Bromodomain Inhibition By OTX015 Regulates c-MYC and HEXIM1 in a Panel of Human Acute Leukemia Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5957-5957
Author(s):  
Marie-Magdelaine Coudé ◽  
Thorsten Braun ◽  
Jeannig Berrou ◽  
Mélanie Dupont ◽  
Raphael Itzykson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Mrna Expression ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Antileukemic Activity ◽  
Protein Levels ◽  
Gene And Protein Expression

Abstract Background: The bromodomain-containing protein 4 (BRD4) activates the transcription elongation factor b (P-TEFb) which regulates RNA polymerase II. Conversely, hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) inactivates P-TEFb. BRD4/HEXIM1 interplay influences cell cycle progression and tumorigenesis. It has been widely demonstrated that BRD4 knockdown or inhibition by JQ1 is associated with c-MYC downregulation and antileukemic activity. We recently reported that the small molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland), currently in clinical development, mimics the effects of JQ1 (Braun et al, ASH 2013). We evaluated the effect of OTX015 on c-MYC, BRD2/3/4, and HEXIM1 in human in vitro leukemic models. Methods: c-MYC, BRD2/3/4 and HEXIM1 expression was assessed in six acute myeloid leukemia (AML; K562, HL-60, NB4, NOMO-1, KG1, OCI-AML3) and two acute lymphoid leukemia (ALL; JURKAT and RS4-11) cell lines after exposure to 500 nM OTX015. Quantitative RT-PCR and Western blotting were performed at different time points (24-72h). A heatmap was computed with R-software. Results: c-MYC RNA levels were ubiquitously downregulated in all AML and ALL cell lines after 24h exposure to OTX015 (Figure 1). c-MYC protein levels decreased to a variable extent at 24-72h in all cell lines evaluated other than KG1. BRD2, BRD3 and BRD4 mRNA expression was significantly decreased in K562 cells (known to be OTX015-resistant) after 48h exposure to OTX015 but was increased in HL60 and NOMO-1 cells, while minimal to no increases were observed in other cell lines. OTX015 induced a decrease in BRD2 protein expression in most cell lines, but not in K562 cells. In contrast, decreased BRD4 protein expression was only seen in the OCI-AML3, NB4 and K562 cell lines. BRD3 protein levels were not modified after OTX015 exposure in all cell lines evaluated other than KG1. HEXIM1 mRNA expression increased after 24h exposure to 500 nM OTX015 in all cell lines except OTX015-resistant K562 cells in which the increase was considered insignificant (less than two-fold). Increases in HEXIM1 protein levels were observed in OCI-AML3, JURKAT and RS4-11 cell lines at 24-72h but not in K562 cells. Conclusion: Taken together, these results show that BRD inhibition by OTX015 modulates HEXIM1 gene and protein expression, in addition to c-MYC decrease and BRD variations. HEXIM1 upregulation seems to be restricted to OTX015-sensitive cell lines and was not significantly affected in OTX015-resistant K562 cells. Further studies are needed to clarify the role of HEXIM1 in antileukemic activity of BRD inhibitors. Figure 1: Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Figure 1:. Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Disclosures Riveiro: OTD: Employment. Herait:OncoEthix: Employment. Dombret:OncoEthix: Research Funding.

scholarly journals A Novel WNT5A-Mimicking Peptide Affects Leukemia Cell Survival in the Bone Marrow Microenvironment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2949-2949
Author(s):  
Fernanda Marconi Roversi ◽  
Maura Lima Pereira Bueno ◽  
Rafael Gonçalves Barbosa Gomes ◽  
Guilherme Rossi Assis-Mendonça ◽  
Paulo Latuf Filho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Actin Polymerization ◽  
Leukemia Cell ◽  
Ros Production ◽  
Protein Levels ◽  
Cd34 Cells

