scholarly journals Novel Histone Deacetilase (HDAC) Inhibitors: In Vitro Effects on Leukemic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4946-4946
Author(s):  
Maria Rosaria Ricciardi ◽  
Roberto Licchetta ◽  
Paola Bergamo ◽  
Stefano Iacovelli ◽  
Andrea Miele ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Mtt Assay ◽  
Dose Response ◽  
Lymphoid Cell ◽  
Myeloid Leukemia ◽  
Response Curve ◽  
Myeloid Cell ◽  
Lymphoid Cell Line

Abstract Abstract 4946 Histone deacetylase inhibitors (HDAC-I) are a class of agents with the capacity to induce acetylation of histone and non-histone proteins. These molecules have been intensively investigated in a variety of malignancies because of their ability to inhibit proliferation, induce differentiation and apoptosis in tumor cells. However, clinical response to clinically available HDAC-I have been obtained only in a proportion of patients, prompting further studies aimed at identifying more active compounds and at defining the molecular mechanisms of response to this class of agents. Acetyl-L-carnitine (ALCAR) is a metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane, thereby contributing to the regulation of energy production and metabolism. ALCAR activity as a modulator of cellular stress response has prompted its use to protect against chemotherapy-induced neurotoxicity. However, ALCAR effects on neoplastic cells are still not defined, especially in combination with chemotherapy. Here we investigated the effects of MS-275, a HDAC-I, on cell proliferation and apoptosis in cell line models of acute myeloid leukemia (AML) acute lymphoblastic leukemia (ALL), and multiple myeloma (MM), in comparison with vorinostat also known as SAHA (suberoyl anilid hydroxamic acid), the most widely used HDAC-I in clinical setting. HDAC-I were tested at doses ranging from 5 to 5000nM. In addition, the effects of simultaneous exposure to 10 mM of ALCAR and selected sub-toxic concentration of HDAC-I were analyzed. The cytotoxic effect of the treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentration inducing 50 % cell killing (IC-50) was calculated from the dose-response curve. Cell cycle inhibition and induction of apoptosis were analyzed by flow cytometry using the Acridine- Orange (AO) technique. Results indicated that the tested compound MS-275 significantly inhibited cell growth, as assessed by MTT assay, when used at of 5000nM. Comparative analysis of the efficacy of the two different HDAC-I compounds indicated that MS-275 was the more effective agent and the only one with clear dose-dependent activity, while SAHA displayed a flat dose-response curve, which dropped only at the highest concentration. In particular, the myeloid cell line Molm-13 was strikingly sensitive to MS-275 (IC50: < 15 nM), U937 and HL60 myeloid cell lines, the lymphoid cell line Jurkat and the MM cell line ARH-77 showed intermediate sensitivity (IC50: < 1000 nM), while the lymphoid cell line CEM R was resistant (IC50 > 10 uM). SAHA showed no activity in U937 cells when used at concentrations ranging from 100 to 1000 nM, with a dramatic reduction of absorbance at 5000 nM (>80% reduction compared to the control). Nevertheless, the combination of 500 nM SAHA with 10mM ALCAR reveled a synergistic interaction, with a 46% reduction in absorbance. We then analyzed the effects on apoptosis induction, as determined by the percentage of cells with a sub-G1 DNA content. MS-275 dose-dependently induced apoptosis in HL-60 cells (4.2%, 17.1%, 60.8%, and 87.5% in the presence of 100, 500, 1000, 5000 nM of MS-275, respectively). Conversely, SAHA induced minimal apoptosis (< 10%) at concentration ranging from 100 to 1000 nM, although > 75% of cells became apoptotic after treatment with the compound at 5000 nM. In summary, our results show that the HDAC-I MS-275 is a potent inhibitor of leukemic cell growth, capable of inducing apoptosis particularly in cell lines derived from myeloid leukemia and MM. Preliminary studies exploring the combined use of ALCAR with the SAHA support a potential anti-neoplastic synergism in selected hematological malignancies. Disclosures: Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Pisano: Sigma-Tau: Employment. Tafuri: Sigma-Tau: Research Funding.

