Transcriptional Profiling and Cytokine Signaling In The Pathogenesis Of Diamond-Blackfan Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2474-2474
Author(s):  
Yoan Konto-Ghiorghi ◽  
Elena Bibikova ◽  
Bertil Glader ◽  
Anupama Narla ◽  
Kathleen Sakamoto
Keyword(s):  
Western Blot ◽  
Cord Blood ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Diamond Blackfan Anemia ◽  
Qrt Pcr ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Diamond-Blackfan Anemia (DBA) is a pediatric bone marrow failure syndrome, characterized by anemia and congenital abnormalities. Ribosomal protein S19 (RPS19) is mutated in approximately 25% of DBA patients, resulting in haploinsufficiency. Eighty percent of patients will initially respond to corticosteroids; however, this drug is associated with significant toxicity, including immunosuppression and growth delay. Identification of novel pathways could lead to new approaches to treat DBA that minimize the toxicities observed with current therapies. To recapitulate the effects of RPS19 deficiency, we transduced CD34+ cells purified from fetal liver or cord blood with two different shRNA lentiviral constructs against RPS19. GFP was used as a selection marker for cells transduced with RPS19 or control scrambled shRNA. RPS19 knockdown was confirmed by Western blot analysis and by quantitative real-time PCR (qRT-PCR) (73%, p< 0.01 for shRNA1, and 87%, p< 0.01 for shRNA2, respectively), compared with scrambled shRNA control. To further characterize the transcriptional landscape of RPS19-deficient cells, we performed RNA-sequencing analysis with mRNA from fetal liver CD34+ cells transduced with lentivirus RPS19 shRNA. Our results showed that genes involved in cytokine/chemokine signaling, including GDF15, CCL1, and CD70, are overexpressed in RPS19-deficient cells. We hypothesize that genes regulating the expression of these cytokines could contribute to red cell progenitor defects observed in DBA patients. qRT-PCR confirmed the three cytokines GDF15, CCL1, and CD70 to be overexpressed in both fetal liver CD34+ RPS19-deficient cells (more than 10-fold for the three genes, p< 0.01, using each of the two RPS19 shRNAs) and cord blood CD34+ RPS19-deficient cells (more than 10-fold for GDF15 and CCL1, and 3-fold for CD70, p< 0.01, using each of the two RPS19 shRNAs). To test whether GDF15 is a modulator of erythropoiesis, we transduced fetal liver CD34+ cells with lentiviral shRNA against GDF15 (50% knock-down, p<0.01) and assessed their erythroid differentiation potential in methylcellulose. Methylcellulose colony assays indicated that the numbers of erythroid colonies (BFU-E and CFU-E) are decreased by 60% in fetal liver CD34+ cells transduced with shRNA-GDF15 compared to fetal liver CD34+ cells transduced with a control scrambled shRNA, thus suggesting a role for GDF15 in erythroid differentiation. To understand the mechanisms that lead to GDF15, CCL1, and CD70 overexpression in RPS19-deficient CD34+ cells, we compared in silico the promoter sequences of these three cytokine genes. Similar putative transcription factor binding sites were identified in all three promoters, for regulators that have already been shown to play major roles in hematopoiesis: GATA1, GATA2, IKAROS1, IKAROS4, and E2F1. We then performed Western Blot analysis to measure the expression of GATA1, IKAROS1, and E2F1 at the protein level in RPS19-deficient CD34+ cells. These results showed that GATA1 expression was decreased by 50% in RPS19-deficient fetal liver CD34+ cells and by 25% in RPS19-deficient cord blood CD34+ cells. Likewise, IKAROS1 expression in Western Blot analysis was decreased by 20% in RPS19-deficient fetal liver CD34+ cells and by 15% in RPS19-deficient cord blood CD34+ cells. We are currently studying the mechanisms by which these pathways contribute to DBA pathogenesis and to identify potentially novel targets for DBA therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 663: Identification of 2 Novel Candidate Genes of Atherosclerosis Susceptibility on Mouse Chromosome 3: Pla2g12a and Elovl6

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Alexandros Nicolaou ◽  
Kristina Sass ◽  
Bernd H Northoff ◽  
Daniel Teupser ◽  
Lesca M Holdt
Keyword(s):  
Western Blot ◽  
Candidate Genes ◽  
Mouse Chromosome ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ldl Receptor ◽  
Atherosclerotic Lesion ◽  
Lesion Size ◽  
Specific Expression ◽  
Qrt Pcr

