Effects of SHIP Over Expression On the Invasion and Migration of K562 Cells and Its Mechanism.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5051-5051
Author(s):  
Lin Yang ◽  
Jianmin Luo ◽  
Xiaojun Liu ◽  
Shupeng Wen
Keyword(s):  
Western Blot ◽  
K562 Cells ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Leukemia Cells ◽  
Migration And Invasion ◽  
Over Expression ◽  
A Cells ◽  
Group A ◽  
Group B

Abstract Abstract 5051 Introduction The phosphatidylinositol-3 kinase (PI3K) pathway plays a central role in regulating numerous biologic processes, including survival, adhesion, migration, metabolic activity, proliferation, differentiation, and end cell activation through the generation of the potent second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P(3)). The SH2 domain-containing inositol polyphosphate 5-phosphatase (SHIP) is known to play an important role in the negative regulation by PI3K-dependent signaling cascades activated by several tyrosine-kinase coupled cytokine receptors. SHIP knockout mice display myeloproliferation and hyper-responsiveness to growth factor stimulation and have short life span with myeloid cell infiltration into vital organs. We have proved SHIP inhibits growth of leukemia cells, and it may have an important role in apoptosis of leukemia cells. However, to date the role of anti-migrated effect of SHIP and its mechanism is unclear. Methods Based on the results of our previous studies, with different human leukemia cell lines K562 as study samples, including K562 transfected with wtSHIP (Group A), K562 transfected with muSHIPC1076T (Group B) and K562 transfected with empty vector (Group C); Expression of SHIP at mRNA and protein level was detected by real-time PCR and Western blot, respectively; By Transwell chamber assay was used to count the numbers of Group A and Group B cells that penetrated the matrigel to the back of PVPF membrane after transfection; Primary migration associated factor FAK, p-FAK and NFκB were screened by Western blot. The expression of MMP-2 and MMP-9 was examined by zymography. Results Compared with Group C and Group B, the migration of Group A cells was decreased (32±6 vs 78±13 and 83±16)(P<0.01); Over expression of wild-type SHIP does not affect the MMP2 secretion in K562, but site-directed mutant in C terminus of SHIP can significantly block MMP-9 secretion in K562 cells, By Western blot analysis, the expression of p-FAK and NFκB protein in Group A cells were down-regulated to 44% and 63% of the Group C, respectively. Conclusions The results confirmed SHIP as a negative regulator for cell migration and invasion in bcr/abl transformed cells through decreasing the MMP-9 expression, which may be induced by reduced phopha-FAK and NFαB expression, and implied that it may function through its PXXP domain. Disclosures No relevant conflicts of interest to declare.

The Effects of Site-Directed Mutation of SHIP Gene On the Regulating Mechanisms of PI3K/Akt Signal Pathway in Human Leukemia Cell Line K562.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5050-5050
Author(s):  
Jianmin Luo ◽  
Lin Yang ◽  
Xiaojun Liu ◽  
Shupeng Wen ◽  
Jingyu Zhang
Keyword(s):  
High Pressure ◽  
K562 Cells ◽  
Signal Pathway ◽  
Negative Regulator ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Wild Type ◽  
Directed Mutation ◽  
Group A ◽  
Group B

