytarabine-dependent eIF4E phosphorylation was blocked when human leukemia cell lines were treated with the pharmacologic Mnk inhibitor, indicating that Mnk regulates eIF4E activity. Such phosphorylation was also found to be defective in Mnk1/Mnk2 double knockout mouse embryonic fibroblast (MEF) cells, as compared to wild-type MEFs. Importantly, cytarabine-induced apoptosis was strongly enhanced in Mnk1−/− Mnk2−/− MEFs, as compared to Mnk1+/+Mnk2+/+ MEFs. To define the role of Mnk kinases in the generation of chemotherapy-induced antileukemic responses, human leukemia cell lines and bone marrow or peripheral blood mononuclear cells from patients with AML were used in clonogenic assays in methylcelluose to determine the effects of Mnk inhibition in the cytarabine-mediated leukemic progenitor (CFU-L) growth. The Mnk inhibitor potentiated the inhibitory effects of cytarabine on U937-derived CFU-L colonies and bone marrow or peripheral blood-derived CFU-L from 3 patients with AML. Interestingly, combinations of the Mnk inhibitor with the mTOR inhibitor, rapamycin, also resulted in more pronounced inhibitory effects on CFU-L colony formation than each agent alone. Altogether, these findings demonstrate that the Mnk pathway is activated during treatment of AML cells with cytarabine and that such activation occurs in a negative feedback regulatory manner to counteract the antileukemic effects of cytarabine. They also raise the possibility that targeting Mnk kinases may provide a novel approach to enhance the effects of chemotherapy on AML cells in vitro and possibly in vivo.

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Expression of retinoic acid receptor alpha mRNA in human leukemia cells

Blood ◽

10.1182/blood.v74.1.99.bloodjournal74199 ◽

1989 ◽

Vol 74 (1) ◽

pp. 99-102 ◽

Cited By ~ 1

Author(s):

C Largman ◽

K Detmer ◽

JC Corral ◽

FM Hack ◽

HJ Lawrence

Keyword(s):

Retinoic Acid ◽

Cell Lines ◽

Retinoic Acid Receptor ◽

Leukemia Cell ◽

Leukemia Cells ◽

Human Leukemia ◽

Retinoic Acid Receptor Alpha ◽

Acid Receptor ◽

Receptor Alpha ◽

Human Leukemia Cells

The expression of the newly described human retinoic acid receptor alpha (RAR alpha) in six nonlymphoid and six lymphoid leukemia cell lines and nine freshly obtained samples of leukemia cells from patients with acute nonlymphoid leukemia was assessed by Northern blot analysis, using a full length cDNA clone of RAR alpha as probe. RAR alpha was expressed in all 12 cell lines and in all fresh leukemia samples as two major transcripts of 2.6 and 3.5 kb in size. Levels of RAR alpha expression and transcript sizes in retinoid-sensitive cells (such as HL60 or fresh promyelocytic leukemia cells) were not different from those in other samples. Moreover, expression of RAR alpha was not significantly modulated by exposure to cis-retinoic acid (cisRA) in either cisRA-responsive or unresponsive cells. By using a 3′ fragment of the RAR alpha gene as a probe, we confirmed that the transcripts visualized did not represent the homologous RAR beta gene. RAR alpha appears to be expressed in most human leukemia cells regardless of the type of biologic response to retinoic acid.

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Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells

PLoS ONE ◽

10.1371/journal.pone.0256708 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256708

Author(s):

Christopher von Beek ◽

Linnéa Alriksson ◽

Josefine Palle ◽

Ann-Marie Gustafson ◽

Mirjana Grujic ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Acute Leukemia ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Tumor Formation ◽

Leukemia Cells ◽

Human Leukemia ◽

Bone Marrow Biopsies

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

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A single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells

Blood ◽

10.1182/blood.v66.3.679.bloodjournal663679 ◽

1985 ◽

Vol 66 (3) ◽

pp. 679-685

Author(s):

CW Distelhorst ◽

BM Benutto ◽

RC Griffith

Keyword(s):

Glucocorticoid Receptor ◽

Cell Lines ◽

Glucocorticoid Receptors ◽

Sodium Dodecyl ◽

Leukemia Cell ◽

Glucocorticoid Resistance ◽

Leukemia Cells ◽

Human Leukemia ◽

Intact Cells ◽

Human Leukemia Cells

We determined the mol wt of glucocorticoid receptors in human leukemia cells in order to detect glucocorticoid receptor defects that might cause glucocorticoid resistance. Glucocorticoid receptors in intact cells were affinity labeled with [3H]dexamethasone-21-mesylate and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Receptors in normal human peripheral blood mononuclear cells and six human leukemia cell lines had mol wt of 97,000. Malignant cells from ten of 25 patients with leukemia contained electrophoretically abnormal glucocorticoid receptors having mol wt of 55,000 in addition to normal size receptors (Mr = 97,000). The receptor abnormality was not restricted to a particular type of leukemia and was seen in cells from both newly diagnosed patients and patients who had received prior chemotherapy, including prednisone. The abnormal receptor was not generated when cells having only normal size receptors were assayed under conditions that favor proteolysis or when cytosol from cells containing the abnormal receptor form was mixed with cytosol from cells containing only normal size receptors. The mol wt of the abnormal receptors in human leukemia cells was the same as the mol wt of receptors in mutant mouse lymphoma cell lines, S49 143R and S49 55R, which have the nuclear transfer-increased phenotype of glucocorticoid resistance. This work describes for the first time a single common electrophoretic abnormality of glucocorticoid receptors in human leukemia cells. Further investigation of glucocorticoid receptor defects in human leukemia cells could lead to an improved understanding of the mechanisms of glucocorticoid resistance in leukemia as well as a method of predicting which patients are likely to be resistant to glucocorticoid therapy.

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Expression of retinoic acid receptor alpha mRNA in human leukemia cells

Blood ◽

10.1182/blood.v74.1.99.99 ◽

1989 ◽

Vol 74 (1) ◽

pp. 99-102 ◽

Cited By ~ 45

Author(s):

C Largman ◽

K Detmer ◽

JC Corral ◽

FM Hack ◽

HJ Lawrence

Keyword(s):

Retinoic Acid ◽

Cell Lines ◽

Retinoic Acid Receptor ◽

Leukemia Cell ◽

Leukemia Cells ◽

Human Leukemia ◽

Retinoic Acid Receptor Alpha ◽

Acid Receptor ◽

Receptor Alpha ◽

Human Leukemia Cells

Abstract The expression of the newly described human retinoic acid receptor alpha (RAR alpha) in six nonlymphoid and six lymphoid leukemia cell lines and nine freshly obtained samples of leukemia cells from patients with acute nonlymphoid leukemia was assessed by Northern blot analysis, using a full length cDNA clone of RAR alpha as probe. RAR alpha was expressed in all 12 cell lines and in all fresh leukemia samples as two major transcripts of 2.6 and 3.5 kb in size. Levels of RAR alpha expression and transcript sizes in retinoid-sensitive cells (such as HL60 or fresh promyelocytic leukemia cells) were not different from those in other samples. Moreover, expression of RAR alpha was not significantly modulated by exposure to cis-retinoic acid (cisRA) in either cisRA-responsive or unresponsive cells. By using a 3′ fragment of the RAR alpha gene as a probe, we confirmed that the transcripts visualized did not represent the homologous RAR beta gene. RAR alpha appears to be expressed in most human leukemia cells regardless of the type of biologic response to retinoic acid.

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