Signal Transducer and Activator of Transcription (Stat)-3 Activates the Receptor Tyrosine Kinase-Like Orphan (ROR)-1 Gene in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 57-57
Author(s):  
Li Ping ◽  
David Harris ◽  
Zhiming Liu ◽  
Michael Keating ◽  
Zeev Estrov
Keyword(s):  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Luciferase Assay ◽  
Mobility Shift ◽  
Protein Levels ◽  
Cell Chip ◽  
Stat3 Phosphorylation ◽  
Mobility Shift Assay ◽  
Activation Sequence ◽  
Gene Mrna

Abstract Abstract 57 ROR1, an embryonic protein involved in organogenesis and Wnt signaling, is expressed in B-cell CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors ψ-interferon activation sequence (GAS)-like elements typically activated by Stat3, we sought to determine whether Stat3 activates ROR1. In MM1 cells interleukin (IL)-6 induced Stat3 phosphorylation and upregulated ROR1 whereas STAT3-siRNA downregulated both Stat3 and ROR1 protein levels, suggesting that Stat3 transcribes ROR1. Therefore, we cloned the human ROR1 promoter, generated a series of truncated promoter constructs and assessed their activity by using the luciferase assay. We found that IL-6 augmented the luciferase activity of ROR1 -195, ROR1 -666, ROR1 -834, and co-transfection with Stat3-siRNA significantly attenuated it, suggesting that IL-6 enhanced ROR1 expression by activating Stat3. Furthermore, we established that a region, located between bp -122 and -134, harbors a GAS-like element and activates the ROR1 promoter upon exposure to IL-6. Binding of Stat3 to that region in IL-6-stimulated MM1 cells was confirmed by the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether Stat3 transcribes ROR1 in CLL, we obtained fresh CLL cells and by using the same GAS-like element-containing probe we performed EMSA. CLL cell nuclear protein bound this probe and anti-Stat3 and -phsophoserine Stat3 antibodies induced a super-shift. CLL cell ChIP confirmed that Stat3 binds to the promoter of ROR1 as well as the promoters of the Stat3-regulated genes STAT3, c-Myc and P21, but not that of the control gene RPL30. Finally, using qRT-PCR and western immunoblotting we determined that STAT3-shRNA downregulated ROR1, STAT3 and STAT3-regulated gene mRNA by 4-6 fold, and Stat3 and ROR1 protein levels by 50%. Taken together, these data suggest that constitutively activated Stat3 binds to the ROR1 promoter, activates transcription, and induces production of ROR1 protein in CLL cells. Disclosures: No relevant conflicts of interest to declare.

Signal Transducer and Activator of Transcription (STAT)-3 Induces Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) Receptor-α Expression in Chronic Lymphocytic Leukemia (CLL) Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3879-3879
Author(s):  
Ping Li ◽  
Zhiming Liu ◽  
David Harris ◽  
Alessandra Ferrajoli ◽  
Yongtao Wang ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Antigen Expression ◽  
Lymphocytic Leukemia ◽  
Janus Kinase ◽  
Luciferase Assay ◽  
Mobility Shift ◽  
Western Immunoblot ◽  
Gm Csf ◽  
Protein Levels ◽  
Stat3 Phosphorylation

