Integrin β7-Mediated Regulation of Multiple Myeloma Cell Adhesion, Migration and Survival.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 949-949
Author(s):  
Paola Neri ◽  
Li Ren ◽  
Kathy Gratton ◽  
Alex Klimowicz ◽  
Adnan Mansoor ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Growth Factors ◽  
Cell Lines ◽  
Function Analysis ◽  
Consensus Sequence ◽  
Gene Array ◽  
Signalling Pathways ◽  
High Dose ◽  
Target Area

Abstract Abstract 949 Background: Integrin β7 (ITGB7) mRNA is detected in primary myeloma (MM) cells and its presence was correlated with maf gene activation. However, little is known about the role ITGB7 plays in MM. Methods and results: We have determined the expression of ITGB7 by flow cytometry in a large library of human MM cell lines and found it to be universily expressed, albeit at different levels. Similarly ITGB7 mRNA was detected by qRT-PCR in 25/27 samples of primary MM CD138+ cells. In order to better investigate the role of ITGB7 in MM adhesion, migration and survival we performed a loss-of-function analysis using the shRNA lentivirus system. Lentiviral transduction particles with validated ITGB7 shRNA, were transduced into three different MM cell lines (MM1S, INA-6 and H929) establishing stable clones with silenced ITGB7. Using adhesion assays we have demonstrated that ITGB7silenced cells are 40-50% less adherent to fibronectin (FN), E-cadherin (E-CDH) and BMSCs coated plates when compared to ITGB7positive cells (scrambeled shRNA), confirming the role ITGB7 in MM cell adhesion to stromal elements. In a transwell migration assay, ITGB7 silencing abrogated MM cells migration in response to SDF-1 gradient implicating ITGB7 in MM cells migration. Next, we investigated whether ITGB7 conferred a survival benefit to MM cells. By MTT assay ITGB7silenced cell are more sensitive to the cytotoxic effect of Bortezomib and Melphalan when cultured on regular plate and/or in the presence of FN and E-CDH, suggesting a role for ITGB7 in conferring drug resistance to MM cells through cell-adhesion dependent and independent mechanisms. Mechanistic studies have shown that ITGB7silenced cells have reduced NF-kB activity with reduced NFkB-p65 binding to the related consensus sequence and decreased nuclear phospho-p65 translocation was confirmed by immunofluorescence staining. In ELISA based assay ITGB7silenced cells co-cultured with BMSCs produced less cytokines and growth factors (VEGF, IL-6, TNF, IL-1, SDF1α) compared to ITGB7positive/BMSCs co-cultures confirming the role of ITGB7 in mediating MM cells intereaction with BMSCs altering the cytokines and growth factors produced by stromal cells. In order to investigate the signalling pathways downstream of ITGB7, ITGB7positive and ITGB7silenced INA-6 and MM1S cells were added into human FN coated plates and total RNA extracted and submitted to gene array profiling using the U133 Plus 2.0 Array. Several NF-kB regulated genes (TNF-α2, syndecan 4, IL-6, IL-8, CDK-6, Max) were downregulated and pathway analysis (Ingenuity Systems Software) identified several MM relevant pathways to be modulated by ITGB7 silencing; among them p53, integrin, IGF-1, IL-6 and VEGF signaling as well as cell cycle, apoptosis and the Wnt/β-catenin signalling pathways. Based on the in vitro data, we next investigated in vivo the effect of ITGB7 silencing on engraftment and homing of MM to bone marrow (BM), using a human plasmacytoma xenograft scid mouse model. ITGB7silenced cells grew mainly locally at the subcutaneous implantation site with delayed engraftment into the BM while ITGB7positive cells homed mostly into the BM, as indicated by the number of human CD138+ cells counted on femoral BM sections (14% in ITGB7positive vs. 4% in ITGB7silenced). In addition, tumor vessels density within the xenografted tumors, as assessed by CD31 staining, was significantly reduced in ITGB7silenced compared to ITGB7positive tumors (CD31 target area %: 4.1 vs 7.7 respectively). Lastly using myeloma tissue microarray (TMA) we have correlated ITGB7 expression with a significantly worse survival outcomes post high dose therapy and stem cell transplantation with a mTTP of 0.9 years in ITGB7 positive patients versus 2.6 years in negative cases (p<0.005). Conclusions: Taken together our results support a role for ITGB7 in MM cells migration, homing and survival and pave the way for a novel therapeutic approach targeting this molecule. Disclosures: Bahlis: Celgene: Honoraria, Speakers Bureau; OrthoBiotech: Honoraria, Speakers Bureau.

