Flow Cytometric Detection of Asparagine Synthetase Protein in Leukemia Cells; Indication for L-Asparaginase Therapy

Abstract Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzyme-based methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. L-asparaginase (L-asp) therapy causes depletion of plasma asparagine followed by the loss of intracellular asparagine. Due to the lack of a rapid up-regulation of asparagine synthetase (ASNS) protein content in ALL cells, they are preferentially killed by L-asp. Elevated expression of ASNS within the leukemia cells causes decreased sensitivity to L-asp. The proof of ASNS deficiency in leukemia cells is considered to predictive for effectiveness of L-asp even in acute myeloid leukemia (AML) other than ALL patients. The establishment of quantitative estimation of ASNS protein content would be useful for the L-asp treatment in leukemia therapy. Objective: Our aim was to set up a flow cytometry system to check ASNS deficiency in leukemia cells and to investigate the sensitivity to L-Asp and the ASNS expression in AML leukemia cells. Methods: AML (KG-1, HL-60, U937) and ALL (MOLT-4, RS4;11) and CML (K562) cell lines were grown in RPMI1640 medium with 10% FCS. Primary leukemic cells from the peripheral blood or bone marrow of 20 AML patients were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was evaluated by cytosolic flow cytometry with Z5808 McAb (Hybridoma 31: 325-332.2012) and expressed as a ΔMFI(Difference of Mean Fluorescence Intensity(MFI) between by Z5808 and isotypic control) or MFI ratio(MFI by Z5808/MFI by isotypic control). When a sufficient amount of leukemic cells was available, sensitivity to L-asp (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of E. coliL-asp with the cells and by measuring the cell viability with a counting kit (WST1 viability assay) at day 3. Results: Determination of IC50 for the HL-60 (⊿MFI 48 ± 8.01, MFI ratio 1.77 ± 0.03) and U937 (⊿MFI 16.7 ± 0.47, MFI ratio 1.19 ± 0.02) demonstrated that these cells were equally sensitive to L-asp than the ALL cell line MOLT-4 in vitro (0.37 and 0.02IU/mL versus 0.15 IU/mL, respectively). K562 and KG-1 (⊿MFI 135.7 ± 5.66, MFI ratio 2.48 ± 0.09) cells with the highest ASNS expression exhibited resistance to L-asp (>10 IU/ml). Both of ASNS Expression by ⊿MFI and MFI ratio was inversely correlated with L-asp sensitivity judging from cell line studies. Judging from cell line study, the threshold for ASNS protein expression effective for L-asp treatment was considered to be <25 for ⊿MFI and <1.8 for MFI ratio respectively. Fresh leukemia cases contained three ALLs, Ph1ALL, and 13 AML; M0, 1; M1, 3; M2, 2; M4, 1; M5, 3; M7, 3; and Acute Mixed lineage leukemia, 2 cases. IC50 determination was possible on 11/20 patients. Four displayed a high sensitivity to L-asp (IC50 < 0.01 IU/mL) whereas two displayed resistant to L-asp (IC50 >10 IU/mL) Remaining 5 patients were a moderate sensitivity (IC50 < 0.5 IU/mL). ASNS Expression in ALL was almost near zero. ASNS expression in fresh AML was low in all cases except for one M2 and one M4 cases. Indeed, at high ASNS expression, these cases were resistant in vitro to L-asp. The patients with blasts sensitive to L-asp had mainly M1, M5 or M7 AML. A good inverse correlation between ASNS expression and sensitivity to L-asp was observed also in primary leukemic cells from AML patients. Conclusions: We demonstrated here that some of AML cell lines with low ASNS expression are more sensitive to L-asp than leukemia cells with high ASNS expression. Also, fresh AML cells with low ASNS expression are more sensitive to L-asp than with high ASNS expression. 11/13AML or two AMLL cases were supposed to be effective for L-asp. Plasma asparagine depletion by L-asp in selected patients having low ASNS may be a promising therapeutic approach even for AML. This work was supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) KAKENHI Grant Number 24590713. Disclosures No relevant conflicts of interest to declare.

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L-Asparaginase Sensitivity and Asparagine Synthetase Expression In Primary Tumor Cells From AML Patients

Blood ◽

10.1182/blood.v122.21.1423.1423 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1423-1423

Author(s):

Willy Berlier ◽

Karine Aguera ◽

Mohamed El Hamri ◽

Marie-Pierre Goutagny ◽

Fanny Gallix ◽

...