Quantitative trait locus (QTL) mapping in an F2 intercross (n=452) of atherosclerosis-susceptible C57BL/6 (B6) and atherosclerosis-resistant FVB mice on the LDL-receptor deficient background revealed a novel atherosclerosis susceptibility locus on mouse chromosome (Chr) 3. In previous work the susceptible genetic region on Chr3 was narrowed to 80 - 160 MB and validated by congenic FVB.Chr3 B6/B6 mice. We hypothesized that underlying genetic variation in this region leads to differential expression of causal genes, thereby affecting atherosclerosis susceptibility. We performed transcriptome-wide expression analyses in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=4/4) using Illumina Ref-8 arrays followed by validation in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=8/9) as well as in livers of B6 and FVB mice (n=5/5) by quantitative real-time PCR (qRT-PCR). C is -regulation was investigated in F2 livers (n=47) by correlating the expression to the genotype. Tissue-specific expression of genes was examined by qRT-PCR in parental B6 and FVB mice. Western blot analysis and immunohistochemical staining (IHC) were performed. Mechanisms of atherogenesis were investigated by RNAi. Pla2g12a and Elovl6 were identified as candidate genes co-segregating with the atherosclerosis QTL at marker rs13464244. Pla2g12a mRNA expression was inversely correlated (r 2 =0.2, p=0.002) with atherosclerotic lesion size in F2 mice while Elovl6 expression was positively correlated (r 2 =0.18, p=0.002). qRT-PCR revealed a strong expression of Pla2g12a in muscle and fat tissues whereas Elovl6 was highly expressed in liver and fat tissues. Western blot analysis revealed significantly decreased protein expression of Pla2g12a in livers of B6 compared to FVB and an increased expression of Elovl6 in B6 mice. IHC staining of Pla2g12a and Elovl6 in aortic roots indicated high expression in macrophages and predominantly in endothelial cells. siRNA knockdown of Elovl6 was associated with reduced adhesion and increased apoptosis. In conclusion, we identified Elovl6 and Pla2g12a as promising candidate genes of atherosclerosis susceptibility on mouse Chr3. Further work is necessary to better understand the influence of these two genes on atherosclerosis development.

Delta-4 Notch ligand promotes erythroid differentiation of human umbilical cord blood CD34+ cells

2006 ◽  
Vol 34 (4) ◽  
pp. 424-432 ◽  
Author(s):  
Akira Sugimoto ◽  
Mayuko Yamamoto ◽  
Motoyuki Suzuki ◽  
Toshiya Inoue ◽  
Shuji Nakamura ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Erythroid Differentiation ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Notch Ligand ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Overcoming TNF-α Induced Suppression of Erythroid Differentiation of Human Cord Blood CD34+ Cells with Arsenic Trioxide.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3451-3451
Author(s):  
Jong-Ho Won ◽  
Hee-Jeong Cheong ◽  
Sook-Ja Kim ◽  
Sang-Byung Bae ◽  
Chan-Kyu Kim ◽  
...  
Keyword(s):  
Cord Blood ◽  
Arsenic Trioxide ◽  
Erythroid Differentiation ◽  
Glycophorin A ◽  
Dependent Manner ◽  
Human Cord Blood ◽  
Colony Assay ◽  
Cd34 Cells ◽  
Tnf Α ◽  
Cord Blood Cd34

Abstract The anemia of chronic disease-which encompasses inflammation, infection, tissue injury, and conditions associated with the release of proinflammatory cytokines (such as cancer)- is one of the most common forms of anemia seen clinically. Symptomatic anemia requires treatment. The two major forms of treatment are transfusions and erythropoietin. Arsenic trioxide (As2O3) used to treat human diseases for centuries in traditional Chinese medicine. Our recent studies suggest that low dose of As2O3 induces erythroid differentiation of K562 human leukemic cells and high dose of As2O3 induce apoptosis. In this study, we have investigated in vitro effect of As2O3 on the erythroid differentiation and it could inhibit TNF-α induced suppression of erythroid differentiation of human cord blood CD34+ cells. Expression of glycophorin A was 35.94 ± 7.94% after 7 days culture of human cord blood CD34+ cells and was decreased to 17.63 ± 7.33% when culture of human cord blood CD34+ cells with 100ng/mL of TNF-α. Expression of glycophorin A was increased in dose dependent manner after 7 days treatment with As2O3 and As2O3 increased percentage of glycophorin A in culture with TNF-α compared to TNF-α alone. The results of colony assay of CFU-MIX and BFU-E after culture with various conditions revealed similar patterns with expression of glycophorin A. These results suggest that As2O3 induces erythroid differentiation of human cord blood CD34+ cells and can reverse TNF-α induced suppression of erythroid differentiation of human cord blood CD34+ cells. The BFU-E colony assay of the human cord blood CD34+ cells after culture with TNF-α or/and Arsenic trioxide. The BFU-E colony assay of the human cord blood CD34+ cells after culture with TNF-α or/and Arsenic trioxide.

