Academic literature on the topic '@Double plasma multipolaire'

Abstract High expression of NEK2 mediated by p53 contributes to progression and relapse of multiple myeloma Xiangling Feng1,2, Jiaojiao Guo1, Bowen Ouyang2, Yinghong Zhu1,Gang An3, Hao Zhen1, Jiliang Xia1, Yongjun Guan1, Xinying Zhao2, Lugui Qiu3, Jiaxi Zhou3, Fenghuang Zhan4,Wen Zhou1 1, Cancer Research Institute,Central South University, Changsha 410078, China. 2Xiang Ya School of Public Health, Central South University, Changsha, Hunan, China. USA. 3State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China. 4Department of Internal Medicine, Division of Hematology, Oncology, and Blood & Marrow Transplantation, University of Iowa, Iowa City, USA. E-mail: wenzhou@csu.edu.cn. Background: Loss of p53 is an independent prognostic factor in patients with multiple myeloma (MM). Our previous studies found abnormal high expression of NEK2 was closely related to the poor prognosis and drug resistance of myeloma patients. However, it's unclear how NEK2 was up-regulated in MM. Through bioinformatics analysis, the binding site of p53 protein is found in NEK2 promoter, but the relationship and function of p53 and NEK2 in MM are poorly understood. Materials and Methods: In this study, p53-/- MM cell lines (ARP1 and KMS11) and p53 p53+/+ MM cell lines (MM1S and H929) were used for investigating the role of NEK2 in p53-/- MM cell. FISH was performed on interphase nuclei of MM primary cells to detect p53 and NEK2 copy numbers. Chromatin immunoprecipitation and fluorescence reporter system were applied for examining the binding site of p53 protein in the distal NEK2 promoter. CGH and RNA-seq were performed to validate copy number changes and variations in the expression of several transcripts. Results: The top 10% of MM patients with the highest NEK2 expression and lowest p53 had a significantly inferior OS (P<0.001) in TT2 and TT3 patients (GSE2658) and the expression of NEK2 increased significantly in myeloma cells during chemotherapy(GSE19554), while p53 decreased with the disease progression, suggesting a strong relationship with drug resistance. Single cell PCR showed increased NEK2 expression correlated with decreased p53 expression in single CD138+ plasma cell. FISH confirmed the loss of p53 in CD138+ plasma cells with amplification of NEK2 copies. Furthermore, NEK2 was also high expressed in p53 low expressed MM cells by Immunofluorescence (IF) (P<0.01). In addition, NEK2 was upregulated in p53-/- MM cell lines and HEK293 cells by deleted p53 gene with CRISPR technique both on mRNA and protein level (P<0.01), suggesting a negative correlation between the p53 and the expression level of NEK2. Meanwhile, when p53 deletion and NEK2 overexpression occur simultaneously, the phenomena of asymmetric mitosis and multipolar division are more obvious (P<0.001), suggested that the double hit of p53 deletion and NEK2 overexpression increases the chromosomal instability. Further in vivo study indicated the subcutaneous tumorigenesis in p53 deletion and NEK2 overexpression group was significantly greater than that of the single overexpression of NEK2 and deletion of p53 group (P<0.001), suggested that NEK2 overexpression and p53 deletion enhances the tumorigenic ability in vivo. While down-regulation of NEK2 by shRNA in p53 deletion cells, cell growth was inhibited in vitro and in vivo.To explore the relationship between p53 and NEK2, chromatin immunoprecipitation and fluorescence reporter system showed that p53 could bind to the promoter region of NEK2 and regulate its transcriptional expression. Further CGH analysis of the deletion of p53 expression in HEK293 cells can cause 1q21.4-44 amplification of the chromosome region of the NEK2 directly, which further confirmed by FISH. Finally, RNA-seq revealed several chromosome instability genes were abnormal expressed in NEK2 overexpression and p53 deletion double-hit group. Conclusion: In summary, p53 deletion and NEK2 overexpression induced cancer cell drug resistance, proliferation and chromosomal instability. p53 could bind to the promoter region of NEK2 and cause NEK2 amplification. Down-regulation of NEK2 by shRNA in p53 deletion cells inhibited cell growth in vitro and in vivo. Thus,The significance of this study will provide the pre-clinical application of the NEK2 inhibitor to overcome the drug resistance induced by p53 in MM. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 2952 Background Centrosome aberrations are common in many types of human malignancies and are associated with aneuploidy. Loss of centrosome duplication control will often create multipolar spindles that in turn could be responsible for incorrect segregation of whole chromosomes leading to aneuploidy. Hyperdiploidy (subtype of aneuploidy) is one of the most frequent cytogenetic abnormalities in multiple myeloma (MM), where molecular changes are among the primary genetic events in disease pathogenesis. But no correlation between centrosome aberrations and aneuploidy in MM has ever been found. Aims The objective of our study was to evaluate association of MM ploidy category with centrosome amplification in both B and plasma cells subpopulations and to investigate structural defects (gain/loss) and gene expression changes in genes controlling centrosome numbers. Methods Immunofluorescent labeling was used for evaluation of centrosome amplification (CA) in B-cells (CD19+) and PCs (CD138+) of MM patients. Centrin (centrosome protein) copy numbers were used to define presence of centrosome amplification (CA) in cells: cells with more than 4 signals of centrin were considered to be positive. Samples with ≥11% of B-cells or ≥10% of PCs with >4 fluorescence signals of centrin were considered