Role of Erythropoietin Receptor in t(12;21) Positive Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2792-2792
Author(s):  
Renate Panzer-Gruemayer ◽  
Gerd Krapf ◽  
Dominik Beck ◽  
Gerhard Fuka ◽  
Christian Bieglmayer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Erythropoietin Receptor ◽  
Molecular Signature ◽  
Leukemic Cells ◽  
Drug Induced ◽  
Induced Apoptosis

Abstract The chromosomal translocation t(12;21)(p13;q22) resulting in the TEL/AML1 (also known as ETV6/ RUNX1) fusion gene is the most frequent translocation in childhood B cell precursor (BCP) ALL. This type of ALL is characterized by a unique molecular signature, which includes the overexpression of the gene for the erythropoietin receptor (EpoR). So far, it is not known what causes the overexpression of the EpoR gene or whether it has any effect on the t(12;21) positive leukemia. We therefore aimed to evaluate potential mechanisms responsible for the upregulation of the EpoR in t(12;21) leukemias and to find out whether signalling via this receptor affects survival or proliferation of leukemic cells. In addition, we planned to explore signalling pathways linked to the respective effects and to elucidate relevant mechanisms that might be essential for cell survival. We first excluded the possibility that the EpoR expression is upregulated as a consequence of high Epo levels in the plasma that are induced by the patients’ low hemoglobin (Hb) levels. While Hb levels from patients with t(12;21)+ ALL were significantly lower compared to those with other subtypes of BCP ALL (median, 6,15g/dL and 7,9g/dL, respectively; p<0.001 Wilcoxon 2- sample test), which correlated with high Epo levels in the plasma, the extent of EpoR mRNA expression of leukemic cells was independent of the respective amount of Epo in the individual patient’s plasma. Next, the influence of Epo on t(12;21) + leukemic cell lines was evaluated and revealed a consistent time and dose dependent increase in proliferation (Epo concentrations 10, 50, 100U/ml for 72 hours) determined by 3H-Thymidine incorporation. This effect was abrogated upon addition of a blocking anti-EpoR antibody thereby confirming the specificity of EpoR signalling. Since Epo may have apoptosis-modulating potential in EpoR expressing malignant cells, we tested its influence on drug-induced apoptosis. For this purpose IC50 concentrations of drugs that are commonly used for the treatment of children with BCP ALL were used. A reduction of glucocorticoid (GC)-induced apoptosis by Epo was demonstrated in t(12;21)+ cell lines while no effect was seen in combination with other drugs or in t(12;21) negative cell lines. Preliminary data indicate that NF-kappa B as well as PI3K/Akt pathways are triggered by Epo, implying that they play a role in this rescue mechanism. Given that cell lines may have intrinsic changes, we are presently evaluating whether the observed results can also be reproduced in primary leukemic cells. In support of this assumption are results in a limited number of primary t(12;21)+ leukemias showing a superior survival (MTT assay) and reduced apoptosis rate to GC when cultured in the presence of Epo. These findings are in contrast to those in t(12;21) negative BCP ALLs. In conclusion, our data indicate that overexpression of EpoR in t(12;21) positive leukemias is not induced by low Hb, a feature that is generally observed in patients with this type of leukemia. Binding of Epo to its receptor in vitro leads to enhanced survival and negatively affects the sensitivity to GCs. Whether these findings have any implications on the treatment and care of patients with t(12;21)+ leukemia needs to be addressed in further studies. Financial support: OENB10720, FWF P17551-B14 and GENAU-CHILD Projekt GZ200.136/1 - VI/1/2005 to RPG.