scholarly journals Inhibition of NHE1 Induced Apoptosis in Cytarabine Resistant Leukemia Cell Lines and Primary Leukemia Cells from AML Patients, Which Showed Increased Intracellular pH and NHE1 Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3614-3614 ◽  
Author(s):  
Shin Young Hyun ◽  
Young Kyung Kim ◽  
Ji Eun Jang ◽  
Yundeok Kim ◽  
Yu Ri Kim ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Qrt Pcr ◽  
Nhe1 Activity ◽  
Induced Apoptosis ◽  
Primary Leukemia

Abstract Background: Na/H exchanger 1 (NHE1), an important participant in the precise regulation system of intracellular pH (pHi), is known to be involved in pathological processes such as cell transformation, maintenance and active progression of the neoplastic process. Some studies have showed that leukemic cells showed higher pHi than normal cells, and NHE1 inhibitor could induce acidification and apoptosis of the leukemic cells. In this study, we tried to elucidate the role of NHE1 in leukemic cells according to cytarabine (AraC) resistance. Materials and Methods: Two human AML cell lines, AraC sensitive (AS)-OCI-AML2 cells and AraC resistant (AR)-OCI-AML2 cells, primary leukemic cells from AML patients, and normal bone marrow mononuclear cells (BMMNC) from healthy donor were analyzed. The pH-sensitive fluorescent dye, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was used to measure pHi and NHE1 activity. The fluorescent ratio of the 490/440 nm was calibrated intracellularly. The expression of NHE1 was measured by qRT-PCR and western blot analysis. To inhibit the NHE1, the amiloride analogue, 5-(N,N-hexamethylene) amiloride (HMA) (10 µM, 20 µM, 30 µM) was used. Results: To confirmed AraC sensitivity, cell lines were treated with 10 µM AraC for 24 hours, and apoptosis fraction in AS-OCI-AML2 cells and AR-OCI-AML2 cells were 53.1±7.2 % and 4.0±0.8 %, respectively. The pHi of AR-OCI-AML2 cells was significantly higher than AS-OCI-AML2 cells (7.839±0.033 vs. 7.589±0.129, P=0.045) and BMMNC (7.839±0.033 vs. 7.578±0.035, P=0.083), and these differences were associated with higher NHE1 activity. Compared AS-OCI-AML2 cells, AR-OCI-AML2 cells showed significantly higher NHE1 expression by western blot analysis (Figure 1), and NHE1 mRNA levels (0.039±0.014 vs. 1.565±0.070, P<.001) by qRT-PCR. Treatment with HMA (20 µM) could induce apoptosis both on AS-OCI-AML2 cells (26.9±2.8%) and AR-OCI-AML2 cells (37.4±18.8%). Interestingly, induction of apoptosis by HMA was dose-dependent both in AS-OCI-AML2 cells and AR-OCI-AML2 cells, and higher concentration of HMA (30 µM) could induce apoptosis on most of AR-OCI-AML2 cells (68.7±20.2%). Co-treatment experiment with 10 µM AraC and 20 µM HMA in AS-OCI-AML2 cells showed additive effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 53.1±12.4 vs. 53.1±12.4 vs. 67.20±4.3%, Figure 2), but in AR-OCI-AML2 cells, co-treatment did not show additional or synergistic effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 4.0±0.1 vs. 27.1±2.2 vs. 28.1±2.0%, Figure 2). As in the cell lines, primary leukemia cells from patients with AraC resistance showing higher pHi and NHE activity than those from patients without. HMA could induce apoptosis on primary cell lines regardless AraC sensitivity. Conclusions: In this study, we first showed that NHE1 inhibition could induce apoptosis in leukemia cells regardless AraC sensitivity. Apoptotic activity was related with higher pHi and NHE activity in AraC resistant cell lines and primary leukemic cells. NHE inhibition induced apoptosis may be independent with AraC induced apoptosis. The heterogeneity in pHi and NHE activity within leukemic cells may be related to alteration in drug delivery machinery or dormant status of leukemia cells. Further experimental and clinical studies are needed to elucidate the therapeutic application of NHE1 inhibitor to AraC resistant AML. Figure 1. Western blot analysis showed higher level of expression of Na/H exchanger I in AR-AML-OCI2 cells than AS-AML-OCI2 cells. Figure 1. Western blot analysis showed higher level of expression of Na/H exchanger I in AR-AML-OCI2 cells than AS-AML-OCI2 cells. Figure 2. Percentage of apoptotic cells after treatment with 20 µM HMA and/or 10 µM AraC. Figure 2. Percentage of apoptotic cells after treatment with 20 µM HMA and/or 10 µM AraC. Disclosures No relevant conflicts of interest to declare.

