Total inhibition of involucrin synthesis by a novel two-step antisense procedure. Further examination of the relationship between differentiation and malignancy in hybrid cells

1992 ◽  
Vol 102 (4) ◽  
pp. 799-805 ◽  
Author(s):  
E.F. Griffin ◽  
H. Harris
Keyword(s):  
Cell Line ◽  
Target Genes ◽  
Hybrid Cell ◽  
Terminal Differentiation ◽  
Complete Inactivation ◽  
Total Inhibition ◽  
Antisense Constructs ◽  
Progressive Growth ◽  
Normal Human

A novel procedure involving the sequential use of two different antisense constructs has been used to inhibit the synthesis of involucrin in a hybrid cell line formed by the fusion of a human cervical carcinoma cell with a normal human keratinocyte (ESH100P6). In this cell line, and other similar hybrids, malignancy, as measured by progressive growth in vivo, is suppressed; and it has been shown that the keratinocyte imposes its own programme of terminal differentiation on the non-malignant hybrid cell. In particular, involucrin, a precursor of one of the major components of the cornified envelope of mature keratinocytes, continues to be produced. When, however, malignant segregants arise in the hybrid cell population, the terminal differentiation programme of the keratinocyte is not expressed and involucrin ceases to be made. It seemed possible that if the synthesis of involucrin, a critical marker of keratinocyte terminal differentiation, could be completely inhibited, this differentiation programme might be disrupted, and the malignant phenotype might then reappear in the non-malignant hybrids. This question was investigated further in the present paper. Total, and specific, inhibition of involucrin synthesis was indeed achieved by a sequential two-step antisense procedure, which might provide a systematic general method for the complete inactivation of other selected target genes.

C-Myb Influences the Proliferation and Commitment of Erythroid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1597-1597
Author(s):  
Alexandros Vegiopoulos ◽  
Jonathan Frampton
Keyword(s):  
Cell Cycle ◽  
Target Genes ◽  
Terminal Differentiation ◽  
Rapid Decline ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Complete Inactivation ◽  
Foetal Liver ◽  
Low Levels ◽  
Factor C

Abstract The transcription factor c-Myb is involved in the regulation of proliferation, survival and differentiation of haemopoietic progenitors. Mouse embryos homozygous for a null mutation in the c-myb gene die of anaemia around 15 dpc, however, the reasons leading to the failure of erythropoiesis are unknown. In order to investigate the precise function of c-Myb in erythroid progenitors we manipulated c-Myb expression using targeted c-myb alleles. To study erythropoiesis with reduced c-Myb expression we used embryos heterozygous for a c-myb null allele (c-myb−) or homozygous for a previously described knockdown allele (c-mybKD). Furthermore, we achieved complete inactivation of c-myb using the IFN-inducible transgene MxCre in erythroid progenitors cultured from foetal liver of embryos carrying a floxed c-myb allele (c-mybF). We demonstrate that low levels of c-Myb lead to an accumulation in the foetal liver of CD71+Ter119− erythroid cells expressing markers of uncommitted progenitors as well as low levels of c-Kit. To prove that this is the consequence of a block in commitment, we cultured foetal liver cells and demonstrated the outgrowth of immature cells unable to progress and terminally differentiate in response to Epo. A requirement for high levels of c-Myb in later stages of erythropoiesis was revealed by studying cultured foetal liver progenitors carrying one functional c-myb allele (c-myb+/−). The number and size of CFU-Es was reduced, while BFU-Es were poorly haemoglobinised. Interestingly, we observed a tendency of cultured c-myb+/− progenitors to spontaneously and rapidly exit the cell cycle and aberrantly differentiate. In agreement with this, Cre-mediated inactivation of c-myb in cultured cells led to a rapid decline in c-Kit expression and the induction of certain erythroid genes, although obvious terminal differentiation was absent. Strikingly, expression of the megakaryocytic marker CD41 emerged on a subpopulation of cells. In contrast to cells expressing low levels of c-Myb, complete inactivation resulted in cell cycle arrest. In order to systematically uncover molecular pathways regulated by c-Myb we have determined the global gene expression changes in response to induced c-myb inactivation using Affymetrix GeneChip arrays. Several differentially expressed genes were identified and are being further analysed as novel c-Myb target genes. In conclusion, we provide evidence for a complex role for c-Myb in the regulation of several stages of erythroid development, including expansion, commitment and terminal differentiation. In particular, we demonstrate that high levels of c-Myb are required for the response of progenitors to erythropoietic stimuli as well as for ordered terminal differentiation. c-Kit, long suspected as a possible c-Myb-regulated gene but never before proven to be at the level of the endogenous gene in primary cells, has emerged as a key target for the actions of c-Myb in erythroid progenitors. Finally, a number of novel target genes have been identified as additional important potential effectors downstream of c-Myb in developing erythroid cells.

