Silencing of TEL/AML1 In Definitive Leukemic Cells Does Not Impair Cell Survival

Abstract Abstract 3229 The most frequent structural chromosomal aberration in childhood acute lymphoblastic leukemia t(12;21) generates TEL/AML1 fusion gene. Resulting TEL/AML1 protein probably acts as an aberrant transcription factor that deregulates AML1-dependent transcription but its target genes and thus also the exact role in leukemic cells remain unknown. In vivo studies showed that TEL/AML1 itself is not sufficient to cause leukemia but may induce a preleukemic state characterized by the increased numbers of multipotent or B-cell progenitors with an incomplete block of differentiation. Despite its role for leukemia establishment the relevance of TEL/AML1 fusion gene for leukemia persistance has not been studied enough.To address this question and to explore the possibility of TEL/AML1-targeted therapy, we studied the effects of RNAi-mediated TEL/AML1 silencing on leukemic cells. As the only siRNA used for TEL/AML1 silencing published so far (Diakos et al, Blood, 2007) targets also the wild type AML1 (46% transcript reduction, our data), our first goal was to identify efficient and TEL/AML1-specific siRNA. We designed eleven different siRNAs spanning the fusion point of TEL/AML1 lacking the total sequence homology to wild type TEL and AML1 alleles to avoid their silencing. These 11 siRNAs were tested in HeLa cells transgenic for TEL/AML1-ires2-EGFP reporter. After lipofection into HeLa cells the efficiency of individual siRNAs was measured as a decrease of EGFP reporter fluorescence by flow cytometry. The best five siRNAs, that induced 50–58% silencing of the EGFP reporter, were tested at the mRNA level in TEL/AML-positive leukemic cell line. 24h after electroporation of siRNAs, when the silencing reached its maximum, two most efficient siRNAs induced 58% and 57% TEL/AML1 transcript reduction, respectively. We achieved 61% TEL/AML1 transcript reduction with the pool of both siRNAs while there was only slight reduction (14%) of wild type AML1 transcript. We used this efficient and specific siRNA pool to silence TEL/AML1 in REH and UOC-B6 TEL-AML1 positive cell lines and studied its effect on cell viability, proliferation and global gene expression. Applying two rounds of siRNA electroporation within 48 hours interval we achieved 74% and 86% TEL/AML1 protein knockdown in REH and UOC-B6 cells, respectively. We used trypan blue staining followed by optical microscopy to monitor cell viability and staining with annexin V and propidium idode to assess apoptosis rate by flow cytometry. Analysis of DNA content using staining with propidium iodide was performed to assess cell-cycle distribution. Incorporation of nucleoside analog was measured by flow cytometry to analyse de novo DNA synthesis as an indicator of proliferation rate. Despite the common expectation derived from studies on other fusion oncogenes (BCR/ABL, AML1/ETO, E2A/PBX), TEL/AML1 silencing neither decreased cell viability, nor induced apoptosis. On the contrary, TEL/AML1 depletion was accompanied by the slight but significant increase in the fraction of S-phase cells and corresponding rise in proliferation rate. Opposite effects on cell cycle distribution and proliferation were induced when we silenced wild type AML1. These findings support our hypothesis that TEL/AML1 may block previously established AML1 function in G1/S progression through the cell cycle. In line with the lack of effect on cell viability and discreet effect on cell-cycle distribution and proliferation we found no significant changes in global gene expression pattern upon TEL/AML1 depletion. Our data indicate, that TEL/AML1 is dispensable for the survival of definitive leukemic cells. This work was supported by grants MSM0021620813 and MZOFNM2005. Disclosures: No relevant conflicts of interest to declare.

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MLN4924, an Investigational Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Inhibits NF-κB Activity and Induces G1 Cell Cycle Arrest and Apoptosis in Pre-Clinical Models of Hodgkin Lymphoma (HL)

Blood ◽

10.1182/blood.v120.21.3717.3717 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3717-3717

Author(s):