Abstract Abstract 5050 Introduction SHIP is an SH2 domain containing inositol-5-phosphatasse that appears to be a negative regulator of hematopoiesis. Our previous researching works have first reported that SHIP gene was genetically altered in leukemia patients and proved that mutation of SHIP gene was closely correlated with the increased phosphorylation of Akt and poor prognosis of AML patients; SHIP gene is a key regulative gene in the PI3K/Akt pathway, and transfection with wild type SHIP gene into K562 cell line can inhibit the proliferation of K562. Up to now, however, there are no any reports about what changes of the PI3K/Akt signal transduction regulation will happened if SHIP has site mutation in leukemia cells. The objectives of our study is to investigate the effects of site-directed mutation of SHIP on the expression level of the protein related to the PI3K/Akt signal pathway in leukemia cells, and to study the effects of site-directed mutation of SHIP on the key PIP3 activity related to the PI3K/Akt signal pathway in leukemia cells. Methods Based on the results of our previous studies, the absence of endogenous SHIP in K562 cells provided a useful systerm to study the role of SHIP in growth and apoptosis. The recombined lentivirus plasmids wtSHIP or muSHIPP28L were transfected stably into human leukemia cells K562. The cell proliferation, cell life cycle and cell apoptosis of K562 transfected with wid-type SHIP or muSHIPP28L were determined by MTT, fluorescent staining and flow cytometry. The expression level and difference of total Akt□Ap-Akt473 and p-Akt308 were reconfirmed by SDS-PAGE western blot. PI(3,4,5)P3 and PI(3,4)P2 was assayed by High pressure liquid chromatography. Results The decreased ability of proliferation and DNA synthesis, cell colony fomation ability and enhanced apoptosis rate were observed in K562 cells transfected with wild-type SHIP (Group A), but the same changes had not been observed in K562 cells transfected with muSHIPP28L (Group B) or empty vector (Group C). Wild-type SHIP can down-regulate phosphorylations of Akt308 and Akt473, but muSHIPP28L can't. High pressure liquid chromatography results showed that the PI3,4,5-P3 level was obviously decreased in Group A,no changes above indicate in Group B and Group C. The PI3,4P2 level in Group A was significantly higher than Group B and Group C. Conclusions The results confirmed SHIP as a negative regulator for cell proliferation in leukemia cells, and implied that it may function through its normal structure. Disclosures No relevant conflicts of interest to declare.

Adaptor Protein Lnk Binds to PDGFRA, PDGFRB and FIP1L1-PDGFRA, but Not to the TEL-PDGFRB Fusion Protein.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2213-2213
Author(s):  
Saskia Gueller ◽  
Sigal Gery ◽  
H. Phillip Koeffler
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
Nih3t3 Cells ◽  
Sh2 Domains ◽  
Juxtamembrane Region

Abstract PDGFRA and PDGFRB (platelet derived growth factor receptors alpha and beta) are frequently expressed on malignant hematopoietic cells and regulate various cellular responses such as development, proliferation, differentiation, cell survival and cellular transformation. Stimulation by either autocrine loops or constitutional activation by chromosomal translocation (i.e. chronic myelomonocytic leukemia [CMML, TEL-PDGFRB] or chronic eosinophilic leukemia [CEL, FIP1L1-PDGFRA]) makes them important factors in development of hematopoietic disorders. Normally, interaction with the ligand PDGF, induces dimerization of two distinct receptor subunits, resulting in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. We hypothesized that one such protein may be the adaptor Lnk, a negative regulator of several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain, a pleckstrin homology domain (PH) and a dimerization domain (DD). The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. We investigated the ability of Lnk to bind to PDGFRA, PDGFRB, FIP1L1-PDGFRA and TEL-PDGFRB. To determine the domain of Lnk that is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including: a mutation in the SH2 domain (R392E); deletion of the SH2 domain; deletion of the PH and SH2 domains and a construct only containing the DD domain. 293T cells were co-transfected with cDNAs encoding either PDGFRA, PDGFRB or one of the translocation products and either wild-type or mutant Lnk. Whole cell lysates were used to perform immunoprecipitation with either V5-tag or PDGFR antibodies. Binding of Lnk and PDGFR was detected by Western blot probed with PDGFR or V5-tag antibodies. NIH3T3 cells were transfected either with empty vector or Lnk cDNA, transfectants were selected for 5 days with G418, serum starved for 16 hours and induced with PDGF for 10 minutes. Phosphorylation of downstream targets of PDGFRA and PDGFRB was detected by Western blot. Our data showed that Lnk bound to PDGFRA and PDGFRB only after exposure of the cells to PDGF and to the FIP1L1-PDGFRA fusion protein independent of PDGF exposure. Mutation or deletion of the Lnk SH2 domain abolished binding completely in PDGFRA and FIP1L1-PDGFRA, but just partly in PDGFRB. Expression of Lnk in NIH3T3 cells inhibited phosphorylation of ERK after treatment with PDGF. In other experiments, we determined that Lnk bound the juxtamembrane region of this class of receptors. Interestingly, the TEL-PDGFRB fusion protein was unable to bind Lnk, although its breakpoint in PDGFRB is distal to the juxtamembrane domain and the whole intracellular region of PDGFRB is included in the fusion protein. Further exploration of the mechanisms by which Lnk affects wild-type or PDGFR fusion product will provide insight into the molecular pathophysiology of myeloid disorders and could help develop new treatments.

