Dramatic Down-Regulation Of MNDA Expression In CP-CML Cells From The LSC Compartment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2717-2717
Author(s):  
Céline Bourgne ◽  
Alexandre Janel ◽  
Juliette Berger ◽  
Agnès Guerci ◽  
Caroline Jamot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Differentiation ◽  
Cell Line ◽  
Cell Lines ◽  
Fold Increase ◽  
Positive Control ◽  
Polymorphonuclear Cells ◽  
Primary Cells ◽  
Down Regulation ◽  
Cd34 Cells

Abstract Introduction Chronic Phase - Chronic Myeloid Leukemia (CP-CML) is a myeloproliferative disorder characterized by malignant proliferation of the granulocytic lineage without the arrest of cell differentiation. Tyrosine Kinase Inhibitors (TKI) have revolutionized CML treatment but several studies showed that a combination of TKI and Interferon alpha (IFNα) provides better clinical response. Myeloid Nuclear Differentiation Antigen (MNDA), which belongs to the hematopoietic interferon-inducible nuclear proteins with the 200-amino-acid repeat (HIN200) gene family, encodes a protein expressed in myeloid cells but whose function remains poorly understood. Because of its high expression in polymorphonuclear cells, its involvement in cell differentiation and apoptosis, and its induction by IFNα, we evaluated MNDA expression in CML cells and its modulation after incubation with IFNα. Material and methods We tested MNDA expression in several cell lines (K562, KCL22, LAMA84, TF1 and U937 (positive control)), in polymorphonuclear cells from healthy donors (HD-PMN, n=13) and in primary cells from patients with CP-CML at diagnosis (CP-CML; n=17). The relative expression of the MNDA transcript was analyzed using the 2-ΔΔCt method and was normalized to the endogenous reference gene GAPDH. HD-PMN were used as calibrator. We developed a multiparametric flow cytometry assay (CD45-V500/CD14-APC-H7/CD15-PerCpCy5.5/CD34-PC7/CD38-V450/MNDA-FITC) to detect MNDA protein in the different cell subsets, particularly in CD34+cells. Results As previously described, MNDA was poorly expressed in the K562 cell line. Similarly, mRNA was detected at low levels in two other CML cell lines (KCL22, LAMA84) and in TF1 cells, but at a high level in the U937 cell line, used as a positive control. In each cell line, the transcript expression was correlated to the protein level, as evaluated by flow cytometry (MFI ratio: 2.04±0.21, 2.36±0.24, 1.59±0.14, 1.88±0.11 and 8.77±0.54 for K562, KCL22, LAMA84, TF1 and U937, respectively (n=3)). In CP-CML primary cells, MNDA expression was greatly diminished as compared with HD-PMN in both mRNA (0.20±0.08 (n=17) vs. 1.32±0.21 (n=10); p=1.52x10-6) and protein (MFI ratio: 6.9±0.98 vs. 16.31±1.25, p=0.001). After having verified that IFNα (2000 U/ml, 16 hours) induced MNDA expression in HD mononuclear cells but not in PMN, we observed that induction of MNDA was moderate in CML cell lines K562 and LAMA84 (2-fold increase, n=3) whereas the level of MNDA mRNA was significantly increased in TF1 cells (28-fold increase, n=4). Induction in primary CML cells was variable (3/5 patients). Aiming to evaluate the expression of MNDA in leukemic stem cells (LSC), we first analyzed MNDA expression in CD34+ and CD34+/CD38- cells from HD. We observed that MNDA is down-regulated in healthy CD34+ and CD34+/CD38- cells compared to mature cells (mRNA: about 4 logs, protein: 8-10 fold lower, n=4), but we always detected a significant signal in CD34+cells (MFI ratio: 2.76±0.46, n=3). However, MNDA was not expressed by CML cells from the LSC compartment (n=4). This inhibition does not seem to be antagonized by nilotinib or IFNα (n=2). Discussion/Conclusion MNDA expression appears to be clearly down-regulated in CP-CML cells and dramatically so in the LSC compartment. In some patients, we observed sustained sensitivity to IFNα, but only in the compartment of more mature cells. This suggests early deregulation of MNDA expression which seems to be only partially dependant on differentiation. The mechanisms involved in this down-regulation remain to be elucidated but could be independent to TK activity of BCR-ABL protein and resistant to IFNα in the LSC compartment. This marked deregulation of MNDA in the LSC compartment is an additional argument in favor of intrinsic changes specific to primitive cells. Disclosures: No relevant conflicts of interest to declare.

