Journal: Cancer Cell International

Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

doi: 10.1186/1475-2867-7-14

Figure Lengend Snippet: Viability and growth kinetics of U2OS in vitro . A) , Growth curves of U2OS cells with growth medium alone (dark line), or growth medium with one dose of decitabine at a final concentration of 1 μM (light graph). The y-axis indicates cell number in millions and the x-axis indicates time points in days. The results are expressed as cell counts at each corresponding time point. Each data point is the Mean of cell counts from 2 experiments (5 passages apart) each consist of 2 independent cultures and the error bars indicate the standard deviation. The findings indicate a slight increase of U2OS cells' doubling time and a decrease of 18% (p = 0.045) in the viability of treated cells compared to untreated control B) , Cell death in U2OS cells caused by decitabine treatment at 1 μM concentration (light column) compared to no-treatment (dark column). The results are expressed as percentage of cell death (fraction of cells with positive PI stain). The y-axis indicates the percentage of cells with PI staining (dead cells). Each column is the Mean of 3 experiments with error bars indicating standard deviation. The asterisk indicates significant increase in cell death (p < 0.05) as a result of decitabine treatment.

Article Snippet: 12 hours after plating they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 μM without changing the medium.

Techniques: In Vitro, Concentration Assay, Standard Deviation, Staining

Journal: Cancer Cell International

Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

doi: 10.1186/1475-2867-7-14

Figure Lengend Snippet: Analysis of differential 5-methylcytidine content of U2OS xenografts using histological sections . Analysis of the relative levels of cellular 5-methylcytidine within xenografts derived from representative control untreated mice (panel A); or decitabine treated mice (panel B) using immunohistochemical staining with 5-mc-Ab. To the right a 20X enlargement of representative U2OS histology is shown. More 5-mc-Ab staining is evident in the nuclei from control sections (panel A-right side) in comparison to nuclear staining in the sections derived from decitabine treated mice (panel B right side). These data are consistent with a reduction in 5-methylcytidine nuclear content in U2OS xenografts derived from mice treated with decitabine. Enlargements of darkly stained host murine kidney cells correspond to heavily methylated differentiated renal tissue (denoted 2 in panels A and B). In panel C the staining intensity from control (dark columns) and decitabine treated (light columns) is quantitated using Aperio scanning image analysis of sections. The graph to the right of panel C confirms that the 5-mc-Ab staining in control untreated (dark columns) U2OS xenograft sections is more intense than the decitabine treated (light columns) U2OS xenografts. This decrease in staining intensity was significant (p < 0.05). In contrast host kidney cells from both control and treated mice do not exhibit any significant difference in staining intensities.

Article Snippet: 12 hours after plating they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 μM without changing the medium.

Techniques: Derivative Assay, Immunohistochemical staining, Staining, Methylation

Journal: Cancer Cell International

Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

doi: 10.1186/1475-2867-7-14

Figure Lengend Snippet: Induction of differentiation and apoptosis by decitabine treatment of U2OS in vivo . A) , Decitabine effects on tumor volumes in vivo . The results compare tumor volumes in control xenografts to decitabine treated xenografts as described in methods. Each column is the Mean of tumor volumes measured in 12 xenograft tumors. There was a significant decrease (p < 0.05) in tumor volumes as a result of decitabine treatment. B) , Osteoid assessment in vivo . Each column is the Mean of osteoid evaluation of 9 sections. There was significant increase (p < 0.0001) in osteoid formation as a result of decitabine treatment. C) , Representation images of H&E sections. Control xenograft tumors (left) show solid sheets of poorly differentiated cells with minimal osteoid (image magnification × 100). Decitabine treated tumors shows less dense cell population and increased areas of osteoid seen as light pink and lacy matrix with the nuclei of osteoblasts sitting closer to the produced matrix (image magnification × 100). The arrows in the enlargement show the osteoid matrix surrounding osteoblasts (defined as eosinophilic osteoid-like material). C), And D) , Mitotic count as identified in the same sections used to asses for osteoid evaluation. Each column is the Mean mitotic count in 9 sections (a minimum of 1100 nuclei were scanned per section). Decitabine treatment resulted in a significantly lower mitotic count (p < 0.0001). The arrows in the enlargement image in C (left) indicate mitotic nuclei. E) , Results for apoptosis analysis by TUNEL assay. Each column is the Mean count of TUNEL positive nuclei seen in 9 images representing 9 sections (≥1000 nuclei were scanned per section). There was a significant increase (p < 0.05) of apoptotic cells as a result of decitabine treatment. F) , Representation images from TUNEL assay of control tumors (left) and decitabine treated tumors [50] (image magnification × 200). The arrows in the enlargement image show the TUNEL-positive nuclei (apoptotic nuclei). Error bars indicate standard deviation from the Mean values and p-values are based on comparison between control and decitabine treated tumors using student t-test.