Activation Of EphrinB2/EphB4 Influences Myeloid Leukemia Cell Migration and Invasion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1360-1360
Author(s):  
Xuan Zhou ◽  
Liu Xiaoli ◽  
Na Xu ◽  
Yajuan Xiao ◽  
Jinfang Zhang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Migration And Invasion ◽  
Cell Migration And Invasion ◽  
Healthy Donors ◽  
Myeloid Leukemia Cell

Abstract Eph receptors and ephrin ligands are cell-surface molecules capable of bidirectional signaling that control cell-cell interactions, migration and invasion. However, their role and regulation in myeloid leukemia cells remain to be defined. To address the hypothesis that Ephrin/EphB is an important regulator of myeloid leukemia cell migration and invasion, we first screened the mRNA levels of 23 eph and ligand ephrin RTK family members in myeloid leukemia cells (K562, HL-60, THP-1) and mononuclear cells from healthy donors, then found that EphB4, EphA5, EfnA1 highly expressed in most myeloid leukemia cells compared to healthy donors(P<0.05). Both the mRNA and protein levels of EphB4 and EphA5 were detected in 13 primary myeloid leukemia cells (5 from patients with extramedullary leukemia among 13 cases) and 10 mononuclear cells from healthy donors by real-time RT-PCR and Immunoblot analysis. The results showed that both the mRNA and protein levels of EphB4 and EphA5 were higher in 13 primary myeloid leukemia cells relative to the 10 healthy donors (P=0.046). Moreover, the EphB4 were highly expressed in 5 patients with extramedullary leukemia compared with 8 patients without extramedullary leukemia. These findings indicated that EphB4 and EphA5 expression were correlated with the development of myeloid leukemia cells, moreover, EphB4 may be closely related with myeloid leukemia cell migration or invasion. To further clarified the question, migration were determined in leukemia cell lines (K562 cells) which were treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins by transwell migration assay. Invasion were also determined by matrigel invasion assay. The results showed that, after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.8 to 2.5-fold, P<0.05), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.2 to 1.8-fold, P<0.05). However, after ephrinA1–Fc stimulation, the numbers of K562 cells migrating through transwell chamber didn’t significantly increase compared to Fc proteins stimulation (P>0.05), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P>0.05). These findings indicated that ephrinB2–Fc could activate EphB4, leading to the change of myeloid leukemia cell migration and invasion. Further study may help to assess a promising potential of this protein to be used as a prognostic marker or as a target for a molecular therapy. Disclosures: No relevant conflicts of interest to declare.

EphrinB2/EphB4 Interaction Promotes Myeloid Leukemia Cell Invasion through RhoA-Mediated Mechanism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1018-1018 ◽  
Author(s):  
Xuan Zhou ◽  
Liu Xiaoli ◽  
Na Xu ◽  
Lin Li ◽  
Qisi Lu ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Immunoblot Analysis ◽  
Receptor Activation ◽  
Leukemia Cells ◽  
Migration And Invasion ◽  
Mmp9 Expression ◽  
Myeloid Leukemia Cell