Abstract Abstract 3879 GM-CSF stimulates proliferation of granulocytes, macrophages, and hematopoietic progenitors. Upon binding to its cellular receptor (R), GM-CSF induces dimerization of the GM-CSFR α and β subunits, phosphorylation of Janus kinase (JAK)-2, and activation of downstream signaling pathways. Because GM-CSF improved the response to Rituximab monotherapy in patients with CLL (Ferrajoli A. Leuk Lymphoma 50:514, 2009) and was found to upregulate CD20 cell surface antigen expression (Vanugopal P. et al. Leuk Res 24:411, 2000), we investigated the effect of GM-CSF on CLL cells. Incubation of peripheral blood (PB) CLL cells with increasing concentrations of GM-CSF (0.05 to 1.0 μM) did not induce the phosphorylation of STAT3, STAT5, AKT, or ERK as assessed by western immunoblot, or phosphorylation of JAK2 as assessed by immunoprecipitation. Therefore, we investigated whether CLL cells express GM-CSFR. Western immunoblot studies revealed that, like normal B lymphocytes, CLL cells do not express GM-CSFRβ but, unlike normal B cells, CLL cells express high levels of GM-CSFRα. Flow cytometry analysis of PB cells from 8 patients with CLL showed that 13 to 59% of CLL cells (CD19+/CD5+) co-expressed CD116 (GM-CSFRα) but not CD131 (GM-CSFRβ). Thus, we wondered what induces GM-CSFRα expression in CLL cells. Because STAT3 is constitutively activated in CLL and sequence analysis revealed that the GM-CSFRα promoter harbors γ-interferon activation sequence (GAS)-like elements typically activated by STAT3, we sought to determine whether STAT3 activates GM-CSFRα. In MM1 cells, interleukin (IL)-6 induced STAT3 phosphorylation and up-regulated GM-CSFRα whereas STAT3-siRNA down-regulated both STAT3 and GM-CSFRα protein levels, suggesting that STAT3 activates transcription of GM-CSFRα. To clarify these findings, we cloned the human GM-CSFRα promoter, generated a series of truncated promoter constructs and assessed their activity using a luciferase assay. We found that IL-6 augmented luciferase activity of GM-CSFRα promoter −4012 – +23, −3018 – +23, −2517 – +23, and −496 – +23, suggesting that IL-6 enhanced GM-CSFRα expression by activating STAT3. Furthermore, we established that regions, located between bp −3581 TTGTTGAAAA −3572, −2984 TTTTCTTAA −2976 and −77 TTTCCCAA −70, harbor GAS-like elements that activated the GM-CSFRα promoter upon exposure to IL-6. Binding of STAT3 to those regions in IL-6-stimulated MM1 cells was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether STAT3 induced transcription of GM-CSFRα in CLL, we obtained fresh PB CLL cells and, by using the same GAS-like element-containing probes, performed EMSA. CLL cell nuclear protein bound these probes and anti-STAT3 and -phosphoserine STAT3 antibodies attenuated the binding. CLL cell ChIP confirmed that STAT3 binds to the promoter of GM-CSFRα as well as the promoters of the STAT3-regulated genes STAT3, c-Myc and P21, but not to that of the control gene RPL30. Finally, using qRT-PCR and western blot analysis we determined that STAT3-shRNA down-regulated GM-CSFRα, STAT3 and STAT3-regulated gene mRNAs, and STAT3 and GM-CSFRα protein levels. Taken together, these data suggest that constitutively activated STAT3 binds to the GM-CSFRα promoter, activates its transcription, and induces production of GM-CSFRα protein in CLL cells. Disclosures: No relevant conflicts of interest to declare.

STAT3-Activated GM-CSFRα Shuttles To The Nucleus and Protects CLL Cells From Apoptosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4118-4118
Author(s):  
Ping Li ◽  
Uri Rozovski ◽  
David Harris ◽  
Lui Zhiming ◽  
Alessandra Ferrajoli ◽  
...  
Keyword(s):  
B Cells ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Mass Spectrometry Analysis ◽  
Luciferase Assay ◽  
Cell Fractionation ◽  
Mobility Shift ◽  
Ligand Interaction ◽  
Spectrometry Analysis ◽  
Gm Csf