scholarly journals HYAL1 Is Downregulated in Idiopathic Pulmonary Fibrosis and Inhibits HFL-1 Fibroblast Proliferation When Upregulated

2020 ◽  
Vol 2020 ◽  
pp. 1-15 ◽  
Author(s):  
Dong Leng ◽  
Xiaoxi Huang ◽  
Jiawen Yi ◽  
Hongying Zhao ◽  
Yuhui Zhang
Keyword(s):  
Data Mining ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Western Blot ◽  
Microarray Data ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Lung Fibroblast ◽  
Fibroblast Proliferation ◽  
Qrt Pcr

Background. Idiopathic pulmonary fibrosis (IPF), the most common interstitial lung disease, arises from transforming growth factor beta 1- (TGFβ1-) induced aberrant fibroproliferation in response to epithelial injury. The TGFβ1 antagonists—hyaluronidases (HYALs)—have been used to clinically treat pulmonary fibrosis. This study focused on characterizing the effect of HYAL1, the main enzyme in hyaluronan degradation, on human lung fibroblast proliferation and apoptosis, and elucidating its potential underlying mechanism of action. Methods. We first performed microarray data mining of previously published gene expression datasets to identify key gene signatures in IPF lung tissues. HYAL1 expression levels in IPF and normal lung tissues were then characterized using immunohistochemistry followed by real-time quantitative reverse transcription-PCR (qRT-PCR) and western blot analysis on isolated fibroblasts from fresh lung tissues of IPF and healthy donors. A human fetal lung fibroblast HFL-1 cell line, which was used in place of primary lung fibroblasts, was used to assess the proliferative or apoptotic effects associated with lentiviral-induced HYAL1 overexpression using CCK-8 cell proliferation assay and Annexin V-APC staining. The identification of potentially associated molecular pathways was performed using microarray analysis followed by qRT-PCR and western blot analysis. Results. Lung tissue microarray data mining and immunohistochemistry revealed significantly downregulation of HYAL1 in IPF lung tissue. However, HYAL1 expression level in IPF fibroblasts was significantly upregulated at the mRNA level, but not altered at the protein level. HYAL1 overexpression in HFL-1 fibroblasts reduced fibroproliferation modestly but did not promote apoptosis. In addition, HYAL1 overexpression led to concomitant transcription factor downregulation, bone morphogenetic protein receptor 2 (BMPR2) signaling activation, but had no effect on TGFβ receptor 2 (TGFβR2) signaling. Conclusions. We showed that HYAL1 overexpression could prevent HFL-1 fibroproliferation. Furthermore, our findings suggest that transcriptional regulators and BMP receptor signaling may be involved in HYAL1 modulation in IPF therapy.

PIP4KIIα Gene Silencing by Lentivirus-Mediated Delivery of shRNA in Human K562 Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3200-3200
Author(s):  
Tânia Regina Zaccariotto ◽  
Daniela Maria Ribeiro ◽  
Joao Machado-Neto ◽  
Magnun N N Santos ◽  
Carolina Lanaro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Qrt Pcr ◽  
Globin Gene Expression