Abstract Background and Objective: Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrinB ligands in a variety of tumors, suggesting a functional relation between EphB/ephrinB signaling and tumor progression. However, how they regulate the invasiveness of myeloid leukemia cells were still unknown. Our previously study suggested that EphB4 were highly expressed in patients with extramedullary leukemia compared with patients without extramedullary leukemia, which indicated that the expression of EphB4 was related with myeloid leukemia cell invasion. To address the molecular mechanism, we aimed to characterize the role of EphB4 and ephrinB2 ligands in the interaction of myeloid leukemia cells. Methods: To clarify the question, myeloid leukemia cell lines (K562 cells and THP-1 cells) treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins were cultured in vitro, then migration and invasion were determined by transwell assay according to different time. Pulldown western immunoblot analysis were used to detect the level of GTP-RhoA and total RhoA; the phosphorylation of EphB4 and MMP9 expression were also determined by immunoblot analysis before and after the treatment of different clustered Fc proteins. Results: The results showed that after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.85-fold, P=0.033), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.46 -fold, P=0.025). However, the numbers of K562 cells migrating through transwell chamber after ephrinA1–Fc stimulation didn’t significantly increase compared to Fc proteins stimulation (P=0.411), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P=0.072) after ephrinA1–Fc stimulation. Moreover, after ephrinB2–Fc stimulation, the numbers of THP-1 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (2.25-fold, P<0.01), meanwhile, the numbers of THP-1 cells invading the matrigel also enhanced (1.66 -fold, P<0.01). However, the numbers of THP-1 cells migrating through transwell chamber and the numbers of THP-1 cells invading the matrigel didn’t significantly enhanced (P>0.05, P>0.05) after ephrinA1–Fc stimulation. Furthermore, EphB4 immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody revealed that EphB4 is phosphorylated on tyrosine in K562 cells after ephrinB2–Fc stimulation. Additionally, the level of active RhoA (GTP-RhoA) and MMP9 in K562 cells were both significantly increased in response to EphB4 receptor activation with its ligand ephrin-B2-Fc ( P<0.05). Conclusions: These findings suggested that EphB4/EprinB2 signaling played an important role in myeloid leukemia cells progression by promoting their migratory ability, activating RhoA activity and increasing MMP9 expression. Our findings reveal a novel regulation of this intriguing receptor/ligand family that contributes to the cell invasiveness of myeloid leukemia cells. Disclosures No relevant conflicts of interest to declare.

JQ1, a Potential Therapeutic Molecule for Myeloid Leukemia with PTEN Deficiency

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5899-5899 ◽  
Author(s):  
Nicholas J Baltz ◽  
Natalia C Colorado ◽  
Yan Yan ◽  
Shelly Lensing ◽  
Delli Robinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Jak Inhibitors ◽  
Facs Analysis ◽  
Leukemia Cell Lines

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy that continues to have high relapse and treatment-related mortality rates, despite recent advances in clinical management and therapy. Janus kinase (JAK) inhibitors inhibit the activity of the JAK/STAT pathway and have demonstrated some clinical responses in AML patients. However, survival analysis suggests that more than half of AML patients do not benefit from treatment with JAK inhibitors. Furthermore, PTEN deficiency is frequently found in patients in the late stages of cancer, which causes hyperactivated AKT and MAPK pathways. However, emerging data suggests that leukemia cells with PTEN deficiency are resistant to MAPK inhibitors. Over the past decade, it has been demonstrated that dysregulated epigenetics play an important role in myeloid leukemogenesis. The bromodomain and extraterminal domain (BET) family includes adaptor proteins Brd2, Brd3, Brd4, and Brdt that regulate gene expression via binding to acetylated chromatin and subsequently activating RNA Polymerase II driven transcriptional elongation, resulting in the promotion of gene expression. BRD4 is a BET protein required for disease maintenance in AML. JQ1 is a small molecule that interferes with transcriptional regulators, such as BRD4, by preventing them from interacting with acetylated regions of the genome and thus inhibiting the transcriptional activation of BRD4 target genes. Prior research in lymphocytic leukemia cell lines suggests that JQ1 also decreases STAT5-dependent gene transcriptional activities. We hypothesize that the inhibition of BET proteins may correct the over-activated transcriptional activities in myeloid leukemia cells and induce disease regression. We tested our hypothesis in PTEN deficient myeloid leukemia cell lines, TF-1a and K562, and used human cord blood mononuclear cells (CB) for normal cell comparison. Methods: 1) To test whether JQ1 can inhibit colony formation, we seeded cells on 0.3% agar and McCoys' 5A medium supplemented with nutrients and 15% fetal bovine serum, without cytokines, and added JQ1 diluents to the cultures at concentrations of 32.5-1000nM overnight after the cultures were established. 2) To test whether JQ1 can inhibit leukemia cell proliferation, we cultured cells in liquid medium with JQ1 for 48-72 hours, and quantified the viable cells using alamarBlue® assay. 3) To investigate whether JAK/STAT5 activity is altered by JQ1 in leukemia cells, we quantified phosphorylated STAT5 (pSTAT5) in cells via flow cytometry and western blot. We treated the cells with JQ1 at various concentrations for 2 hours and then stimulated the cells for 15 minutes in medium with 0.5% BSA and 10ng/mL GM-CSF prior to staining the cells with anti-pStat5 (pY694) antibody conjugated with Alexa Fluor® 647 for FACS analysis or lysing the cells for western blot analysis. Results: In the colony formation assay, we found that TF-1a cells were more sensitive to JQ1 than the CB cells and K562, with an IC50 of 62.5-125 nM for TF-1a cells (p<0.0001), and 250-500nM for both CB and K562 cells, respectively. Proliferation assay results also supported that TF1a cells are sensitive to JQ1 with an IC50 of 125-250nM, whereas neither CB nor K562 reached the IC50 in the tested concentration range. This suggests that the IC50 of JQ1 for TF1a cells is achievable at concentrations that are mostly nontoxic to normal CB cells, but K562 cells are not sensitive to JQ1. FACS analysis revealed that pSTAT5 is constitutively activated in K562 cells but not in TF-1a cells. Interestingly, the levels of pSTAT5 in both TF-1a and K562 cells were not altered by JQ1 treatment at tested concentrations, which was confirmed by western blot. Conclusions: Our data suggest: 1) JQ1 and other bromodomain inhibitors could be potential therapeutic molecules for selected myeloid leukemias; 2) JQ1 inhibition on colony formation and proliferation in TF-1a cells is not pSTAT5 related. Further studies are underway to test whether JQ1 is effective in primary mouse leukemia cells with Pten deficiency. Disclosures No relevant conflicts of interest to declare.