Abstract Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates the survival, proliferation, differentiation, and activation of hematopoietic cells, dendritic cells, and T cells. Upon GM-CSF binding, the α and β subunits of the GM-CSF receptor (GM-CSFR) dimerize and activate signaling. While exploring a possible role for GM-CSF in treating chronic lymphocytic leukemia (CLL), we found that GM-CSF did not enhance the phosphorylation of STAT3, Akt, or ERK, suggesting that GM-CSF does not directly affect CLL cells. Indeed, as in normal B cells, flow cytometry analysis of CLL cells did not detect GM-CSFRβ. However, unlike in normal B cells, GM-CSFRα (CD116) was present in CLL cells. Using confocal microscopy, cell fractionation studies, and GM-CSFRα antibody epitope mapping, we detected GM-CSFRα not only on the surfaces but also in the cytosol and nuclei of CLL cells, suggesting that GM-CSFRα has functions that are unrelated to receptor-ligand interaction. Because STAT3 is constitutively activated in CLL cells and because the promoter of the GM-CSFRα gene harbors putative STAT3 binding sites, we hypothesized that STAT3 activates GM-CSFRα in CLL cells. Both chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) analyses revealed that STAT3 binds to the promoter of GM-CSFRα. We therefore cloned the GM-CSFRα promoter in multiple myeloma cell line MM1 and used luciferase assay we identified STAT3 binding activity. To investigate the function of GM-CSFRα, we induced the overexpression of GM-CSFRα in 293FT cells and analyzed the RNA and protein that co-immunoprecipitated with GM-CSFRα. We identified 7200 RNA transcripts that co-immunoprecipitated with GM-CSFRα, including SKI and MAFA oncogenes, two serine/threonine kinase genes, and DEFT1P2 and DEFP1P, which encode proteins that belong to members of the death effector domains family, known to have a role in modulating apoptosis. Overall, the transcripts that co-immunoprecipitated with GM-CSFRα belong to survival pathways, the JAK/STAT pathway, and hematopoietic lineage pathways. Mass spectrometry analysis revealed that housekeeping proteins, chaperon proteins, KAP1, and ISG-15 co-immunoprecipitated with GM-CSFRα. We found that GM-CSFRα–bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFRα–bound ISG-15 inhibited the nuclear factor-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα-siRNA induced the apoptosis of CLL cells. Taken together, our data suggest that STAT3 induces the transcription of GM-CSFRα, which has distinct, ligand-independent, anti-apoptotic activities. Disclosures: No relevant conflicts of interest to declare.

STAT3 Activates Anti-Apoptotic Genes But When In Access Induces Apoptosis Of CLL Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5294-5294 ◽  
Author(s):  
Rozovski Uri ◽  
David Harris ◽  
Ping Li ◽  
Zhiming Liu ◽  
Ji Yuan ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Luciferase Assay ◽  
Spontaneous Apoptosis ◽  
Mobility Shift ◽  
Apoptotic Pathway ◽  
Wide Range ◽  
In Cells

Abstract In chronic lymphocytic leukemia (CLL) cells, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 induces the transcription and proliferation of anti-apoptosis genes including c-Myc, BCL2, Bcl-XL, and Mcl-1 (Hazan-Halevy, 2010). Therefore, we hypothesized that the CLL cells of patients with high peripheral blood absolute lymphocyte counts (ALCs) have a high level of STAT3 protein expression and a low rate of spontaneous apoptosis. Using Western blotting and flow cytometry, we quantitated STAT3 levels and apoptosis rates in CLL cells from 64 patients with high (N=32) and low (N=32) ALCs. As expected, the levels of STAT3 expression were significantly higher in cells from patients with high ALCs (145,000 ± 49,738/µL) than in cells from patients with low ALCs (12,800 ± 4,654/µL). However, contrary to our hypothesis, Annexin V/PI staining revealed that the levels of procaspase-3, its activated (cleaved) form caspase-3, and cleaved PARP as well as the apoptosis rates of cells from patients with high ALCs were significantly higher than those in cells from patients with low ALCs. These findings suggest that cells from patients with high ALCs are prone to spontaneous apoptosis. An RNA microarray analysis revealed that the levels of apoptotic pathway genes were upregulated in cells from patients with high ALC (P = 0.002), and Reverse-transcriptaction PCR of Caspase-3, Calpain 9, MAPK 8, KRAS, PLCγ-2, and PKC validated the array data. Intrigued by these findings, we sought to determine whether high levels of intracellular STAT3 induce apoptosis. Indeed, overexpression of STAT3 in interleukin-6–stimulated MM1 cells upregulated caspase-3 and caspase-3 protein levels and induced apoptosis. Similarly, in CLL cells levels of caspase3 and procaspase3 remained stable across a wide range of STAT3 levels, but when STAT3 reached a threshold, level of caspase3 and procaspase3 markedly increased. Because sequence analysis revealed that the caspase-3 promoter harbors γ-interferon activation sequence-like elements, we cloned the caspase-3 promoter in MM1 cells and, using a luciferase assay, identified regions with putative STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electrophoretic mobility shift essay (EMSA) confirmed that STAT3 binds to the promoter of caspase-3. However, caspase-3 was activated only in cells with high STAT3 expression levels, suggesting that STAT3 binds to caspase-3 with low affinity. To assess STAT3’s binding affinity to the caspase-3 promoter, we prepared serial dilutions of CLL cell DNA and, using ChIP and EMSA, found that STAT3’s binding affinities to p21 and c-Myc were 8 and 4 times as high, respectively, as its binding affinity to caspase-3, suggesting that high levels of STAT3 protein are required to activate caspase-3. Taken together, these findings suggest that activated STAT3 has a previously unknown pro-apoptotic function. At high intracellular levels, rather than providing CLL cells with survival advantage, STAT3 induces apoptosis by activating caspase-3. Thus, because CLL cell proliferation is coupled with spontaneous CLL cell apoptosis, the number of circulating CLL cells based on the cells’ proliferation rate is often lower than expected. Disclosures: No relevant conflicts of interest to declare.

Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4152-4152
Author(s):  
Rozovski Uri ◽  
George Calin ◽  
Tetsoro Setoyama ◽  
Abundu Lucilla ◽  
Harris David ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Indirect Evidence ◽  
Micro Rna ◽  
Lymphocytic Leukemia ◽  
Signal Transducer ◽  
Mobility Shift ◽  
Protein Coding ◽  
Mobility Shift Assay ◽  
Transcription 3

Abstract MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL. Disclosures: No relevant conflicts of interest to declare.

Signal Transducer and Activator of Transcription (STAT)-3 Induces the Expression of Microrna-155 in Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2885-2885
Author(s):  
Ping Li ◽  
Srdana Grgurevic ◽  
Zhiming Liu ◽  
David Harris ◽  
Uri Rozovski ◽  
...  
Keyword(s):  
Research Funding ◽  
Luciferase Activity ◽  
Lymphocytic Leukemia ◽  
Luciferase Assay ◽  
Mobility Shift ◽  
Activity Data ◽  
Immune Precipitation ◽  
Mobility Shift Assay ◽  
Si Rna ◽  
Γ Interferon

Abstract Abstract 2885 MicroRNAs (miRs) are involved in the initiation, progression and dissemination of CLL cells (Calin GA, Croce CM. Blood 114:4761, 2009). Recent studies showed that high levels of miR-155, previously shown to regulate hematopoietic cell development, are expressed in CLL cells. Because transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation, it is thought that miR-155 plays a role in the pathogenesis of CLL (Calin GA et al. N Engl J Med 353:1793, 2005). STAT3 is constitutively activated in CLL and induces the transcription of several STAT3-regulated genes. A recent study demonstrated that STAT3 activates miR-21 and miR-181b-1 (Iliopolus D. et al. Mol Cell 39:493, 2010). Therefor we wondered whether STAT3 enhances the expression of miR-155 in CLL cells. Because a sequence analysis revealed that the promoter of miRNA-155 harbors γ-interferon activation sequence-like elements typically activated by STAT3, we sought to determine whether STAT3 directly activates miR-155 expression. We generated truncated constructs of the miR-155 promoter, co-transfected them into MM1 cells together with STAT3 small interfering (si) RNA (siRNA), and assessed their luciferase activity. The luciferase activity data suggested that of the two putative STAT3 binding sites only one site is involved in STAT3 induced transcription because STATR3-siRNA reduced the activity of miRNA-155 promoter of constructs that harbor this site. To confirm these data we performed an electrophoretic mobility shift assay (EMSA) and chromatin immune-precipitation (ChIP). The EMSA confirmed that STAT3 bound the miR-155 promoter in fresh CLL cells, and ChIP confirmed that STAT3 bound one putative STAT3-binding site in the miR-155 promoter but not to the other, as demonstrated by the luciferase assay; STAT3 co-immuno-precipitated only one putative STAT3 binding region of miR-155 promoter and other STAT3-regulated genes. Finally, STAT3-small hairpin RNA (shRNA) downregulated miR-155 and other STAT3-regulated genes, suggesting that constitutively activated STAT3, binds miR-155 promoter and induces miR-155 transcription in CLL cells. Disclosures: Keating: Celgene Corporation: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Xcenda: Consultancy, Speakers Bureau.