Hydrocortisone does Not Influence in Vitro Glucocorticoid Sensitivity of Acute Lymphoblastic Leukemia Blasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5228-5228
Author(s):  
Lidewij T. Warris ◽  
Marry M. van den Heuvel-Eibrink ◽  
Ingrid M. Ariës ◽  
Rob Pieters ◽  
Erica L.T. van den Akker ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Side Effects ◽  
Cell Lines ◽  
Fluorescence Intensity ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Glucocorticoid Sensitivity ◽  
Leukemic Cell Lines

Abstract Introduction: Dexamethasone-induced neuropsychological side effects on mood, behavior and cognition seriously affect quality of life in children with acute lymphoblastic leukemia (ALL) during a long treatment period. Based on recent studies in animals, we hypothesized that these neuropsychological side effects are mediated by dexamethasone-induced cortisol depletion of the mineralocorticoid receptor (MR) in the brain. Therefore, we hypothesize that these side effects could be ameliorated by an intervention with hydrocortisone. For clinical application settings however, an absolute prerequisite is that MR activation does not interfere with the efficacy of the glucocorticoids, dexamethasone and prednisolone, on ALL cells. Materials and Methods: To investigate responsiveness of leukemic cell lines and fresh patients’ leukemic cells to dexamethasone and prednisolone in the presence of hydrocortisone, MTT-assays were performed. In addition MR and the glucocorticoid receptor (GR) expression on leukemic cells of different ALL subtypes was studied with a microarray-based gene expression profiling and validated by quantitative real-time PCR. Results: Leukemic cells expressed the MR at a very low level with a significantly higher (P≤0.001) expression in ETV6-RUNX1+ patients (median: 160.7 [AU] of fluorescence intensity, range: 38.1 - 760.6 [AU]) versus other ALL subtypes (median: 41.8 [AU] of fluorescence intensity, range: 25.1 - 276.2 [AU]). MR expression did not differ between glucocorticoid resistant and sensitive patients’ cells. Hydrocortisone addition did not affect glucocorticoid sensitivity of leukemic cell lines and patients’ leukemic cells of different leukemic subtypes also including ETV6-RUNX1+. Glucocorticoid sensitive patients’ cells became significantly more sensitive by hydrocortisone addition (prednisolone: P≤0.01, dexamethasone: P≤0.05). Conclusion: This present study shows that hydrocortisone does not interfere with efficacy of dexamethasone and prednisolone in vitro. These findings support a clinical randomized trial to study whether addition of hydrocortisone decreases the neuropsychological side effects of dexamethasone in children with ALL. Acknowledgments: The financial support of the KiKa® (Kinderen Kankervrij) foundation is highly appreciated. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel ELISA-Based Peptide Biosensor Assay for Screening ABL1 Activity in vitro: A Challenge for Precision Therapy in BCR-ABL1 and BCR-ABL1 Like Leukemias

2021 ◽  
Vol 12 ◽  
Author(s):  
Oksana Montecchini ◽  
Stefania Braidotti ◽  
Raffaella Franca ◽  
Giulia Zudeh ◽  
Christian Boni ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Phosphorylation Site ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Elisa Assay ◽  
Cell Lysates ◽  
Jak2 Inhibitor ◽  
Tk Inhibitors

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased (basal vs. PABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value &lt; 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to PPHOSPHO-ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on PABL; a lower signal was instead observed for NALM6 and REH (p &lt; 0.001 and p &lt; 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib (p &lt; 0.001 for K562, NALM6, ALL-SIL and p &lt; 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-ELISA assay on leukemic cells isolated from patient’s bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of PABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the PABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.

scholarly journals FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors

2007 ◽  
Vol 204 (8) ◽  
pp. 1813-1824 ◽  
Author(s):  
Jennifer O'Neil ◽  
Jonathan Grim ◽  
Peter Strack ◽  
Sudhir Rao ◽  
Deanne Tibbitts ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Homozygous Deletion ◽  
Notch Receptor ◽  
Leukemic Cells ◽  
Mrna Levels

γ-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

scholarly journals Imaging Flow Cytometric Analysis of Stilbene-Dependent Apoptosis in Drug Resistant Human Leukemic Cell Lines

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1896 ◽  
Author(s):  
Marcin Czop ◽  
Anna Bogucka-Kocka ◽  
Tomasz Kubrak ◽  
Karolina Knap-Czop ◽  
Anna Makuch-Kocka ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Drug Resistant ◽  
Resistant Phenotype ◽  
Stilbene Derivatives ◽  
Induced Apoptosis