as CA positive. A total of 140 patients were evaluated for CA in PCs and/or B-cells, including 50 patients where both cell types were analyzed. The patient population characteristics were as follows: males/females 67/73, median age of 66 years (range, 40–92 years). Newly diagnosed (52/140) and relapsed (88/140) patients were included in this study; most of them had advanced stage of MM (DS II/III n = 107; ISS II/III n = 92). Interphase FISH with cytoplasmic immunoglobulin light chain staining (cIg FISH), oligonucleotide-based arrayCGH (20 patients) and qRT-PCR (5 CA positive vs 5 CA negative patients) were performed on plasma cells. Hyperdiploidy analysis was done using Multi-Color Probe Panel (LSI D5S23/D5S721, CEP 9 and CEP 15) for chromosome 5, 9 and 15. Only cells with three or more signals from at least two of three investigated chromosomes were classified as hyperdiploid. ArrayCGH and qRT-PCR were focused on chosen list of mitotic genes, according to their role in normal centrosome duplication process. Results The frequency of MM cases positive for CA was 35% (35/100) and 39% (32/82), based on the analysis of PC samples and B-cell samples, respectively. Overall, 18% (9/50) of MM patients were double-positive. Presence of centrosome amplification in B-cells of MM patients was established in our previous study. Significant correlation of centrosome amplification in PCs with hyperdiploidy was not found. But association of CA in B-cells with PCs hyperdiploidy using phi 4-point correlation was proven (phi=0.358, p<0.05). In group of newly diagnosed patients (52/140), this correlation was much stronger (phi=0.555, p<0.05). ArrayCGH analysis of genes controlling centrosome duplication did not show correlation between their copy number defects and hyperdiploidy in myeloma cells. As for gene expression analysis, significant differences were found in levels of ARKA and PCNT genes (p<0.05). Relative quantification coefficient R of these genes was two times higher in CA positive patients when compared to CA negative patients. No significant correlation between amount of CA positive PCs and B-cells was found (p>0.05). But after splitting patients based on CA threshold, significant correlation was identified (r=-0,763, p=0.017) in double-positive group. Conclusion In our study, we show association of CA in B-cells with PCs hyperdiploidy. This finding relates to the role of B-cell mitotic disruption in MM aneuploidy and cell carcinogenesis. It gives us a possibility to suspect the impact of abnormal B cells in myeloma cells development. B-cells with CA probably enter mitosis but do not finish it properly resulting in aneuploid cells; these cells may represent an aneuploid pool of MM cells. Acknowledgments This study was supported by grants MSM 0021622434 and IGA 10207-3 from the Departments of Education and Health of the Czech Republic. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2827 Poster Board II-803 Centrosome amplification (CA) has been previously detected in hematological malignancies including multiple myeloma (MM) and is usually associated with disease progression. CA leads to the formation of multipolar mitotic spindles that may lead to chromosome segregation errors and genomic instability. In this pilot study, we have evaluated the occurrence of CA in two populations of B-lineage cells including B-lymphocytes and plasma cells (PCs) of MM patients. We have analyzed possible associations of CA with established prognostic factors including the most common chromosomal abnormalities in malignant PCs. Immunofluorescence labeling was used for the evaluation of centrosome amplification (CA) in B-cells (CD19+) and PCs (CD138+) of MM patients. The centrin (centrosome protein) copy numbers were used to define three cellular subpopulations: (1) no centrin signal (Non-CS), (2) 1-4 centrin signals (1-4CS) or (3) more than 4 signals of centrin (CA). Samples with ≥11% of B-cells or ≥10% of PCs with >4 fluorescence signals of centrin were considered CA positive. A total of 70 patients were evaluated for CA in PCs and/or B-cells, including 18 patients who had analysis of both cell types. The patient population characteristics were as follows: males/females 34/36, median age of 65 years (range, 40-84 years). Most patients had advanced stage of MM (DS II/III n = 48; ISS II/III; n = 21). Peripheral blood samples from 20 healthy donors were used as controls and for the estimation of CA positivity threshold for B-cells (Mean + 3SD). There was a statistically significant difference between the percentage of B-cells subpopulations with centrosome amplification in MM patients and that in healthy donors ([Mean ± SD] 9.9 ± 7.9% versus 3.2 ± 2.5%; P<0.0001). The frequency of MM cases positive for CA was 34% (17/50) and 37% (14/38) based the analysis of PC samples and B-cell samples, respectively. Overall, 22% (4/18) MM patients were double-positive. No significant correlation was detected between B-cells and PCs (r=0.387; P=0.113) obtained from patients with both available samples. No significant associations were identified between CA status and the following common cytogenetic abnormalities in PCs: del(13)(q14) (p= 1.000); del(17)(p13) (p=0.132); gain(1)(q21) (p= 1.000), hyperdiploidy (p= 1.000). In summary, we have confirmed the presence of centrosome amplification in B-cells of MM patients. Immunofluorescence staining is a sensitive method for the detection of abnormal subpopulations of B-cells that probably represent a reservoir of clonogenic cells in MM. This study was supported by grants NR 8945-4/2006, MSM 0021622434, MZ LC 06027 and IGA NR 9317 from the Departments of Education and Health of the Czech Republic. Disclosures: No relevant conflicts of interest to declare.

