Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: H(2)O(2)-mediated cytotoxicity of pharmacologic ascorbate concentrations to neuroblastoma cells: potential role of lactate and ferritin.

doi: 10.1159/000315098

Figure Lengend Snippet: Fig. 1. Cytotoxicity of ascorbate (A) and H2O2 (B) to the human neuroblastoma cell line Kelly (upper part) and SK-N-SH (lower part). Correlation between cyto- toxicity and cell number. Various numbers of Kelly or SK-N-SH cells were incubated with ascorbate (0.6-5 mM f.c.) or H2O2 (25-100 µM f.c.) for 48 h. Viability was assessed by MTT assay. n ≥ 5 for ascorbate and n ≥ 4 for H2O2, each performed 6-fold. Untreated controls set as 100%, mean ± SD.

Article Snippet: Cell lines and cell culture The human neuroblastoma cell line Kelly was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).

Techniques: Incubation, MTT Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: H(2)O(2)-mediated cytotoxicity of pharmacologic ascorbate concentrations to neuroblastoma cells: potential role of lactate and ferritin.

doi: 10.1159/000315098

Figure Lengend Snippet: Fig. 3. (A): Release of ferritin by human neuroblastoma cells. Kelly and SK-N-SH cells (5x106 cells/ml) were incubated in PBS++ and ferritin release into the supernatant was monitored for up to 10 h. n=3 for both cell lines, each performed in duplicate, mean ± SD. (B): Release of ferritin from Kelly and SK-N-SH cells in comparison to fibroblasts (each 2.5 x 106

Article Snippet: Cell lines and cell culture The human neuroblastoma cell line Kelly was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).

Techniques: Incubation, Comparison

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: H(2)O(2)-mediated cytotoxicity of pharmacologic ascorbate concentrations to neuroblastoma cells: potential role of lactate and ferritin.

doi: 10.1159/000315098

Figure Lengend Snippet: Fig. 5. Model for the generation of H2O2 in the tumour microenvironment, where ascorbate is present. Neuroblastoma cells are known to generate and release ferritin. Iron (Fe2+) can be set free from ferritin by O2 •- and probably also directly by ascorbate. In the presence of O2, Fe2+ is oxidized to Fe3+ and O2 •-

Article Snippet: Cell lines and cell culture The human neuroblastoma cell line Kelly was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).

Techniques:

Journal: Haematologica

Article Title: Depletion of SIRT6 enzymatic activity increases acute myeloid leukemia cells’ vulnerability to DNA-damaging agents

doi: 10.3324/haematol.2017.176248

Figure Lengend Snippet: SIRT6 is highly enriched in acute myeloid leukemia (AML) and its expression confers poor prognosis. (A) Protein lysates from a panel of AML cell lines (left), primary patient-derived AML cultures or normal peripheral blood mononuclear cells (PBMCs) (right) were analyzed for SIRT6 expression by Western blot. GAPDH was used as loading control. The quantification of SIRT6/GAPDH ratios is shown below. One experiment of two is shown. (B) Six AML cell lines were analyzed for SIRT6 (green) by immunofluorescence. 4′,6-diamidino-2-phenylindole nuclear stain is shown in blue. Original magnification ×20. (C) Box plot distributions of SIRT6 gene expression levels in normal hematopoietic cells from healthy donors and leukemic CD34 + blasts from AML patients (n=300), combining data from GSE1159, GSE9476 and GSE30377 (using the probe set 219613_s_at). Normal hematopoietic samples included CD34 + selected cells [n=18: 8 from bone marrow (BM) and 10 from PB], CD34 + CD38 – cells (n=10), unselected bone marrows (n=10), and unselected peripheral blood (n=6). Numbers below the graph are the number of samples per group. ns: not significant; ** P <0.05; *** P <0.001 [one-way analysis of variance (ANOVA) with expression data log 2 transformed]. (D) Increased SIRT6 mRNA expression is observed in AML patients (CD34 + blast cells) relative to normal controls (CD34 + cells collected from PB or BM of healthy donors; **=0.006; * P =0.02, unpaired t -test). GAPDH mRNA expression was used as an internal control. (E) Survival curves relative to SIRT6 expression in 100 individuals affected by AML diagnosed at our clinic. The patient group with higher SIRT6 expression (red line) had shorter overall survival than the patient cohort with lower SIRT6 expression (blue line) (log rank test). Median SIRT6 mRNA value was used to divide AML patients.

Article Snippet: The AML cell lines U937, MOLM-14, MV4-11, HL60, HEL, THP-1, NOMO-1, OCI-AML2, OCI-AML3 and NB4 were provided by collaborators or were purchased from ATCC or DSMZ (Braunschweig, Germany).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Immunofluorescence, Staining, Gene Expression, Transformation Assay