Abstract Background: The crosstalk between hematopoietic cells and bone marrow (BM) microenvironment in hematological malignancies is related to disease initiation, maintenance and relapse. BM niche sustains a protective response against currently available treatments that have shown unwanted adverse effects and high levels of toxicity for patients. WNT5a is a glycoprotein secreted by mesenchymal stromal cells (MSC) that activates the WNT non-canonical pathway in hematopoietic cells, modulating important biological processes related to neoplasia development. Aims: To investigate WNT5a mRNA expression, protein levels and methylation pattern in Myelodysplastic Syndrome (MDS) and de novo Acute Myeloid Leukemia (AML) patients and their impact on clinical outcomes, and to analyze effects of Foxy-5 (WNTResearch), a new WNT5a-mimicking compound. Methods: WNT5a mRNA expression was analyzed in mononuclear cells (MC) from 371 AML patients (212 male, median age 61 years [range: 2-87]) (Ohsu, Nature 2018). BMMSC and BM biopsies from 5 healthy donors (HD), 6 MDS and 13 AML patients at diagnosis were submitted to analysis of WNT5a mRNA expression and methylation after Azacytidine (1μM) treatment by real-time PCR, and WNT5a protein levels by immunohistochemistry and immunofluorescence (IF). A panel of myeloid leukemia cell lines (U937, HL60, THP1, KG1a, K562) were treated with Foxy-5 (1 to 100μM) for 72h in monoculture, coculture and 3D-coculture (with MSC) and evaluated for ROS production (DCFDA dye), cell proliferation (Ki-67 stain), autophagy (acridine orange dye), chemotaxis (Transwell), actin polymerization (phalloidin stain), cell cycle (PI/RNAse stain), cell viability (MTT assay), apoptosis (Annexin-V stain) and protein expression (Western blot, WB). MC and CD34+ cells from HD were submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA or Mann-Whitney tests, as appropriate. Results: WNT5a gene expression was reduced in MC from AML patients with adverse cytogenetic risk compared to favorable and intermediate cytogenetic risk (fold-decrease [FD]: 42.9; 18.8, respectively) (P<.05) and in BMMSC from AML compared to HD (FD: 53.3) (P<.05). Accordingly, WNT5a gene expression in MDS and AML BMMSC treated with Azacytidine was restored (fold-increase [FI]: 3.99; 1.50, respectively). WNT5a protein expression were diminished in BMMSC from MDS and AML patients compared to HD onto a 3D-coculture (IF)(P<.05) and immunohistochemically detected in all BM hematopoietic lineages. Foxy-5 reduced ROS production in U937 (FD of mean fluorescence intensity [MFI]: 48.2; 46.6), HL60 (FD: 47.1; 115.0), KG1a (FD: 34.9; 20.7) and K562 (FD: 19.0; 24.3) at 100μM in monoculture and coculture, respectively (P<.05). Foxy-5 also significantly decreased proliferation in U937 (FD: 41.0), HL60 (FD: 18.0), THP1 (FD: 36.0) and K562 (FD: 68.0) at 100μM (P<.05), confirmed by a 3D-coculture containing these cell lines and MSC. Foxy-5 reduced monocyte differentiation and inhibited CD11b expression in U937 (FD: 16.4) and THP1 (FD: 14.4). Cell cycle progression was blocked in sub G0/G1 phase in all cell lines (P<.05) after Foxy-5 treatment, probably mediated by the reduction of cyclin D1 protein levels, as verified by WB. Further, Foxy-5 reduced AKT1/2/3 and ERK1/2 phosphorylation levels, possibly by beta-catenin inhibition, with disruption of actin polymerization (U937 (FD: 65.3), HL60 (FD: 35.9), THP1 (FD: 58.5), K562 (FD: 15.0)) at 100μM (P<.05) and consequent impairment of CXCL12-induced chemotaxis (U937 (FD: 27.9), HL60 (FD: 42.5), THP1 (FD: 82.4), K562 (FD: 45.1)) at 100μM (P<.05). In coculture, cell autophagy was reduced in U937 (FD: 27.8), HL60 (FD: 35.9), KG1a (FD: 16.4) and K562 (FD: 35.8) when treated with Foxy-5 at 100μM (P<.05). Finally, Foxy-5 treatment did not affect cytotoxicity in MC and CD34+ cells from HD. Conclusion: WNT5a downregulation in MDS and AML patients occurs probably by methylation and contributes to poor prognosis. Foxy-5, by restoring WNT5a levels, could represent a strategy to counterbalance several oncogenic processes present in leukemia by reducing ROS production and, consequently, inhibiting cell growth and differentiation, downregulating PI3K and MAPK pathways, disrupting actin polymerization and decreasing autophagy. Thus, Foxy-5 treatment may be an important approach to impair leukemia growth and maintenance and arises as a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Aberrant Stabilization of c-Myc Protein in Lymphoblastic and Myelogenous Leukemia Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1532-1532
Author(s):  
Suman Malempati ◽  
Rosalie C. Sears
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Half Life ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Proteasome Inhibition ◽  
Protein Stabilization ◽  
Hematopoietic Malignancies ◽  
Protein Levels ◽  
Leukemia Cell Lines

