scholarly journals Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1241-1245 ◽  
Author(s):  
Giuseppe S.A. Longo ◽  
Richard Gorlick ◽  
William P. Tong ◽  
Emine Ercikan ◽  
Joseph R. Bertino
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Partial Purification ◽  
Acute Myelocytic Leukemia ◽  
Human Leukemia ◽  
Folylpolyglutamate Synthetase ◽  
Leukemia Cell Lines ◽  
Short Term Exposure ◽  
Long Chain

Abstract Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.

scholarly journals Disparate Affinities of Antifolates for Folylpolyglutamate Synthetase From Human Leukemia Cells

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1241-1245
Author(s):  
Giuseppe S.A. Longo ◽  
Richard Gorlick ◽  
William P. Tong ◽  
Emine Ercikan ◽  
Joseph R. Bertino
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Partial Purification ◽  
Acute Myelocytic Leukemia ◽  
Human Leukemia ◽  
Folylpolyglutamate Synthetase ◽  
Leukemia Cell Lines ◽  
Short Term Exposure ◽  
Long Chain

Previous work showed that acute myelocytic leukemia blasts accumulate less long chain polyglutamates of methotrexate (MTX) than acute lymphocytic leukemia blasts when incubated with this radiolabeled antifolate. This difference likely explains the increased sensitivity of lymphoid leukemias to short-term exposure of MTX as compared with myeloid leukemias. In this study, we examined the basis for differences between long chain MTX polyglutamate accumulation between different leukemia cell types using both leukemia cell lines and blasts freshly isolated from blood of leukemic patients. The major difference found between leukemia cells that accumulate long chain polyglutamates and those that do not were differences in Km values for the enzyme folylpolyglutamate synthetase. Km values did not change with partial purification of this enzyme, indicating that interfering substances in crude lysates were not responsible for this difference. We postulate that there may be differences in the properties of this enzyme related to tissue specific expression. In contrast to MTX, both Tomudex (Zeneca Pharmaceuticals, Wilmington, DE) and 1843U89, potent inhibitors of thymidylate synthetase, have low Kms for folylpolyglutamate synthetase, and polyglutamate forms of these inhibitors are accumulated to the same degree in both myeloid and lymphoid acute leukemia cells, paralleling the equivalent cytotoxicity found between myeloid and lymphoid leukemia cell lines. Based on these results, we believe a clinical trial of Tomudex in patients with acute myeloid leukemia is warranted.

Disparate Mechanisms of Antifolate Resistance Provoked by Methotrexate and Its Metabolite 7-Hydroxymethotrexate in Leukemia Cells: Implications for Efficacy of Methotrexate Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4369-4369
Author(s):  
Freidoun Albertioni ◽  
Gerrit Jansen ◽  
Kambiz Fotoohi ◽  
Yehuda G. Assaraf ◽  
Lilah Rothem ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Parent Drug ◽  
Underlying Mechanism ◽  
Human Leukemia ◽  
Folylpolyglutamate Synthetase ◽  
Leukemia Cell Lines ◽  
Short Term Exposure ◽  
Extensive Metabolism ◽  
Antifolate Resistance

Abstract Methotrexate (MTX) is one of the leading drugs in the treatment of leukemia, but extensive metabolism to 7-hydroxymethotrexate (7-OHMTX) can limit its therapeutic efficacy. In this study we investigated whether 7-OHMTX itself can provoke antifolate resistance that may further disrupt MTX efficacy. For this purpose we developed resistance to 7-OHMTX as well as MTX in two human leukemia cell lines (CCRF-CEM and MOLT-4) by stepwise exposure to increasing concentrations of 7-OHMTX and MTX. Consequently, both leukemia cell lines displayed marked levels of resistance to 7-OHMTX (>10 fold) and MTX (>75 fold), respectively. The underlying mechanism of resistance in the MTX-exposed cells was a marked decrease (>10-fold) in reduced folate carrier (RFC)-mediated cellular uptake of MTX. This was associated with transcriptional silencing of the RFC gene in MTX-resistant CCRF-CEM cells. In contrast, the molecular basis for the resistance to 7-OHMTX was solely due to a marked decreased (> 95%) in folylpolyglutamate synthetase (FPGS) activity which conferred >100-fold MTX resistance upon a short term exposure to this drug. This is the first demonstration that 7-OHMTX can provoke distinct modalities of antifolate resistance as compared to the parent drug MTX.

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

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3837-3843 ◽  
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.

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

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2845 ◽  
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.

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

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1821-1821 ◽  
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.

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

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2683-2689 ◽  
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.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1512-1512
Author(s):  
Gabriel Gracia-Maldonado ◽  
Jason Clark ◽  
James C. Mulloy ◽  
Ashish R Kumar
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Human Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Novel Target

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.

scholarly journals Ampelopsin Inhibits Cell Proliferation and Induces Apoptosis in HL60 and K562 Leukemia Cells by Downregulating AKT and NF-κB Signaling Pathways

2021 ◽  
Vol 22 (8) ◽  
pp. 4265
Author(s):  
Jang Mi Han ◽  
Hong Lae Kim ◽  
Hye Jin Jung
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
White Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Ros Generation ◽  
Anticancer Activities ◽  
Nuclear Condensation ◽  
Leukemia Cell Lines ◽  
Antileukemic Effect

Leukemia is a type of blood cancer caused by the rapid proliferation of abnormal white blood cells. Currently, several treatment options, including chemotherapy, radiation therapy, and bone marrow transplantation, are used to treat leukemia, but the morbidity and mortality rates of patients with leukemia are still high. Therefore, there is still a need to develop more selective and less toxic drugs for the effective treatment of leukemia. Ampelopsin, also known as dihydromyricetin, is a plant-derived flavonoid that possesses multiple pharmacological functions, including antibacterial, anti-inflammatory, antioxidative, antiangiogenic, and anticancer activities. However, the anticancer effect and mechanism of action of ampelopsin in leukemia remain unclear. In this study, we evaluated the antileukemic effect of ampelopsin against acute promyelocytic HL60 and chronic myelogenous K562 leukemia cells. Ampelopsin significantly inhibited the proliferation of both leukemia cell lines at concentrations that did not affect normal cell viability. Ampelopsin induced cell cycle arrest at the sub-G1 phase in HL60 cells but the S phase in K562 cells. In addition, ampelopsin regulated the expression of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors differently in each leukemia cell. Ampelopsin also induced apoptosis in both leukemia cell lines through nuclear condensation, loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), and regulation of Bcl-2 family members. Furthermore, the antileukemic effect of ampelopsin was associated with the downregulation of AKT and NF-κB signaling pathways. Moreover, ampelopsin suppressed the expression levels of leukemia stemness markers, such as Oct4, Sox2, CD44, and CD133. Taken together, our findings suggest that ampelopsin may be an attractive chemotherapeutic agent against leukemia.

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