Keyword(s):

Cell Viability ◽

Cell Line ◽

Tumor Cells ◽

Primary Tumor ◽

High Sensitivity ◽

Principal Investigator ◽

Asparagine Synthetase ◽

Leukemic Cells ◽

Lymphoid Leukemia

Abstract Introduction The rational of use of L-asparaginase (L-Aspa) is based on asparagine synthetase (ASNS) deficiency in leukemic cells. Its efficacy is due to its capacity to deplete plasmatic asparagine, starving the leukemic cells and subsequently inhibiting protein synthesis. It is a mainstay in the treatment of acute lymphoid leukemia (ALL), where it is established that resistance to treatment is in part related to detectable expression of ASNS (Aslanian et al., 2001; Su et al., 2008). In acute myeloid leukemia (AML), promising results have been obtained in clinical trials (Capizzi et al., 1988), with an improvement of complete remission rates from 18% to 54% in refractory patients <60 year old and from 0% to 31% in refractory patients >60 year old. More recently, it has been reported that leukemic cells from AML patients with M1, M4 and M5 FAB subtypes were more sensitive to L-Aspa (Okada et al., 2003).The aim of this study was to investigate the sensitivity to L-Aspa and the ASNS expression in an AML cell line (HL-60) and in primary leukemic cells from newly diagnosed AML patients. Materials and methods AML (HL-60) and ALL (MOLT-4) cell lines were grown according to ATCC recommendations. Primary leukemic cells from the bone marrow of 24 AML patients (median age: 69 years; range: 2-83) were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was determined by western-blot on isolated leukemic cells and expressed as a ratio ASNS/cyclophilin A. When a sufficient amount of leukemic cells was available, sensitivity to L-Aspa (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of L-Aspa (0.001 to 10 IU/mL) with the cells and by measuring the cell viability with a counting kit (CCK-8 viability assay) at day 4. Results Determination of IC50 for the HL-60 cell line demonstrated that these cells were equally sensitive to L-Aspa than the ALL cell line MOLT-4 in vitro (0.23 IU/mL versus 0.19 IU/mL, respectively). The expression of ASNS in the HL-60 cell line was low but higher than in MOLT-4 which is a well known ASNS deficient cell line. IC50 determination was possible on 17/24 patients. Seven displayed a high sensitivity to L-Aspa (IC50 < 0.01 IU/mL) whereas 5 displayed a moderate sensitivity (IC50 < 0.5 IU/mL). Remaining 5 patients were resistant to L-Aspa. The cells from the healthy subject were resistant (IC50 > 10 IU/mL). To date, ASNS expression has been evaluated on 16 patients with ratio ranges from 0 to 2.27: six were negative, 4 low positive (< 0.2), and 6 positive (> 0.5, amongst them 4 were > 1). No correlation was observed between ASNS expression and FAB grade. However, patients with blasts sensitive to L-Aspa had mainly M1 or M5 AML. Conclusion AML cell line HL-60 and 71% of primary cells from AML patients were found sensitive to L-Aspa. The sensitivity of cells from M1 and M5 AML patients is consistent with the findings of Okada et al. (2003). Globally, these results suggest that L-Aspa is effective for killing AML cells. Based on the epidemiology of AML subtypes (Selter et al., 2011) and our results, L-Aspa therapy may be beneficial for 50-70% of patients with AML. However, L-Aspa has only been used scarcely in the treatment of AML, mainly because of the commonly observed adverse effects that impair its use. A new formulation of L-Aspa encapsulated in homologous red blood cells was reported with a better safety profile, allowing its use even in elderly patients (Hunault et al., ASH 2012). A clinical study is currently recruiting patients unfit for intensive chemotherapy in order to evaluate its efficacy in combination with low-dose cytarabine (NCT01810705) in AML. In this study, L-Aspa sensitivity and ASNS expression in primary tumor cells at diagnosis will be explored to investigate the relationship with clinical response. Disclosures: Berlier: ERYTECH Pharma: Employment. Aguera:ERYTECH Pharma: Employment. Gallix:ERYTECH Pharma: Employment. Bertrand:ERYTECH Pharma: Principal Investigator Other. Thomas:ERYTECH Pharma: Principal Investigator Other; Orphan Europe: Consultancy. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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Blast Asparagine Synthetase Deficiency in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.4333.4333 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4333-4333