Background: The natural compounds have been researched extensively as an alternative to the conventional chemotherapy and radiation. Stilbene derivatives appear as a group of therapeutics which deserves special attention. The present study was designed to analyze the effects of stilbene derivatives on drug resistant human leukemic cells. The aim of this work was to evaluate the apoptotic effect of stilbene derivatives in various concentrations on leukemic cells (LC) with and without resistant phenotype. Methods: Human acute promyelocytic leukemia (APL) cell lines (HL60, HL60/MX1, HL60/MX2) and acute lymphoblastic leukemia (ALL) cell lines (CEM/C1, CCRF-CEM) were studied. T-resveratrol, piceatannol, rhaponticin, deoxyrhaponticin, pterostilbene were used to stimulate apoptosis. Mitoxantrone (MIT) was applied to induce drug resistance. Results: t-Resveratrol (RES), deoxyrhaponticin (D-RHAP), rhaponticin (RHAP), pterostilbene (PTER), and piceatannol (PIC) influenced viability and induced apoptosis in all investigated cell lines. Conclusions: Our results confirmed that RES, PIC, RHAP, D-RHAP, and PTER are essential therapeutic compounds with anticancer activity exhibited by induction of apoptosis in leukemic cells with and without resistant phenotype. Stilbene-induced apoptosis in HL60/MX1, HL60/MX2, CEM/C1, and CCRF-CEM leukemia cell lines have been presented in very few studies so far and our research is an important contribution to the investigation of these substances.

Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
G Manfioletti ◽  
V Gattei ◽  
E Buratti ◽  
A Rustighi ◽  
A De Iuliis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Specific Cell ◽  
B Cell Differentiation ◽  
Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

Pioglitazone Inhibits the Growth of Human Leukemic Cell Lines and Primary Leukemic Cells in Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4493-4493 ◽  
Author(s):  
Yoshihiro Hatta ◽  
Minoru Saiki ◽  
Yuko Enomoto ◽  
Shin Aizawa ◽  
Umihiko Sawada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Peroxisome Proliferator ◽  
Leukemic Cell Lines ◽  
Ppar Γ

Abstract Troglitazone and pioglitazone are one of thiazolidinediones that are high affinity ligand for the nuclear receptor called peroxisome proliferator-activated receptor gamma (PPAR-γ). Troglitazone is a potent inhibitor of clonogenic growth of acute myeloid leukemia cells when combined with a retinoid. However, the effect of pioglitazone to neoplastic cells and normal hematopoietic cells has not been studied yet. Adult T-cell leukemia (ATL), prevalent in western Japan, is a highly aggressive malignancy of mature T lymphocyte. Therefore, we studied antitumor effect of pioglitazone against leukemic cells including ATL as well as normal hematopoietic cells. With 300 μM of pioglitazone, colony formation of ATL cell lines (MT1, MT2, F6T, OKM3T, and Su9T01) was completely inhibited. Colony formation of HUT102, another ATL cell line, was 12 % compared to untreated control. Clonogenic cells of other leukemic cell lines (K562, HL60, U937, HEL, CEM, and NALM1) was also inhibited to 0–30% of control. Colony formation of primary leukemic cells from 5 AML patients was decreased to 15 %. However, normal hematopoietic cells were weakly inhibited with 300 μM pioglitazone; 77 % of CFU-GM, 70 % of CFU-E, and 33 % of BFU-E survived. Cell cycle analysis showed that pioglitazone decreased the ratio of G2/M phase in HL60 cells, suggesting the inhibition of cell division. By Western blotting, PPAR-γ protein level was similar in all leukemic cells and normal bone marrow mononuclear cells. Taken together, pioglitazone effectively eliminate leukemic cells and could be used as an antitumor agent in vivo.