Matthew J. Barth ◽

Cory Mavis ◽

Francisco J. Hernandez-Ilizaliturri ◽

Myron S. Czuczman

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Western Blot ◽

Cell Viability ◽

Tumor Cells ◽

Cell Lines ◽

Small Molecule ◽

Wild Type ◽

Cycle Arrest ◽

Molecule Inhibitor

Abstract Abstract 3717 The incorporation of combined-modality therapy, risk-stratified chemotherapy selection, high-dose chemotherapy and autologous stem cell support (HDC-ASCS), and monitoring treatment response by functional imaging are factors that have contributed to the improvement in clinical outcomes in HL patients. Unfortunately, those patients not eligible for or that have failed HDC-ASCS remain a challenge for the treating oncologist, stressing the need for novel therapeutic strategies. Significant improvements in the understanding of the biology of HL have been achieved, including cellular pathways altered in HL (e.g. the ubiquitin-proteasome system) and the role of the tumor microenvironment. MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an enzyme responsible for activating NEDD8, an ubiquitin-like molecule in the neddylation cascade that is responsible for cullin-ring ligase (CRL) mediated polyubiquitination of proteins targeting them for proteasomal degradation. In order to better understand the activity of MLN4924 in HL, we performed pre-clinical testing in IkB wild type (L-1236), IkB mutated (KM-H2 and L-428) HL cell lines, and in primary tumor cells derived from a HL patient. Malignant cells were exposed to escalating doses of MLN4924 and changes in cell viability were quantified at different time periods by alamar Blue reduction assay. Patient tumor cells were incubated with MLN4924 for 48 hrs and cell viability was determined using the CellTiterGlo assay. Induction of apoptosis in HL cell lines following exposure to MLN4924 was determined by flow cytometry for Annexin-V and propidium iodide (PI) staining and western blot for caspase-3 and PARP cleavage. Cell cycle analysis was performed by flow cytometry using PI staining. Inhibition of NAE by MLN4924 in HL cell lines was measured by western blot for NEDD8-cullin. Finally, changes in NF-kB activity following MLN4924 exposure were determined by p65 nuclear localization using Image stream technology. MLN4924 exhibited a dose- and time-dependent decrease in cell viability in all HL cell lines at nM concentrations. No differences in anti-tumor activity were observed between IkB-wild type (L-1236 IC50 = 250nM) and IkB–mutated HL cell lines (KM-H2 IC50 = 250nM and L-428 IC50 = 300nM). MLN4924 induced apoptosis in a dose-dependent manner in all cell lines tested. In addition, MLN4924 induced cell cycle arrest in G1 phase and inhibition of NAE was demonstrated by a decrease in NEDD8 conjugated CRL. L1236 cells exposed to MLN4924 also demonstrated a decrease in degradation of IκBα as evidenced by increased levels of p-IκBα following exposure to MLN4924 with a corresponding decrease in p65 nuclear translocation. Surprisingly KMH-2 cells, which carry a mutated IκBα protein that is truncated and non-functional, had a decrease in nuclear p65 following exposure to MLN4924, suggesting an alternative mechanism of NF-kB inhibitory activity by MLN4924. In summary, MLN4924 demonstrates activity against HL cells in vitro through inhibition of NF-kB, and is a promising novel agent for the treatment of HL. We continue to investigate the pre-clinical activity of MLN4924 both as a single-agent and in combination with traditional chemotherapy and other novel agents. Disclosures: No relevant conflicts of interest to declare.

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Usnea Acid as Multidrug Resistance (MDR) Reversing Agent against Human Chronic Myelogenous Leukemia K562/ADR Cells via an ROS Dependent Apoptosis

BioMed Research International ◽

10.1155/2019/8727935 ◽

2019 ◽

Vol 2019 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Wenjing Wang ◽

Shubin Niu ◽

Luxin Qiao ◽

Feili Wei ◽

Jiming Yin ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Multidrug Resistance ◽

Cell Viability ◽

Leukemia Cell ◽

Myelogenous Leukemia ◽

Leukemia Cell Line ◽

Phase Cell ◽

Cell Cycle Distribution ◽

Promising Therapeutic Approach

Purpose. Multidrug resistance (MDR) is a major obstacle in chemotherapy of leukemia treatments. In this paper, we investigated Usnea Acid (UA) as MDR reversal agent on hematologic K562/ADR cells via ROS dependent apoptosis. Methods. CCK8 assay was used to measure cell viability rate of K562/ADR. Intracellular reactive oxygen species (ROS) generation, cell cycle distribution, cell apoptosis were measured with flow cytometry, respectively. Proteins related to apoptosis were measured by Western blot. Intracellular Adriamycin accumulation was observed by confocal microscopy and measured by flow cytometry. Results. In vitro study showed intracellular Adriamycin accumulation was remarkably increased by UA. Cell viability treated with Adr (4 μM) was decreased from 89.8% ± 4.7 to 32% ± 8.9 by combined with UA (4 μM). Adr-induced apoptosis and G1/G0 phase cell cycle arrest were remarkably increased by UA, as well as, intracellular ROS level. However, MDR reversing activity of UA was inhibited by N-acetyl cysteine (NAC), a ROS scavenger. Conclusion. These data provide compelling evidence that UA is a promising agent against MDR in leukemia cell line and suggest a promising therapeutic approach for leukemia.