Abstract Abstract 3200 Background: Phosphatidylinositol-phosphate-kinase type II alpha (PIP4KIIα) belongs to a family of lipid kinases responsible for the production of a variety of lipid second messengers, such as PI4,5P2 (phosphatidylinositol 4,5-biphosphate), and appears to be implicated in the regulation of gene expression, pre-mRNA processing and mRNA export. In a previous study, two transcripts, PIP4KIIα and β-globin, were found to be overexpressed in reticulocytes from two siblings with Hb H disease, suggesting a possible relationship between this enzyme and the production of globins, particularly β-globin. Recently, we established a gene expression pattern for PIP4KIIα in healthy individuals during in vitro erythropoiesis and observed a gradual increase in the expression of this gene during erythroid differentiation similar to that observed for globin genes, reinforcing the hypothesis of a relationship between PIP4KIIα and globin expression. Aim: To investigate the effects of PIP4KIIα gene silencing on the expression of α- and γ-globin genes in human K562 cells. Methods: Two different human K562 cells cultures were transduced with a lentiviral vector encoding PIP4KIIα-specific shRNA or non-relevant control shRNA. After transduction the positive cells were selected by adding puromycin to the culture and collected 2, 6, 8 and 10 days later to analyze gene and protein expression. PIP4KIIα and α- and γ-globin gene expression was assessed by qRT-PCR and quantified using the equation RQ=2−ΔΔCt. Western blot analysis was performed to determine PIP4KIIα protein expression. β-actin and GAPDH were used as endogenous controls in the qRT-PCR, and β-actin in the Western blot. Results: Analysis of the results showed that there was a statistically significant reduction in PIP4KIIα mRNA levels in knockdown cells (79%) (0.208 ± 0.048; p<0.0001) compared with the control culture. Western blot analysis corroborated these findings. PIP4KIIα silencing resulted in an 18% (0.927 ± 0.244; p=0.09) and 44% (0.625 ± 0.124; p=0.03) reduction in the expression of α- and γ-globin genes, respectively, compared with the control. Conclusion: Although the reduction in α-globin gene expression did not achieve statistical significance, our results revealed alterations in α- and γ-globin gene expression in PIP4KIIα knockdown cells, suggesting a parallelism between the expression of PIP4KIIα and globin genes and reinforcing the hypothesis that the former may be involved in regulation of the latter. This work was supported by FAPESP, CNPq and INCTS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pomalidomide Modulates Transcription Networks Regulating Human Erythropoiesis and Globin Switching: Implications for Treatment of Hemoglobinopathies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1375-1375
Author(s):  
Brian M. Dulmovits ◽  
Abena O. Appiah-Kubi ◽  
Julien Papoin ◽  
Michael Gould ◽  
Xiuli An ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Side Effects ◽  
Western Blot ◽  
Culture System ◽  
Erythroid Differentiation ◽  
Qrt Pcr ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Phase Culture