Article Snippet: 12 hours after plating they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 μM without changing the medium.

Techniques: In Vivo, Produced, TUNEL Assay, Standard Deviation

Journal: Cancer Cell International

Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

doi: 10.1186/1475-2867-7-14

Figure Lengend Snippet: Genes up-regulated (p < 0.0025 and ≥2 fold-change) after decitabine treatment identified using AffyChip.

Article Snippet: 12 hours after plating they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 μM without changing the medium.

Techniques: Activity Assay, Binding Assay, Synthesized

Journal: Cancer Cell International

Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

doi: 10.1186/1475-2867-7-14

Figure Lengend Snippet: Real-time gene expression of 6 pro-apoptotic genes U2OS in vitro & in vivo and normal osteoblasts . Total RNA was extracted from U2OS cells in vitro and in vivo and reverse-transcribed as detailed in methods. TaqMan assays were used to determine relative expression using the cDNA from control (no treatment) as base lines and ACTB for a reference gene by applying the ΔΔCt method. Each column is the Mean of three replicas and error bars indicate standard deviation from the Mean. The data is expressed as fold change relative to control (no-treatment). Xeno-4, Xeno-5, and Xeno-6 = decitabine treated xenografts. NHOst = normal human osteoblasts.

Article Snippet: 12 hours after plating they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 μM without changing the medium.

Techniques: Expressing, In Vitro, In Vivo, Standard Deviation

Journal: Cancer Cell International

Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

doi: 10.1186/1475-2867-7-14

Figure Lengend Snippet: Comparison of percentage of methylation across 5 of the tested CpG positions in vitro and in vivo . Each data point is the average of methylation percentage for each CpG position in three experiments. Y-axis indicates the percentage of methylation and the samples are indicated on the x-axis. The location of CpG positions relative to the gene start site and to each other is shown in Figure 5. The results of 5 CpG positions are shown to represent the methylation percentage in the four genes across the samples. There was a significant decrease (p < 0.001) in methylation quantity for each CpG position after decitabine treatment both in vitro and in vivo for the four genes in all sample but not in the NHOst (normal human osteoblasts). p-values were calculated by comparing the percentage of methylation for each individual CpG position in control cells with the same CpG position in the treated cells using student t-test and they all resulted in p < 0.001.

Article Snippet: 12 hours after plating they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 μM without changing the medium.

Techniques: Methylation, In Vitro, In Vivo

Journal: Cancer Cell International

Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

doi: 10.1186/1475-2867-7-14

Figure Lengend Snippet: Summary of Pyro-Q-CpG findings . Illustrated are the summary findings by Pyro-Q-CpG analysis of U2OS cells in vitro without treatment (control) and with 1 μM decitabine treatment (treated). The illustration also summarizes Pyro-Q-CpG findings in control U2OS xenograft tumors (Xeno-1, Xeno-2, and Xeno-3), and 2.5 mg/kg decitabine treated U2OS xenograft tumors (Xeno-4, Xeno-5, and Xeno-6). DNA from NHOst (normal low-passage human osteoblasts) was also analyzed for experiment control. DNAs from early embryonic DNA (Emb-DNA) and universally methylated DNA (Met-DNA) were used for negative and positive control respectively. CpG-islands are denoted by grey rectangles relative to the gene start site. The region further enlarged below corresponds to each tested CpG sequence. The tick marks denotes the individual CpG dinucleotides. The transcription start site is indicated by a directional arrow with the base pair numbers annotated for each tested sequence. The extent of methylation is represented by the scale bar (bottom right). GADD45A , PAWR , and PDCD5 , had a high level of methylation before decitabine treatment while HSPA9B had an intermediate level of methylation before treatment. In all cases the methylation was decreased significantly (p < 0.001) as a result of decitabine treatment. The detailed results for all genes are shown in [Additional file ].

