scholarly journals Drug priming enhances radiosensitivity of adamantinomatous craniopharyngioma via downregulation of survivin

2016 ◽  
Vol 41 (6) ◽  
pp. E14 ◽  
Author(s):  
Christina Stache ◽  
Christiane Bils ◽  
Rudolf Fahlbusch ◽  
Jörg Flitsch ◽  
Michael Buchfelder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Cell Death ◽  
Cell Cultures ◽  
Survivin Expression ◽  
Egfr Signaling ◽  
Qrt Pcr ◽  
Tki Treatment ◽  
Adamantinomatous Craniopharyngioma

OBJECTIVE In this study, the authors investigated the underlying mechanisms responsible for high tumor recurrence rates of adamantinomatous craniopharyngioma (ACP) after radiotherapy and developed new targeted treatment protocols to minimize recurrence. ACPs are characterized by the activation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR), known to mediate radioresistance in various tumor entities. The impact of tyrosine kinase inhibitors (TKIs) gefitinib or CUDC-101 on radiation-induced cell death and associated regulation of survivin gene expression was evaluated. METHODS The hypothesis that activated EGFR promotes radioresistance in ACP was investigated in vitro using human primary cell cultures of ACP (n = 10). The effects of radiation (12 Gy) and combined radiochemotherapy on radiosensitivity were assessed via cell death analysis using flow cytometry. Changes in target gene expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Survivin, identified in qRT-PCR to be involved in radioresistance of ACP, was manipulated by small interfering RNA (siRNA), followed by proliferation and vitality assays to further clarify its role in ACP biology. Immunohistochemically, survivin expression was assessed in patient tumors used for primary cell cultures. RESULTS In primary human ACP cultures, activation of EGFR resulted in significantly reduced cell death levels after radiotherapy. Treatment with TKIs alone and in combination with radiotherapy increased cell death response remarkably, assessed by flow cytometry. CUDC-101 was significantly more effective than gefitinib. The authors identified regulation of survivin expression after therapeutic intervention as the underlying molecular mechanism of radioresistance in ACP. EGFR activation promoting ACP cell survival and proliferation in vitro is consistent with enhanced survivin gene expression shown by qRT-PCR. TKI treatment, as well as the combination with radiotherapy, reduced survivin levels in vitro. Accordingly, ACP showed reduced cell viability and proliferation after survivin downregulation by siRNA. CONCLUSIONS These results indicate an impact of EGFR signaling on radioresistance in ACP. Inhibition of EGFR activity by means of TKI treatment acts as a radiosensitizer on ACP tumor cells, leading to increased cell death. Additionally, the results emphasize the antiapoptotic and pro-proliferative role of survivin in ACP biology and its regulation by EGFR signaling. The suppression of survivin by treatment with TKI and combined radiotherapy represents a new promising treatment strategy that will be further assessed in in vivo models of ACP.

Downregulation of CD30, Resistance to MMAE, and Upregulation of MDR1 Are All Associated with Resistance to Brentuximab Vedotin

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3643-3643
Author(s):  
Robert Chen ◽  
Jessie Hou ◽  
Edward Newman ◽  
Young Kim ◽  
Cecile Donohue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Gene Expression Profiling ◽  
Western Blotting ◽  
Primary Tumor ◽  
Expression Profiling ◽  
Primary Tumors ◽  
Qrt Pcr ◽  
Mts Assay