Author(s):

Willy Berlier ◽

Mohamed El Hamri ◽

Karine Aguera ◽

Yannick Campion ◽

Fabien Gay ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Viability ◽

Cell Line ◽

Myeloid Leukemia ◽

High Sensitivity ◽

Asparagine Synthetase ◽

Leukemic Cells ◽

Acute Myeloid

Abstract Abstract 4333 Introduction L-asparaginase (L-Aspa) is one of the standard components of acute lymphoid leukemia (ALL) therapy. Its efficacy as an antineoplasic agent is due to its capacity to deplete plasmatic asparagine, starving the leukemic cells and subsequently inhibiting protein synthesis. In vitro studies have already shown the sensitivity to L-Aspa of leukemic cells from patients with acute myeloid leukemia (AML), depending on the French-American-British (FAB) subtype (Zwaan et al., 2000; Okada et al., 2003). Despite promising results in clinical trials (Capizzi et al., 1988; Wells et al., 1993), L-Aspa has only been used scarcely in the treatment of AML, mainly because of the adverse effects observed commonly that impair its use. The existence of a new formulation of L-Aspa (L-Aspa encapsulated in heterologous red blood cells) with a better safety profile allows considering its use for the treatment of AML (Godfrin et al., 2012). The sensitivity to L-Aspa may be inversely correlated to their expression of asparagine synthetase (ASNS, an enzyme catalyzing the intracellular synthesis of asparagine). The aim of this study was to investigate the potential of L-Aspa for AML treatment by determining the leukemic cell expression of ASNS and their sensitivity to L-Aspa. Materials and methods Studies were performed on an AML cell line (HL-60) and on leukemic cells isolated from the bone marrow of AML patients by Ficoll density gradient. The IC50 (concentration inhibiting 50% of cell viability) was determined in vitro by incubating various concentrations of L-Aspa with the cells and measuring the cell viability with a cell counting kit (CCK-8 viability assay). ASNS expression was determined by western-blot. Results As a preliminary, determination of IC50 for the HL-60 cell line demonstrated that these cells were equally sensitive to L-Aspa than the ALL cell line MOLT-4 in vitro(0.23 IU/mL versus 0.19 IU/mL, respectively). The expression of ASNS in the HL-60 cell line was low in comparison with other cancer cell lines. Concerning blasts isolated from AML patients, 2/3 patients displayed an IC50 < 0.01 IU/mL whereas 1/3 displayed an IC50 as low as 0.13 IU/mL, which means a high sensitivity to L-Aspa. All these patients were negative for ASNS expression. On 6 patients tested for ASNS expression, 5 had their blasts negative whereas one was positive. Patients with blasts negative for ASNS expression were all affected by FAB M5 or M1 AML whereas patient with blasts positive was diagnosed with a M2 AML. Conclusion Both AML cell line and cells isolated from AML patients were sensitive to L-Aspa. In all clinical cases, high sensitivity and/or lack of ASNS expression were linked to a FAB M5 or M1 AML, consistent with data from literature suggesting a higher sensitivity of M1, M4 and M5 AML subtypes. The only clinical case positive for ASNS expression was a FAB M2 AML, consistent with literature that indicates an in vitro resistance of the M2 AML subtype to L-Aspa. Globally, these results suggest that L-Aspa is effective for killing AML cells with low/no ASNS expression. Thus, thanks to its low toxicity profile, L-Aspa may be considered as a potential therapy for FAB subtypes or individuals characterized by low/no ASNS expression. Based on the epidemiology of AML subtypes (Selter et al., 2011), L-Aspa therapy may be beneficial for 50% of patients with AML. These results finally highlight the necessity of the determination of the grade and/or the expression of ASNS in leukemic cells to select AML patients at potential for L-Aspa therapies. Disclosures: Berlier: ERYTECH Pharma: Employment. Aguera:ERYTECH Pharma: Employment. Campion:ERYTECH Pharma: Employment. Gay:ERYTECH Pharma: Employment. Godfrin:ERYTECH Pharma: COO Other.