High Number of Copy Number Alterations and Over-Expression of Genes Involved in the Response Mechanisms to Genotoxic Stress Both Characterize Newly Diagnosed Multiple Myeloma (MM) Patients Carrying Amplified MDM4 and/or Deleted p53,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3935-3935
Author(s):  
Carolina Terragna ◽  
Marina Martello ◽  
Sandra Durante ◽  
Lucia Pantani ◽  
Elena Zamagni ◽  
...  
Keyword(s):  
Stem Cell ◽  
Copy Number ◽  
Plasma Cells ◽  
Genotoxic Stress ◽  
Newly Diagnosed ◽  
Copy Number Alterations ◽  
Over Expression ◽  
Expression Of Genes ◽  
Group A ◽  
Group B

Abstract Abstract 3935 Background The p53 tumor suppressor pathway is tightly kept in check, or completely silenced in cancer cells. A potent inhibitor of p53 is represented by MDM4, which is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. TP53 mutations are rare in newly diagnosed MM, while occur more frequently as late event in the course of the disease and are related to survival. Recently, the adverse prognostic impact of chr. 1q amplification, described in almost 40% of newly diagnosed MM pts, has been reported. The minimal amplified region on chr. 1q harbors MDM4. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) incorporated into autologous stem cell transplantation, in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. Methods Thirty eight pts treated with VTD incorporated into autologous stem cell transplantation were analysed by means of gene expression profile (Affymetrix U133 Plus2.0 array) and unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array). Both GEP and SNP arrays experiments were performed on highly purified CD138+ bone marrow plasma cells obtained at diagnosis from each pts. The presence of CNAs in chr.1 and 17 was evaluated to identify pts carrying amp(1q) and del(17p). Results Eighteen out of 38 pts (42%) carried a minimal amplification region of 1,1 Mb on chr.1q, which harbors MDM4. Five out of 38 pts (13%) carried a minimal deletion region of 482 Kb on chr.17, which harbors TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 18 pts) or the absence of both these abnormalities (group B, 20 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≥ 3 bone lesions in pts carrying amplified MDM4 and/or deleted p53. The rate of best complete or near complete response was 89% in group A and 75% in group B. With a median follow-up of 36 months, the risk of relapse or progression was 50% for pts in group A and 25% for those in group B. The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03); indeed, the presence of amplified MDM4 and/or deleted p53 was significantly associated with a list of 95 CNAs (clustered on chr. 1, 2, 6, 8, 11, 13, 16 and 18), which included del16q (with a minimal area of deletion including WWOX), observed in 39% vs. 5% cases from group A and B, respectively (p<0.05) and chr.8 aberrations (with amplifications and or deletions, both including TRPS1) observed in 61% vs. 15% cases from group A and B, respectively (p<0.05). A comparison of expression profiles of patients carrying or not amplified MDM4 and/or deleted p53 confirmed the over-expression of MDM4 in the former group of pts and highlighted in these pts an overall activation of genes involved in the response mechanisms to genotoxic stress: indeed a significant over-expression of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) was observed in pts with amplified MDM4 and/or deleted p53 (one-way ANOVA plus multiple-test correction with FDR <0,05). Finally, this group of pts significantly over-express the transcription factor YY1, which is known to interact with p53, thus inhibiting its transcriptional activity. Conclusions Pts carrying amplified MDM4 and/or deleted p53 showed a significantly higher number of CNAs and the significant over-expression of genes involved in the response mechanisms to genotoxic stress, as compared to pts lacking these chromosomal aberrations. This might account for the worse outcome of patients harboring del(17p) and/or amp(1q). The amplification of MDM4 locus and the over-expression of YY1 might contribute to maintain p53 in an OFF state by an indirect mechanism. Additional data on the role of both direct and indirect control of p53 pathway on VTD-treated MM pts prognosis, extended to an higher number of pts, will be presented during the meeting. Supported by: Fondazione Del Monte di Bo e Ra, Ateneo RFO grants (M.C.) BolognAIL. Disclosures: Cavo: Genzyme: Honoraria.