scholarly journals The High Affinity CXCR4 Inhibitor, BL-8040, Induces Apoptosis of AML Blasts and Their Terminal Differentiation By Blocking AKT/ERK Survival Signals and Downregulating BCL-2, MCL-1 and Cyclin-D1 through Regulation of Mir-15a/16-1 Expression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 767-767
Author(s):  
Michal Abraham ◽  
Shiri Klein ◽  
Baruch Bulvik ◽  
Hanna Wald ◽  
Dvora Holam ◽  
...  
Keyword(s):  
Cell Death ◽  
Cyclin D1 ◽  
Target Genes ◽  
Combined Treatment ◽  
Terminal Differentiation ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Survival Signals

Abstract Background: Acute Myeloid Leukemia (AML) is a heterogeneous group of diseases characterized by uncontrolled proliferation and survival of hematopoietic stem and progenitor cells. The chemokine CXCL12 and its receptor CXCR4 are key players in the survival, bone marrow (BM) retention and the maintenance of AML blasts in their stemness state. CXCR4 overexpression is associated with poor prognosis in AML patients. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy. Objective: To study the effect of the CXCR4-antagonist, BL-8040, on the survival of human AML blasts and to investigate the molecular mechanism by which inhibition of CXCR4 signaling leads to leukemia cell death. Methods: Human AML cell lines and human primary AML samples were used for in vitro studies. The in-vivo effect of BL-8040 was tested using the MV4-11, U-937, THP-1 cells and human primary AML cells engrafted in NOD scid gamma (NSG) mice. Results: We found that BL-8040 directly induced apoptosis of AML cells both in FLT3-ITD and FLT3-WT AML, in-vitro and in-vivo. BL-8040 treatment triggered mobilization of AML blasts from their protective BM microenvironment and induced their terminal differentiation, in-vitro and in-vivo. The apoptosis of AML cells induced by BL-8040 was attributed to miR-15a/miR-16-1 up-regulation resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin-D1. The increase in miR-15a/miR-16-1 levels directly induced AML cell death. Moreover, CXCR4 blockade by BL-8040 also inhibited survival signals by the ERK/AKT kinases enhancing the apoptosis effect. Survival of AML cells was found to be dependent on BCL-2 as demonstrated by the ability of the BCL-2 inhibitor, ABT-199, to induce dose dependent apoptosis in vitro. It was reported that the MCL-1 protein plays a key role in acquiring resistance to ABT-199. We found that BL-8040 synergizes with ABT-199 in inducing AML cell death. This could be attributed to the reduction of both, AKT/ERK and MCL-1 levels, by treatment with BL-8040. In addition, BL-8040 synergizes with the FLT3 inhibitor AC220 in the induction of AML cell death both in-vivo and in-vitro. The combined treatment of BL-8040 and AC220 was found to prolong survival and reduce minimal residual disease in-vivo. Interestingly, the combined treatment was also associated with a significant reduction in the expression of BCL-2 and ERK signaling. Conclusions: BL-8040 can be a potential therapeutic option in AML by targeting not only AML anchorage in the BM but also AML survival and differentiation. Our results demonstrate that BL-8040 in AML regulates the expression of miR-15a/16-1 and their target genes BCL-2, MCL-1 and cyclin-D1. Furthermore, these results indicate that the CXCR4 antagonist, BL-8040 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 pathway and inhibition of the ERK/AKT survival signaling cascade in AML cells. Our results provide rational for combination of BL-8040 with ABT-199 to overcome potential acquired resistance to ABT-199 in AML patients. The synergistic effect of BL-8040 with AC220 could provide a rational basis for the combination of BL-8040 with FLT3 inhibitors in FLT3-ITD AML patient population. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Abraham: Biokine Therapeutics Ltd: Employment. Bulvik:Biokine Therapeutics Ltd: Employment. Wald:Biokine Therapeutics Ltd: Employment. Eizenberg:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Peled:Biokine Therapeutics Ltd: Consultancy, Employment.