Abstract Background: Both Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) express surface CD30. Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), specifically to cells expressing surface CD30. Although BV elicits a high response rate (75% in HL and 86% in ALCL), the majority of patients who do not attain complete response (CR) will eventually develop resistance to BV. It is not known whether resistance to BV is through a) CD 30 alterations b) resistance to cytotoxic agent MMAE or c) overexpression of drug exporters. We developed 2 BV-resistant cell models and obtained primary lymphoma samples from patients with relapsed/progressive disease post BV therapy. We examined CD30 expression, MMAE resistance, drug exporter expression, and gene expression profiles in vitro and in vivo to determine mechanisms of resistance to BV. Methods: HL cell line(L428) and ALCL cell line (KARPAS 299) were used for in vitro experiments. The selection of BV resistant cell model (L428R and KARPAS 299R) used two different approaches (pulsatile or constant exposure). Both BV resistance and MMAE resistance were confirmed by MTS assays. CD30 expression was measured by flow cytometry,qRT-PCR, and Western blotting. Drug exporter expression was measured using qRT-PCR to MDR1, MRP1, and MRP3. In vivo experiments utilized primary tumor samples from 15 HL and 4 ALCL patients who had developed relapsed/progressive disease post BV treatment. CD30 expression was assessed by immunohistocytochemistry (IHC). Gene expression profiling was performed in both parental and resistant HL and ALCL cells, and in 4 ALCL primary tumor samples using Affymetrix whole genome GeneChip® Human Genome U133 2.0 Plus. Results: MTS assay showed the IC50 of KARPAS 299R to BV shifted from 24 +/- 10 ng/ml to 28 +/- 9 ug/ml, an 1183-fold increase. MTS assay also showed the IC50 of KARPAS 299R to MMAE only increased 2-fold when compared to KARPAS 299. Flow cytometry showed downregulation of surface CD30 expression in KARPAS 299R as compared to KARPAS 299 parental (59% vs. 96%, median intensity 78 +/- 17 vs. 591 +/- 51). This downregulation was confirmed by qRT-PCR and Western blotting for CD30. As KARPAS 299R is a mixed cell population, we sorted them into CD30+ and CD30- subpopulations. We then analyzed for BV sensitivity based on CD 30 expression status in KARPAS 299R. MTS assay showed that KARPAS 299R CD30+ cells were equally as resistant to BV as KARPAS 299R CD30- cells (figure 1A). IHC performed in 4 ALCL primary tumor samples showed persistent CD 30 expression in relapsed/progressive tumor specimens post BV treatment. Gene expression profiling on KARPAS 299R showed downregulation of CD30 as compared to KARPAS 299. Gene expression profiling on pre- and post-treatment ALCL samples (8) did not show significant differences in CD30 expression. The top four upregulated genes in relapsed/progressive samples as compared to pretreatment samples were LCE3D, WNT3, TNNT, CITED2. The top four downregulated genes in relapsed/progressive samples as compared to pretreatment samples were CXCL13, C4orf7, MS4A1, and IGJ. MTS assay showed that the IC50 of L428R to BV has shifted from 32 +/- 11 ug/ml to 391 +/- 92 ug/ml, a 12-fold increase. MTS assays showed the IC50 of L428R to MMAE has increased 99-folds when compared to L428 (figure 1B). No difference was seen in CD 30 expression by flow cytometry, qRT-PCR, or western blotting between L428R vs. L428. IHC performed in 15 HL primary tumors show persistent CD30 expression in relapsed/progressive tumor specimen post-BV treatment. qRT-PCR showed upregulation of MDR1mRNA in L428R as compared to L428. Gene expression profiling on L428R showed upregulation of MDR1 as compared to L428. Conclusion: Downregulation of CD30 is seen in BV-resistant ALCL cell model. However, sensitivity to BV did not depend solely on the level of CD30 expression as CD30+ cell subpopulations still exhibited resistance to BV in vitro. Upregulation of MDR-1 and resistance to MMAE were seen in BV-resistant HL cells, rather than downregluation of CD30. Downregulation of CD30 was not seen in HL or ALCL primary tumors. Further work is ongoing to explore/validate potential targets derived from gene expression profiling in ALCL primary tumors. Figure 1A Sensitivity to BV is not related to CD30 expression Figure 1A. Sensitivity to BV is not related to CD30 expression Figure 1B. Figure 1B. Viability of L-428 parental versus BV-resistant cells Disclosures Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau, Travel expenses Other.