scholarly journals Specific and Cross-reacting Antibodies in ABO Heterospecific Twin Pregnancy

Blood ◽  
1957 ◽  
Vol 12 (10) ◽  
pp. 883-906 ◽  
Author(s):  
WOLF W. ZUELZER ◽  
FLOSSIE COHEN ◽  
ABNER R. ROBINSON ◽  
KATHRYN BEATTIE
Keyword(s):  
B Cells ◽  
Maternal Serum ◽  
Hemolytic Disease ◽  
A Cells ◽  
Neutralization Techniques ◽  
Group A ◽  
Abo Hemolytic Disease ◽  
Group B

Abstract A "study in depth" is reported concerning the case of hemolytic disease of a group B infant born to a group O mother, whose group A twin was apparently unaffected. It was shown that the hemolytic disease of the affected infant was due to a specific anti-B antibody. The study included parallel examinations of the antibodies in the sera of the three individuals. The specificity of the B-anti-B reaction was demonstrated in vitro and in vivo. The powerful anti-B antibody of the mother had no effect on the group A twin, in whose serum anti-B was present in large amounts. In vitro, studied by the usual technics of cross absorption the maternal and the fetal anti-A and anti-B serum antibodies behaved as if strictly specific. By applying successive absorption, elution and neutralization techniques, however, it was possible to demonstrate additional cross-reacting antibodies in the maternal serum which could be separated from one another and from the specific anti-A and anti-B antibodies. From the erythrocytes of the normal group A twin such cross-reacting antibody could be eluted. The cross-reacting anti-B antibody, isolated in pure form in eluates, could be shown to be loosely attached to group A erythrocytes without producing visible agglutination reactions while after elution from A cells it did visibly agglutinate group B cells. It could be eluted from A cells, absorbed by fresh A cells and reeluted while retaining its anti-B effect. It was neutralized by group B saliva only. A separate anti-A antibody with similar properties was eluted from B cells and specifically neutralized by group A saliva. A partial affinity of these antibodies for heterologous erythrocytes but not for specific soluble substances was thus demonstrated. These findings support neither the linkage hypothesis of cross reactions between anti-A and anti-B nor the C-anti-C hypothesis of hemolytic disease. They are in keeping with the view that group O sera contain variable complexes of anti-A and anti-B antibodies, composed of multiple fractions with different partial specificities. It is suggested that the occurrence or non-occurrence of cross-reacting antibodies found in sera of group O mothers whose infants develop hemolytic disease is best explained on this basis. It is further stressed that the demonstration of an antibody in mother or child in ABO hemolytic disease does not necessarily indicate its pathologic significance.