Hematopoietic Cell Lines and Patient Samples Show a Correlation Between Upregulated MSI2 and BCR-ABL Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2618-2618
Author(s):  
Jaspal S. Kaeda ◽  
Rolf Schwarzer ◽  
Robert K. Slany ◽  
Thomas Burmeister ◽  
Elisabetta Vagge ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Blast Crisis ◽  
Myeloid Cell ◽  
Hematopoietic Stem ◽  
Lymphoid Cell Lines

Abstract Increased expression of Musashi 2 (also known as MSI2), a mRNA binding protein, is reported to trigger progression of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC), which is frequently fatal. The data imply that MSI2 is upregulated by HOXA9 leading to disruption of the critical hematopoietic stem cell (HSC) fate decisions via the NUMB-NOTCH signaling pathway. These events lead to HSC proliferation, impaired myeloid differentiation and are associated with worse prognosis in CML and acute myeloid leukemia (Kharas, et al. Nat Med 2010; 16:903; Ito, et al. Nature 2010; 466:765). Previously, we confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in CP, irrespective of lymphoid or myeloid transformation (Kaeda, et al. Blood. 2011;118;supplement). To further elucidate this regulatory pathway we assessed whether HOXA9 expression correlated with increased MSI2mRNA levels and upregulation of NOTCH. Because of the rarity of BC samples and finding MSI2 increased in lymphoid and myeloid BC CML patients, we studied lymphoid [n=12 (Lymphoma:8; Myeloma:3; ALL:1)] and myeloid [n=12 (AML:4; CML:6; HES:1; ET:1)] cells lines. We also included 29 BCR-ABL positive (e1a2: 18; e13a2: 6; e14a2: 5) diagnostic samples from acute lymphoblastic leukaemia (ALL) patients (M:18; F:11); median age 50 years (range: 26-74). We quantified MSI2 and HOXA9 mRNA transcripts by quantitative PCR and expressed the data as % ratio of the control gene, GUSβ.Western blotting of cell line protein extracts was performed to assess expression of NOTCH 1, 2, 3 receptors and delta like ligand 3. We found MSI2 [median: 7.10% (range:0.06-38.71)] and HOXA9 [median: 2.40% (range:0.00-51.13)] expression was generally restricted to myeloid cell lines. Of the 12 lymphoid cell lines, MSI2 was only detectable in SUPB15 with 8.44%. The latter is a B-lymphocyte ALL cell line, known to express BCR-ABL e1a2 transcript. Similarly, HOXA9 [median: 0.00 (range:0.00-27.0%)] expression ranged from undetectable to 0.08%, among this group, apart from HD-MY-Z with 27.0%. In contrast, HOXA9 [median: 0.02% (range:0.01-2.18)] and MSI2 [median: 3.58% (range: 1.24-22.38)] transcripts were detectable in all 29 BCR-ABL positive ALL patients, irrespective of the transcript type expressed. Among the ALL samples 7 (24%) had increased MSI2 levels, i.e. >6.7% (Kaeda, et al. Blood. 2011;118;supplement) and of these 6 expressed e1a2 transcript and the other e13a2. In summary, upregulated MSI2 expression was observed in 17 (32.0%) of the 53 samples screened. But only 4 [KG1, EOL1, HEL and MEG-01 (all myeloid cell lines)] of the 17 also had increased HOXA9 levels. Remarkably, 5 (62%) of the 8 myeloid cell lines with increased MSI2 are known to express BCR-ABL. NOTCH1 receptor was detectable in all the lymphoid cell lines. But, NOTCH expression was highly variable in myeloid cell lines. Overall, an upregulated MSI2 mRNA expression was not reflected in the NOTCH receptor levels nor in the HOXA9levels. Our observations are consistent with MSI2 being limited to myeloid linage. However, in contrast to earlier reports our data imply that MSI2 functions via a pathway other than NOTCH signaling and is not regulated by HOXA9 alone. But the cell lines and ALL patients’ data provide further evidence of correlation between MSI2 and BCR-ABL expression, suggesting they interact, directly or indirectly, to arrest cell differentiation and trigger BC. These findings, together with our reported data, show increased MSI2 levels to be an important biomarker of poor prognosis and are likely to have an impact in optimizing clinical management. It also represents a potential novel therapeutic target, especially for the BCR-ABL positive stem cells resistant to tyrosine kinase inhibitors. Disclosures: Kaeda: Novartis: Research Funding. le Coutre:Novartis: Research Funding.

scholarly journals The ether lipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine induces expression of fos and jun proto-oncogenes and activates AP-1 transcription factor in human leukaemic cells

1994 ◽  
Vol 302 (2) ◽  
pp. 325-329 ◽  
Author(s):  
F Mollinedo ◽  
C Gajate ◽  
M Modolell
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Ether Lipid ◽  
Myeloid Cell ◽  
Mrna Levels ◽  
Lymphoid Cell Line ◽  
Enhancer Element ◽  
Leukaemic Cells