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NOTCH1 Mutation in CLL Enhances Cell Cycle G1/S Transition through Specific Cyclin Overexpression: Preclinical Ground for CDK4/6 Targeted Inhibition

Blood ◽

10.1182/blood-2021-152931 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1544-1544

Author(s):

Salvador Carrillo-Tornel ◽

Tzu Hua Chen-Liang ◽

María Zurdo ◽

Anna Puiggros ◽

Andrea Gómez-Llonín ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Flow Cytometry ◽

Next Generation Sequencing ◽

S Phase ◽

Wild Type ◽

Trisomy 12 ◽

Anti Cd20 ◽

Generation Sequencing

Abstract Introduction: The partially understood biological consequences of the NOTCH1 acquired lesion, seems to be distinctive enough among chronic lymphocytic leukemia (CLL) patients, as clinical studies have repeatedly found specific features: intermediate prognosis, anti-CD20 poorer responses, and a higher frequency of trisomy 12 and Richter transformation. Though located in a different domain, the activating nature of NOTCH1 mutation in T lymphoblastic leukemia relies on cell cycle regulators. In fact, pivotal studies, from the pre-next generation sequencing era, showed dysregulation of cyclins-gene expression, as driver of the unique CLL features. Thus, our goal was to revisit the cell cycle in CLL, but focusing now in the NOTCH1 mutated subset (NOTCH1MUT), hypothesizing that biological differences versus wild type cases (NOTCH1WT) would explain the clinical ones, and exploiting potential differences with targeted molecules in vitro. Methods: From 2010 to 2019, presentation bone marrow aspirates or blood samples DNA was collected during the diagnostic workout from 378 CLL patients, all of them annotated by next generation sequencing. G 0/early-G 1 effectors gene expression was measured by RT-qPCR in negatively immunoselected circulating CLL cells. A siRNA approach was selected for silencing by electroporation 7 NOTCH1WT and 2 NOTCH1MUT cases. Cell cycle and apoptosis flow cytometry assays were performed on cultured fresh primary cells from n? NOTCH1MUT and 4 NOTCH1WT cases, before and after exposure to different concentrations of palbociclib, a CDK4/6 inhibitor. Results: We found that 37/378 (9.8%) of patients harbored a NOTCH1 mutation. NOTCH1MUTcases presented with higher lymphocyte counts [NOTCH1MUT 17.2 x10 9/L vs. NOTCH1WT 9.7 x10 9/L; p=0.042], trisomy 12 (35.1% vs. 11.4%; p<0.001) and a higher frequency of an unmutated IGHV status (70% vs. 21%; p<0.001). Of note, NOTCH1MUT patients had poorer responses to anti-CD20 based schemes than NOTCH1WT patients (35.7 vs. 69.8% complete response; p). We found that NOTCH1MUT cases showed a relevant increase of 38-fold change (FC) for CCND3, 27-FC for CDK4 and CCND2, 11-FC for CCND1 and 9-FC for CDK6 gene expression in negatively immunoselected circulating CLL cells at diagnosis. In addition, NOTCH1MUT cases displayed a statistically significant higher percentage of cells in the S phase than the wild type cases (21% vs. 1%, p=0.004). Though significance was not met, NOTCH1MUT cases showed a higher percentage of events within G 2-M (28% vs. 26 %). Next, we incorporated the flow cytometry assay to in vitro palbociclib treated CLL cells from 3 NOTCH1MUT and 4 NOTCH1WT cases. Five days after culture stimulation, cells were exposed to 38 and 76 μM (dose range for reaching maximum CLL cells sensitivity plateau) of the drug for 48 hours. As stated above, NOTCH1MUT cases were characterized by a much higher proportion of cells in S phase at baseline (21%), which was reduced in a dose dependent manner to an 8% and a 6% after exposure to palbociclib, respectively. The standard 48-72 hours drug assay may not be the most suitable for slow growth tumors as CLL and, in particular, for testing cell cycle inhibitors. Thus, we designed an assay for two cell cycles based on the average population doubling time of the primary cell culture experiments (0.6 in 72 hours), and using the mean steady state plasma concentration of palbociclib achieved clinically: 1 μM. After 120 hours, the baseline 15% of cells in S phase was reduced to an 0.64% after exposure to palbociclib 1 μM in NOTCH1MUT cases and a 1.8x-increase in the percentage dead cells was noted, compared with NOTCH1WT cases. Conclusions: Compared with NOTCH1WT CLL cases, we describe an overexpression of effectors of early phase in NOTCH1MUT. This profile made NOTCH1MU cells more suited to enter and traverse through the cell cycle and could explain, in part, the proliferative clinical-biological features of this subset of patients and opening a window for exploiting therapeutically these differences. Ours experiments in vitro with palbociclib sets the ground for the clinical research. Figure 1 Figure 1. Disclosures Jerez: BMS: Consultancy; Novartis: Consultancy; GILEAD: Research Funding.