Abstract Sickle cell disease (SCD) represents a major challenge in hematology, with approximately 100,000 Americans afflicted and the annual number of newborns with SCD set to rise over the next 40 years worldwide. Current treatment approaches rely on increasing levels of fetal hemoglobin (HbF) to prevent painful vaso-occlusive crises and hemolysis secondary to red cell sickling. Hydroxyurea remains the only pharmacologic intervention approved for SCD; however, it has limited efficacy and carries significant side effects such as myelosuppression. Thus, there is a critical need to develop drugs that enhance HbF production without similar dose limiting side effects. Second generation immunomodulatory drugs, such as pomalidomide, are a class of emerging HbF inducers both in vitro and in vivo. Recent work from our laboratory revealed that hydroxyurea and pomalidomide differentially regulate HbF production in CD34+ cells undergoing erythroid differentiation using a 3-phase culture system. Pomalidomide, but not hydroxyurea, was found to decrease BCL11A expression through a yet to be defined mechanism. In the present study, we sought to characterize erythropoiesis and the expression of key transcription factor networks in this 3-phase culture system to determine the mechanisms underlying pomalidomide’s effect. Following a four day expansion period, isolated CD34+ cells from the peripheral blood of SCD or normal individuals were differentiated along erythroid lineage in the presence of pomalidomide (1μM) or DMSO (vehicle) for 14 days. As an additional control, CD34+ cells were also treated with hydroxyurea (10μM). Proliferation and erythroid differentiation were assessed at 7, 11 and 14 days of culture. Although a 50% decrease in cell growth was noted in cells treated with hydroxyurea, no such decrement was found in control, DMSO and pomalidomide-treated cells. Moreover, pomalidomide produced a transient delay in erythroid differentiation between days 6 and 11 of culture phenotypically documented by flow cytometric analysis using glycophorin-A, α-4 integrin and band 3 as surface markers monitoring erythroid differentiation as well as morphologically by May-Grunwald Giemsa staining. In contrast, cells treated with hydroxyurea demonstrated accelerated differentiation, compared to the control cultures. However, by day 14 of culture, no significant difference was observed under any condition, suggesting that the delayed cells eventually finished terminal differentiation. In terms of HbF induction, we confirmed elevated production in the cultures with pomalidomide by measuring the number of F-cells by flow cytometry. We also evaluated the production of γ-globin chains by qRT-PCR and western blot at D4 and D11 and found a dramatic increase in the production of γ-globin, in both SCD and normal samples treated with pomalidomide. We posited that pomalidomide might foster changes in transcription factors known to play a role in both erythropoiesis and globin switching. To this end, we evaluated the expression kinetics of BCL11A, SOX6, KLF1, MI2β, GATA1 and FOG1 via qRT-PCR and western blot analyses. In DMSO-treated cultures the above transcription factors were maximally expressed between days 6-8, and their levels diminished during the remainder of the culture. Conversely, pomalidomide markedly decreased BCL11A, SOX6, KLF1 and MI2β between days 4 and 6 in cultures of both SCD and normal samples. In line with our results, MI2β acts as a positive regulator of BCL11A and KLF1, and previous studies have shown that its knock down in CD34 cells leads to decreased levels of BCL11A and KLF1. Western blot analyses confirmed the qRT-PCR data. Further, the divergent expression patterns correlated temporally with the differentiation delay suggesting that pomalidomide modulates expression of members of the BCL11Atranscription factor complex, thereby augmenting γ-globin production. Taken together, these data provide evidence that pomalidomide influences erythropoiesis by modulating transcription factor expression in CD34+ cells differentiated in the 3-phase culture system, leading to a decrease in BCL11A and activation of γ-globin production. Importantly, further exploration of these pathways that function to regulate erythropoiesis and promote HbF silencing, may help elucidate the mechanism of action of pomalidomide as well as identify additional druggable molecules. Disclosures No relevant conflicts of interest to declare.

Functional Analysis of Cyclin D1 in Mantle Cell Lymphoma (MCL) by Specific Lentiviral shRNA Mediated Knockdown.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1584-1584
Author(s):  
Margit Klier ◽  
Natasa Anastasov ◽  
Daniela Angermeier ◽  
Mark Raffeld ◽  
Falko Fend ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Infection Rates ◽  
Facs Analysis ◽  
Cyclin D2 ◽  
Qrt Pcr

Abstract Introduction: Cyclin D1 overexpression is the hallmark of MCL. However, the importance of cyclin D1 for the maintenance of MCL still remains to be defined. Therefore, the aim of this study is to elucidate the role of cyclin D1 overexpression using the siRNA technology in well-characterized MCL cell lines, as a model system. Material and Methods: A highly efficient cyclin D1-shRNA (96% knockdown) was identified using a lacZ-cyclin D1 fusion gene reporter system in HEK-293T cells. This shRNA was cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Seven MCL cell lines were analyzed (Granta 519, Jeko-1, Rec-1, Z-138, UPN-1, Hbl-2 and JVM-2), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. The effect of cyclin D1 knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: The infection rates varied among the different MCL cell lines. Rec-1 and Hbl-2 showed low infection rates (50%) even at high MOI’s (multiplicity of infection), whereas UPN-1 and JVM-2 had moderate infection rates (80%). Jeko-1, Granta 519 and Z-138 showed high infection rates (almost 100% of the cells). Despite the good tranfection rate, the downregulation of cyclin D1, as measured by Western Blot and qRT-PCR, was about 80% in Granta 519, and 65% in Jeko-1 and Z-138. No IFN response, as secondary effect was identified. Interestingly, no apoptosis was observed, and there was only a moderate retardation of growth (60% of control cells) with 10% shift from the S phase to G1 phase of the cell cycle when compared to the controls, suggesting that other cell cycle proteins might compensate, at least partially, for the loss of cyclin D1. Accordingly, cyclin D2 showed upregulation in Western blot analysis and qRT-PCR, whereas the phosphorylation status of retinoblastoma protein on Ser780 was reduced and the expression of the CDK inhibitor p27Kip1 increased. No changes were observed in the expression of cyclin D3, Cyclin E, CDK4 and CDK2. Conclusions: In this study, a system that enables the specific downregulation of cyclin D1 in MCL cell lines was established. Surprisingly, the downregulation of cyclin D1 in MCL cell lines resulted in only a moderate inhibition on cell growth with no apoptosis. The reasons for this might be 1) that the upregulation of cyclin D2 compensates for cyclin D1 downregulation, and/or 2) that the chromosomal translocation leading to cyclin D1 overexpression is an initiating event in MCL lymphomagenesis followed by secondary genetic events at later stages of the disease, which make cyclin D1 dispensable. This finding has important implications for MCL therapy, as strategies targeting only cyclin D1 might be hampered by the redundancy of the system, resulting in a low probability of treatment response.