A Small Molecule Stat Inhibitor Blocks Stat3 and Stat5 Phosphorylation and Demonstrates Cytotoxicity In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2904-2904
Author(s):  
Robyn M. Dennis ◽  
Brandon Ballard ◽  
David John Tweardy ◽  
Karen Rabin
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Positive Control ◽  
Wild Type ◽  
Atp Assay ◽  
Antibody Staining

Abstract Abstract 2904 Survival has improved dramatically in acute lymphoblastic leukemia (ALL), but further gains are unlikely using conventional chemotherapy alone. Several recently discovered, novel cytogenetic lesions with adverse prognostic impact, JAK2 activating mutations and CRLF2 rearrangements, occur in up to 15% of adult and pediatric ALL. These lesions are associated with activation of Jak2 and Stat5, and hold promise as targets for novel therapies affecting these signaling pathways. We performed in vitro testing of a novel small molecule Stat inhibitor, C188-9, in B-lineage ALL cell lines and patient samples with and without JAK2/CRLF2 alterations. C188-9 treatment for one hour at 10 μM inhibited Stat3 and Stat5 phosphorylation in ALL cell lines with JAK2 and CRLF2 alterations, but not in cell lines with wild-type JAK2 and CRLF2, as measured by phospho-flow cytometry (Fig. 1A). Only the cell lines with JAK2 and CRLF2 alterations demonstrated basal Stat5 phosphorylation on Western blot analysis, and this was inhibited by C188-9 treatment (Fig. 1B). C188-9 demonstrated cytotoxicity in ALL cell lines regardless of JAK2/CRLF2 status, with IC50s in the low micromolar concentration range (Fig. 1C). While C188-9 is undergoing investigation currently as a potent inhibitor of Stat3 in acute myeloid leukemia (AML), it also merits further investigation as an agent with Stat5 inhibitory activity and cytotoxicity in ALL. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Disclosures: No relevant conflicts of interest to declare.

Flow Cytometry Detection of Intra-Cellular Tyrosine Kinase Inhibitors (TKI) Showed Variable Uptake in CML CD34+ Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2747-2747
Author(s):  
Céline Bourgne ◽  
Mahchid Bamdad ◽  
Alexandre Janel ◽  
Frédéric Libert ◽  
Agnès Guerci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Blood Cells ◽  
Second Generation ◽  
Molecular Response ◽  
Primary Cells ◽  
Uv Fluorescence ◽  
Cd34 Cells