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Altered Expression Profile of Global Genes in U937 Cells Stably Transfected with a Leukemia Fusion Gene: MOZ-TIF2.

Blood ◽

10.1182/blood.v104.11.4284.4284 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4284-4284

Author(s):

Hong Yin ◽

Kerry L. Blanchard ◽

Jonathan Glass

Keyword(s):

Gene Expression ◽

Cell Line ◽

Expression Profile ◽

Histone Acetyltransferase ◽

Fusion Gene ◽

Global Gene Expression ◽

Established Cell Line ◽

U937 Cells ◽

Wild Type ◽

Established Cell

Abstract Chromosome translocations involving the monocytic leukemia zinc finger (MOZ) gene occurs often in patients with acute myeloid leukemia (AML) of monocytoid phenotype (FAB types M4 or M5). The resulting MOZ-related fusions include MOZ-CBP, MOZ-P300, and MOZ-TIF2. MOZ has been identified as a histone acetyltransferase of unknown function. MOZ has been demonstrated to interact directly with RUNX1 to regulate RUNX1-mediated transcription and the MOZ-CBP fusion disrupted RUNX1-mediated transcription activity. The MOZ fusion partners CBP and p300 are transcription coactivators with histone acetyltransferase activity; TIF2 is a nuclear receptor coactivator which recruits CBP/P300 in receptor activation. In an animal model, the MOZ-TIF2 fusion successfully induced the occurrence of AML. The leukemia transformation depended on a nucleosome binding motif in MOZ and two CBP binding motifs in TIF2 partner. However, the detailed molecular events relevant to leukemogenesis by MOZ-TIF2 have not been elucidated. We have constructed a MOZ-TIF2 fusion and investigated the effects of this fusion on global gene expression in U937 cells. The U937 cell line with expression of MOZ-TIF2 or wild type MOZ gene were established by stable transfection with pcDNA3-MOZ-TIF2 and pcDNA3-MOZ. RNA was isolated from the early passage of stably transfected U937 cells with TRI reagent® - RNA/DNA/protein isolation reagent according to the manufacturer’s protocol. The expression of MOZ-TIF2 in the established cell line was verified by RT-PCR with specific primers for the fusion gene. The examination of global gene expression with Affymetrix gene chip was conducted on the human U95A array. Ten micrograms of total RNA was used. Synthesis of cRNA and subsequent hybridization was completed by the Core Facility at LSUHSC-S according the standard Affymetrix protocol. The human U95A array represents 12,256 oligonucleotides of known genes or expression tags. The gene chip experiment was repeated once for each established cell line with the forced expressed fusion or wild type gene. The raw data was collected and analyzed for the detection and log ratio of each gene or expression tag with the Affymetrix Microarray Suite with the scale set at 2500. The results were further analyzed with GeneSifter.Net. Compared to the expression profile of control cells stably transfected with pcDNA3 vector alone, a > 5-fold change in expression was seen with 49 genes increasing and 32 genes decreasing expression in MOZ-TIF2 expressing cells (p = 0.01). In cells overexpressing wild type MOZ, gene expression increased >5-fold in 411 genes and decreased >5-fold in 48 genes. In a comparison of MOZ expressing cells to MOZ-TIF2 expressing cells a >5-fold change of expression was seen in 296 genes with increased expression in 256 and decreased expression in 40 genes. Among the differentially expressed genes, the c-Myc oncogene expression was increased a strikingly with a 29-fold in MOZ-TIF2 expressing cells over the control cells. Our results suggest that MOZ-TIF2 fusion may interfere with the function of wild type MOZ during the development of myeloid cells by inhibiting MOZ-mediated transcription. In addition, an alternative pathway involving c-Myc may also play an important role in MOZ-TIF2 related leukemogenesis.