IPI-504, a Novel, Orally Active HSP90 Inhibitor, Prolongs Survival of Mice with BCR-ABL T315I CML and B-ALL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2183-2183 ◽  
Author(s):  
Cong Peng ◽  
Julia Brain ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
David Grayzel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Median Survival ◽  
Combination Treatment ◽  
Lymphoblastic Leukemia ◽  
Hsp90 Inhibitor ◽  
Leukemic Cells ◽  
Prolonged Survival ◽  
Hsp90 Inhibition ◽  
T315i Mutation

Abstract Development of mutations within the kinase domain is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. In CML (chronic myeloid leukemia), a disease driven by the constitutively active BCR-ABL oncoprotein, no available TKIs have been effective in treating patients with the BCR-ABL T315I mutation. Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins like BCR-ABL, and affects the stability of these proteins. Several labs have shown that Hsp90 inhibition in vitro results in the degradation of BCR-ABL T315I and induces potent killing of these cell lines. However, these results have not been demonstrated in animal models for BCR-ABL-induced CML and B-ALL (B-cell acute lymphoblastic leukemia, a disease that does not respond well to TKIs including imatinib and dasatinib). Thus, IPI-504, an orally administered Hsp90 inhibitor, was evaluated in murine models of CML and B-ALL. Treatment of mice with wild type (WT)- or T315I BCR-ABL-induced CML with IPI-504 resulted in BCR-ABL protein degradation and a decrease in circulating BCR-ABL positive cells. In response to treatment with vehicle the median survival time of WT and T315I CML mice is approximately 20 days. While the T315I CML mice were resistant to imatinib with a median survival of 21 days, IPI-504 (50 and 100 mg/kg, PO TIW) demonstrated dose-dependent prolonged survival of these mice by 30 and 70 days, respectively (p<0.001 for both doses). Both imatinib and IPI-504 similarly prolonged survival of mice with BCR-ABL-WT-induced CML. In the T315I CML mice prolonged survival of the IPI-504 treated cohort was associated with decreased peripheral blood BCR-ABL positive leukemia cells during treatment, less splenomegaly and improved pulmonary histopathlogy at necropsy. In CML mice receiving mixed BCR-ABL-WT- or T315I-transduced donor bone marrow cells, Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the WT clones, consistent with in vitro studies where T315I BCR-ABL was more sensitive to IPI-504 induced degradation in cell lines than WT BCR-ABL. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice bearing both WT and T315I leukemic cells. IPI-504 also significantly prolonged survival of B-ALL mice bearing the T315I mutation (p<0.001). These results provide a rationale for use of an Hsp90 inhibitor as a novel approach to overcoming resistance to TKIs as well as the potential for first line combination treatment in CML patients. The potential for IPI-504 to eliminate mutant kinases via Hsp90 inhibition provides a new therapeutic strategy for treating BCR-ABL-induced CML, ALL as well as other cancers resistant to treatment with TKIs.

Priming Effect with G-CSF Enhances In Vitro Apoptosis by Treatment with Ara-C and VP-16 in Leukemic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4333-4333
Author(s):  
Jun-ichi Kitagawa ◽  
Takeshi Hara ◽  
Hisashi Tsurumi ◽  
Nobuhiro Kanemura ◽  
Masahito Shimizu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Priming Effect ◽  
Low Dose ◽  
Combined Treatment ◽  
Annexin V ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Significant Difference