Abstract Abstract 2747 Introduction Despite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML), patient response is heterogeneous and it is generally accepted that residual disease and relapse are due to persistent CML cells, considered as leukemic stem cells. Their resistance has been related to lower TKI uptake. The amount of drug penetrating the targeted cells is most likely a major parameter of targeted therapy efficacy since it is essential that the therapeutic molecule be as close as possible to the target molecule. We developed a flow cytometry technique to analyze primary cells. Method To evaluate intracellular imatinib (ICIM) uptake, we developed a patented method based on natural UV fluorescence related to chemical structure. Consequently, since the difference in UV fluorescence units between treated and control cells is proportional to the amount of intra-cellular drugs, we validated this method after incubating K562 and KCL22 cell lines with TKI. The flow cytometry technique was standardized by using Flow-Check Fluorosphere calibrated beads immediately before, and at the end of, each series of analyses with a Coulter Epics Elite™ flow cytometer (Beckman Coulter) equipped with an Innova I90C-4 UV laser (Coherent). Then we analyzed primary blood cells from CML patients in chronic phase before any treatment. After lysis of erythrocytes, nucleated cells were incubated at 1.106 cells/ml with different doses of imatinib (IMA) (n=22), Nilotinib (NIL) (n=20) and Dasatinib (DAS) (n=20) at different times. Whenever possible, CML stem cells were analyzed using CD34-FITC staining. Results In preliminary assays, we checked that there was a significant correlation between additional fluorescence measured by flow cytometry and the amount quantified by physico-chemical analysis after lysing a known number of cells (n=57, r2=0.73, p<0.001), which enabled us to convert UV fluorescence into pg of IMA per cell. Then we confirmed that IMI rapidly penetrated K562 and KCL22 cells (from 5 minutes of incubation) and reached a stable influx in viable cells from 1 hour (T1h). We chose this incubation time for further experiments. Similarly, we choose T2h for second generation TKI. We observed a dose-dependent accumulation in the two cells lines, but with differences at the lowest extra-cellular concentrations (1–5 μM) and not correlated with any membrane pump expression (OCT-1, ABCG2, ABCB1 and ABCC1). ICIM at T1h was correlated with cell sensitivity to IM at T24h expressed by the proportion of dead cells (r2=0.93 and 0.88 for K562 and KCL22 cells, respectively). We then applied our method to primary CML blood cells in comparison with normal blood cells. TKI penetrated all cell subsets, but amounts varied depending on cell sizes (FS/SS characteristics). The first data obtained with IM showed ICIM levels in CML cells that were relatively heterogeneous from one patient to another, ranging from 0.9 to 4 pg/cell for an extracellular concentration of 5 μM, i.e. a higher concentration (x 300) than in culture medium. The ability of the granulocyte cell lineage to store IMA was related to the Sokal prognostic index (p=0.05). We detected variable ICIM levels in CML CD34+ cells from 10/16 patients (0.04–0.7 pg/cell) and no signal for 6/16 patients. Surprisingly, the ability of CD34+ to store second generation TKIs is variable and not necessarily correlated to IMA uptake. Discussion We developed a simple, rapid flow cytometry method directly applicable to primary cells and requiring only few cells which makes it possible to identify target cell subsets, such as CML stem cells. The strong correlation between the ICIM amount and the sensitivity of CML cell lines to TKIs validated the method and suggested that ICIM could be a relevant biomarker for predicting the sensitivity of the CML clone. In our CML series, we observed striking inter-patient variability of the capacity of primary CML cells to store TKI. A correlation with the Sokal score suggests possible predictive value with regard to in vivo CML response to IMA, which could be taken into account when choosing TKI for first-line therapy. Furthermore, we observed marked heterogeneity between CML CD34+ cells for storing TKI that could partially explain the heterogeneity of in vivo response. The relationship between the ability of untreated CML CD34+ cells to store TKI and complete molecular response has to be established. Disclosures: No relevant conflicts of interest to declare.

Expression and Functional Differences of CBFA2T (ETO, MTG) Gene Family Members in Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4208-4208
Author(s):  
Fariborz Mortazavi ◽  
ShriHari Kadkol ◽  
Annette Bruno ◽  
Kristine Baraoidan ◽  
Steven Ackerman ◽  
...  
Keyword(s):  
Gene Family ◽  
Cell Line ◽  
Cell Lines ◽  
Family Members ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Macrophage Differentiation ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract The CBFA2T (ETO, MTG) family has three similar family members - CBFA2T1-T3. CBFA2T1 (ETO, MTG8) and CBFA2T3 (ETO2, MTG16) are targeted by chromosomal translocations in acute myeloid leukemia. To better understand the usual hematopoietic function of this gene family, we examined the expression of CBFA2T RNA using RQ-PCR in cell-lines and human CD34+ hematopoietic cells during macrophage and erythroid differentiation. RQ-PCR on extracted RNA was performed with an icyclerQ instrument (Bio-Rad) using the Quantitect SYBR Green RT-PCR kit (Qiagen) and in vitro transcribed RNA to construct standard curves. CBFA2T3 was the most highly expressed family member in human CD34+ cells, the erythro-leukemia line K562, the myeloid line MPD, the T cell line Jurkatt and the B-cell line LCL-11. However, CBFA2T3 expression decreased by &gt;50% during both macrophage and erythroid differentiation of human CD34+ cells. In contrast, CBFA2T1 expression was almost undetectable in human CD34+ cells and all cell lines except K562 but increased more than 20 fold during erythroid (but not macrophage) differentiation of human CD34+ cells. Extrinsic over-expression of CBFA2T1, but not CBFA2T2, significantly increased glycophorin-A and hemoglobin A expression in K562 cells, consistent with a regulatory role for CBFA2T1 in erythroid differentiation. CBFA2T2 (MTGR1) was moderately expressed in human CD34+ cells and all the cell lines and demonstrated a 2.5 fold increase in expression with macrophage differentiation but essentially no change with erythroid differentiation of human CD34+ cells. These findings suggest that despite their similarity, the CBFA2T family members have distinctive regulatory roles in hematopoietic differentiation.