scholarly journals Regulation of Cell Growth

1987 ◽  
Vol 80 (9) ◽  
pp. 591-593
Author(s):  
A J Barrett
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Growth Regulation ◽  
Platelet Derived Growth Factor ◽  
Haemopoietic Stem Cells

At this meeting of the RSM's Section of Pathology, the regulation of haemopoietic stem cells and growth factors regulating various cell lines were described, and the role of oncogenes, platelet-derived growth factor and nerve growth factor in growth regulation was discussed.

scholarly journals Anticancer activity of chicken cathelicidin peptides against different types of cancer

2021 ◽  
Author(s):  
Maged Mostafa Mahmoud ◽  
Ahmed M. Al-Hejin ◽  
Turki S. Abujamel ◽  
Modhi Alenezi ◽  
Fadwa Aljoud ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Breast Cancer Cell Line ◽  
Cancer Cell Line ◽  
High Dose ◽  
Mcf 7

Abstract This study served as the pioneer in studying the anti-cancer role of chicken cathelicidin peptides. Chicken cathelicidins were used as anticancer agent against the breast cancer cell line (MCF-7) and human colon cancer cell line (HCT116). An in vivo investigation was also achieved to evaluate the role of chicken cathelicidin in Ehrlich ascites cell (EAC) suppression as a tumor model after subcutaneous implantation in mice. In addition, the mechanism of action of the interaction of cationic peptides with breast cancer cell line MCF-7 was also investigated. It was found during the study that exposure of cell lines to higher concentration of chicken cathelicidin for 72 h reduced cell lines growth rate by 90%-95%. These peptides demonstrated down-regulation of (cyclin A1 and cyclin D genes) which are essential for G1/S phase transient and S/G2 phase and consequently causes “prometaphase arrest” ultimately leading to death of MCF-7 cells. The study showed two- and three-times higher expression of the caspase-3, and − 7 genes respectively in MCF-7 cells treated with chicken peptides (especially cathelicidin-2 and − 3) relative to untreated cells which encouraged pro-apoptotic pathway, autophagy, and augmentation of the anti-proliferative activity. Our data showed that chicken ( CATH-1 ) enhance releasing of TNFα, INF-γ and upregulation of granzyme K in treated mice groups, in parallel, the tumor size and volume was reduced in the treated EAC-bearing groups after cathelicidin administration compared to untreated EAC-bearing group. Additionally, animals received high dose of cathelicidin-1 (40 µg/ml) displayed an apical survival rate compared to untreated carcinoma control and animals which received low dose of cathelicidin (10 and 20 µg/ml). Tumor of mice groups treated with chicken cathelicidin displayed high area of necrosis compared to untreated EAC-bearing mice. Based on histological analysis and immunohistochemical staining revealed that the tumor section in Ehrlich solid tumor exhibited a strong Bcl2 expression in untreated control compared to mice treated with 10 & 20 µg/ml of cathelicidin. Interestingly, low expression of Bcl2 were observed in mice taken 40 µg/ml of CATH-1. This study drive intention in treatment of cancer through the efficacy of anticancer efficacy of chicken cathelicidin peptides.

scholarly journals The Role of NRAS G12D Mutations in the Response to Conventional Chemotherapy and 5-Azacitidine in Secondary AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5148-5148
Author(s):  
Andoni Garitano-Trojaola ◽  
Eva Teufel ◽  
Matteo Claudio Da Via' ◽  
Ana Sancho ◽  
Nadine Rodhes ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Sleeping Beauty ◽  
High Dose ◽  
Prognostic Impact ◽  
Conventional Chemotherapy ◽  
Mek Inhibitors ◽  
Response To Chemotherapy