JAK2V617F-Mediated Phosphorylation of PRMT5 Down-Regulates Its Methyltransferase Activity and Promotes Myeloproliferation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 794-794
Author(s):  
Fan Liu ◽  
Xinyang Zhao ◽  
Fabiana Perna ◽  
Lan Wang ◽  
Priya Koppikar ◽  
...  
Keyword(s):  
Cord Blood ◽  
Colony Formation ◽  
Erythroid Differentiation ◽  
Arginine Methylation ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Cd34 Cells ◽  
Cord Blood Cd34 ◽  
Arginine Residues ◽  
Histone Arginine Methylation

Abstract Abstract 794 Background: The cytoplasmic, non-receptor JAK2 tyrosine kinase is mutated at amino acid residue 617 (from valine to phenylalanine) in most patients with myeloproliferative neoplasms (MPNs), resulting in a constitutively activated kinase that phosphorylates STAT proteins in the absence of upstream signals. Overexpression of JAK2V617F leads to cytokine-independent growth of Ba/F3 cells and the JAK2V617F transgenic and knockin mice develop a disease phenotype resembling human polycythemia vera. Results: We hypothesized that the JAK2V617F occurs so consistently in MPNs because it gains some functional property. The type II arginine methyltransferase PRMT5 was initially identified because of its interaction with JAK2 in a yeast two hybrid screen. We examined the interaction between JAK2 and PRMT5 and found that JAK2V617F and JAK2K539L (another active JAK2 kinase) bound PRMT5 more strongly than did wild-type JAK2. PRMT5 mediates the symmetrical dimethylation of arginine residues within histones H2A and H4 and methylates other cellular proteins as well, such as p53. The oncogenic forms of JAK2 acquire the ability to phosphorylate PRMT5, which greatly impaired its methyltransferase activity. We have shown the in vivo importance of this post-translation modification as treating JAK2V617F-positive cells (but not the wild-type JAK2-harboring cells) with different JAK2 inhibitors significantly increased histone arginine methylation levels. To define the effect of inhibiting PRMT5 activity on hematopoiesis, we knocked down PRMT5 in human cord blood derived CD34+ cells using shRNA and observed increased colony formation and erythroid differentiation; In contrast, PRMT5 overexpression in these cells led to reduced colony formation and inhibition of erythroid differentiation. Furthermore, overexpression of PRMT5, especially a phosphorylation site mutant form of PRMT5 (PRMT5M6), diminishes the proliferative and erythroid generating capacity of JAK2V617F+ CD34+ cells isolated from MPN patients to a greater degree than normal cord blood CD34+ cells. Importantly, we found marked increase in PRMT5 phosphorylation in JAK2V617F-positive MPN patents relative to normal cord blood CD34+ cells, suggesting that this phosphorylation is important for the myeloproliferation phenotype. Conclusion: we show that the oncogenic mutant forms of JAK2 kinase, such as JAK2V617F and JAK2K539L, are not simply constitutively active forms of wild-type JAK2, rather they have specific gains-of-function that allow them to phosphorylate PRMT5 and down-regulate its enzymatic activity. Inhibition of PRMT5 contributes to the myeloproliferation and erythroid differentiation promoting effects of JAK2V617F. This gain-of –function mutation results in cross-talk between oncogenic kinases and histone arginine methylation. Taken together, we demonstrate a novel link between the mutant JAK2 kinases and PRMT5 methyltransferase activity, which contributes to MPN pathogenesis. Further insights about the shared gene expression profile of JAK2 inhibition vs. PRMT5 knockdown will be presented to understand the basics for the behavior change in hematopoietic stem/progenitor cells brought about by these two interventions. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document