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High Glucose Affects the Cytotoxic Potential of Rapamycin, Metformin and Hydrogen Peroxide in Cultured Human Mesenchymal Stem Cells

Current Molecular Medicine ◽

10.2174/1566524019666190722115842 ◽

2019 ◽

Vol 19 (9) ◽

pp. 688-698 ◽

Cited By ~ 1

Author(s):

Azam Roohi ◽

Mahin Nikougoftar ◽

Hamed Montazeri ◽

Shadisadat Navabi ◽

Fazel Shokri ◽

...

Keyword(s):

Cell Cycle ◽

Hydrogen Peroxide ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Viability ◽

High Glucose ◽

Cell Cycle Distribution ◽

Efficient Treatment ◽

Chronic Hyperglycemia ◽

Placental Mesenchymal Stem Cells

Background: Oxidative stress and chronic hyperglycemia are two major side effects of type 2 diabetes affecting all cell types including mesenchymal stem cells (MSCs). As a cell therapy choice, understanding the behavior of MSCs will provide crucial information for efficient treatment. Methods: Placental mesenchymal stem cells were treated with various concentrations of glucose, metformin, rapamycin, and hydrogen peroxide to monitor their viability and cell cycle distribution. Cellular viability was examined via the MTT assay. Cell cycle distribution was studied by propidium iodide staining and apoptosis was determined using Annexin Vpropidium iodide staining and flow cytometry. Involvement of potential signaling pathways was evaluated by Western blotting for activation of Akt, P70S6K, and AMPK. Results: The results indicated that high glucose augmented cell viability and reduced metformin toxic potential. However, the hydrogen peroxide and rapamycin toxicities were exacerbated. Conclusion: Our findings suggest that high glucose concentration has a major effect on placental mesenchymal stem cell viability in the presence of rapamycin, metformin and hydrogen peroxide in culture.

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Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520617666170912133054 ◽

2018 ◽

Vol 18 (2) ◽

pp. 210-215 ◽

Cited By ~ 3

Author(s):

Mona Diab-Assaf ◽

Josiane Semaan ◽

Marwan El-Sabban ◽

Soad K. Al Jaouni ◽

Rania Azar ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

T Cell ◽

Flow Cytometric Analysis ◽

Leukemic Cells ◽

Cell Cycle Distribution ◽

Dependent Manner ◽

Apoptosis Induction ◽

Inhibition Of Proliferation ◽

Human T Cell

Introduction: Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. Objective: The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. Materials and Methods: Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. Results: At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. Conclusion: Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

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COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/β-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells

BMC Cancer ◽

10.1186/s12885-021-08164-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Aleksandra Majchrzak-Celińska ◽

Julia O. Misiorek ◽

Nastassia Kruhlenia ◽

Lukasz Przybyl ◽

Robert Kleszcz ◽

...

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Methylation Level ◽

Molecular Mechanisms ◽

Methylation Status ◽

Receptor Expression ◽

Cell Cycle Distribution ◽

Ep4 Receptor ◽

Cox 2 ◽

The Impact

Abstract Background Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/β-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/β-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. Methods Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of β-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. Results Wnt/β-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. Conclusions Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

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BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgVH unmutated chronic lymphocytic leukemia (CLL) cells

Blood ◽

10.1182/blood-2007-12-127688 ◽

2008 ◽

Vol 112 (3) ◽

pp. 782-792 ◽

Cited By ~ 90

Author(s):

Anna Guarini ◽

Sabina Chiaretti ◽

Simona Tavolaro ◽

Roberta Maggio ◽

Nadia Peragine ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Cross Linking ◽