Abstract The c-Myc oncoprotein is a key regulator of cell fate decisions including proliferation, differentiation, and apoptosis. Orderly control of c-Myc protein levels is important in maintaining regulated cell proliferation in normal cells. While c-Myc overexpression is seen in many hematopoietic malignancies, the reason for high protein levels in most cases is unknown and, in general, is not the result of translocations or gene amplification. C-Myc levels vary with cell cycle and are kept very low in quiescent cells. Protein half-life is controlled by phosphorylation at two specific N-terminal sites, Serine 62 and Threonine 58, which regulate c-Myc degradation by the ubiquitin proteasome pathway. Two Ras-dependent signaling pathways (Raf/MEK/ERK and PI(3)K/Akt) modulate phosphorylation at Serine 62, which stabilizes the protein, and Threonine 58, which targets Myc for ubiquitination and subsequent degradation. We recently reported that a stabilized form of c-Myc (c-Myc T58A) contributes to oncogenic transformation of human cells in culture (Yeh et al, Nat. Cell Bio.6:308–318, 2004). Here we describe the role of c-Myc protein stabilization in 2 pediatric ALL cell lines (REH and Sup-B15), 1 AML cell line (HL-60), and 1 CML cell line (K562). Markedly higher expression of c-Myc protein was seen in all 4 cell lines as compared to normal peripheral blood mononuclear cells (PBMCs). FISH analysis demonstrated amplification of the c-myc gene in HL-60 cells as has been previously reported, but not in REH, Sup-B15, or K562 cells. Using [35S]methionine pulse-chase analysis we demonstrate that the half-life of c-Myc in REH (55 minutes), Sup-B15 (47 minutes), and K562 (40 minutes) cells is longer than in normal PBMCs (9 to 15 minutes), but is not significantly prolonged in HL60 cells. We provide additional functional evidence for aberrant protein stabilization based on greater elevation of c-Myc protein after proteasome inhibition in PBMCs and HL-60 cells than in REH, Sup-B15, or K562 cells. These results suggest that that abnormalities in c-Myc degradation exist upstream of ubiquitination in the ALL and CML cell lines. Consistent with this hypothesis, experimental inhibition of the PI(3)K pathway knocked down c-Myc levels in REH and Sup-B15 cells, an effect that was abrogated by concomitant proteasome inhibition. This result suggests that abnormal activation of the PI(3)K pathway could participate in c-Myc stabilization in these cells. In addition, destabilization of c-Myc by PI(3)K inhibition correlated with a significant decrease in cell proliferation. In conclusion, we demonstrate that aberrant stabilization of c-Myc protein occurs in human leukemia cell lines. Affecting the c-Myc degradation pathway in hematopoietic malignancies that have stabilized c-Myc may constitute a novel therapeutic target. Additional experiments are ongoing to assess c-Myc stability in primary cells from leukemic bone marrow samples.

Development of a Gene Expression Panel, for the Prediction of Protein Abundances in Cancer Cell Lines

2021 ◽  
Vol 16 ◽  
Author(s):  
Gunhee Lee ◽  
Yeun-Jun Chung ◽  
Minho Lee
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Prediction Models ◽  
Cancer Cell Lines ◽  
Support Vector ◽  
Rna Expression ◽  
Expression Levels ◽  
Protein Levels

Background: Due to the ease of quantifying mRNA expression in comparison with that of protein abundances, many studies have utilized it to infer protein product quantification. However, the mRNA expression values for a gene and its protein products are not known to have a strong relationship, because of the complex mechanisms required to regulate the amounts of protein levels, from translation to post-translational modifications. Methods: We have developed, in this study, models to predict protein levels from mRNA expression levels using the transcriptome and reverse phase protein arrays (RPPA)-based on protein levels in pan-cancer cell lines. When predicting the abundance of a protein expression, in addition to using RNA expression of the corresponding gene, we also used RNA expression levels of a particular set of other genes. By applying support vector regression, we have identified a 47-gene expression panel that contributes to the improved performance of the prediction, and its optimal subsets specific to each protein species. Result and Conclusion: Eventually, our final prediction models doubled the number of predictable protein expressions (r > 0.7). Due to the weaknesses of RPPA, our model had some limitations, however, we expect that these prediction models and the panel can be widely used in the future to infer protein abundances.