Z-Guggulsterone Downregulates Survivin and Induces Cell Death in Large B Cell Lymphoma Cells In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4752-4752
Author(s):  
Maria K. Angelopoulou ◽  
Konstantinos Lilakos ◽  
Vassilios Salpeas ◽  
Sotirios Sachanas ◽  
Penelope Korkolopoulou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Survivin Expression ◽  
B Cell Lymphoma ◽  
Lymphoma Cells

Abstract Introduction: Survivin is a member of the Inhibitor of Apoptosis Proteins and has recently gained attention as a possible therapeutic target in malignancies, due to its dual role both as an antiapoptotic protein and as a cell cycle regulator. It is overexpressed in malignant cells and confers resistance to chemotherapy and other stimuli triggering apoptosis. Z-Guggulsterone (Z-GGS) is a plant sterol, which has been used in inflammatory conditions and has been recognized as a potent NF-kB suppressor. Since Survivin, as well as other antiapoptotic proteins, are under NFkB regulation, we studied the effect of Z-GGS on two B-cell lymphoma cell lines. Methods: DB and HT cell lines were treated with increasing concentrations (10μM, 20μM and 30μM) of Z-GGS, for 24, 48 and 72 hours. Survivin expression was tested with Flow Cytometry and Survivin transcripts were measured with quantitative real time PCR using the Universal Probe Library hydrolysis probes and expressed as Survivin/abl ratio. Cell viability was assessed with the MTT assay. Results: Both cell lines were positive for Survivin at baseline by flow cytometry (66% of total cells for DB and 95% for HT). Treatment of DB cells with 10, 20 and 30μM Z-GGS resulted in a 44%, 49% and 68% reduction of Survivin expression at 24 hours, respectively, whereas the effect on HT was less prominent with a 10% reduction at 24 hours with 30μM Z-GGS. Survivin transcripts decreased as well, with the maximum effect observed at 72 hours with 30μM Z-GGS for both cell lines: Survivin/abl was 0.009 for untreated cells vs 0.0008 with 30μM Z-GGS for DB cells and 0.0135 vs 0.0005 for HT cells. Linearity was observed for increasing concentrations of Z-GGS at 72 hours. Cell viability was practically unaffected at any time point with 10 and 20μM Z-GGS for both cell lines, whereas 30 μM Z-GGS resulted in a 63% and 78% cell death at 48 and 72 hours respectively for DB cells and 67% and 83% for HT cells. Conclusions: The steroid Z-GGS downregulates Survivin expression in B-lymphoma cells in vitro and induces cell death at 30μM concentration. Further experiments will clarify its possible role in the treatment of B-cell malignancies.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

scholarly journals A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 37 ◽  
Author(s):  
Jennifer Linden ◽  
Kiel Telesford ◽  
Samantha Shetty ◽  
Paige Winokour ◽  
Sylvia Haigh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Clostridium Perfringens ◽  
Specificity And Sensitivity ◽  
Epsilon Toxin ◽  
Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

scholarly journals Programmed Cell Death-1: Programmed Cell Death-Ligand 1 Interaction Protects Human Cardiomyocytes Against T-Cell Mediated Inflammation and Apoptosis Response In Vitro

2020 ◽  
Vol 21 (7) ◽  
pp. 2399
Author(s):  
Woan Ting Tay ◽  
Yi-Hsien Fang ◽  
Suet Theng Beh ◽  
Yen-Wen Liu ◽  
Ling-Wei Hsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
T Cell ◽  
T Lymphocytes ◽  
Programmed Cell Death ◽  
T Cell Response ◽  
Tumor Bearing ◽  
Cd8 T Lymphocytes ◽  
Programmed Cell Death 1

Aim: Immunological checkpoint therapy is considered a powerful method for cancer therapy and acts by re-activating autologous T cells to kill the cancer cell. Myocarditis cases have been reported in cancer patients after immunological therapy; for example, nivolumab treatment is a monoclonal antibody that blocks programmed cell death-1/programmed cell death ligand-1 ligand interaction. This project provided insight into the inflammatory response as a benchmark to investigate the potential cardiotoxic effect of T cell response to the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis in regulating cardiomyocyte injury in vitro. Methods and Results: We investigated cardiomyopathy resulted from the PD-1/PD-L1 axis blockade using the anti-PD-1 antibody in Rockefeller University embryonic stem cells-derived cardiomyocytes (RUES2-CMs) and a melanoma tumor-bearing murine model. We found that nivolumab alone did not induce inflammatory-related proteins, including PD-L1 expression, and did not induce apoptosis, which was contrary to doxorubicin, a cardiotoxic chemotherapy drug. However, nivolumab was able to exacerbate the immune response by increasing cytokine and inflammatory gene expression in RUES2-CMs when co-cultured with CD4+ T lymphocytes and induced apoptosis. This effect was not observed when RUES2-CMs were co-cultured with CD8+ T lymphocytes. The in vivo model showed that the heart function of tumor-bearing mice was decreased after treatment with anti-PD-1 antibody and demonstrated a dilated left ventricle histological examination. The dilated left ventricle was associated with an infiltration of CD4+ and CD8+ T lymphocytes into the myocardium. PD-L1 and inflammatory-associated gene expression were significantly increased in anti-PD-1-treated tumor-bearing mice. Cleaved caspase-3 and mouse plasma cardiac troponin I expressions were increased significantly. Conclusion: PD-L1 expression on cardiomyocytes suppressed T-cell function. Blockade of PD-1 by nivolumab enhanced cardiomyocyte inflammation and apoptosis through the enhancement of T-cell response towards cardiomyocytes.