Abstract Introduction: We have recently reported that the effectiveness of low dose Ara-C, VP-16 and G-CSF (AVG therapy) for elderly AML patients who were ineligible for intensive chemotherapy (Hematol Oncol, in press). G-CSF has been reported to potentiate in vitro anti-leukemic effect of Ara-C. The mechanism of the potentiation is assumed to recruit quiescent G0 leukemic cells into cell cycle. We hypothesized that the enhanced cytotoxicity was due to the apoptosis by the effect of the priming of G-CSF, and the effect was depended on the cell cycle. In order to afford proof of this hypothesis, we assayed proliferation, apoptosis, and cell cycle in leukemic cell lines. Materials: Ara-C, VP-16, G-CSF was provided by Nippon Shinyaku, Nihonkayaku, Chugai pharmacy, respectively, Tokyo, Japan. 32D and HL-60 were obtained from RIKEN Bioresource Center Cell Bank (Ibaragi, Japan), Ba/F3 was generous gifts from Dr. Kume, Jichi medical school, Tochigi, Japan. Methods: 5 x 105/ml HL60, 32D and Ba/F3 were cultured with various concentrations of Ara-C and/or VP-16 in the presence or absence of G-CSF 50ng/ml for 3 days. At the end of the culture, cell proliferation and viability were determined by using the trypan blue. The Annexin V-binding capacity of treated cells was examined by flow cytometry using ANNEXIN V-FITC APOPTOSIS DETECTION KIT I purchased from BD Pharmingen™. Cell cycle analysis was done with BrdU Flow KIT purchased from BD Pharmingen™. The incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies, and the levels of cell-associated BrdU are then measured by flow cytometory. Result: Ara-C and VP-16 inhibited proliferation and decreased viability of leukemic cell lines dose-dependently. Half killing concentration (IC50) was redused in combination of Ara-C and VP-16 than Ara-C or VP-16 alone. In G-CSF dependent cell line (32D), IC50 was redeced in the presence of G-CSF than absence of G-CSF at G-CSF, and there was no significant difference between with and without G-CSF in G-CSF independent cell lines (HL-60, Ba/F3) (p<0.05). In combined treatment of low dose Ara-C (10−7M) and VP-16 (10−7M), the percentage of apoptotic cells were increased to 20.67% from 13.04% by addition of G-CSF in 32D, and there was no significant differencebetween with and without G-CSF in HL-60 and Ba/F3 (p<0.05). At combined treatment of low dose Ara-C and VP-16, the percentage of G0/G1 phase cells were decreased to 43.94% from 35.63% and S phase cells were increased to 29.50% from 24.05% in 32D by addition of G-CSF, and there was no significant difference between with and without G-CSF in HL-60 and Ba/F3 (p<0.05). Discussion: We first showed a combination effect of Ara-C and VP-16. Next we demonstrated that the potentiation of the cytotoxicity was mediated through the mechanism of apoptosis, and apoptosis played an important role for eradicating leukemic cells by low dose Ara-C and VP-16. And G-CSF recruited cells G0/G1 phase into S phase in G-CSF dependent cells by addition of G-CSF. These results suggest that priming effect of G-CSF significantly potentiate the cytotoxicity mediated by AVG chemotherapy. Conclusion: The priming effect of G-CSF might be admitted at least of a part in AML cells.

Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2423-2423
Author(s):  
Sergej Konoplev ◽  
Hongbo Lu ◽  
Michael A Fiegl ◽  
Zhihong Zeng ◽  
Wenjing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

Chemoprevention Against Hepatocellular Carcinoma of Cornus officinalis in vitro

2004 ◽  
Vol 32 (05) ◽  
pp. 717-725 ◽  
Author(s):  
Jung-San Chang ◽  
Lien-Chai Chiang ◽  
Fen-Fang Hsu ◽  
Chun-Ching Lin
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Xtt Assay ◽  
Leukemic Cell Lines ◽  
Cornus Officinalis ◽  
Anti Oxidant ◽  
Dose Dependent

The water extracts of Cornus officinalis Sieb. et Zuce against hepatocellular carcinoma (HCC) was studied for its chemopreventive potential. Three HCC cell lines (HepG2, SK-Hep1 and PLC/PRF/5) and three leukemic cell lines (U937, K562 and Raji) were tested with XTT assay. Extracts of C. officinalis inhibited all these HCC cells and leukemic cells at a concentration of 100 μg/ml (P<0.05) and was dose-dependent (P<0.0001). P53 (P<0.0001) and Ras (P=0.001) significantly affected its activity against HCC. Extracts of C. officinalis also possessed the anti-oxidant activity through free radicals scavenging activity at a concentration of 50 μg/ml (P<0.05). In summary, our experiment implied that C. officinalis might be a candidate for chemopreventive agent against HCC through the antioxidant and anti-neoplastic effects.

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