Down-Regulation of BMI-1 Is a New Marker of Sensitivity to Mdm2 Inhibition in B-Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2522-2522 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Daniela Erriquez ◽  
Anna Ferrari ◽  
Cristina Papayannidis ◽  
Claudia Venturi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Cell Viability ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Annexin V ◽  
Sensitive Cell ◽  
Protein Levels ◽  
Leukemia Cell Lines ◽  
Reh Cells

Abstract Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Usp9x Silencing and Enzyme Inhibition Suppress Myeloma Cell Survival and in Vivo Tumor Growth.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2936-2936
Author(s):  
Luke F. Peterson ◽  
Hanshi Sun ◽  
Yihong Liu ◽  
Malathi Kandarpa ◽  
Diane Giannola ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Protein Expression ◽  
Tumor Growth ◽  
Cell Survival ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Protein Levels

Abstract Abstract 2936 Due to clinical success with proteasome inhibitors and E3 ligase modulators, proteins of the ubiquitin/proteasome system have emerged as novel therapeutic targets in several hematological malignancies. Deubiquitinases (DUBs) play a key role in regulating all aspects of this pathway and are aberrantly expressed or activated in several hematological malignancies. Usp9x is a high MW DUB, which has previously been shown to alter many signaling pathways, and its overexpression has been associated with drug resistance and poor prognosis in myeloma patients. Usp9x is reported to control cell survival through deubiquitination of Mcl-1 and other substrates, thereby reducing their proteosomal degradation. Since Mcl-1 plays a major role in myeloma cell survival and drug resistance, we compared Usp9x gene expression in myeloma cell lines to primary myeloma specimens. Usp9x gene expression varied among myeloma cell lines by 1.5 to 2.5-fold and endogenous Usp9x protein levels were even more variable (range 1.5 to 8-fold). Elevated Usp9x protein expression was not consistently associated with elevated Usp9x enzymatic activity as myeloma cells with the highest Usp9x gene and protein expression (KMS-11) had minimal Usp9x DUB activity. Moreover, Mcl-1 levels did not consistently correlate with Usp9x gene, protein or DUB activity, suggesting that a more complex mechanism regulates Usp9x activity and Mcl-1 stability in myeloma cells. To further examine the relationship between Usp9x and Mcl-1, we suppressed Usp9x expression (with siRNA and shRNA) and inhibited Usp9x activity with small molecule DUB inhibitors, WP1130 and VM030. Silencing Usp9x expression in H929 and MM1.S cells with shRNA vectors resulted in a reduction in Mcl-1 levels and induction of apoptosis, which approached the level of cell death achieved with direct Mcl-1 silencing. Similar results were obtained with siRNA-based Usp9x silencing in RMPI-8226 and KMS-11 cells; however, this approach led to the activation of a homologous DUB, Usp24, primarily through an increase in its protein stability. Usp24 silencing led to a decrease in myeloma cell survival, suggesting that Usp9x and Usp24 are coordinately regulated and play a role in myeloma cell survival. Measurement of Usp9x and Usp24 gene expression levels in primary myeloma samples and cell lines demonstrated that myeloma cell lines express 2 to 100-fold lower levels of these DUBs compared to primary tumors. Immuno-depletion studies illustrated that both Usp9x and Usp24 were activated in myeloma cells, and targeting both DUBs with WP1130 or VM030 led to a rapid reduction in Mcl-1 protein levels and the onset of apoptosis in both primary myeloma cells and cell lines. Further, treatment of NSG mice bearing MM1.S or RPMI-8226 tumors with VM030 resulted in Usp9x and Usp24 inhibition, reduction in Mcl-1 protein levels and suppression of myeloma tumor growth with limited toxicity. Together, these results suggest that Usp9x and Usp24 are highly expressed and activated in myeloma cells and both DUBs contribute to cell survival, but through different mechanisms. These results support the use of DUB inhibitors with specificity for Usp9x and Usp24 in the treatment of myeloma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals WIPI1, BAG1, and PEX3 Autophagy-Related Genes Are Relevant Melanoma Markers