3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 82
Author(s):  
M. Paczkowski ◽  
C. Bidwell ◽  
D. Spurlock ◽  
J. Waddell ◽  
R. L. Krisher
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Dna Methyltransferase ◽  
In Vitro Maturation ◽  
Fold Increase ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Dna Methyltransferase 1

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P < 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (>7-fold increase in vivo), caspase 8 (>4-fold increase in vivo), and eukaryotic translation initiation factor 1B (>4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (>3-fold and >5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

scholarly journals P06.01 Delta24-ACT oncolytic adenovirus as a therapeutic approach for DIPG

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii36-iii36
Author(s):  
V Laspidea ◽  
M Puigdelloses ◽  
M García-Moure ◽  
I Iñigo-Marco ◽  
J Gallego ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Lines ◽  
In Vivo Studies ◽  
Immunogenic Cell Death ◽  
Murine Models ◽  
Antitumoral Effect ◽  
Infected Cells

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. MATERIALS AND METHODS We use the NP53 and XFM murine DIPG cell lines. Flow cytometry was used to assess cell infectivity and ligand expression. We analyzed viral replication using a method based in hexon detection, and viral cytotoxic effect using the MTS assay. For immunogenic cell death analysis, we measured ATP secretion by a luminometric assay and calreticulin location by flow cytometry and immunofluorescence. For in vivo studies, cells and virus were injected in the pons of the mice, using the screw-guided system. RESULTS In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. CONCLUSIONS Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

scholarly journals Activation of Utrophin Promoter by Heregulin via theets-related Transcription Factor Complex GA-binding Protein α/β

1999 ◽  
Vol 10 (6) ◽  
pp. 2075-2086 ◽  
Author(s):  
Tejvir S. Khurana ◽  
Alan G. Rosmarin ◽  
Jing Shang ◽  
Thomas O. B. Krag ◽  
Saumya Das ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Transcription Factors ◽  
Neuromuscular Junction ◽  
Muscle Cell ◽  
Cell Cultures ◽  
Binding Protein ◽  
Duchenne's Muscular Dystrophy ◽  
Ga Binding Protein

Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate thatets-related GA-binding protein α/β transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein α/β complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal–regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne’s muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.

Correlates of Lenalidomide Induced Immune Stimulation and Response in CLL: Analysis in Patients on Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 979-979 ◽  
Author(s):  
Georg Aue ◽  
Stefania Pittaluga ◽  
Delong Liu ◽  
Larry Stennett ◽  
Susan Soto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Expression Profiling ◽  
Research Funding ◽  
Immune Stimulation ◽  
Intramural Research Program ◽  
Pre Treatment ◽  
Program Research