Increased Vitreous Chemerin Levels Are Associated with Proliferative Diabetic Retinopathy

2016 ◽  
Vol 236 (2) ◽  
pp. 61-66 ◽  
Author(s):  
Jun Li ◽  
Wen-chao Hu ◽  
Hui Song ◽  
Jing-na Lin ◽  
Xin Tang
Keyword(s):  
Diabetic Retinopathy ◽  
Western Blot ◽  
Proliferative Diabetic Retinopathy ◽  
Vitreous Hemorrhage ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Macular Holes ◽  
Group A ◽  
Group B ◽  
Endothelial Growth Factor

Purpose: To investigate chemerin in the vitreous bodies of patients with proliferative diabetic retinopathy (PDR) and determine the correlation between the levels of vitreous chemerin and vascular endothelial growth factor (VEGF). Methods: This study included 17 patients suffering from PDR and vitreous hemorrhage (VH) (group A), 21 patients with PDR and tractional retinal detachment (TRD) (group B) and 25 patients with idiopathic macular holes or preretinal membranes (control group). All vitreous samples were obtained through pars plana vitrectomy. Enzyme-linked immunosorbent assay and Western blot analysis were performed to evaluate the levels of vitreous chemerin and VEGF. Results: Vitreous concentrations of chemerin were significantly higher in PDR patients with VH and TRD than those in the controls [4.82 ng/ml (3.91-6.13) vs. 5.03 ng/ml (4.01-6.15) vs. 2.53 ng/ml (1.53-5.66), p = 0.025]. The ratio of vitreous chemerin to plasma chemerin concentration significantly differed between groups A and B and the control group [4.93% (4.69-5.34) vs. 4.98% (4.63-5.19) vs. 2.58% (1.78-4.58), p < 0.001]. Western blot results indicated that the levels of vitreous chemerin protein in PDR patients significantly increased compared with those in the controls. Spearman correlation analysis further showed that vitreous chemerin levels in patients with PDR were positively correlated with vitreous VEGF levels (r = -0.542, p < 0.001). Conclusions: Increased vitreous chemerin levels are associated with the development of PDR.

Abstract 5867: The Beneficial Effect of Raloxifene in Aortic Valve Calcification is Associated with Up-Regulation of Gas6-Axl Pathway

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Mony Shuvy ◽  
Suzan Abedat ◽  
Sameh Daher ◽  
Michael Valitsky ◽  
Ronen Beeri ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Western Blot ◽  
Beneficial Effect ◽  
Aortic Valve Calcification ◽  
Down Regulation ◽  
Valve Calcification ◽  
Control Valves ◽  
Group A ◽  
Group B

Aortic valve calcification (AVC) is an inflammatory regulated process. Growth arrest-specific 6 (Gas6) is involved in atherosclerosis and inflammation; in addition the Gla domain of Gas6 directly inhibits mineralization. We sought to assess the role of Gas6 in AVC using our unique animal model of reversible AVC based on a uremia-inducing diet. We also evaluated the possible role of raloxifene (a selective estrogen modulator), which has demonstrated anti inflammatory effects on blood vessels, in the pathogenesis of AVC. Aortic valves were obtained from four groups of rats (n=10 each): calcified valves- from rats fed with the uremia-inducing diet for 7 weeks (group A), valves after calcification resolution following diet cessation (group B), valves from rats fed with the same diet who also received subcutaneous raloxifen (1 mg/kg/day) (group C), and control valves. Valves were examined using multislice computed tomography (MSCT), histology, RT-PCR and western blot. Histological evaluation of calcified valves (group A) revealed positive staining for calcium deposits and osteoblast’s markers (osteopontin, Runx-2). Gene expression analyses of these valves revealed 2.5 times decrease in Gas6 level compared to control valves (p<0.01). Western blot confirmed these results, and further showed a significant decrease of 33 % in Axl (Gas6 receptor) level along with down-regulation of the AKT survival pathway. Resolution of AVC (group B) was demonstrated using MSCT and histology; it was associated with down-regulation of osteoblast features, and up-regulation of both Gas6-Axl and AKT pathways. MSCT of raloxifene treated rats (group C) showed only minimal valve calcification compared with group A (Agatston score 23 ± 7 vs. 105± 15; p< 0.05). The beneficial effect of raloxifene was also confirmed by histology and was associated with up-regulation of Gas6 and Axl (30% and 40% increase respectively compared with group A; p<0.05) and of the AKT pathway. Down-regulation of the Gas6-Axl pathway is a critical step in inducing AVC. The reversibility of AVC was associated with up-regulation of the Gas6-Axl and AKT pathways. These pathways were significantly restored by raloxifene, which may be a promising treatment for AVC.