The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) has been recently shown to induce apoptosis in the human leukaemic HL-60 and U937 myeloid cell lines [Mollinedo, Martinez-Dalmau and Modolell (1993) Biochem. Biophys. Res. Commun. 192, 603-609]. We have found that ET-18-OCH3 is also able to promote apoptosis in the human leukaemic Jurkat T lymphoid cell line. This lymphoid cell line as well as the two myeloid HL-60 and U937 cell lines incorporated significant amounts of exogenously added radiolabelled ET-18-OCH3. Addition of ET-18-OCH3 to these human leukaemic cells induced an increase in the steady-state mRNA levels of fos and jun proto-oncogenes, components of the transcription factor AP-1. These increases in fos and jun mRNA levels were associated with the activation of the AP-1 transcription factor after addition of ET-18-OCH3 to human leukaemic cells, as assessed by an enhanced binding activity of transcription factor AP-1 to its cognate DNA sequence as well as by stimulation of transcription from an AP-1 enhancer element. These data demonstrate that the ether lipid ET-18-OCH3 can affect gene expression by inducing expression of fos and jun proto-oncogenes and by modulating the activity of transcription factor AP-1.

scholarly journals Adhesion of NFS-60 myeloid leukemia cells to MC3T3-G2/PA6 stromal cells induces granulocyte colony-stimulating factor production

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 415-420 ◽  
Author(s):  
T Yoshikubo ◽  
K Ozawa ◽  
K Takahashi ◽  
M Nishikawa ◽  
N Horiuchi ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Myeloid Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
Csf Production

Abstract To examine the interaction between immature myeloid cells and stromal cells in the induction of granulocyte colony-stimulating factor (G-CSF) production, stromal cells of the MC3T3-G2/PA6 (PA6) murine cell line, which has preadipocyte characteristics and can support hematopoiesis, were cocultured with various myeloid cell lines and G-CSF mRNA expression was examined by Northern and reverse transcriptase- polymerase chain reaction analyses. A significant amount of G-CSF mRNA was induced by the culture of an interleukin-3/G-CSF-dependent murine myeloid leukemia cell line, NFS-60, on PA6 stromal cells for 16 hours. Using a G-CSF-dependent subline of DA-1 (DA-1N), the biologic activity of G-CSF was also detected in PA6/NFS-60 coculture supernatants, but not in the culture supernatant of PA6 or NFS-60 alone. Direct contact of NFS-60 cells with the PA6 stromal layer was essential for the induction of G-CSF mRNA, as indicated by the following observations: (1) NFS-60 cells efficiently adhered to PA6 cells; (2) medium conditioned by NFS-60 cells did not contain the activity to induce G- CSF mRNA in PA6 cells; and (3) induction of G-CSF mRNA was not observed when NFS-60 cells were separated from PA6 cells by a microporous membrane (0.45-microns pore size). Several other myeloid cell lines, including FDC-P2, 32Dcl3, WEHI-3, and DA-1, did not induce G-CSF mRNA expression after the coculture with PA6 cells, although significant numbers of these cells adhered to PA6 cells. Therefore, NFS-60 cells may express or overexpress a molecule that is involved in adhesion- mediated induction of G-CSF production.

Iron transport in a lymphoid cell line with the hemochromatosis C282Y mutation

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2734-2740 ◽  
Author(s):  
Christopher R. Chitambar ◽  
Janine P. Wereley
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Iron Uptake ◽  
Iron Transport ◽  
Lymphoid Cell Line ◽  
C282y Mutation ◽  
B Lymphoid ◽  
The Impact

Abstract The gene for hemochromatosis (HFE) is expressed in a variety of cells, including those not thought to be affected by this disease. The impact of HFE on iron transport was examined in B-lymphoid cell lines developed from a patient with hemochromatosis with the HFE C282Y mutation (C282Y cells) and an individual with the wild-type HFE gene (WT cells). Whereas both cell lines expressed HFE protein, C282Y cells displayed less HFE protein at the cell surface. Transferrin receptor (TfR) number was 2- to 3-fold greater in WT cells than in C282Y cells, while TfR affinity for transferrin (Tf) was slightly lower in C282Y cells. TfR distribution between intracellular and cell-surface compartments was similar in both cell lines. Iron uptake per cell was greater in WT cells but was not increased proportional to TfR number. When considered relative to cell-surface TfR number, however, iron uptake and Tf internalization were actually greater in C282Y cells. Surprisingly, Tf-independent iron uptake was also significantly greater in C282Y cells than in WT cells. The ferritin content of C282Y cells was approximately 40% that of WT cells. Exposure of cells to pro-oxidant conditions in culture led to a greater inhibition of proliferation in C282Y cells than in WT cells. Our results indicate that in this B-lymphoid cell line, the HFE C282Y mutation affects both Tf-dependent and -independent iron uptake and enhances cell sensitivity to oxidative stress. The role of HFE in iron uptake by B cells may extend beyond its known interaction with the TfR.

