[Show abstract][Hide abstract] ABSTRACT: Background: Signaling induced by binding of erythropoietin-producing hepatoma-amplified sequence (EPH) receptors to their cell-surface ephrin ligands is implicated in hematopoiesis and growth of various cancer cells. However, the roles of EPH-ephrin signaling in leukemia have not been elucidated. We investigated the effects of EPHB4 and ephrin B2 on the growth of leukemia cells. Materials and Methods: Seven human leukemia cell lines were used to examine the effects of recombinant ephrin B2 and EPHB4 on cell proliferation by colorimetric WST-1 assay and colony assays; on protein tyrosine phosphorylation; and on mRNA expression by reverse transcription-polymerase chain reaction and microarray analysis. Results: In an erythroid leukemia-derived cell line AA, exogenous ephrin B2 induced proliferation and colony formation; in addition, it up-regulated protein tyrosine phosphorylation and the expression of growth-related genes such as FBJ murine osteosarcoma viral oncogene homolog B and v-src avian sarcoma viral oncogene homolog. Conclusion: Growth-promoting effects of ephrin B2 were observed in an erythroid leukemia cell line, suggesting that the EPH-ephrin signaling may be involved in the pathology of leukemia.
[Show abstract][Hide abstract] ABSTRACT: Aim: The effects of small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2 on cell proliferation and downstream signaling pathways in leukemia cells were examined.
Two T-lymphoblastic leukemia (T-ALL) cell lines and two acute myeloblastic leukemia (AML) cell lines were transfected with siRNAs targeting NOTCH1 and NOTCH2. The effects of knockdown on cell proliferation and protein expression were examined by colorimetric WST-8 assay and immunoblotting, respectively.
In T-ALL cell lines, NOTCH1 knockdown as well as NOTCH2 knockdown suppressed cell proliferation and induced apoptosis. v-Myc avian myelocytomatosis viral oncogene homolog (MYC) protein expression was down-regulated in NOTCH1-knockdown cells but not affected in NOTCH2-knockdown cells. In AML cell lines, cell proliferation was not significantly affected by NOTCH siRNAs. NOTCH2 knockdown increased the level of cleaved NOTCH1 fragment without increasing NOTCH1 expression. NOTCH knockdown reduced the level of mechanistic target of rapamycin (mTOR) protein in the monoblastic leukemia cell line THP-1. Contrastingly, NOTCH activation by NOTCH ligand stimulation increased the expression of mTOR in THP-1 cells.
These novel findings on NOTCH signaling may contribute to the development of effective NOTCH-targeted therapies against leukemia.
[Show abstract][Hide abstract] ABSTRACT: Aim: To examine the effects of echinomycin, a compound that inhibits DNA-binding activity of hypoxia-inducible factor-1 (HIF1), on leukaemia cell growth.
Three acute myeloid leukaemia cell lines and three T-lymphoblastic leukaemia cell lines were cultured with echinomycin. Cell growth, mRNA and protein expression levels were examined by WST-1 assay, reverse-transcription polymerase chain reaction and immunoblotting, respectively.
HIF1α protein was expressed in all cell lines under normoxia. Treatment with echinomycin suppressed cell growth and induced apoptosis in association with decreased mRNA expression of HIF1 targets, glucose transporter-1 (GLUT1) and B-cell CLL/lymphoma-2 (BCL2). Echinomycin also suppressed the protein expression of NOTCH1, cleaved NOTCH1, v-myc myelocytomatosis viral oncogene homolog (MYC), v-akt murine thymoma viral oncogene homolog-1 (AKT), phosphorylated AKT, mechanistic target of rapamycin (mTOR), and phosphorylated mTOR and increased that of cleaved caspase-3 in some cell lines.
Echinomycin suppresses leukaemia cell growth in association with reduced NOTCH1 expression. This is the first report to show that HIF inhibitor treatment suppresses NOTCH1 signalling. HIF inhibitors could be novel candidates for a molecular-targeted therapy against leukaemia.
[Show abstract][Hide abstract] ABSTRACT: Aim: PP242 is a compound which inhibits both mammalian target of rapamycin complex-1 (mTORC1) and mTORC2. We examined the effects of PP242 and rapamycin on mTOR signalling and evaluated potential crosstalk with the NOTCH signalling in eight leukemia cell lines. MATERIALS AND METHODS: We examined the effects of treatment with these inhibitors on cell growth and protein expression. RESULTS: PP242 suppressed growth more potently than did rapamycin. In two cell lines poorly sensitive to PP242, PP242 failed to inhibit v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation. Suppression of mTOR phosphorylation was weaker in myeloid cell lines. Rapamycin induced eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation in three cell lines. Phosphorylation of both isoforms (p70 and p85) of S6 kinase (S6K) was suppressed in three cell lines; only p70 was suppressed in the others. NOTCH1 expression and activation were up-regulated by PP242 in one cell line but down-regulated in another. CONCLUSION: PP242 is a candidate for molecular-targeted leukemia therapy, although its effects must be evaluated on a case-by-case basis. Crosstalk was found between the mTOR and NOTCH signalling pathways.