Journal: Haematologica

Article Title: Depletion of SIRT6 enzymatic activity increases acute myeloid leukemia cells’ vulnerability to DNA-damaging agents

doi: 10.3324/haematol.2017.176248

Figure Lengend Snippet: SIRT6 affects proliferation and vulnerability to DNA-damage agents in acute myeloid leukemia (AML) cells. (A) SIRT6 silencing in THP-1 and U937 cells using a lentiviral delivery system. (Left) Western blot analysis of pGIPz-infected cells after 48 hours (h) of selection with 1 μg mL-1 puromycin. (Center) Cell numbers evaluated by cell counting with trypan blue exclusion. (Right) AML-engineered cells were assessed for cell number and (B) cell-cycle progression. All data throughout the panel are shown as mean±Standard Deviation (s.d.) of triplicates. ns: not significant; * P <0.01; ** P <0.001, Student t -test. (C) Representative western blots showing DDR pathway deregulation in THP-1 cells depleted of SIRT6 compared with control. GAPDH was used as loading control. One representative blot of two is shown. (D) OCI-AML-2 and OCI-AML-3 cells were transduced with a scrambled shRNA (CTR) or with an anti-SIRT6 shRNA (#911). Cells were used for immunoblotting detection of SIRT6 or γ-tubulin expression (top) or in viability experiments. For the latter, 2×10 4 cells/well were plated in 96-well plates and incubated for 48 hours (h) with or without DNR or ARA-C at the indicated concentration. Thereafter, dead cells were detected by propidium iodide staining and flow cytometry. 2×10 4 OCI-AML2 (E) and primary AML (F) cells/well were plated in 96-well plates and incubated for 72 h with (w) / or without (w/o) DNR/ARA-C (at the indicated concentration) w / or w/o compound 1, as in Sociali et al . Thereafter, dead cells were detected by propidium iodide staining and flow cytometry. *0.04< P <0.01; **0.009< P <0.001; ***<0.0001.

Article Snippet: The AML cell lines U937, MOLM-14, MV4-11, HL60, HEL, THP-1, NOMO-1, OCI-AML2, OCI-AML3 and NB4 were provided by collaborators or were purchased from ATCC or DSMZ (Braunschweig, Germany).

Techniques: Western Blot, Infection, Selection, Cell Counting, Standard Deviation, Control, Transduction, shRNA, Expressing, Incubation, Concentration Assay, Staining, Flow Cytometry

Journal: Haematologica

Article Title: Depletion of SIRT6 enzymatic activity increases acute myeloid leukemia cells’ vulnerability to DNA-damaging agents

doi: 10.3324/haematol.2017.176248

Figure Lengend Snippet: Ongoing DNA damage and high CIN signature are associated with intense replicative stress and SIRT6 overexpression in acute myeloid leukemia (AML) cells. (A) Expression levels in a panel of 32 human AML cell lines for the probe sets corresponding to the chromosomal instability signature described by Carter et al. using GSE59808. Red: gene expression over the median; blue: expression under the media. (B) Immunofluorescence staining of γ-H2A.X in AML cell lines and primary tumor cells; magnification ×40. (C) Western blot analysis (1 representative blot of 3) of γ-H2A.X in AML cell lines (top), AML patients’ cells, and peripheral blood mononuclear cells (PBMCs) from healthy donors (bottom). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (D) 53BP1 and RAD51 number of foci in a panel of AML cells presenting with high (red bracket) and low (blue bracket) DNA damage. (E) SIRT6 expression was compared to CIN signature among AML cell lines in the GSE59808 data set; * P =0.04. (F) GSEA enrichment profile for AML cell lines (included in GSE59808) divided in high and low SIRT6 expression groups of chromosomal instability signature, as reported by Carter et al . The analysis pointed to an association between high SIRT6 levels and CIN in AML cells.

Article Snippet: The AML cell lines U937, MOLM-14, MV4-11, HL60, HEL, THP-1, NOMO-1, OCI-AML2, OCI-AML3 and NB4 were provided by collaborators or were purchased from ATCC or DSMZ (Braunschweig, Germany).

Techniques: Over Expression, Expressing, Gene Expression, Immunofluorescence, Staining, Western Blot

Journal: Haematologica

Article Title: Depletion of SIRT6 enzymatic activity increases acute myeloid leukemia cells’ vulnerability to DNA-damaging agents

doi: 10.3324/haematol.2017.176248

Figure Lengend Snippet: SIRT6 inhibition makes acute myeloid leukemia (AML) blasts more sensitive to DNR treatment in NSG mice. (A) Growth of U937 control and SIRT6-depleted xenografts in mice treated with vehicle or DNR (3 mg/kg i.p. day 1 and day +4) at day 20 after tumor engraftment. * P =0.036. Data are mean tumor volume±Standard Deviation (s.d.). (B) 1×10 6 of scramble or shSIRT6-expressing HL-60 cells were engrafted into NSG mice (n=20) by tail-vein injection. Once a systemic xenograft was confirmed, mice were randomized to receive DNR (1.5 mg/kg for 3 days) (treated group) or vehicle (control group). Histogram represents percentage of human CD45 + cells in mice, at day 31 post engraftment. Data are represented as mean±Standard Error of Mean (SEM); ** P =0.006. (C) Representative flow cytometric dot plots representing tumor engraftment evaluated at day 40 post injection. (D) Kaplan-Meier survival plot showing median survival of mice injected with tumors with (w) / without (wo) SIRT6 before and after treatment with vehicle or DNR.

Article Snippet: The AML cell lines U937, MOLM-14, MV4-11, HL60, HEL, THP-1, NOMO-1, OCI-AML2, OCI-AML3 and NB4 were provided by collaborators or were purchased from ATCC or DSMZ (Braunschweig, Germany).

Techniques: Inhibition, Control, Standard Deviation, Expressing, Injection