scholarly journals A Functional SNP in the Promoter of LBX1 Is Associated With the Development of Adolescent Idiopathic Scoliosis Through Involvement in the Myogenesis of Paraspinal Muscles

2021 ◽  
Vol 9 ◽  
Author(s):  
Leilei Xu ◽  
Zhenhua Feng ◽  
Zhicheng Dai ◽  
Wayne Y. W. Lee ◽  
Zhichong Wu ◽  
...  
Keyword(s):  
Adolescent Idiopathic Scoliosis ◽  
Idiopathic Scoliosis ◽  
Muscle Development ◽  
Luciferase Assay ◽  
Paraspinal Muscles ◽  
Mobility Shift ◽  
Putative Promoter Region ◽  
Mobility Shift Assay ◽  
First Time ◽  
Functional Snp

Previous studies have shown that LBX1 is associated with adolescent idiopathic scoliosis (AIS) in multiple populations. For the first time, rs1322330 located in the putative promoter region of LBX1 was found significantly associated with AIS in the Chinese population [p = 6.08 × 10–14, odds ratio (OR) = 1.42, 95% confidence interval of 1.03–1.55]. Moreover, the luciferase assay and electrophoretic mobility shift assay supported that the allele A of rs1322330 could down-regulate the expression of LBX1 in the paraspinal muscles of AIS. In addition, silencing LBX1 in the myosatellite cells resulted in significantly inhibited cell viability and myotube formation, which supported an essential role of LBX1 in muscle development of AIS. To summarize, rs1322330 may be a novel functional SNP regulating the expression of LBX1, which was involved in the etiology of AIS possibly via regulation of myogenesis in the paraspinal muscles.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

2008 ◽  
Vol 87 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Y. Wittrant ◽  
B. Sriniketan Bhandari ◽  
H. Abboud ◽  
N. Benson ◽  
K. Woodruff ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Binding Motif ◽  
Rt Pcr ◽  
Mobility Shift ◽  
Protein Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Cellular Pathways ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between −1.7 and −0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

Aspirin Induces Apoptosis in Human Leukemia Cells Independently of NF-κB and MAPKs through Alteration of the Mcl-1/Noxa Balance.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4825-4825
Author(s):  
Ana M Cosialls ◽  
Daniel Iglesias-Serret ◽  
Maria Piqué ◽  
Montserrat Barragán ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Leukemia Cells ◽  
Induced Apoptosis

Abstract Abstract 4825 Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in most cell types. We examined the mechanism of aspirin-induced apoptosis in human leukemia cells. Our results show that aspirin induced apoptosis in leukemia Jurkat T cells independently of NF-κB. Although aspirin induced p38 MAPK and c-Jun N-terminal kinase (JNK) activation, selective inhibitors of these kinases did not inhibit aspirin-induced apoptosis. We studied the regulation of Bcl-2 family members in aspirin-induced apoptosis. The mRNA levels of some pro-apoptotic members, such as BIM, NOXA, BMF or PUMA, were induced by aspirin. However, none of these pro-apoptotic proteins increased and the levels of Mcl-1 protein were reduced. Interestingly, in the presence of aspirin the protein levels of Noxa remained high. This alteration of the Mcl-1/Noxa balance was also found in other leukemia cell lines and primary chronic lymphocytic leukemia cells (CLL). Furthermore, in CLL cells aspirin induced an increase in the protein levels of Noxa. Knockdown of Noxa or Puma significantly attenuated aspirin-induced apoptosis. These results indicate that aspirin induces apoptosis through alteration of the Mcl-1/Noxa balance. Disclosures No relevant conflicts of interest to declare.