Abstract Secondary Acute Myeloid Leukemia (sAML) accounts for 10-30% of all AML. It arises from a preexisting clonal disorder of hematopoiesis, such as myelodysplastic syndromes (MDS) or chronic myeloproliferative neoplasia (cMPN) in most cases (60-70%) or from exposure to a leukemogenic agent e.g. chemotherapy. sAML is generally considered to be of unfavorable prognosis, as treatment sensitivity is reduced, compared to de novo AML (dnAML) and overall survival is shortened. The incidence of AML associated NRAS are similar between sAML and dnAML (10 to 15%, Jelena D. Milosevic et al.). Prognostic impact of such mutations have been controversially discussed, but have been linked to favorable response to high dose cytarabine treatment in dnAML patients (Andreas Neubauer et al.), thus providing the first example of an oncogenic mutation impacting drug response in dnAML. This effect, however, has not yet been shown in sAML, therefore the aim of this work is to study the role of mutated NRAS in the response to chemotherapy and the hypomethylating agent (HMA) 5-azacitidine in sAML. We utilized two sAML cell lines SET-2 and HEL (both NRAS wildtype) in which we stably introduced the NRAS WT and the known activating hotspot mutation NRAS G12D using the sleeping Beauty technology. The dose-response assays of conventional chemotherapy and 5-azacitidine were carried out in the parental cell lines (SET-2/HEL) compared to NRAS WT (SET-2 NRAS WT/HEL NRAS WT) and NRAS G12D (SET-2 NRAS G12D/HEL NRAS G12D). In contrast to our expectations, both NRAS G12D mutation harboring cell lines, SET-2 and HEL developed resistance to cytarabine, idarubicin and 5-azacytidine, whereas the ones with wildtype NRAS remained susceptible to the drugs. To reverse the resistance we tested the MEK inhibitors Binimetinib and Trametinib in our SET-2 NRAS G12D cell line model according to recent reports about preclinical efficacy of MEK inhibition in NRAS mutant dnAML cells (Michael R. Burgess et al.). And in fact, single agent Binetimib and Trametinib treatments reduced cell viability by 20% at 48 hours. Strikingly, in combination with 5-azacitidine, Binimetinib and Trametinib treatments led to a viability reduction by 90%. Next we induced necroptosis in our NRAS mutant cell line models through the combination of Birinapant (SMAC mimetics) and Emricasan (Inhibitor of Caspase 8), as recently described by Brumatti et al. and were, in addition, able to reduce the cell viability by 60 %. In summary, we provide first evidence, that in contrast to dnAML, activating NRAS mutations may promote resistance to conventional chemotherapy and 5-azacitidine in sAML cell lines. Furthermore we were able to demonstrate, that the combination of MEK inhibitors (Binimetinib and Trametinib) and 5-azacitidine as well as the induction of necroptosis such as the combination birinapant and emricasan, may provide a potential strategy to overcome the resistance. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals LEDGF/p75 is Required for an Efficient DNA Damage Response

2020 ◽  
Author(s):  
Victoria Liedtke ◽  
Christian Schröder ◽  
Dirk Roggenbuck ◽  
Romano Weiss ◽  
Ralf Stohwasser ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Protein A ◽  
Signalling Pathways ◽  
Cancer Type ◽  
Knock Out ◽  
Signalling Molecules ◽  
Protein Levels

Abstract BackgroundLens epithelium derived growth factor splice variant of 75 kDa (LEDGF/p75), is overexpressed in different solid cancers and cancer cell lines and various autoinflammatory diseases. Due to its ability to bind chromatin, it acts as a transcriptional co-activator and promotes anti-apoptotic signalling pathways that lead to increased tumour aggressiveness and resistance to chemotherapy. The role of LEDGF/p75 in DNA-damage repair (DDR) is still not completely elucidated particularly regarding the ubiquitin-dependent regulation and degradation of DDR signalling molecules.MethodsDifferent LEDGF model cell lines were generated, a complete knock-out of LEDGF (KO) as well as the re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Then, various assays were performed to determine their proliferation and migration capacity as well as their chemosensitivity. Moreover, DDR signalling pathways were investigated by western blot and immunofluorescence.ResultsLEDGF-deficient cells exhibited a decreased proliferation (dt (WT) = 21 h, dt (KO) = 26 h) , 60 % decreased migration, as well as an 30-50 % increased sensitivity towards the topoisomerase II inhibitor etoposide. Moreover, LEDGF depleted cells showed a significant reduction by 65 % in the recruitment of downstream DDR-related proteins like replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. Re-expression of LEDGF/p75 rescued all knock-out effects, while re-expression of LEDGF/p52 had no effect.Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and Breast cancer type 1 susceptibility protein (BRCA1). In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. ConclusionsThis study provides evidence that LEDGF is not only an important player in the DDR after chemotherapeutic treatments but is also involved in the maintenance of the general genome integrity. Moreover, this study provides for the first time an insight into the possible role of LEDGF in the ubiquitin-dependent regulation of DDR signalling molecules and highlights the involvement of LEDGF/p75 in homology-directed DNA repair.