The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4063-4063 ◽  
Author(s):  
Shiva Bamezai ◽  
Jing He ◽  
Deniz Sahin ◽  
Fabian Mohr ◽  
Fabio Ciccarone ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Parp Inhibitor ◽  
Leukemic Cell ◽  
Aberrant Expression ◽  
Acute Myeloid

Abstract DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML. Disclosures Mulaw: NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

scholarly journals Metabolism of platelet-activating factor in human haematopoietic cell lines. Differences between myeloid and lymphoid cells

1991 ◽  
Vol 273 (3) ◽  
pp. 573-578 ◽  
Author(s):  
M C Garcia ◽  
C Garcia ◽  
M A Gijon ◽  
S Fernandez-Gallardo ◽  
F Mollinedo ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Platelet Activating Factor ◽  
Myeloid Cell ◽  
Hl60 Cell ◽  
Dependent Manner ◽  
Granulocytic Differentiation ◽  
Lymphoid Cell Lines

The binding and metabolism of platelet-activating factor (PAF) was studied in human cell lines resembling myeloid cells (HL60 and U937) and B and T lymphocytes (Daudi and Jurkat). All of the cell lines were found to bind and catabolize exogenous [3H]PAF in a time- and temperature-dependent manner. PAF binding could also be demonstrated in isolated membrane fractions, which provides further evidence of the existence of true membrane receptors. Myeloid cell lines contained numbers of receptors at least 10-fold higher than in lymphoid cell lines. Biosynthesis of PAF upon challenge by ionophore A23187 could be demonstrated in HL60 and U937 cells. In contrast, lymphoid cell lines were unable to produce PAF. Incubation with [14C]acetate showed incorporation of the label into three main fractions: neutral lipids, phosphatidylcholine and PAF, but the distribution of the label varied depending on the cell line. Significant incorporation into phosphatidylcholine was observed in uninduced myeloid cell lines. A phospholipase A2 acting on 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine and an acetyl-CoA:lyso-PAF acetyltransferase were expressed in the HL60 cell line and showed variations in specific activity with granulocytic differentiation. In contrast, these enzyme activities were not expressed in Daudi and Jurkat cell lines. These data indicate (1) the occurrence of PAF binding and catabolism in both myeloid and lymphoid cell lines; (2) the restriction of PAF biosynthesis to myeloid cell lines, especially HL60 cells; (3) the occurrence of differentiation-elicited changes in the specific activities of the enzymes involved in PAF biosynthesis by the remodelling pathway; and (4) the central role played by the disposal of lyso-PAF, a product of the phospholipase A2 reaction, in PAF biosynthesis.

Use of different human myeloid leukemia cell lines to determine the specificity of 3,3’-diaminobenzidine cytochemistry for the ultrastructural localization of myeloperoxidase and catalase

1995 ◽  
Vol 53 ◽  
pp. 1060-1061
Author(s):  
J. E. Boyd ◽  
C. S. Gilbert ◽  
B. Pinnix ◽  
C. A. Ballinger ◽  
J. M. Kinkade ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Ultrastructural Localization ◽  
Ultrastructural Cytochemistry ◽  
Leukemia Cell Lines ◽  
Biochemical Assays ◽  
Myeloid Leukemia Cell

Myeloid cells are known to contain myeloperoxidase (MPO) and catalase. This study has used MPO and catalase replete and deficient myeloid cell lines to clarify the localization of these components using 3,3’-diaminobenzidine (DAB) ultrastructural cytochemistry. Conditions of DAB incubation can be modified to preferentially stain catalase (alkaline at pH 9.7) or MPO (neutral at pH 7.0-7.6), but crossreactivity persists, preventing the discrimination between catalase and peroxidase. Biochemical assays demonstrated both MPO and catalase in HL60 cells; similar amounts of catalase but no MPO activity in the A7 cell line; increased amounts of catalase but no MPO activity in the HP50 and HP100 cell lines; and neither MPO nor catalase in the KG1 cell line. Neutral DAB stained MPO (pH 7.4; [DAB] 5 or 20 mg/10 mL 0.05 M Tris; 30 min or 120 min; 24° or 37°C; 0.01% H2O2) in HL60 (Fig. 1), but not in A7. Alkaline DAB intensely stained catalase (pH 9.7; 20 mg/10 mL; 120 min; 37°C; 0.01% or 0.03%) in A7.

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