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Daniela D’Arcangelo ◽  
Claudia Giampietri ◽  
Mario Muscio ◽  
Francesca Scatozza ◽  
Francesco Facchiano ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Survival Data ◽  
Enrichment Analysis ◽  
Expression Change ◽  
Protein Levels ◽  
Functional Relationships ◽  
Oxidative Damages ◽  
Gene And Protein Expression ◽  
Human Protein Atlas

ROS and oxidative stress may promote autophagy; on the other hand, autophagy may help reduce oxidative damages. According to the known interplay of ROS, autophagy, and melanoma onset, we hypothesized that autophagy-related genes (ARGs) may represent useful melanoma biomarkers. We therefore analyzed the gene and protein expression of 222 ARGs in human melanoma samples, from 5 independent expression databases (overall 572 patients). Gene expression was first evaluated in the GEO database. Forty-two genes showed extremely high ability to discriminate melanoma from nevi (63 samples) according to ROC (AUC≥0.85) and Mann-Whitney (p<0.0001) analyses. The 9 genes never related to melanoma before were then in silico validated in the IST online database. BAG1, CHMP2B, PEX3, and WIPI1 confirmed a strong differential gene expression, in 355 samples. A second-round validation performed on the Human Protein Atlas database showed strong differential protein expression for BAG1, PEX3, and WIPI1 in melanoma vs control samples, according to the image analysis of 80 human histological sections. WIPI1 gene expression also showed a significant prognostic value (p<0.0001) according to 102 melanoma patients’ survival data. We finally addressed in Oncomine database whether WIPI1 overexpression is melanoma-specific. Within more than 20 cancer types, the most relevant WIPI1 expression change (p=0.00002; fold change=3.1) was observed in melanoma. Molecular/functional relationships of the investigated molecules with melanoma and their molecular/functional network were analyzed via Chilibot software, STRING analysis, and gene ontology enrichment analysis. We conclude that WIPI1 (AUC=0.99), BAG1 (AUC=1), and PEX3 (AUC=0.93) are relevant novel melanoma markers at both gene and protein levels.

scholarly journals Patient-derived gene and protein expression signatures of NGLY1 deficiency

2021 ◽  
Author(s):  
Benedikt Rauscher ◽  
William F Mueller ◽  
Sandra Clauder-Münster ◽  
Petra Jakob ◽  
M Saiful Islam ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Web Application ◽  
Ribosome Biogenesis ◽  
Genetic Disorder ◽  
Cell Types ◽  
Cell Type ◽  
Protein Levels ◽  
Gene And Protein Expression ◽  
Complex Genetic Disorder

Abstract N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

scholarly journals Genomic and non-genomic effects of progesterone and pregnenolone on the function of bovine endometrial cells

2009 ◽  
Vol 54 (No. 5) ◽  
pp. 205-214 ◽  
Author(s):  
M.K. Kowalik ◽  
D. Slonina ◽  
J. Kotwica
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Mrna Expression ◽  
Expression Analysis ◽  
Membrane Component ◽  
Endometrial Cells ◽  
Gene And Protein Expression ◽  
Independent Mechanism ◽  
Genomic Effects ◽  
Cox 2