Vitamin D as Radiosensitizer: A Review in Cell Line -

2020 ◽  
Vol 10 (6) ◽  
pp. 315-324
Author(s):  
Fahmi Radityamurti ◽  
Fauzan Herdian ◽  
Tiara Bunga Mayang Permata ◽  
Handoko Handoko ◽  
Henry Kodrat ◽  
...  
Keyword(s):  
Vitamin D ◽  
Cell Differentiation ◽  
Cell Line ◽  
Cell Lines ◽  
Anticancer Agent ◽  
Medical Literature ◽  
In Vitro Studies ◽  
Anti Cancer

Introduction: Vitamin D has been shown to have anti-cancer properties such as antioxidants, anti-proliferative, and cell differentiation. The property of vitamin D as an anticancer agent triggers researchers to find out whether vitamin D is useful as a radiosensitizer. Multiple studies have been carried out on cell lines in various types of cancer, but the benefits of vitamin D as a radiosensitizer still controversial. This paperwork aims to investigate the utilization of Vitamin D3 (Calcitriol) as radiosensitizer in various cell line through literature review.Methods: A systematic search of available medical literature databases was performed on in-vitro studies with Vitamin D as a radiosensitizer in all types of cell lines. A total of 11 in-vitro studies were evaluated.Results: Nine studies in this review showed a significant effect of Vitamin D as a radiosensitizer agent by promoting cytotoxic autophagy, increasing apoptosis, inhibiting of cell survival and proliferation, promoting gene in ReIB inhibition, inducing senescene and necrosis. The two remaining studies showed no significant effect in the radiosensitizing mechanism of Vitamin D due to lack of evidence in-vitro settings.Conclusion: Vitamin D have anticancer property and can be used as a radiosensitizer by imploring various mechanism pathways in various cell lines. Further research especially in-vivo settings need to be evaluated.

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

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.

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity

1983 ◽  
Vol 3 (6) ◽  
pp. 1053-1061
Author(s):  
W H Lewis ◽  
P R Srinivasan
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Plating Efficiency ◽  
Wild Type Cell ◽  
Wild Type ◽  
Metaphase Chromosomes

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

Lack of PRDM1α Expression in Hodgkin/Reed-Sternberg Cells Is Likely Mediated by a Translational or Post-Translational Mechanism.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4367-4367
Author(s):  
Wayne Tam ◽  
Leonard Tan ◽  
Mario Gomez ◽  
Shaoan Fan ◽  
Yifang Liu ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Western Blotting ◽  
Plasma Cell ◽  
Zinc Finger Protein ◽  
Cell Phenotype

Abstract Hodgkin/Reed Sternberg (H/RS) cells are the neoplastic cells in classical Hodgkin lymphoma (HL). They are thought to resemble post-germinal center (GC) B cells with expression of markers associated with late stage of B-cell differentiation, for example, interferon regulatory factor -4/multiple myeloma-1 (IRF4/MUM1) and syndecan 1 (CD138). The PR (PRDI-BF1-RIZ) domain zinc finger protein 1 (PRDM1), a transcription repressor with a master regulatory role in plasma cell differentiation, is normally co-expressed with IRF-4/MUM-1 in plasma cells and in a subset of activated GC cells committed to plasma cell fate. We studied expression of PRDM1α, the functional isoform of PRDM1, in 14 classical HL cases [including 3 positive for Epstein-Barr-virus (EBV)] and 4 HL cell lines by immunohistochemistry and Western blotting, respectively. H/RS cells in primary HL cases are negative for PRDM1α, implying a desynchrony in expression between IRF-4/MUM1 and PRDM1. While the myeloma cell line U266 expresses relatively abundant PRDM1α, it was undetectable by Western Blotting in all HL cell lines tested, except for the EBV-positive HL cell line L591 which, unlike in vivo H/RS cells, has a Type III EBV latency pattern. PRDM1α expression in L591 but not in vivo H/RS cells suggests that PRDM1 expression may be modulated by latency type-specific EBV-encoded gene products or the B-cell phenotype exhibited by the cell line. The lack of PRDM1α protein in H/RS cells is not due to impaired gene transcription, since real-time quantitative PCR revealed similarly abundant PRDM1α transcripts in the HL cell lines as U266. In the absence of mutation in the PRDM1 coding region, these results suggest that failure to accumulate PRDM1α protein in H/RS cells is likely due to abnormal translation repression or protein turnover. Loss of functional PRDM1 as a result of translational or post-translational deregulation may represent a novel molecular lesion in HL.