Cell Cycle Distribution ◽

Moderate Increase ◽

Functional Changes ◽

Cytoskeleton Organization ◽

Different Response

Abstract Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgVH mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G1 phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG0/1 cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgVH M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

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Role of Sigma Factors in Controlling Global Gene Expression in Light/Dark Transitions in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Journal of Bacteriology ◽

10.1128/jb.01036-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7829-7840 ◽

Cited By ~ 30

Author(s):

Tina C. Summerfield ◽

Louis A. Sherman

Keyword(s):

Gene Expression ◽

Global Gene Expression ◽

Growth Conditions ◽

Day Length ◽

Regulation Of Transcription ◽

Wild Type ◽

Protein Levels ◽

In The Wild ◽

Altered Gene ◽

Pcc 6803

ABSTRACT We report on differential gene expression in the cyanobacterium Synechocystis sp. strain PCC 6803 after light-dark transitions in wild-type, ΔsigB, and ΔsigD strains. We also studied the effect of day length in the presence of glucose on a ΔsigB ΔsigE mutant. Our results indicated that the absence of SigB or SigD predominately altered gene expression in the dark or in the light, respectively. In the light, approximately 350 genes displayed transcript levels in the ΔsigD strain that were different from those of the wild type, with over 200 of these up-regulated in the mutant. In the dark, removal of SigB altered more than 150 genes, and the levels of 136 of these were increased in the mutant compared to those in the wild type. The removal of both SigB and SigE had a major impact on gene expression under mixotrophic growth conditions and resulted in the inability of cells to grow in the presence of glucose with 8-h light and 16-h dark cycles. Our results indicated the importance of group II σ factors in the global regulation of transcription in this organism and are best explained by using the σ cycle paradigm with the stochastic release model described previously (R. A. Mooney, S. A. Darst, and R. Landick, Mol. Cell 20:335-345, 2005). We combined our results with the total protein levels of the σ factors in the light and dark as calculated previously (S. Imamura, S. Yoshihara, S. Nakano, N. Shiozaki, A. Yamada, K. Tanaka, H. Takahashi, M. Asayama, and M. Shirai, J. Mol. Biol. 325:857-872, 2003; S. Imamura, M. Asayama, H. Takahashi, K. Tanaka, H. Takahashi, and M. Shirai, FEBS Lett. 554:357-362, 2003). Thus, we concluded that the control of global transcription is based on the amount of the various σ factors present and able to bind RNA polymerase.

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Cytotoxic Potential of the Coelomic Fluid Extracted from the Sea Cucumber Holothuria tubulosa against Triple-Negative MDA-MB231 Breast Cancer Cells

Biology ◽

10.3390/biology8040076 ◽

2019 ◽

Vol 8 (4) ◽

pp. 76 ◽

Cited By ~ 4

Author(s):

Claudio Luparello ◽

Debora Ragona ◽

Dalia Maria Lucia Asaro ◽

Valentina Lazzara ◽

Federica Affranchi ◽

...

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Sea Cucumber ◽

Triple Negative ◽

Metastatic Breast ◽

Water Soluble ◽

Coelomic Fluid ◽

Cell Cycle Distribution ◽

Widespread Species ◽

Tnbc Cell

Growing evidence has demonstrated that the extracts of different holothurian species exert beneficial effects on human health. Triple negative breast cancers (TNBC) are highly malignant tumors that present a poor prognosis due to the lack of effective targeted therapies. In the attempt to identify novel compounds that might counteract TNBC cell growth, we studied the effect of the exposure of the TNBC cell line MDA-MB231 to total and filtered aqueous extracts of the coelomic fluid obtained from the sea cucumber Holoturia tubulosa, a widespread species in the Mediterranean Sea. In particular, we examined cell viability and proliferative behaviour, cell cycle distribution, apoptosis, autophagy, and mitochondrial metabolic/cell redox state. The results obtained indicate that both total and fractionated extracts are potent inhibitors of TNBC cell viability and growth, acting through both an impairment of cell cycle progression and mitochondrial transmembrane potential and a stimulation of cellular autophagy, as demonstrated by the increase of the acidic vesicular organelles and of the intracellular protein markers beclin-1, and total LC3 and LC3-II upon early exposure to the preparations. Identification of the water-soluble bioactive component(s) present in the extract merit further investigation aiming to develop novel prevention and/or treatment agents efficacious against highly metastatic breast carcinomas.

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