Progesterone (P<sub4</sub>) decreases oxytocin (OT)-stimulated prostaglandin (PG)F<sub>2&alpha;</sub>, but not PGE<sub>2</sub> secretion from bovine endometrial cells and this effect is partly elicited via a non-genomic route. The aim of this study was to determine whether P<sub>4</sub> and pregnenolone (P<sub>5</sub>), in the presence or absence of OT, influence: (a) the gene expression of enzymes responsible for PG<sub>s</sub> synthesis: cyclooxygenase-2 (COX-2), synthase of PGF<sub>2&alpha;</sub> (PGFS) and PGE=sub>2</sub> (PGES), (b) protein expression of COX-2, PGFS and PGES, and (c) P<sub>4</sub> receptor membrane component 1 (PGRMC1) gene expression in bovine endometrial cells. The epithelial endometrial cells (2.5 × 10<sup>5</sup>/ml) from Days 14–16 of the oestrous cycle were incubated for 72–96 h to attach the cells to the bottom of a well. Next, the cells were preincubated for 30 min with P<sub>4</sub> and P<sub>5</sub> (10<sup>–5</sup>M each) and incubated for 4 h and 6 h alone or with OT (10<sup>–7</sup>M). Thereafter, the medium was collected for PGE<sub>2</sub> and PGFM determination, while cells were harvested for gene and protein expression analysis. The used steroids: (a) inhibited OT-stimulated PGF<sub>2&alpha;</sub>, but not PGE<sub>2</sub> secretion from endometrial cells, (b) did not affect the expression of mRNA for COX-2, PGFS, PGES and PGRMC1 in endometrial cells after 4 and 6 h, (c) they decreased OT-stimulated COX-2 mRNA expression only after 6 h incubation, and (d) did not influence COX-2, PGFS and PGES protein expression after 6 h. These results indicate that P<sub>4</sub> and P<sub>5</sub> inhibit OT-stimulated secretion/production of luteolytic PGF<sub>2&alpha;</sub> by a transcription-independent mechanism and partly by down-regulation of COX-2 mRNA.

scholarly journals Patient-derived gene and protein expression signatures of NGLY1 deficiency

2021 ◽  
Author(s):  
Benedikt Rauscher ◽  
William F. Mueller ◽  
Sandra Clauder-Muenster ◽  
Petra Jakob ◽  
M. Saiful Islam ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Web Application ◽  
Genetic Disorder ◽  
Cell Types ◽  
Cell Type ◽  
Protein Levels ◽  
Gene And Protein Expression ◽  
Proteasomal Genes ◽  
Complex Genetic Disorder

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. While gene and protein expression levels agreed well with each other, expression differences were more pronounced at the protein level. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parent controls, depending on the genotype, NGLY1 protein was still detectable in all patient- derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosomal mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cystein ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

scholarly journals Interleukin-6, -8, and TGF-β Secreted from Mesenchymal Stem Cells Show Functional Role in Reduction of Telomerase Activity of Leukemia Cell Via Wnt5a/β-Catenin and P53 Pathways

2020 ◽  
Vol 10 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Ezzatollah Fathi ◽  
Behnaz Valipour ◽  
Zohreh Sanaat ◽  
Hojjatollah Nozad Charoudeh ◽  
Raheleh Farahzadi
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Real Time Pcr ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Telomerase Activity ◽  
Htert Gene ◽  
Gene And Protein Expression

Purpose: The effect of mesenchymal stem cells (MSCs) on the immortality features of malignant cells, such as hematologic cancerous cells, are controversial, and the associated mechanisms are yet to be well understood. The aim of the present study was to investigate the in vitro effect of bone marrow-derived MSCs (BMSCs) on the chronic myeloid leukemia cell line K562 through telomere length measurements, telomerase activity assessments, and hTERT gene expression. The possible signaling pathways involved in this process, including Wnt-5a/β-catenin and P53, were also evaluated. Methods: Two cell populations (BMSCs and K562 cell line) were co-cultured on transwell plates for 7 days. Next, K562 cells were collected and subjected to quantitative real-time PCR, PCR-ELISA TRAP assay, and the ELISA sandwich technique for telomere length, hTERT gene expression, telomerase activity assay, and cytokine measurement, respectively. Also, the involvement of the mentioned signaling pathways in this process was reported by real-time PCR and Western blotting through gene and protein expression, respectively. Results: The results showed that BMSCs caused significant decreases in telomere length, telomerase activity, and the mRNA level of hTERT as a regulator of telomerase activity. The significant presence of interleukin (IL)-6, IL-8, and transforming growth factor beta (TGF-β) was obvious in the co-cultured media. Also, BMSCs significantly decreased and increased the gene and protein expression of β-catenin and P53, respectively. Conclusion: It was concluded that the mentioned effects of IL-6, IL-8, and TGF-β cytokines secreted from MSCs on K562 cells as therapeutic agents were applied by Wnt-5a/β-catenin and P53 pathways

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