Protein kinase A mediates novel serine-584 phosphorylation of HDAC4

2019 ◽  
Vol 97 (5) ◽  
pp. 526-535 ◽  
Author(s):  
Shanmukha K. Doddi ◽  
Githavani Kummari ◽  
Jagannadham M.V. ◽  
Arunasree M. Kalle
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Target Genes ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Wild Type ◽  
Kinase A

Given the well-established diversified signaling pathways for histone deacetylase 4 (HDAC4) and the regulation of HDAC4 by several post-translational modifications (PTMs), including phosphorylation, sumoylation, and ubiquitination, an unbiased and detailed analysis of HDAC4 PTMs is needed. In this study, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) to describe phosphorylation at serine 584 (Ser584) along with already-known dual phosphorylation at serines 265 and 266 (Ser265/266), that together regulate HDAC4 activity. Overexpression of site-specific HDAC4 mutants (S584A, S265/266A) in HEK 293T cells, followed by HDAC activity assays, revealed the mutants to be less active than the wild-type protein. In vitro kinase assays have established that Ser584 and Ser265/266 are phosphorylated by protein kinase A (PKA). Luciferase assays driven by the myocyte enhancer factor 2 (MEF2) promoter and real-time PCR analysis of the MEF2 target genes show that the S584A and S265/266A mutants are less repressive than the wild-type. Furthermore, treatment with PKA activators such as 8-Bromo-cAMP and forskolin, and silencing either by shRNA or its inhibitor H-89 in a mouse myoblast cell line (C2C12) and in a non-muscle human cell line (K562), confirmed in vivo phosphorylation of HDAC4 in C2C12 but not in K562 cells, indicating the specific functional significance of HDAC4 phosphorylation in muscle cells. Thus, we identified PKA-induced Ser584 phosphorylation of HDAC4 as a yet unknown regulatory mechanism of the HDAC4–MEF2 axis.

An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo

1994 ◽  
Vol 107 (4) ◽  
pp. 813-825 ◽  
Author(s):  
M.R. Shanks ◽  
D. Cassio ◽  
O. Lecoq ◽  
A.L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Membrane Proteins ◽  
Cell Line ◽  
Hybrid Cell ◽  
The Other ◽  
Hepatocyte Polarity ◽  
Rat Hepatoma

Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for > 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761–1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

scholarly journals Phosphorylation status of the Kep1 protein alters its affinity for its protein binding partner alternative splicing factor ASF/SF2

2006 ◽  
Vol 400 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Cécile Robard ◽  
Alex Daviau ◽  
Marco Di Fruscio
Keyword(s):  
Alternative Splicing ◽  
Protein Binding ◽  
Cell Line ◽  
Topoisomerase I ◽  
Target Genes ◽  
Rna Binding ◽  
Splicing Factor ◽  
Alternatively Spliced ◽  
Alternative Splicing Factor