Article Snippet: 12 hours after plating they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 μM without changing the medium.

Techniques: In Vitro, Methylation, Positive Control, Sequencing

Journal: Frontiers in Oncology

Article Title: The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37-Targeted Radioimmunotherapy in Activated B Cell Like Diffuse Large B Cell Lymphoma Cell Lines

doi: 10.3389/fonc.2019.01301

Figure Lengend Snippet: Experimental layout. U-2932 and RIVA cells were treated with 1 or 0.5 μg/mL 177 Lu-lilotomab satetraxetan (600 MBq/mg) for 18 h, excess 177 Lu-lilotomab satetraxetan removed, and cells seeded onto 384-well plates pre-printed with the 384-compound Cambridge Cancer compound library sourced from Selleckchem at final concentrations of 10 nM, 100 nM, or 1 μM. Untreated control cells were seeded on parallel plates. Viability measurements, using RealTime-Glo, were carried out between day 3 and 6 after seeding. Inhibitory compounds were considered as candidate hits if they: (1) in combination with 177 Lu-lilotomab satetraxetan inhibited cell proliferation over two consecutive days to a degree greater than the expected additive effect of the mono-treatments alone (BLISS theorem, see Materials and Methods for details), and (2) drug treatment alone did not reduce viability to <90% of that of untreated control.

Article Snippet: Cells treated with 177 Lu-lilotomab satetraxetan and untreated control cells were seeded onto 384-well plates pre-printed with the 384-compound Cambridge Cancer Compound library sourced from SelleckChem.

Techniques:

Journal: Frontiers in Oncology

Article Title: The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37-Targeted Radioimmunotherapy in Activated B Cell Like Diffuse Large B Cell Lymphoma Cell Lines

doi: 10.3389/fonc.2019.01301

Figure Lengend Snippet: JNJ-7706621 synergistically reduces viability of U-2932 cells when combined with 177 Lu-lilotomab satetraxetan. U-2932 cells were treated with 0.5, 1, and 2 μg/mL 177 Lu-lilotomab satetraxetan as in and seeded in 384-well plates pre-printed with JNJ-7706621, Alisertib, and GSK461364 in an 11 step concentration gradient (0–1,280 nM). Viability was measured as in . In left panels the fraction of viable cells relative to untreated control at day 5 are plotted for cells exposed to single treatment or combinations for each drug dose (blue: drug alone, red: drug combined with 0.5 μg/mL 177 Lu-lilotomab satetraxetan, green: drug combined with 1 μg/mL 177 Lu-lilotomab satetraxetan and orange: drug combined with 2 μg/mL 177 Lu-lilotomab satetraxetan) (A) JNJ-7706621, (B) Alisertib, and (C) GSK461364. Right panels show Fa/CIs plots obtained by the Chou-Talaly method using the CompuSyn software. The fraction affected (Fa) is the fraction of non-viable cells relative to untreated control. For each combination with a specific Fa value the CI indicates whether the combined treatment is synergistic (<1) or antagonistic (>1). In blue are data from the same experiment as that shown in left panels, whereas red and yellow circles represent two independent experiments performed in U-2932 with JNJ-7706621. In experiment 2, 100, 266, 707, 1,880, and 5,000 nM of JNJ-7706621 were combined with the same doses of 177 Lu-lilotomab satetraxetan as in experiment 1, and in experiment 3 the doses of JNJ-7706621 were; 200, 532, 1,410, 3,760, and 10,000 nM. Error bars: STDEV of triplicate samples.

Article Snippet: Cells treated with 177 Lu-lilotomab satetraxetan and untreated control cells were seeded onto 384-well plates pre-printed with the 384-compound Cambridge Cancer Compound library sourced from SelleckChem.

Techniques: Concentration Assay, Software