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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3305-3305 ◽  
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.

Establishment and Characterization of a Novel Human Myeloid Leukemia Cell Line, AMU-AML1, Carrying t(12;22)(p13;q11) with No Fusion of MN1-TEL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4375-4375
Author(s):  
Mayuko Goto ◽  
Ichiro Hanamura ◽  
Motohiro Wakabayashi ◽  
Hisao Nagoshi ◽  
Tomohiko Taki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Chromosome Band ◽  
Structural Abnormality ◽  
Myeloid Leukemia Cell

Abstract Abstract 4375 Leukemia cell lines are ubiquitous powerful research tools that are available to many investigators. In balanced chromosomal aberration in leukemia, a chimeric fusion gene formed by genes existing on breakpoints is frequently related to leukemogenesis. Cytogenetic abnormalities of chromosome band 12p13 are detected non-randomly in various hematological malignancies and usually involved TEL, which encodes a protein of the ETS transcription factor family. Chromosome band 22q11-12 is one of partners of translocation 12p13 and t(12;22)(p13;q11-12) results in fusion of TEL and MN1 or in just the partial inactivation of TEL. It is important to analyze precisely the breakpoint in a non-random translocation such as t(12;22)(p13;q11-12) and in addition it contributes to the better understanding of the molecular pathogenesis of leukemogenesis. In this study, we established a novel human myeloid leukemia cell line, AMU-AML1, having t(12;22) from a patient with acute myeloid leukemia with multilineage dysplasia and analyzed its characters. Mononuclear cells were isolated by Ficoll-Hypaque sedimentation from patient's bone marrow before initiation of chemotherapy and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 3 months, cell proliferation became continuous. The cell line, named AMU-AML1, was established. In AMU-AML1, the following pathogens were negative for EBV, CMV, HBV, HCV, HIV-1, HTLV-1 and mycoplasma. A doubling time of AMU-AML1 cells was about 96 hours. Proliferation of the cells was stimulated by rhG-CSF (10 ng/ml), rhGM-CSF (10 ng/ml), M-CSF (50 ng/ml), rhIL-3 (10 ng/ml) and rhSCF (100 ng/ml) but not by IL-5 (10 ng/ml), rhIL-6 (10 ng/ml), and rhEPO (5 U/ml). AMU-AML1 was positive for CD13, CD33, CD117 and HLA-DR, negative for CD3, CD4, CD8 and CD56 by flow cytometry analysis. G-banding combined with SKY analysis of AMU-AML1 cells showed single structural abnormality; 46, XY, t(12;22)(p13;q11.2). Double-color FISH using PAC/BAC clones listed in NCBI website and array CGH analyses indicated that the breakpoint in 12p13 was within TEL or telomeric to TEL and it of 22q11 was centromeric to MN1. A chimeric MN1-TEL transcript and fusion protein of MN1-TEL could not be detected by RT-PCR and western blot analysis. The wild type of MN1 protein was strongly expressed in AMU-AML1 compared with other leukemic cell lines with t(12;22), MUTZ-3 and UCSD/AML1. Our data suggest that AMU-AML1 had a t(12;22)(p13;q11.2) without fusion of MN1-TEL and the expression level of MN1 protein was relatively high, which might have some effects on leukemogenesis. In conclusion, AMU-AML1 is a useful cell line to analyze the biological consequences of the leukemic cells with t(12;22)(p13;q11.2) but no fusion of MN1-TEL. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2233-2233
Author(s):  
Toshiyuki Kitoh ◽  
Kou Suchan Gao Siqiang ◽  
Hidefumi Kato ◽  
Kenji Miyata ◽  
Yasuto Shimomura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Protein Content ◽  
Cell Line ◽  
Cell Lines ◽  
Asparagine Synthetase ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Promising Therapeutic Approach

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.