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Temozolomide sensitivity of malignant glioma cell lines – a systematic review assessing consistencies between in vitro studies

BMC Cancer ◽

10.1186/s12885-021-08972-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Michael T. C. Poon ◽

Morgan Bruce ◽

Joanne E. Simpson ◽

Cathal J. Hannan ◽

Paul M. Brennan

Keyword(s):

Cell Viability ◽

Malignant Glioma ◽

Cell Line ◽

Cell Lines ◽

Glioma Cell ◽

Glioma Cell Line ◽

In Vitro Studies ◽

Experimental Conditions ◽

Glioma Cell Lines

Abstract Background Malignant glioma cell line models are integral to pre-clinical testing of novel potential therapies. Accurate prediction of likely efficacy in the clinic requires that these models are reliable and consistent. We assessed this by examining the reporting of experimental conditions and sensitivity to temozolomide in glioma cells lines. Methods We searched Medline and Embase (Jan 1994-Jan 2021) for studies evaluating the effect of temozolomide monotherapy on cell viability of at least one malignant glioma cell line. Key data items included type of cell lines, temozolomide exposure duration in hours (hr), and cell viability measure (IC50). Results We included 212 studies from 2789 non-duplicate records that reported 248 distinct cell lines. The commonest cell line was U87 (60.4%). Only 10.4% studies used a patient-derived cell line. The proportion of studies not reporting each experimental condition ranged from 8.0–27.4%, including base medium (8.0%), serum supplementation (9.9%) and number of replicates (27.4%). In studies reporting IC50, the median value for U87 at 24 h, 48 h and 72 h was 123.9 μM (IQR 75.3–277.7 μM), 223.1 μM (IQR 92.0–590.1 μM) and 230.0 μM (IQR 34.1–650.0 μM), respectively. The median IC50 at 72 h for patient-derived cell lines was 220 μM (IQR 81.1–800.0 μM). Conclusion Temozolomide sensitivity reported in comparable studies was not consistent between or within malignant glioma cell lines. Drug discovery science performed on these models cannot reliably inform clinical translation. A consensus model of reporting can maximise reproducibility and consistency among in vitro studies.

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883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.883 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A925-A925

Author(s):

Alessandra Romano ◽

Nunziatina Parrinello ◽

Sara Marino ◽

Enrico La Spina ◽

Massimo Fantini ◽

...

Keyword(s):

Flow Cytometry ◽

Functional Analysis ◽

Epithelial Cells ◽

Cell Line ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Phase 1 ◽

Adcc Activity ◽

Phenotypic Analysis

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb’s target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples.

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In Vitro Cytotoxicity of Oleanolic/Ursolic Acids-Loaded in PLGA Nanoparticles in Different Cell Lines

Pharmaceutics ◽

10.3390/pharmaceutics11080362 ◽

2019 ◽

Vol 11 (8) ◽

pp. 362 ◽

Cited By ~ 20

Author(s):

Amélia M. Silva ◽

Helen L. Alvarado ◽

Guadalupe Abrego ◽

Carlos Martins-Gomes ◽

Maria L. Garduño-Ramirez ◽

...

Keyword(s):

Cell Viability ◽

Cell Line ◽

Cell Lines ◽

Specific Activity ◽

Polymeric Nanoparticles ◽

Hepatoma Cell ◽

Plga Nanoparticles ◽

Hepatoma Cell Line ◽

Human Hepatoma Cell

Oleanolic (OA) and ursolic (UA) acids are recognized triterpenoids with anti-cancer properties, showing cell-specific activity that can be enhanced when loaded into polymeric nanoparticles. The cytotoxic activity of OA and UA was assessed by Alamar Blue assay in three different cell lines, i.e., HepG2 (Human hepatoma cell line), Caco-2 (Human epithelial colorectal adenocarcinoma cell line) and Y-79 (Human retinoblastoma cell line). The natural and synthetic mixtures of these compounds were tested as free and loaded in polymeric nanoparticles in a concentration range from 2 to 32 µmol/L. The highest tested concentrations of the free triterpene mixtures produced statistically significant cell viability reduction in HepG2 and Caco-2 cells, compared to the control (untreated cells). When loaded in the developed PLGA nanoparticles, no differences were recorded for the tested concentrations in the same cell lines. However, in the Y-79 cell line, a decrease on cell viability was observed when testing the lowest concentration of both free triterpene mixtures, and after their loading into PLGA nanoparticles.