Comparison of Three Different NOD/SCID-Related Strains in Preclinical Models of Acute Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2361-2361
Author(s):  
Alice Agliano ◽  
Ines Martin-Padura ◽  
Paola Merighetti ◽  
Patrizia Mancuso ◽  
Cristina Rabascio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Blood Cells ◽  
Lymphoblastic Leukemia ◽  
Primary Cells ◽  
Preclinical Models ◽  
Immunodeficient Mice ◽  
Nsg Mice ◽  
Different Strains

Abstract Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in a variety of different strains of immunodeficient mice has led to preclinical models extensively used to investigate AML and ALL stem cells, biology, and drug sensitivity. We investigated the engraftment kinetics of two AML cell lines (HL-60 and KG-1), two ALL cell lines (MOLT-16 and 697) and AML primary cells from an AML M4 patient (QD1-EIO, described in Fusetti et al, Cancer Res 2000) in 3 different strains of NOD/LtSz-Prkdcscid (NOD/SCID, NS)-related immunodeficient mice. NS, NS/beta2 null (NSB) and NOD/SCID/IL-2Rgamma null (NSG) mice were injected ip with 10x106 AML or ALL cells. Mice were observed daily and sacrificed when leukemia-related symptoms were evident. Overall, leukemia-related symptoms were observed in 71, 84 and 86% of NS, NSB and NSG mice, respectively (n=42, p<0.01), after a median of 53, 49 and 35 days (p<0.001). Leukemia engraftment was investigated in the marrow, the blood and the spleen by means of morphology, flow cytometry and quantitative PCR for human genes. AML HL-60 and KG-1 cells accounted for 10-6, 5-1 and 7-3 % of peripheral blood cells in NS, NSB and NSG mice, respectively. ALL MOLT-16 and 697 cells accounted for 2-12, 22-27 and 1-27 % of blood cells in NS, NSB and NSG mice, respectively. AML primary cells QD1-EIO accounted for <1, <1 and 3% of blood cells in NS, NSB and NSG mice, respectively. Similar engraftment results were observed in the marrow and in the spleen. Leukemia cell-injected NSG mice, compared to NS and NSB, showed a significantly higher increase of circulating endothelial mature cells (CEC, enumerated by flow cytometry as CD45−, CD13+ VEGFR2+ cells) and progenitors (CEP, enumerated by flow cytometry as CD45−, CD13+ VEGFR2+ CD117+ cells, see Shaked et al, Cancer Cell 2005). This CEC and CEP increase paralleled leukemia engraftment. Taken together, our data indicate that the 3 different strains have significantly different leukemia engraftment behavior and kinetics. Engraftment in NSG mice is significantly faster compared to the other two strains, leukemia-related microenvironment is differently modulated, and less leukemic burden might be needed to observe leukemia-related symptoms. These data might have major implications to design future studies on leukemia-initiating stem cells, leukemia biology and preclinical leukemia treatment studies.

Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4547-4547
Author(s):  
Huanling Zhu ◽  
Ting Liu ◽  
Yongqian Jia
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Sensitive Cell ◽  
Imatinib Resistance ◽  
Low Expression

Abstract Objective To establish an imatinib resistance cell line and to study its resistant principia. Methods K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. Clone imatinib resistance cell lines by limited dilution culture. MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance. Results Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib. The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell. HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold). MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines. Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.

Sign in / Sign up

Export Citation Format

Share Document