scholarly journals PS2 - 167 CIC Deficiency is Associated with Dysregulation of Genes Involved in Cell Adhesion and Developmental Processes

2016 ◽  
Vol 43 (S4) ◽  
pp. S13-S13
Author(s):  
V.G. LeBlanc ◽  
S. Chittaranjan ◽  
M. Firme ◽  
S.Y. Chan ◽  
J. Song ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Somatic Mutations ◽  
Control Cell ◽  
The Cancer Genome Atlas ◽  
Mitogen Activated Protein Kinase ◽  
Low Grade ◽  
Type I ◽  
Developmental Processes

Somatic mutations in the Capicua (CIC) gene were first identified in Type I low-grade gliomas (LGGs), which are characterized by 1p/19q co-deletions and IDH mutations. They are found at frequencies of ~50-70% in this glioma subtype, and have since been identified in ~40% of stomach adenocarcinomas (STADs) of the microsatellite instability (MSI) subtype; however, the role of these somatic mutations in malignancy has yet to be established. In Drosophila, CIC functions as a transcriptional repressor whose activity is inhibited upon activation of the mitogen-activated protein kinase (MAPK) signalling pathway. Though mammalian CIC appears to retain these functions, only three of its target genes have been established in human cells: ETV1, ETV4, and ETV5 (ETV1/4/5). To further probe CIC’s transcriptional network, we developed CIC knockout cell lines and performed transcriptomic and proteiomic analyses in these and in control cell lines expressing wild type CIC, identifying a total of 582 differentially expressed genes. We also used RNA-seq data from The Cancer Genome Atlas (TCGA) for Type I LGGs and STADs to perform additional differential expression analyses between CIC-deficient and CIC-expressing samples. Though gene-level overlap was limited between the three contexts, we found that CIC appears to regulate the expression of genes involved in cell-cell adhesion, metabolism, and developmental processes in all three contexts. These results shed light on the pathological role of CIC mutations and may help explain why these have been associated with poorer outcome within Type I LGGs.

Determinating the role of the E3 Ubiquitin Ligase Ariadne 2 in mammalian systemss

2007 ◽  
Vol 30 (4) ◽  
pp. 87
Author(s):  
A. E. Lin ◽  
A. Wakeham ◽  
A. You-Ten ◽  
G. Wood ◽  
T. W. Mak
Keyword(s):  
Function Analysis ◽  
Critical Role ◽  
Ring Finger ◽  
E3 Ligase ◽  
Signalling Pathways ◽  
Loss Of Function ◽  
In Vivo Models

Ubiquitination is a eukaryotic process of selective proteolysis, where a highly conserved ubiquitin protein is selectively added as a chain to the targeted to a protein for degradation. In recent years, the process of ubiquitination has been shown to be a critical mechanism that can affect essential signalling pathways, including apoptosis, cell cycle arrest and induction of the inflammatory response. Thus, alterations in the ubiquitination process can alter signalling pathways pivotal to numerous disease pathologies. This is clearly demonstrated in perturbations of ubiquitination in the NFκB giving rise to cancer and other immunological disease processes. To gain insight into pathways that require regulation by ubiquitination, our lab has directed focus on the highly conserved E3 ligase, Ariadne 2. Ariadne 2 is characterized as a putative RING finger E3 ligase and is part of the family of highly conserved RBR (RING-B-Box-RING) superfamily. The role of Ariadne 2 has been well studied in Drosophila melanogaster, however, little is known of the function of Ariadne 2 in mammalian systems. Therefore, the main objectives of the project are as follows: To determine the biological role of Ariadne 2, the role of Ariadne 2 in development and differentiation, and the consequences of in vivo loss of Ariadne 2 expression. We are currently investigating the role of Ariadne 2 as an E3 ligase and its involvement in the immune response. To date, we have shown that Ariadne 2 is ubiquitously expressed, especially in the brain, heart, spleen and thymus. For in vivo loss of function analysis, mice were generated by homologous recombination to be deficient for Ariadne 2. These deficient mice die prematurely soon after birth, suggesting a critical role for Ariadne 2 in development and survival. We are currently focusing on the role of Ariadne 2 in development and it’s role in immune pathologies, in particular, spontaneous autoimmunity, using both in vitro studies and in vivo models.

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