The Effects Of IL-9 On CLL Correlate With Activation Of The JAK/STAT Pathways and Micrornas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1627-1627
Author(s):  
Na Chen ◽  
Xin Wang ◽  
Li Peipei ◽  
Xiao Lv ◽  
Kang Lu
Keyword(s):  
Western Blot ◽  
Time Course ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Elevated Serum ◽  
Lymphocytic Leukemia ◽  
Janus Kinase ◽  
Clinical Staging ◽  
Signal Transducer ◽  
Protein Levels

Abstract Background Chronic lymphocytic leukemia (CLL) is a common leukemia in adults, but its pathogenesis is still poorly understood. Interleukin-9(IL-9) has pleiotropic functions in hematological malignancies. Phosphorylation (p) of signal transducer and activator of transcription (STAT) occurs in hematologic malignancies, including STAT3 and STAT6. Studies from cell culture and animal models have revealed that the Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling pathway, which can be activated by sorts of cytokines involved IL-9. Circulating microRNAs(miRNAs) can be sensitive biomarkers for CLL and STAT3 stimulation induced miR-155 and miR-21 upregulation in CLL cells. Objective The expressions of IL-9, pSTAT3, pSTAT6, miR-155 and miR-21 were detected in CLL patients. We strived to prove that STAT3 and STAT6 phosphorylation may play a key regulating role in the secretion of IL-9, which may affect the proliferation and apoptosis of CLL cells. We also examined whether the overexpressions of miR155 and miR21 genes promote IL-9 production and STAT3 affects the transcription of miR155 and miR21 in CLL cells. Methods We measured serum levels of IL-9 in 47 patients with CLL and healthy controls using ELISA. Expressions of pSTAT3 and pSTAT6 were evaluated by Western blot in peripheral blood mononuclear cells (PBMCs) from 20 CLL patients with upregulation of IL-9 and B cells from healthy samples. Analyses of miRNA155 and miRNA21 expressions were conducted on 20 patient samples and healthy samples through qPCR. IL-9 production was detected by Western blot and qPCR in both IL-9 stimulated CLL cells (MEC-1) and miR-transfected MEC-1 cells at different time point. And then the IL-9 secreting was detected after these cells exposed to WP1066 or A77-1726 for 48 hours. Finally, we observed that cell proliferation with CCK-8 incorporation assay and cell apoptosis using AnnexinV-FITC/PI staining followed by flow cytometry. Results An elevated serum level of IL-9 was detected in 20 of 47 sera from CLL patients (Figure 1A). Sera IL-9 level was correlated with the clinical staging, ZAP-70 expression, B2M expression and IgVH status of CLL patients. (P<0.05,Table 1). Overexpressions of pSTAT3, pSTAT6 in protein levels were detected in 20 CLL patients with upredulation of IL-9(Figure 1B-C), while expressions of miRNA 155 and miRNA21 were elevated in 20 CLL patients. The tyrosine STAT3 and STAT6 phosphorylation in MEC-1 cells stimulated by 20ng/mL IL-9 with time course was associated with an increase of the IL-9 secreting with time dependence. MEC-1 cells stimulated with IL-9 by a time course followed by treatment of Wp1066 (10ng/mL) and A77-1726(10ng/mL) eliminated the IL-9 secreting (Figure 1D). IL-9 protein levels were increased in miR-155 transfected MEC-1 cells and miR21 transfected MEC-1 cells treated with 10ng/mL IL-9 at 120min compared with untransfected cells. However Wp1066 could inhibit these facts (Figure 1E). The MEC-1 apoptoses inhibited and the proliferation enhanced by IL-9(20ng/ml) could be blocked by Wp1066 and A77-1726 (Figure 2A-B). MEC-1 cells transfected with miR-155 and miR21 could promote all these effects of IL-9, which inhibited by Wp1066 (Figure 2C-D). Conclusion Our findings suggest that a new explanation about the possible molecular mechanism of regulation of IL-9 production in CLL. It may provide useful insights in understanding the cross-talking pathways among IL-9, STAT3, STAT6, miR-155 and miR-21, which promote the pathogenesis of CLL. Disclosures: No relevant conflicts of interest to declare.

A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells

2008 ◽  
Vol 416 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Manjunath B. Joshi ◽  
Danila Ivanov ◽  
Maria Philippova ◽  
Emmanouil Kyriakakis ◽  
Paul Erne ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Protein Levels ◽  
Nuclear Extracts ◽  
Nucleoprotein Complex ◽  
Thioredoxin 1 ◽  
Mobility Shift Assay ◽  
Reporter Gene Analysis

T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H2O2) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from −99 to −2304 bp, located the minimal promoter region of T-cad within −285 bp from the translation start site. Reporter activity in ECs transfected with the −285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to −156 to −203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to −156 to −203 bp after streptavidin–agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.

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