Mutations in the Drosophila kep1 gene, encoding a single maxi KH (K homology) domain-containing RNA-binding protein, result in a reduction of fertility in part due to the disruption of the apoptotic programme during oogenesis. This disruption is concomitant with the appearance of an alternatively spliced mRNA isoform encoding the inactive caspase dredd. We generated a Kep1 antibody and have found that the Kep1 protein is present in the nuclei of both the follicle and nurse cells during all stages of Drosophila oogenesis. We have shown that the Kep1 protein is phosphorylated in ovaries induced to undergo apoptosis following treatment with the topoisomerase I inhibitor camptothecin. We have also found that the Kep1 protein interacts specifically with the SR (serine/arginine-rich) protein family member ASF/SF2 (alternative splicing factor/splicing factor 2). This interaction is independent of the ability of Kep1 to bind RNA, but is dependent on the phosphorylation of the Kep1 protein, with the interaction between Kep1 and ASF/SF2 increasing in the presence of activated Src. Using a CD44v5 alternative splicing reporter construct, we observed 99% inclusion of the alternatively spliced exon 5 following kep1 transfection in a cell line that constitutively expresses activated Src. This modulation in splicing was not observed in the parental NIH 3T3 cell line in which we obtained 7.5% exon 5 inclusion following kep1 transfection. Our data suggest a mechanism of action in which the in vivo phosphorylation status of the Kep1 protein affects its affinity towards its protein binding partners and in turn may allow for the modulation of alternative splice site selection in Kep1–ASF/SF2-dependent target genes.

scholarly journals Attenuation of Pancreatic Cancer In Vitro and In Vivo via Modulation of Nrf2 and NF-κB Signaling Pathways by Natural Compounds

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3556
Author(s):  
Marta Cykowiak ◽  
Robert Kleszcz ◽  
Małgorzata Kucińska ◽  
Jarosław Paluszczak ◽  
Hanna Szaefer ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Signaling Pathways ◽  
Cell Line ◽  
Target Genes ◽  
Pancreatic Cancer Cell Line ◽  
Cancer Cell Line ◽  
In Vivo Studies ◽  
Phenethyl Isothiocyanate

Pancreatic cancer is a disease in which deregulation of signaling pathways plays a key role, thus searching for their novel modulators is a promising therapeutic strategy. Hence, in this study, the effect of phytochemical combinations on the canonical and non-canonical activation of Nrf2 and its interaction with the NF-κB pathway was evaluated in extensively proliferating pancreatic cancer cell line, PSN-1, in comparison to non-cancerous MS1 cells. The activation of Nrf2 and NF-κB, expression of their target genes, and effect on cell survival were assessed in PSN-1 cells. The tumor burden was evaluated in mice carrying xenografts. PSN-1 cells were more sensitive to the tested compounds as compared to the MS1 cell line. Combination of xanthohumol and phenethyl isothiocyanate was more effective than single compounds at decreasing the canonical and non-canonical activation of Nrf2 in PSN-1 cancer cells. Decreased activation of NF-κB, and subsequent reduced cytosolic COX-2 and nuclear STAT3 level indicated their anti-inflammatory and pro-apoptotic activities. In vivo studies showed the partial response in groups treated with xanthohumol or the combination of xanthohumol and phenethyl isothiocyanate. Overall, these results suggest that the combination of xanthohumol and phenethyl isothiocyanate may be a promising therapeutic candidate against pancreatic cancer.

scholarly journals WT1 and DNMT3A Play an Essential Function and Represent Therapeutic Vulnerabilities in Certain AML Samples, As Shown By CRISPR/Cas9 Mediated Knockout in PDX Models In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 377-377
Author(s):  
Maryam Ghalandary ◽  
Yuqiao Gao ◽  
Martin Becker ◽  
Diana Amend ◽  
Klaus H. Metzeler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Cell Lines ◽  
Target Gene ◽  
Target Genes ◽  
Nsg Mice ◽  
Essential Function ◽  
Pdx Models

Abstract Background: The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo. Methods: Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline. Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry. Human AML cell lines were studied in vitro for comparison. Results: In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays. Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models. In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A. Conclusion: We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. Disclosures No relevant conflicts of interest to declare.

