ac133 mutz 2 cells  (DSMZ)

Journal: Cancers

Article Title: Cordycepin (3′dA) Induces Cell Death of AC133 + Leukemia Cells via Re-Expression of WIF1 and Down-Modulation of MYC

doi: 10.3390/cancers15153931

Figure Lengend Snippet: Effects of exposure to increasing concentration of cordycepin on MUTZ−2 and primary AML cell viability. ( A ) Cell viability was analyzed through Trypan blue cell count in MUTZ−2 cells after cordycepin treatment at increasing concentrations (50, 100, 200 µM) for 24 and 48 h. ( B ) Dose- and time-response cytotoxicity of the drug were analyzed by the methyl thiazole tetrazolium (MTT) assay in MUTZ−2 cells treated with increasing concentrations of cordycepin (20, 50, 100 and 200 μM) for 24, 48 and 72 h. Values are expressed as the percentage of viable cells for each condition relative to controls. ( C ) MTT assay on mononuclear cells (MNCs) from an AML patient (AML#526) with increasing concentrations of cordycepin (50, 100 and 200 μM) for 24 h. ( D ) MTT assay on primary selected cells (AC133 + ) from an AML patient (AML#523) with increasing concentrations of cordycepin (25, 50 and 100 μM) for 24 h. The relative IC 50 for MUTZ−2 is shown as the mean ± SD (standard deviation) of at least three independent experiments; the p -values are indicated in the graphs: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ANOVA and Tukey’s multiple comparisons test.

Article Snippet: AC133 + -MUTZ-2 cells (DSMZ ACC 271) were grown in 60% alpha-MEM (Gibco) medium, complemented with 20% fetal bovine serum (FBS), 20% vol conditioned medium by 5637 cell line (DSM ACC 35), 1% penicillin/streptomycin, 1% of glutamine and 50 ng/mL SCF (STEM CELL Technologies).

Techniques: Concentration Assay, Cell Counting, MTT Assay, Standard Deviation

Journal: Blood

Article Title: Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response

doi: 10.1182/blood-2017-10-810986

Figure Lengend Snippet: AX is a potent inhibitor in leukemic cell lines without inducing any HSR. (A) K562, KCL22, and HL60 were treated with the indicated (cytotoxic) concentration of AX, NB and AUY922 for 48 hours, and protein lysates were later subjected to immunoblot analysis. AX and NB (C-terminal HSP90 inhibitors) do not induce expression of HSP70, HSP40, and HSP27, whereas AUY922 (an N-terminal HSP90 inhibitor) demonstrates HSR induction by triggering their expression. HSP60 (primarily present in mitochondria) and PDI (primarily present in endoplasmic reticulum) served as controls for the HSR in the cytoplasm, in response to inhibition of HSP90 dimerization via the CTD. (B) K562, KCL22, and HL60 (Mutz-2; data not shown) were treated with AX for 48 hours, and enzymatic activity of caspase-3/7 was later examined by caspase-3/7–dependent Glo assay (absorbance at 405 nm). (C) K562, HL60, KCL22 cells were seeded in methylcellulose medium containing respective compounds at indicated concentration after treatment in liquid medium for 24 hours. Colonies were counted after 14 days. (D) 5 × 105 luciferase-expressing K562 cells were subcutaneously transplanted into NSG mice. Starting the day after transplantation, animals were treated by peritumoral injection (15 µg) of compound AX (0.5 mg/kg dose) or solvent only (DMSO). One control DMSO-treated mouse was sacrificed earlier (on day 16) because of large tumor size. Luminescence was monitored every 3 or 4 days after intraperitoneal injection of 100 µL luciferin, and the final analysis was performed on day 17 (n = 5 mice per group). (E) AX reduced tumor burden with respect to tumor weight 0.24 ± 0.01 g vs vehicle 1.6 ± 0.6 g (P = .04; 1-tailed t test). (F) Immunoblot analysis of tumor samples derived from mice treated with AX revealed downregulation of BCR-ABL1 kinase activity and its associated downstream signaling pathways involving Stat5a and Crkl. (G) Immunoblot analysis of tumor samples derived from mice after treatment with AX. Samples displayed no HSR, as evaluated by expression of HSF-1, HSP70, and HSP27; PDI and HSP60 were used as controls. Columns depict the mean of 3 independent experiments (n = 3). Significance analyses of normally distributed data with variance similar between groups used paired, 2-tailed Student t test. *P < .05, **P < .005, ***P < .001.

Article Snippet: K562, KCL22, HL60, Kasumi, 697, SEM, and Mutz-2 leukemic cell lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and maintained at 37°C with 5% CO 2 , except for the Mutz-2 37 and SUP-B15 (BCR-ABL1) BCP-ALL cell lines, which were cultured in McCoy 5A supplemented with 20% FCS (DSMZ, Braunschweig, Germany).

Techniques: Concentration Assay, Western Blot, Expressing, Inhibition, Activity Assay, Glo Assay, Luciferase, Transplantation Assay, Injection, Derivative Assay