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All-Trans-Retinoic Acid (ATRA) Causes Extensive Differentiation In the NPM Mutant, Non-APL Leukemic Cell Line OCI-AML3

Blood ◽

10.1182/blood.v116.21.3305.3305 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3305-3305 ◽

Cited By ~ 1

Author(s):

Matthew A. Kutny ◽

Steven J. Collins ◽

Keith Loeb ◽

Roland B. Walter ◽

Soheil Meshinchi

Keyword(s):

Cell Viability ◽

Cell Count ◽

Cell Line ◽

Cell Lines ◽

Time Course ◽

Conflicts Of Interest ◽

Live Cell ◽

Leukemic Cells ◽

Viable Cell Number ◽

Nuclear Segmentation

Abstract Abstract 3305 The differentiating agent ATRA has been used successfully in the treatment of acute promyelocytic leukemia (APL). By comparison, non-APL AML has not shown similar sensitivity to ATRA induced differentiation. Recent data has suggested that a subset of de novo AML patients with nucleophosmin (NPM1) mutations may benefit from addition of ATRA to conventional therapy. The NPM1 gene has several functions affecting cell cycle proliferation including regulation of ribosome biogenesis and centrosome duplication and it acts as a histone chaperone. Mutation of the NPM1 gene leads to differentiation arrest contributing to AML pathogenesis. We hypothesized that leukemia cells with NPM1 mutations could be induced to undergo differentiation. We tested this hypothesis with the NPM1 mutant AML cell line OCI-AML3 and compared the results to identical assays using the AML cell line HL-60 which has been previously well documented to differentiate in response to ATRA therapy. OCI-AML3 and HL-60 cell lines were treated for 5 days with control media and four ATRA doses including 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell viability was assessed by flow cytometry. Compared to the control condition, OCI-AML3 cells treated with the lowest dose of ATRA (0.2 μM) had a live cell count 21.6% of the control. HL-60 cells treated at even the highest ATRA dose (25 uM) had a live cell count 79.3% of the control. Due to the sensitivity of OCI-AML3 cells to the toxic effects of ATRA, the experiment was repeated with lower doses of ATRA including 0.001 μM, 0.01 μM and 0.1 μM. At the lowest dose of ATRA (0.001 μM), OCI-AML3 cells demonstrated a cell viability of 49% with further decrease to 26% at 0.1 μM dose of ATRA. At similar ATRA doses, cell viability for HL-60 cells was 91% and 85%, respectively (see table 1). Table 1: Cell viability as a percent of control cells after 5 days of treatment at three different doses of ATRA in OCI-AML3 and HL-60 cell lines. Cell Line: ATRA 0.001 μM ATRA 0.01 μM ATRA 0.1 μM OCI-AML3 49% 33% 26% HL-60 91% 91% 85% We subsequently determined the time course of changes in cell growth and the extent of differentiation at each point was determined by morphologic assessment. Both cell lines were treated with ATRA at doses of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM for a total of 4 days. Each day viable cell number was determined. In contrast to the HL-60 cells which had continued growth in lower ATRA doses, OCI-AML3 cells demonstrated exquisite sensitivity to growth arrest at the lowest doses of ATRA. Cell morphology was assessed daily with modified Wright-Giemsa staining of cells. Cells were examined for signs of myeloid differentiation including decrease in nuclear to cytoplasmic (N/C) ratio, nuclear segmentation, and cytoplasmic granules and vacuoles. At the lowest dose of ATRA (0.001 μM), after 4 days of exposure, significant number of OCI-AML3 cells demonstrated morphologic evidence of differentiation. At this ATRA dose and exposure interval, HL-60 cells showed no evidence of differentiation. At an ATRA dose of 1 μM (considered a standard dose used for differentiation of HL-60 cells), the OCI-AML3 cells showed differentiation changes as early as day 2 with nuclear segmentation and decreased N/C ratio while HL-60 cells did not show any change at this time point. After 4 days of ATRA exposure, most OCI-AML3 cells showed segmented nuclei and vacuolated cytoplasm, whereas HL-60 cells showed less distinct signs of differentiation with some cytoplasm granules and cup shaped nuclei. This data suggests that leukemic cells with NPM mutations may be susceptible to the pro-differentiating properties of ATRA. Further substantiation of this data with primary human specimens may ultimately provide the rationale for a novel therapeutic option using ATRA-based differentiation therapy for subsets of non-APL AML. Disclosures: No relevant conflicts of interest to declare.