Roles of Phosphorylated p210bcr/abl and Hsp90 Protein in Apoptosis Induced by Berbamine in K562 Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4418-4418
Author(s):  
Xiaohong Zhang ◽  
Jianrong Sun ◽  
Rongzhen Xu ◽  
Zhiwen He ◽  
Yin Gu
Keyword(s):  
Western Blot ◽  
Caspase 3 ◽  
K562 Cells ◽  
Hsp90 Inhibitor ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Protein Levels ◽  
Hsp70 Protein ◽  
Hsp90 Protein ◽  
Chaperone Hsp70

Abstract Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance to apoptosis. The dysregulated activity of the bcr/abl oncoprotein tyrosine kinase, which is encoded by the bcr-abl fusion gene generated from the chromosomal translocation of t(9; 22) and present in approximately 95% of CML patients, has been shown to be responsible for these malignant phenotypes. Numerous studies have demonstrated that only being phosphorylated that p210bcr/abl oncoprotein can promote cell proliferation and survival, and block apoptosis of tumor cells. Bcr/abl tyrosine kinase has chaperone association with heat shock protein 90 (Hsp90), which plays an essential role in stabilization of bcr/abl protein. Here we describe the activity of a natural small molecular compound, berbamine from plant Berberis amurensis that can selectively induce cell death of both Gleevecsensitive and -resistant Ph+ CML cells, but little is known about its exact mechanisms. We investigated the the expression levels of phosphorylated p210bcr/abl protein and Hsp90 protein in apoptosis induced by berbamine in K562 cells by means of various technologies, including cell culture system, flow cytomerty, western blot and immunoprecipitation (IP). Methods: Human Ph+ CML leukemia K562 cells, which express endogenous p210bcr/abl protein, were cultured in RPMI 1640 and treated with berbamine as indicated time and dose. Flow cytometry (FCM) and Annexin V-FLUOS/PI staining kit were used to evaluate apoptosis of leukemic cells; FCM and cytoperm/cytofix plus Caspase-3-McAb-PE were employed to measure leukemic cells with activated Caspase-3. Phosphorylation of p210bcr/abl protein in leukemic cells were assessed by a combination of immunoprecipitation (IP) with c-abl antibody and Western blot with p-Tyr(pY99)antibody. The protein levels of P210bcr/abl, Hsp90 and Hsp70 in leukemic cells were determined by Western blot with antibodies to c-abl, Hsp90 and Hsp70, respectively. Results: After treatment with berbamine at 8 μg/ml for 48 h, the percentages of leukemic cells expressing activated caspase-3 and apoptotic cells were 45.69% and 48.43%, respectively. IP and WB results showed that berbamine at low concentration markedly inhibited phosphorylation of p210bcr/abl protein in leukemia cells, and the amount of phosphorylated p210bcr/abl in leukemia cells exposured to berbamine at 8 μg/ml for 6 h were only 8.41% of that of untreated leukemia cells without the protein levels of P210bcr/abl down-regulated. Berbamine also down-regulated chaperone Hsp90 protein, and the amount of Hsp90 protein in leukemia cells treated with berbamine at 8 μg/ml for 48 h accounted for 18.37% of that of untreated leukemia cells. Interestingly, berbamine at 8 μg/ml had no obvious effects on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis. Conclusions: Berbamine could induce caspase-3-mediated apoptosis of Ph+ leukemia cells through inhibiting phosphorylation of p210bcr/abl protein and down-regulating its chaperone Hsp90 protein. Unlike Hsp90 inhibitor GA that could upregulate Hsp70, berbamine had no obvious effects on chaperone Hsp70 protein expression in leukemia cells, suggesting that berbamine may be a novel class of Hsp90 inhibitor, and further study is required.

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