A monoclonal antibody that detects expression of transferrin receptor in human erythroid precursor cells

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 671-678 ◽  
Author(s):  
D Lebman ◽  
M Trucco ◽  
L Bottero ◽  
B Lange ◽  
S Pessano ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Line ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Transferrin Receptor ◽  
Time Course ◽  
Surface Membrane ◽  
Peripheral Blood Lymphocytes ◽  
Leukemic Cells ◽  
Erythroid Precursor

Abstract A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12--O- tetradecanoyl-phorbol-13-acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.

scholarly journals A monoclonal antibody that detects expression of transferrin receptor in human erythroid precursor cells

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 671-678
Author(s):  
D Lebman ◽  
M Trucco ◽  
L Bottero ◽  
B Lange ◽  
S Pessano ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Line ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Transferrin Receptor ◽  
Time Course ◽  
Surface Membrane ◽  
Peripheral Blood Lymphocytes ◽  
Leukemic Cells ◽  
Erythroid Precursor

A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12--O- tetradecanoyl-phorbol-13-acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.

scholarly journals The Identification of Novel Epigenetic Therapies for ALK-Driven Haematological Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1483-1483
Author(s):  
Stephen P. Ducray ◽  
Ricky Trigg ◽  
Andrew J. Bannister ◽  
Raymond Lai ◽  
Gerda Egger ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Haematological Malignancies ◽  
Front Line ◽  
Dnmt Inhibitor ◽  
Anaplastic Lymphoma

Introduction Through conserved signalling pathways, Anaplastic Lymphoma Kinase (ALK) is well-described in driving haematological malignancies including Anaplastic Large Cell Lymphoma (ALCL) and Diffuse Large B-Cell Lymphoma (DLBCL) and as such presents itself as an amenable therapeutic target. Hence, directed therapeutics (ALK tyrosine kinase inhibitors; TKI) are being used in the treatment of ALK-driven cancers. Unfortunately, findings in the clinic and clinical research studies have taught us that resistance to ALK inhibitors can develop through the activation of ALK signalling bypass tracks. As such there is a need for the development of novel front-line, dual-combination, as well as second-line therapies. Methods A large-scale epigenetic targeted drug library consisting of approximately 300 FDA-approved drugs and novel agents was applied to a number of cell lines representing ALK-driven haematological malignancies: ALCL cell lines (DEL, JB-6, KARPAS-299, SU-DHL-1, SUP-M2) and the DLBCL cell line LM-1. Drugs which caused a &gt;75% decrease in cell viability were classified as 'candidate drugs' and studied further. Results Several of the validated drugs have previously been used/trialled in the clinic for the treatment of various cancers, e.g. aurora kinase (XL-228), topoisomerase (Mitoxantrone HCl) and HDAC (Romidepsin) inhibitors - these functioned as internal controls for the drug screens. However, an assortment of novel drugs was also identified that have not previously been described in the context of the treatment of ALK-driven haematological malignancies; including the FLT3 inhibitor KW2449 which caused a &gt;75% decrease in viability in all the tested cell lines and as such may serve as a novel front line therapy. In addition, a novel DNA methyltransferase (DNMT) inhibitor was identified which is efficacious and resulted in a &gt;90% decrease in viability in all cell lines treated across both disease entities. Furthermore, we investigated the combinatorial potential of the identified DNMT inhibitor with ALK TKIs such as Brigatinib and observed the inhibitor acting synergistically (as per Bliss-Independence calculations) resulting in a further decrease in cell viability. Several cell lines that are resistant to ALK TKIs were also assessed for their sensitivity to the DNMT inhibitor and were shown to be susceptible to this drug, as demonstrated by a significant decrease in cell viability. Figure 1: (A) Viability following drug screen in a representative cell line. Those drugs which led to a &gt;75% change in viability were taken forward for validation, as shown in (B). (C) Candidate drugs identified for each of the cell lines tested, grouped according to their molecular target. Conclusion In conclusion, an epigenetic drug library has been employed to identify novel therapeutic agents for the treatment of ALK-driven haematological malignancies including ALCL and DLBCL. Data reveal a potent inhibitor of DNA methylation as a candidate drug that suppresses the growth of ALK-driven malignancies both alone and in combination with ALK TKIs. Significantly, this identified drug also inhibits the growth of cell lines resistant to directed therapeutics such as ALK TKIs suggesting it has potential clinical use. Disclosures No relevant conflicts of interest to declare.