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Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽

10.1182/blood.v66.2.447.447 ◽

1985 ◽

Vol 66 (2) ◽

pp. 447-455 ◽

Cited By ~ 66

Author(s):

D Zipori ◽

J Toledo ◽

K von der Mark

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Cell Line ◽

Cell Lines ◽

Stromal Cell ◽

Leukemic Cells ◽

Matrix Composition ◽

Mouse Bone Marrow ◽

Type I

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

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MiR-624-5p enhances cell resistance against cisplatin via PDGFRA/Stat3/PI3K axis in ovarian cancer

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v19i4.3 ◽

2020 ◽

Vol 19 (4) ◽

pp. 691-698

Author(s):

Lin I-Ju ◽

Tian YongJie

Keyword(s):

Ovarian Cancer ◽

Cell Viability ◽

Cell Line ◽

Cell Lines ◽

Clinical Stage ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay

Purpose: The purpose of this study was to evaluate the role of miR-624-5p in ovarian cancer.Methods: MiR-624-5p expression in ovarian cancer {OC) cell lines and normal cells (NCs) was evaluated and compared the differential miR-624-5p in OC A2780 cells and cisplatin-resistant OC cell line (A2780/DDP). CCK-8 was used to evaluate changes in cell viability of the A2780 and A2780/DDP cell lines as well as silenced miR-624-5p. Western Blot examined the Stat3 and phosphorylated Pi3k. The binding between PDGFRA and miR-624-5p was predicted on Targetscan and verified through Luciferase Reporter Assay. The role of PDGFRA in A2780/DDP by overexpressing PDGFRA was evaluated by RT-qPCR and CCK-8 assays. RT-qPCR assay also measured miR-624-5p expression responsive to different dosages of cisplatin and CCK8 examined viability levels correspondingly. In addition, the interplay of PDGFRA and miR-624-5p by combined downregulation of both miR-624-5pand PDGFRA were evaluated.Results: OC cells had higher miR-624-5p expression than NCs but lower compared to cisplatinresistant A2780/DDP cells. A2780/DDP cells had higher viability than OC cell line A2780. Stat3 and phosphorylated PI3K were activated in A2780/DDP cells. Silencing miR-624-5p led to lower viability inA2780/DDP cells. miR-624-5p expression dropped as the cisplatin concentration increased, resulting in decreasing viability respectively. Luciferase Reporter assay validated the binding of miR-624-5p and PDGFRA in A2780/DDP cells. Overexpressed PDGFRA induced lower cell viability in A2780/DDP cells. Downregulation of PDGFRA partially restored the lowered viability and inhibited Stat3 as well as phosphorylated Pi3k induced by miR-624-5p inhibitor.Conclusion: MiR-624-5p could add to the cellular resistance to cisplatin in OC in-vitro model, which indicated that it might help unveil the mystery of drug-resistance in clinical stage of ovarian cancer. Keywords: MiR-624-5p, resistance, cisplatin, PDGFRA/Stat3/PI3K, ovarian cancer

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Association of Asparagine Synthetase Expression and Sensitivity to L-Asparaginase in Cell Lines and Primary Pediatric Acute Lymphoblastic Leukemia Samples.

Blood ◽

10.1182/blood.v114.22.2738.2738 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2738-2738

Author(s):

Ivana Hermanova ◽

Jan Trka ◽

Julia Starkova

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Amino Acid ◽

Mrna Expression ◽

Cell Line ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Asparagine Synthetase ◽