Abstract Abstract 979 Lenalidomide's mechanism of action in chronic lymphocytic leukemia (CLL) is not well understood. In vitro data suggest that anti-leukemic immune responses are important. Tumor flare reactions during treatment have been associated with response in some but not other studies. In vivo data that mechanistically link immune stimulation to clinical responses are lacking. We designed an independent, single center, phase II trial of lenalidomide in relapsed/refractory CLL (clinicaltrials.gov: NCT00465127). Here we report final clinical data and results of multiple translational analyses that indicate that an IFNy centered immune response is critical for response. A 3 week on, 3 weeks off treatment scheme (42 day cycles) was chosen to pulse immune stimulation while trying to minimize myelosuppression. The starting dose was 20 mg daily for the first 10 patients and 10 mg for the subsequent 23. Response was measured at 24 weeks. 5 patients, 4 with del 17p, achieved a PR by IWCLL criteria (16%) and were eligible to continue drug for 4 more cycles; the PFS in these patients was 16 months compared to 7 months for all other (p<0.001). Myelosupression remained the limiting side effect. A cytokine release syndrome often accompanied by tumor flare reactions was seen in 78% of patients in cycle 1 and often recurred in subsequent cycles. Compared to other studies it appears that the long treatment free period increased the inflammatory reaction upon restarting of L. All correlative analyses reported here were performed on PBMCs, lymph node (LN) core biopsies and serum obtained from patients during cycle 1 and 2 and included flow cytometry, gene expression profiling (Affymetrix arrays), and cytokine measurements. Nine patients with decreased lymphadenopathy ≥10% (10–85%) on CT after 4 cycles were considered responders (R) for correlative studies. There was a significant decrease in CLL count (median 14% on day 8 and 49% on day 22, p<0.01) and in the number of circulating T (CD3, CD4, CD8) and NK-cells (n=22, p<0.05) with no difference between R and non-responders (NR). In contrast, the CD3 count in LN core biopsies increased 1.4 fold in R compared to matched pre-treatment biopsies (p<0.05) with no change in NR (0.95 fold). In the L free interval CLL cells rebounded to pre-treatment levels. A rapid rebound of CLL counts during treatment interruptions has been previously described but its mechanism is not well understood. In migration assays we observed a 3-fold increased migration towards SDF-1 for L compared to control cells (p=0.03), indicating that increased homing of lymphocytes to tissue sites may be responsible for the rapid decrease in peripheral counts. The cell surface molecules CD40, 54, 86, 95, DR5 were upregulated (p<0.05) while CD5 and 20 were downregulated (p<0.001) on circulating CLL cells. Effects on CD54 and CD5 were stronger in R than NR (p<0.05). Next we performed gene expression profiling on purified PB-CLL cells and LN core biopsies obtained on day 8. L induced upregulation of 95 genes, many of which are known to be regulated by interferon gamma (IFNγ). The comparison with a gene expression signature induced by recombinant IFNγ in CLL cells cultured in vitro confirmed the significant induction of a typical IFNγ response by L in vivo (n=24, p<0.0001). The IFNγ response in PB-CLL cells was no different in R vs NR (n=12, p=0.78), but in LN biopsies it was more prominent in R (n=7) than NR (n=5) (p<0.05). Consistently the IFNG gene was upregulated in LN biopsies of R but actually decreased in NR (p=0.001). Serum IFNγ levels were elevated on L (n=14 at all time points, day 4 p=0.03, day 8 p=0.01, day 22 p=0.02, day 49 p<0.01), but off drug returned to pretreatment levels. Next we sought to determine the source of IFNγ. The tumor cells are ruled out as IFNG was not expressed in purified CLL cells. By flow cytometry the number of IFNγ secreting CD4 T-cells increased on day 8 from 0.8% to 1.5%, p=0.006), an effect that was stronger in R had than NR (p<0.05). IFNγ positive NK cells did not increase on L. These data provide a first mechanistic link between the degree of Lenalidomide induced immune activation to clinical response in CLL. Based on our experience we suggest that continued dosing of L may be superior to dose interruptions. Disclosures: Aue: NHLBI, Intramural Research Program: Research Funding. Off Label Use: Lenalidomide is not FDA approved for CLL. Wiestner:NHLBI, Intramural Research Program: Research Funding.