Downregulation of MiR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells through Modulation of NOTCH1 and BCL2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 361-361
Author(s):  
An De Weer ◽  
Pieter Mestdagh ◽  
Katleen De Preter ◽  
Joni Van der Meulen ◽  
Pieter Van Vlierberghe ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Chromosomal Rearrangements ◽  
Ectopic Expression ◽  
Leukemic Cell ◽  
Model Systems ◽  
Leukemic Cells

Abstract Abstract 361 Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p<0.05) were shown to be related to the EVI1 expression status. Amongst these, miR-449a was selected for further study based on its homology to the known cancer associated miRNA miR-34a. Downregulation of miR-449a by EVI1 was further confirmed in the leukemic cell line U937 with tetracycline controllabel (tet-off) EVI1 overexpression. Next, direct transcriptional regulation of miR-449a expression by EVI1 was demonstrated by chromatin immunoprecipitation (ChIP). To test the functional consequences of downregulation of miR-449a in AML cells, reconstitution of the expression of miR-449a in the Kasumi-3 and UCSD-AML1 cell lines was performed, which resulted in significantly decreased cell viability, increased apoptosis and differentiation towards the megakaryocytic and monocytic lineages. Interestingly, siRNA mediated knockdown of EVI1 expression in Kasumi-3 or UCSD-AML1 almost completely abrogated the miR-449a induced reduction in cell viability, while electroporation of both cell lines with EVI1 siRNAs alone had essentially no effect on cell viability. These data strongly suggest that repression of miR-449a expression is essential for the survival and growth of EVI1 overexpressing cells and that this requirement is specifically imposed by EVI1 itself. We next demonstrated that the predicted miR-449a targets NOTCH1 and BCL2 were bona fide miR-449a targets using promoter reporter assays. To asses the contribution of these target genes to the observed phenotype upon miR-449a upregulation, knockdown of NOTCH1 and BCL2 was performed, revealing similar effects on cell viability and apoptosis. These results indicated that the effects seen upon treatment of cells with a precursor miR-449a are at least partly mediated through NOTCH1 and BCL2. In conclusion, we provided for the first time evidence that EVI1 mediated downregulation of miR-449a leads to NOTCH1 and BCL2 upregulation and is required for sustained proliferation and survival of EVI1 overexpressing cells. These data also open new perspectives for therapeutic intervention through modulation of miR-449a and/or its target genes. Disclosures: No relevant conflicts of interest to declare.

A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines

Planta Medica ◽  
2021 ◽  
Author(s):  
Chawalit Chatupheeraphat ◽  
Sittiruk Roytrakul ◽  
Narumon Phaonakrop ◽  
Kamolchanok Deesrisak ◽  
Sucheewin Krobthong ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Raji Cell ◽  
Leukemic Cell ◽  
B Cell Lymphoma ◽  
Leukemic Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Peptide Fraction

AbstractDespite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

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

BMC Cancer ◽  
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.

scholarly journals A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance Via Blocking Receptor Auto-Induction and BIM/Bmf Expression in ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3758-3758
Author(s):  
Jianping Li ◽  
Catalina Troche ◽  
Julia Hlavka Zhang ◽  
Jonathan Shrimp ◽  
Jacob S. Roth ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Mutant Allele ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Chemotherapeutic Agents ◽  
Rna Seq ◽  
Mesenchymal Transition ◽  
Pediatric All

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

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

Pharmaceutics ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 362 ◽  
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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