Leukemic Cells ◽

Mrna Levels ◽

Basal Expression

Abstract Abstract 2738 Poster Board II-714 L-Asparaginase (L-Asp) is an important component in the combined chemotherapy for childhood acute lymphoblastic leukemia (ALL). Administration of L-Asp leads to depletion of plasmatic asparagine and consequently causes loss of intracellular asparagine. As a non-essential amino acid, asparagine is synthesized from aspartate and glutamine by asparagine synthetase (ASNS). Primary ALL cells are believed to have low ASNS expression and therefore to be sensitive to asparagine depletion. Although increased ASNS level was shown to be connected with L-Asp resistance the exact relationship between ASNS expression and L-Asp sensitivity is not clear. We and others have previously shown TEL/AML1[+] ALL blasts express more ASNS mRNA than TEL/AML[-] do although primary TEL/AML[+] cell are in vitro more sensitive to treatment with L-Asp. Hutson et al (1997) showed that amino acid deprivation led to increased expression of ASNS on mRNA and protein level as well as to increased biological activity. On the other hand, Nan Su et al described negative correlation between L-Asp sensitivity and ASNS protein rather than mRNA levels. Therefore, in our studies we concentrated on protein expression of ASNS in patients' samples. So far, there has been no reproducible published data on ASNS protein detection by Western blot in primary patients' samples. Despite using 3 different antibodies and precise optimization we were not able to detect ASNS protein in patients' samples in contrast to cell lines. Transcripts' levels confirmed significantly lower (2 log) expression of ASNS in patients' leukemic cells compared to leukemic cell lines. Therefore, for further studies on gene and protein relation we had to rely on cell lines as a model. We detected ASNS gene expression and ASNS protein content in four ALL cell lines: REH (TEL/AML1[+]), UOCB6 (TEL/AML1[+]), NALM6 (TEL/PDGFRB[+]) and RS4;11 (MLL/AF4[+]). ASNS mRNA levels were in accord with sensitivity to L-Asp. UOCB6 as the most resistant cell line (IC50=0.04U/ml) had the highest expression of ASNS (normalized ASNS, nASNS=4.946), then NALM6 (IC50=0.01U/ml; nASNS=1.8), REH (IC50=0.6.10−4; nASNS=1.176) and RS4;11 (IC50<0.3.10−4; nASNS=0.024). ASNS protein levels significantly differed through passages in REH cells, likely due to rapid turnover. For the remaining three cell lines L-Asp sensitivity correlated also with protein content. We have previously shown that different basal expression levels do not affect short-term dynamics of ASNS expression after L-Asp administration. Here we were interested to see the changes of sensitivity to L-Asp using gradient silencing of ASNS by RNAi in two cell lines with different basal expression: REH cell line with intermediate ASNS mRNA expression and RS4;11 cell line with very low mRNA expression. Gradient silencing revealed that L-Asp sensitivity correlated with ASNS expression till 50% decrease; further silencing did not potentiate the effect. The same response was seen in both cell lines despite different basal ASNS expression and sensitivity to L-Asp. The ASNS is glutamine dependent enzyme therefore we also studied expression of glutamate dehydrogenase (GDH), an enzyme necessary for glutamine synthesis. We found significantly lower GDH mRNA expression in primary TEL/AML1[+] blasts in comparison with TEL/AML[-] blasts (p=0.019), which might lead to deficiency of glutamine in these cells and consequently higher sensitivity to L-Asp. Accordingly, silencing of ASNS in REH tended to increase GDH expression levels. Our data confirm that generally, both ASNS mRNA and protein expression inversely correlate with the sensitivity to L-Asp in the cell lines. However, it may be misleading to draw conclusions for the patients' cells directly from the results obtained in cell line models. The expression patterns of ASNS in primary leukemic cells differ even from those of genotypically identical cell lines. The control of basal levels of ASNS in leukemic cells remains to be elucidated. Our results implicate an important role of GDH and glutamine metabolic pathway in the regulation of ASNS activity. This work was supported by MSM0021620813 and GAUK 7835. Disclosures: No relevant conflicts of interest to declare.