AbstractIn this work, Langmuir probe measurements were used to characterize a multipolar electron cyclotron resonance (ECR) plasma source. This system has many controllable parameters including microwave power, rf power, gas, pressure, flow rate, and source distance. Both double and triple Langmuir probes were used for the plasma characterization. The results from the Langmuir probe measurements were correlated to the etch characteristics of photoresist. Ion density and photoresist etch rate were found to increase with microwave power but decrease with source distance. However, rf power does not have significant influence on ion density although the photoresist etch rate increases substantially with if power. Ion density first increases then decreases at higher pressure. Maximum ion density occurs at lower pressure for larger distance below the ECR source. Ion density uniformity for an O2 plasma is ±2% across a 16 cm diameter region at 23 cm below the source. For photoresist etched at 10 cm source distance, etch rate uniformity is ±2% for a 15 cm diameter wafer. The results from the Langmuir probe measurements indicate that photoresist etching is enhanced by ion density and ion energy.

L'étude expérimentale du système faisceau d'ions-plasma a montré que la formation de tourbillons dans l'espace des phases ionique représente le régime non-linéaire de l'instabilité du système faisceau d'ions-plasma. Des expériences, réalisées dans une machine double plasma multipolaire, montrent la formation d'une structure de type "bosse et creux" derrière le front de choc induit par une perturbation en échelon de la vitesse du faisceau. L'excitation localisée de l'instabilité acoustique ionique est également enregistrée. On étudie ensuite les régimes instables du système faisceau d'ions-plasma et on caractérise la turbulence acoustique ionique à basse fréquence qui est engendrée par l'instabilité acoustique ionique au voisinage de la fréquence plasma ionique. L'analyse spectrale et l'étude des corrélations permettent de mieux appréhender la situation physique. Enfin, on analyse la turbulence par une méthode statistique, la méthode de la moyenne conditionnelle, qui permet la mise en évidence et l'analyse de l'évolution spatio-temporelle de la dynamique des structures électrostatiques stables se propageant dans le système faisceau-plasma. Ces structures sont associées à des fluctuations négatives du potentiel et de la densité et sont identifiées aux trous d'ions correspondant à la formation de tourbillons dans l'espace des phases ionique. Pour finir, les moyennes conditionnelles sont comparées aux enregistrments synchrones des fluctuations sur un réseau de sondes.

The formation of extravasal fibrin deposits in various tumors has been recognized a long time ago and it has been implicated in various aspects of tumor growth. However, no adequate information is available on the nature of intratumoral fibrin. In this study we attempted to find out if fibrin deposit in human lymph nodes with Hodgkin’s disease is stabilized and made resistant to fibrinolysis by factor XIII /FXIII/ of blood coagulation. The two main tasks for FXIII in fibrin stabilization is to attach a^antiplas-min the main phyiological inhibitor of fibrinolysis to fibrin strands and crosslink fibrin chains. By double immunofluorescent labeling for fibrin and α2-antiplasmin a complete colocalization of the two antigens could be observed. A part of fibrin strands also stained for α2-antiplasmin-plasmin-complex-neoantigen revealing that α2-antiplasmin covalently linked to fibrin inhibited intratumoral fibrinolysis. The finding that immunolabeling for fibrin was preserved following the treatment of sections by concentrated urea solution clearly demonstrates that fibrin chains became crosslinked by FXIII. These results were further supported by SDS PAGE analysis of intratumoral fibrin deposits. There are two theoretical possibilities for the appearance of FXIII in the interstitial space: 1/ plasmatic FXIII can get acrossthe vessel wall when increased permeability is induced 2/ FXIII can be produced and released by certain cells of the tumorous tissue. We explored the secondpossibility by various immunomorphological and enzymcytochemical techniques. Alarge number of FXIII positive cells were detected by immunoperoxidase technique in the follicular and interfollicular region of malignant, but not in normal lymph nodes. These relatively large, multipolar, mononuclear cells possesseda macrophage-like appearance and showedANAE-positivity,i.e.,they belong to thegroup of tumor associated macrophages. FXIII containing cells were labeled by monoclonal anti-Leu M3 (a monocyte/macrophage marker), but not by anti-HLA-DR.They were often found in the immediate vicinity of malignant Hodgkin’s cells and also showed an intimate relationshipwith extravasal fibrin formation.lt is suggested that FXIII secreted byintactor released from damaged macrophages might be involved in the stabilization offibrin in the tumor stroma or around tumor cells.