Mutation Of The Divalent Metal Transporter (Dmt1) Gene Results In Inefficient Induction Of The Erythroid Transcriptional Program Due To Latter Onset Of GATA-1 and Epor Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2197-2197
Author(s):  
Nikola Curik ◽  
Pavel Burda ◽  
Tomas Zikmund ◽  
Filipp Savvulidi ◽  
Monika Horvathova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Iron Uptake ◽  
Stage Iv ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Divalent Metal Transporter ◽  
Epor Expression

Abstract Introduction Divalent metal transporter 1 (DMT1; SLC11A2) encodes trans-membrane protein variants that execute either non-heme iron absorption through apical duodenal membrane of enterocytes or transferrin-bound iron uptake through endosomal membrane of erythroid (and other) cells. Since 2005 several DMT1 mutations affecting accurate protein folding and localization were identified in patients with defective iron uptake leading to microcytic anemia, abnormal growth of erythroid progenitors in vitro, and iron overload in the liver. We have previously demonstrated that defective growth of DMT-1-mutatnt BFU-Es in vitro and anemia associated with ineffective erythropoiesis in vivo can be improved with high-dose erythropoietin (EPO) supplementation (Horvathova et al., 2012). Data from Dmt1-mutant mk/mk mice (Gunshin et al., 2005) suggested that the anemic phenotype is a result of ineffective erythropoiesis within bone marrow and spleen. Hypothesis DMT1-mutant erythropoiesis inhibits EPO receptor (EPOR) signaling whose end-point target as well as upstream activator is the key transcription factor GATA-1. This results in defective erythroid development characterized by impaired survival capacity of erythroid progenitors, increased apoptosis of erythroblasts, and dysregulation of erythroid gene expression leading to ineffective erythropoiesis. Materials Bone marrow cells of murine mk/mk mice were sorted using flow cytometry to obtain differentiating erythroid fractions based on antigens Cd71 and Ter119 (Sokolovsky et al., 2001) which were then subjected to gene expression analysis. Results Firstly, four consecutive fractions (proerythroblasts and early basophilic erythroblasts - stage I, late basophilic erythroblasts – stage II, chromathophillic and orthochromathophillic erythroblasts – stage III, and late chromathophillic erythroblasts and reticulocytes – stage IV) were isolated from mk/mk mice and control littermates. Flow cytometry showed enrichment in stages I and II and depletion in stage IV in the mk/mk bone marrow when compared to wild type controls, consistent with our previous data. In the spleen the major cellular enrichment was seen in stage III accompanied by cell depletion in stage IV. Gene expression of GATA-1 was markedly decreased at the onset (in stages I and II) of erythropoiesis while it was increased in terminal stage IV. The expression pattern of the GATA-1 target gene Epor was similar to that of GATA-1 expression, while expression of b-maj globin was significantly reduced indicating developmental delay of the erythroid Dmt1-mutant compartment (compared to wild type). Whereas GATA-1 and Epor expression is low in early erythropoiesis, a compensatory increase in their expression at later stages is not capable to efficiently upregulate b-maj globin. These data together with flow cytometry analysis identify a developmental blockade of erythropoiesis between stages II(III) and IV. To better understand whether Dmt1 levels regulate GATA-1 and Epor expression we used murine erythroleukemia (MEL) cells containing conditional (estrogen-regulated) transgene encoding GATA-1 fused with estrogen receptor ligand-binding domain (GER). MEL cells are cytologically characterized as proerythroblasts and early basophilic erythroblasts, stage I. Firstly, we established that GATA-1 upregulates Epor expression and directly binds to the Epor gene using RT-PCR, Immunoblotting, and chromatin immunoprecipitation (ChIP) in activated GER cells. Using ChIP-sequencing analysis of GATA-1 (and a panel of histone modifications) the GATA-1 enrichment was clearly identified at three distinct Epor regions in murine erythroblasts and differentiating MEL cells. Next, we downregulated Dmt1 using siRNA and observed that GATA-1-mediated upregulation of Epor in activated GER cells became inhibited. In addition, the knockdown of Dmt1 also inhibited steady state levels of GATA-1 in MEL cells by 25%. Conclusions Ineffective erythropoiesis in Dmt1-mutant mice is blocked at stages II (III) and display deregulation of the Epor signaling cascade involving GATA-1 and its targets. Our data thus interconnect iron uptake and the Epor/GATA-1 pathways and suggest their roles during erythroid pathogenesis upon DMT1 mutations. Grants: P305/11/1745, P301/12/P380, P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, GAUK 251135 82210 Disclosures: No relevant conflicts of interest to declare.

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