Genome-Wide Chromatin Immunopreciptiation and Gene Expression Analysis Indicates That MMSET Is a Transcriptional Repressor in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 627-627
Author(s):  
Dong-Joon Min ◽  
Julia Meyer ◽  
Eva Martinez-Garcia ◽  
Josh Lauring ◽  
Jonathan D. Licht
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Signaling Pathway ◽  
Target Genes ◽  
Transcriptional Repressor ◽  
Low Levels

Abstract Multiple myeloma (MM) is a malignancy of plasma cells characterized by frequent chromosomal translocations of the immunoglobulin heavy chain (IgH) locus. The multiple myeloma SET domain (MMSET) gene is a recurrent chromosomal partner in the t(4;14) translocation, and MMSET levels are elevated in these patients relative to other myeloma cases and normal cells. Previously, we showed that MMSET is a histone methyltransferase with specific activity for lysine 20 on histone H4 and acts as a transcriptional repressor when tethered to a model target gene. To reveal the function of MMSET in t(4;14) MM in vivo, we identified MMSET target genes in the KMS11 t(4;14) MM cell line. Chromatin from these cells was subjected to immunoprecipitation with a polyclonal anti-MMSET antibody in biological replicate, amplified by ligation-mediated PCR and hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,659 human promoters. Data analysis using the MaxFour algorithm ranked putative binding sites based upon intensities of 4 consecutive probes. The top 2000 promoters identified from each experiment were combined to yield a list of 1,412 putative MMSET target genes. This list was analyzed using the DAVID program (david.abcc.ncifcrf.gov/). Genes bound by MMSET includes those implicated in antigen processing and presentation (p<8.7×10-4), cell cycle (p<2.2×10-3), the p53 signaling pathway (p<0.03) apoptosis (p<1.6×10-6) and DNA repair (p<3.3×10-4) Among genes bound by MMSET were XBP1, IRF2, and BCL6, all important transcription factors regulating B cell development. Real-time quantitative PCR validated MMSET binding in 6/6 promoters tested so far. To investigate the role of MMSET in transcriptional regulation, we profiled gene expression in KMS11 cells using Illumina arrays to determine the expression of MMSET bound genes. Nearly 50% of genes bound by MMSET had very low levels of expression (≤100, Range on arrays 10–18,000) while only 13% of genes bound by MMSET were expressed at high levels (>1000). This supports the notion that MMSET represses target genes in vivo. Functional annotation of genes bound by MMSET and expressed at low levels showed over-representation of genes implicated in toll-like receptor signaling pathway (p<2.8×10-3), cytokine-signaling (p<3.3×10-3) and JAK2/STAT signaling (p<0.08), transmembrane receptor function (p<4×10-8), and apoptosis (p<0.01), while those bound yet expressed at high levels were implicated in oxidative phosphorylation (p<3.9×10-4) and protein synthesis (p<4.1×10-6). The effects of MMSET on gene expression were further investigated using KMS11KO cells in which the rearranged MMSET allele was ablated by homologous recombination. RNA from KMS11 and KMS11KO cells was profiled by Illumina arrays and genes showing a significant change in gene expression were determined by significance analysis of microarray (SAM) testing with 1% of false discovery rate. Among the 720 genes bound by MMSET and expressed at a level of >100 in the wild-type KMS11 cells, 35 genes were up-regulated and 20 genes were down-regulated (>1.5 fold) in the KMS11KO cell line. Among the 692 genes bound by MMSET and expressed at a level of ≤100, 9 genes were up-regulated in KMS11KO cells. The up-regulated genes (presumably bound and repressed by MMSET) were categorized in cytokine receptor (p<0.02) and JAK2/STAT signaling pathway (p<0.05), nucleosome assembly (p<6×10-4), apoptosis (p<0.01), and cell differentiation (p<0.05). Collectively these data suggest that MMSET may interfere with signal transduction, chromatin modulation and apoptosis pathways involved in the terminal differentiation of the plasma cell. Intriguingly MMSET also bound and was associated with repression of RB1 and RBL2 suggesting a role of MMSET in cell cycle control. Chromatin immunoprecipitation analysis of a MMSET bound gene (ARHGAP25) revealed that MMSET binding was correlated with increased tri-methylation of H4K20, a repression-associated chromatin mark. MMSET binding of this promoter was decreased but still detectable in the KMS11KO cells. Collectively these data suggest that MMSET binds and represses many target genes in vivo. However MMSET could still bind to genes expressed at a high level and MMSET ablation was associated with activation of some MMSET target genes, suggesting that its role in gene regulation may be complex and potentially gene-specific.