[Show abstract][Hide abstract] ABSTRACT: Notch1 and its ligand Jagged1 are proteins with important roles in the growth of leukemia cells. Although the detection of Notch1 protein in acute lymphoblastic leukemia cells using immunoblot analysis has been previously reported, the expression patterns of Notch1 and Jagged1 detected by flow cytometry (FCM) in normal blood cells and various leukemia cells have not been well-characterised. In the present study, we examined the expression patterns of Notch1 and Jagged1 in 10 normal blood samples, 8 bone marrow samples, 11 leukemia/lymphoma cell lines and leukemia cells from 22 patients with acute myeloid leukemia (AML), mature T-cell neoplasms or B-cell chronic lymphocytic leukemia (B-CLL) using FCM. The results showed that Notch1 expression is relatively strong in monocytes and granulocytes but weak in lymphocytes. The expression of Notch1 is stronger in bone marrow cells than in the equivalent cells in blood. All the cell lines examined strongly expressed Notch1, and eight cell lines expressed Jagged1. In leukemia cells from patients, four AML samples expressed Notch1 and/or Jagged1. However, three samples expressed neither Notch1 and/or Jagged1 and none of the mature T-cell neoplasm samples expressed either protein. However, all B-CLL samples expressed high levels of both Notch1 and Jagged1. We found that the expression of Notch1 and Jagged1 is detected in various hematological malignancies by FCM. The examination of these proteins is likely to be useful in the characterisation of diseases and individual cases. Examination of these proteins may also be useful in the selection of patients most likely to benefit from novel molecular-targeted therapies using Notch inhibitors in the future.
Experimental and therapeutic medicine 09/2012; 4(3):397-400. DOI:10.3892/etm.2012.633
[Show abstract][Hide abstract] ABSTRACT: The detection of a V617F mutation (G to T exchange at nucleotide 1,849) in the JAK2 gene is crucial for the diagnosis of myeloproliferative neoplasms (MPN) such as polycythemia vera. Although sequence analysis is the standard method for detection, it is not suitable for clinical examinations due to the requirement of expensive equipment. In this study, we evaluated the efficiencies of four PCR-based methods to detect JAK2 V617F: allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRM) and the quenching probe method (QP). The HEL cell line, which harbors a homozygous JAK2 V617F mutation, as well as bone marrow samples from 16 MPN patients and normal control samples, were used in this assessment. The sensitivity of the detection limit of all four methods was also examined using samples of HEL cells mixed in a variety of ratios with cells containing wild-type JAK2. The results of all four methods were found to be concordant. AS-PCR was shown to be the most sensitive; however, it produced false positive results. Although PCR-RFLP demonstrated high specificity, it was time consuming. By contrast, results were obtained using HRM and QP in only 2 h. It was easier to recognize the curves derived from the mutant allele obtained using QP. QP is also suitable for the rough estimation of allele burden. JAK2 V617F assays are mainly used for diagnosis at presentation in clinical settings. We therefore conclude that in situations where high sensitivity is not required, QP is the preferable method for the detection of JAK2 V617F. To the best of our knowledge, this is the first report to demonstrate the efficiency of the QP method for the detection of JAK2 V617F using a standard thermal cycler.
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic protein 4 (BMP4) signaling is involved in the maintenance of hematopoietic stem cells. However, the effects of BMP4 on leukemia and lymphoma cells are unknown.
The effects of recombinant BMP4 on the in vitro growth of 12 leukemia and lymphoma cell lines were examined.
BMP4 treatment promoted the short-term growth of three cell lines and suppressed the growth of one. Induction of differentiation was not observed. BMP4 treatment suppressed the clonogenicity of four out of the six examined cell lines. BMP4 treatment promoted the growth of Jurkat cells but suppressed their ability to form colonies. BMP4 treatment up-regulated the phosphorylation of SMAD1/5/8 complex, indicating that BMP4 mediated signal transduction in the cells.
BMP4 suppressed the clonogenicity of selected leukemia and lymphoma cell lines. The regulation of BMP4 signaling may be a useful therapeutic approach for leukemia and lymphoma, if appropriate cases are selected.
[Show abstract][Hide abstract] ABSTRACT: The Notch inhibitors, γ-secretase inhibitors (GSIs), are promising candidates for molecular targeted therapy against leukemia. However, they show only limited effectiveness. We thought that the efficacy of GSIs might be improved by their combination with Hedgehog inhibitors and Wnt inhibitors because these signaling pathways are also important for the growth of leukemia cells.