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PI3K/Akt/mTOR Pathway Inhibition in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Blood ◽

10.1182/blood.v126.23.1558.1558 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1558-1558 ◽

Cited By ~ 1

Author(s):

Maria Bhatti ◽

Thomas Ippolito ◽

Cory Mavis ◽

Matthew J. Barth

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Viability ◽

Cell Line ◽

Cell Lines ◽

Propidium Iodide ◽

Mtor Pathway ◽

Propidium Iodide Staining ◽

Induction Of Apoptosis ◽

Pre Treatment

Abstract Introduction: Burkitt lymphoma (BL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) in children. Despite significant improvements in survival with de novo disease, treatment of relapsed or refractory BL remains a significant hurdle with survival in only about 20% of patients. Novel therapeutic approaches are necessary to improve outcomes in this group of childhood B-NHL patients with the worst prognosis. Recent literature has identified a high rate of recurrent mutations that result in activation of the PI3K/Akt pathway in BL and have implicated activation of PI3K/Akt in coordination with Myc in BL lymphomagenesis. Our laboratory has developed rituximab and chemotherapy resistant cell line models and subsequently found that these cell lines exhibit increased activation of Akt. We hypothesized that increased activation of Akt may be contributing to chemoresistance and that targeting the PI3K/Akt/mTOR pathway may increase chemoresponsiveness. To that end, we have investigated the effect of inhibiting the PI3K/Akt/mTOR pathway with either the PI3K-delta inhibitor idelalisib or the pan-PI3K/mTOR inhibitor BEZ-235 in cell line models of BL. Methods: The in vitro effect of idelalisib or BEZ-235 was investigated in BL cell lines including Raji, Raji 2R and Raji 4RH (rituximab-chemotherapy resistant), Raji 7R and Raji 8RH (rituximab resistant), Ramos and Daudi. Cell viability following inhibitor exposure was assessed by Alamar blu and cell-titer glo assays. The effect of inhibitor exposure on cell cycle progression was determined by flow cytometry using propidium iodide staining. Inhibition of Akt activation following inhibitor exposure was determined using phospho-flow cytometry. The activity of cytotoxic chemotherapeutic agents following inhibition by idelalisib or BEZ-235 was assessed using Alamar blu and cell titer glo assays. Results: In vitro exposure of BL cell lines to idelalisib in concentrations from 0.1-100µM for 24, 48 or 72 hours resulted in a dose and time-dependent decrease in viable cells in all cell lines tested with IC50 concentrations of 60-300uM. Pre-treatment with the pan-caspase inhibitor QVD resulted in a small reversal in the decrease in cell viability suggesting only a minimal portion of the activity was caspase dependent. When induction of apoptosis was measured using annexin V-propidium iodide staining, little induction of apoptosis was observed with single agent idelalisib at concentrations up to 100uM. Determination of cell cycle progression following exposure to idelalisib at 1, 10, 50 or 100 uM for 24, 48 or 72 hours indicated a time and dose dependent cell cycle arrest in all cell lines. In chemotherapy-sensitive cell lines the arrest was primarily noted in G1, while the chemotherapy-resistant Raji 2R and Raji 4RH cell lines exhibited arrest primarily in G2/M. A significant reduction in cell viability following chemotherapy exposure for 48 hours was noted in chemotherapy resistant Raji 2R cells following pre-treatment for 48 hours with idelalisib 10uM compared to non-idelalisib exposed cells (doxorubicin 10uM 55% vs 77%, p<0.001; vincristine 0.05uM, 48% vs 61%, P<0.001). At higher idelalisib pre-treatment concentrations (50uM) additional synergistic activity was observed in Raji 2R cells (cisplatin 48% vs 61%, p<0.001; dexamethasone 67% vs 87%, p<0.01). To further assess the effect of dual inhibition of PI3K and mTOR, cell lines were exposed to the dual inhibitor BEZ-235. BEZ-235 exhibited a more potent decrease in cell viability compared to idelalisib with activity at nM concentrations. Unlike idelalisib, exposure to BEZ-235 resulted in significant induction of apoptosis by Annexin V-propidium iodide staining. BEZ-235 also exhibited synergistic activity in combination with chemotherapy in all cell lines. At equivalent dosing, BEZ-235 exposure resulted in a more significant decrease in Akt phosphorylation compared to idelalisib as determined by flow cytometry for p-Akt at Ser and Thr phosphorylation sites. Conclusions: Chemotherapy sensitive and resistant BL cell line models are susceptible to inhibition of the PI3K/Akt/mTOR pathway. Targeted inhibition of this pathway leads to a decrease in AKT activation, decrease in cell viability, cell cycle arrest and an increase in sensitivity to cytotoxic chemotherapeutic agents. Broader inhibition of both PI3K and mTOR is more effective than more targeted inhibition of PI3K-delta alone. Disclosures No relevant conflicts of interest to declare.

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