scholarly journals Glucocorticoid effects on chondrogenesis, differentiation and apoptosis in the murine ATDC5 chondrocyte cell line

2002 ◽  
Vol 175 (3) ◽  
pp. 705-713 ◽  
Author(s):  
T Mushtaq ◽  
C Farquharson ◽  
E Seawright ◽  
SF Ahmed
Keyword(s):  
Cell Line ◽  
Bone Growth ◽  
Apoptotic Cell ◽  
Recovery Period ◽  
Terminal Differentiation ◽  
Cell Number ◽  
Proteoglycan Synthesis ◽  
Alp Activity ◽  
Significant Impairment

Glucocorticoids (GC) are used extensively in children and may cause growth retardation, which is in part due to the direct effects of GC on the growth plate. We characterised the ATDC5 chondrocyte cell line, which mimics the in vivo process of longitudinal bone growth, to examine the effects of dexamethasone (Dex) and prednisolone (Pred) during two key time points in the chondrocyte life cycle - chondrogenesis and terminal differentiation. Additionally, we studied the potential for recovery following Dex exposure. During chondrogenesis, Dex and Pred exposure at 10(-8) M, 10(-7) M and 10(-6) M resulted in a significant mean reduction in cell number (28% vs 20%), cell proliferation (27% vs 24%) and proteoglycan synthesis (47% vs 43%) and increased alkaline phosphatase (ALP) activity (106% vs 62%), whereas the incidence of apoptosis was unaltered. Minimal effects were noted during terminal differentiation with both GC although all concentrations of Dex lowered apoptotic cell number. To assess catch-up growth the cells were incubated for a total of 14 days which included 1, 3, 7, 10 or 14 days exposure to 10(-6) M Dex, prior to the recovery period. Recovery of proteoglycan synthesis was irreversibly impaired following just one day exposure to Dex. Although cell number showed a similar pattern, significant impairment was only achieved following 14 days exposure. Irreversible changes in ALP activity were only noticed following 10 days exposure to Dex. In conclusion, GC have maximal effects during chondrogenesis; Dex is more potent than Pred and cells exposed to Dex recover but this may be restricted due to differential effects of GC on specific chondrocyte phenotypes.

scholarly journals The oncomir face of microRNA-206: A permanent miR-206 transfection study

2018 ◽  
Vol 243 (12) ◽  
pp. 1014-1023 ◽  
Author(s):  
Dóra Mihály ◽  
Gergő Papp ◽  
Zsolt Mervai ◽  
Andrea Reszegi ◽  
Péter Tátrai ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Soft Tissue Sarcomas ◽  
Fibroblast Cell ◽  
Fibroblast Cell Line ◽  
Normal Human Fibroblast ◽  
Human Fibroblast Cell ◽  
Normal Human

MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future. Impact statement Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.

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