The effects of the combination of GSI-XXI plus the Hedgehog inhibitor, cyclopamine (Cy), or the Wnt inhibitor, quercetin (Qu), on the in vitro cell growth, colony formation and Notch1 protein expression of three T-cell acute lymphoblastic leukemia (T-ALL) cell lines with NOTCH1 mutations and three acute myeloid leukemia cell lines were examined.
The addition of Cy or Qu to GSI suppressed the growth of DND-41 T-ALL cells additively or synergistically, respectively. Interestingly, Cy treatment and Qu treatment reduced Notch1 protein and its active fragment in DND-41 cells, which suggests a relationship between Notch signaling and Hedgehog or Wnt signaling. The addition of Cy or Qu to GSI promoted the decrease of Notch1 activation and expression.
The anti-leukemic effects of a GSI could be promoted by its combination with Cy or Qu, in appropriate cases selected by in vitro drug sensitivity test.
[Show abstract][Hide abstract] ABSTRACT: Hedgehog (Hh) signaling is involved in cancer cell growth. However, the effects of Hh stimulation on leukemia cells are unknown.
The effects of recombinant sonic Hedgehog (Shh) protein on the in vitro growth of one B-lymphoma and four myeloid leukemia cell lines were examined.
Shh stimulation had no significant effect on the short-term growth of whole cell populations in any of the five cell lines. However, Shh promoted clonogenic cell recovery after suspension culture, suggesting promotion of leukemia stem or progenitor cell amplification in three cell lines. The lack of Hh receptors in one cell line and endogenous Shh expression in another were possible reasons for the lack of effects of Shh in these cases.
These results suggest that Shh stimulation promotes the self-renewal capacity of leukemia stem cells in some cell lines. Inhibition of Hh signaling could represent a novel therapeutic approach in leukemia.
[Show abstract][Hide abstract] ABSTRACT: Notch signaling regulates the fate of hematopoietic stem cells and leukemia cells. However, the role of Notch in erythroid differentiation remains unclear.
We examined the effects of three γ-secretase inhibitors (GSI-IX, GSI-XII and GSI-XXI) that inhibit Notch signaling on the in vitro growth and differentiation of HEL and AA erythroid leukemia cell lines.
GSI treatment induced morphologic erythroid differentiation and promoted hemoglobin production. GSI treatment suppressed short-term growth and colony formation, while treatment with GSI-XXI promoted the growth of AA cells. The degree of differentiation induced by each GSI roughly correlated with the reduction in HES1 mRNA expression.
GSIs have potential uses in differentiation induction therapy for erythroid leukemia in the future. Before clinical use, in vitro sensitivity tests should be performed because the effects of GSIs are diverse depending upon the combination of leukemia cells and GSIs.
[Show abstract][Hide abstract] ABSTRACT: Hedgehog (Hh) and Wnt signaling pathways are involved in the stimulation of growth of leukemia and lymphoma cells. In the present study, whether or not the Hh inhibitor, cyclopamine, and the Wnt inhibitor, quercetin, suppress cell growth was investigated.
The effects of cyclopamine and quercetin on the in vitro growth and protein expression of ten acute leukemia and B-cell lymphoma cell lines were examined.
Cyclopamine and quercetin suppressed cell growth and induced apoptosis in seven and eight cell lines respectively. Cyclopamine decreased the level of Gli1 protein, a target gene product of Hh signaling. Quercetin decreased the level of Notch1 protein and its active fragment in the DND-41 T-lymphoblastic leukemia cell line with constitutive Notch activation.
Cyclopamine and quercetin suppress the growth of a number of leukemia and lymphoma cells. This finding suggests the potential use of these compounds in molecularly-targeted therapy for leukemia and lymphoma.
[Show abstract][Hide abstract] ABSTRACT: There are conflicting reports regarding the effects of Notch activation on nuclear factor-kappaB (NF-kappaB) activity. The relationships are cell type-dependent and have not been fully elucidated. We examined the effects of Notch activation induced by a recombinant Notch ligand, Delta-like1 (Dll1), on the NF-kappaB activity in two acute myeloid leukemia (AML) cell lines. We found that Delta1-induced Notch activation activated the NF-kappaB pathway in THP-1 cells. Regarding the possible mechanisms, Dll1 stimulation increased the mRNA and protein expression levels of some components of the NF-kappaB pathway and induced phosphorylation of IKKalpha/beta, IkappaB and RelA proteins after 24 or 48 h of stimulation. Since the phosphorylation required a long time, it did not appear to be caused by physical interactions between Notch and NF-kappaB proteins, but rather by indirect effects. One possible mechanism for the indirect effects was the observed induction of IL-1beta expression by Dll1 stimulation. On the other hand, Notch activation did not affect NF-kappaB activity in TMD7 cells. RelA was phosphorylated without stimulation, indicating that NF-kappaB was constitutively activated in TMD7 cells. To the best of our knowledge, this is the first study to investigate AML cells and use a recombinant Notch ligand to activate Notch. The present findings lead to better understanding of Notch functions, which have not been fully elucidated.