In vitro assessment of the efficiency of the PIM-1 kinase pharmacological inhibitor as a potential treatment for Burkitt's lymphoma.

Burkitt's lymphoma is an aggressive form of lymphoma affecting B lymphocytes. It occurs endemically in Africa and sporadically in the rest of the world. Due to the high proliferation rate of this tumor, intensive multi-drug treatment is required; however, the risk of tumor syndrome lysis is high. Overexpression of the proto-oncogene proviral integration of the Moloney murine leukemia virus (PIM-1) kinase is associated with the development of hematological abnormalities, including Burkitt's lymphoma (BL). PIM-1 primarily exerts anti-apoptotic activities through BAD phosphorylation. The aim of the present study was to investigate the in vitro efficiency of a PIM-1 kinase pharmacological inhibitor (PIM1-1) in BL. The impact of PIM1-1 was evaluated in terms of the viability and apoptosis status of the BL B cell lines, Raji and Daudi, compared with K562 leukemia cells, which highly express PIM-1. Cell viability and apoptotic status were assessed with western blotting, and PIM-1 gene expression was assessed with reverse transcription-quantitative PCR. After 48 h of treatment, PIM1-1 inhibited the Daudi, Raji and K562 cell viability with a half-maximal inhibitory concentration corresponding to 10, 20 and 30 µM PIM1-1, respectively. A significant decrease of ERK phosphorylation was detected in PIM1-1-treated Daudi cells, confirming the antiproliferative effect. The addition of 10 µM PIM1-1 significantly decreased the PIM-1 protein and gene expression in Daudi cells. An inhibition of the pro-apoptotic BAD phosphorylation was observed in the Daudi cells treated with 0.1-1 µM PIM1-1 and 10 µM PIM1-1 decreased BAD phosphorylation in the Raji cells. The apoptotic status of both PIM1-1-treated cells lines were confirmed with the detection of cleaved capase-3. However, no change in cell viability and PIM-1 protein expression was observed in the 10 µM PIM1-1-treated K562 cells. In conclusion, the findings indicated that the PIM1-1 pharmacological inhibitor may have therapeutic potential in BL, but with lower efficiency in leukemia.

Phyllanthus fraternus manifests potent anti-proliferative activity on cultured Daudi cells.

OBJECTIVE: The aim of this study is to observe the apoptosis of Phyllanthus fraternus Webster against Daudi cells and to study its primary mechanism. MATERIALS AND METHODS: Antiproliferative activity of cultured Daudi cells was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in a dose- and time-dependent manner after treatment with the hydroalcoholic extract of P. fraternus . Trypan blue viability assay was also performed. Apoptosis induction in the cells posttreatment was determined by DNA fragmentation assay, Agarose gel electrophoresis, and Acridine orange/Ethidium bromide dual staining. Protein isolation and analysis was carried out using the standard polyacrylamide gel electrophoresis protocols. RESULTS: The extracts inhibited the growth and proliferation of Daudi cells through induced cell death, which was dose-dependent and time-dependent. The IC50 value was found to be 220 µg/ml after 72 h of treatment. The induction of DNA fragmentation and increase in a number of apoptotic cells posttreatment suggest the possibility of apoptosis induction. A significant decrease in protein level was also observed. CONCLUSION: The results raise the possibility that the hydroalcoholic extract of P. fraternus could be a potent chemotherapeutic agent for the treatment of various cancers. Further evaluation of its potency as a chemotherapeutic agent is imperative.

Interaction of Platelet-Derived Microparticles with a Human B-Lymphoblast Cell Line: A Clue for the Immunologic Function of the Microparticles.

BACKGROUND: Platelets are blood cells with extensive capabilities in hemostasis. They also play a central role in the development of innate and adaptive immune responses. Little information exists about the immunostimulatory role of platelet-derived microparticles (Plt-MPs). To further elucidate this issue, we conducted this study using the B-lymphoblast cell line 'Daudi' as an available surrogate cell line for peripheral blood B lymphocytes. This cell line does not produce immunoglobulins (Igs) and has low expression of activation markers. METHODS: Plt-MPs were isolated from platelet concentrate (PC) using a multi-step centrifugation method. Daudi cells were treated with Plt-MPs in the culture medium while no treatment was given to the control cells. During 5-day co-culture, Daudi cells were evaluated for the Ig production and the expression of the cell surface markers CD86, CD27, and IgD. RESULTS: An increase was observed for the production of IgG and the expression of CD27 and CD86 on Daudi cells in response to Plt-MPs, whereas the IgD level was decreased. The response of Daudi cells was dependent on the concentration of Plt-MPs and the time of their isolation from PCs during storage. The differences of the variables were significant between the treatment and control groups. CONCLUSION: Plt-MPs could induce the activation and differentiation of immortalized cells of B-cell origin. Thus it is conceivable that Plt-MPs may play a significant role as immortalized cell activators in human monoclonal antibody technology in near future.

CD95-mediated apoptosis in Burkitt's lymphoma B-cells is associated with Pim-1 down-regulation.

B-cells of the high-grade non-Hodgkin lymphoma Burkitt's lymphoma (BL) overexpress survival oncoproteins, including the proviral integration site for Moloney murine leukaemia virus kinase (Pim)-1, and become apoptosis resistant. Activated death receptor CD95 after ligation with anti-CD95 monoclonal antibody (mAb) resulted in the regression of BL via induction of apoptosis, suggesting a decrease of survival protein expression. Here, CD95-mediated apoptotic pathways in BL B-cell lines (Raji and Daudi) following treatment with anti-CD95 mAb was investigated with the cause-and-effects on pim-1 gene expression, in comparison with leukemic cell line (K562) used as CD95-negative cells. Immunohistochemical staining for CD95 and Pim-1 was performed, and the effects of anti-CD95 mAb on apoptotic signalling using western blotting, on caspase activity and cell survival of BL B-cell and leukemic cell lines were determined. We showed that Raji cells expressed more CD95 receptors than Daudi cells. Half of each population underwent apoptosis accompanied by decreased cell viability after anti-CD95 mAb treatment. Distinct extrinsic and intrinsic CD95-mediated apoptotic pathways in Raji and Daudi cells were revealed by high caspase activity and mitochondrial outer membrane permeabilization, respectively. We observed decreased Pim-1 transcript and protein expression levels with increased heat-shock protein (Hsp)70 and decreased Hsp90 expression in anti-CD95 mAb-treated cells. Throughout the study, K562 cells did not undergo apoptosis upon anti-CD95 mAb treatment. Pim-1 knockdown following to stable transfection with plasmid vectors induced apoptosis and decreased viability of BL and K562 cells. Therefore, CD95-mediated apoptosis induces Pim-1 down-regulation in BL B-cells, but Pim-1 down-regulation cannot fully eradicate BL and leukaemia.

Molecular effects of the phosphatidylinositol-3-kinase inhibitor NVP-BKM120 on T and B-cell acute lymphoblastic leukaemia.

BACKGROUND: Constitutive activation of the PI3K pathway in T cell acute lymphoblastic leukaemia (T-ALL) has been reported and in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. We investigated the effects of NVP-BKM120, a potent pan-class I PI3K inhibitor, in lymphoblastic leukaemia cell lines. METHODS: Effects of NVP-BKM120 on cell viability, clonogenicity, apoptosis, cell cycle, cell signalling and autophagy were assessed in vitro on T-ALL (Jurkat and MOLT-4) and BL (Daudi and NAMALWA) cell lines. RESULTS: NVP-BKM120 treatment decreased cell viability and clonogenic growth in all tested cells. Moreover, the drug arrested cell cycling in association with a decrease in Cyclin B1 protein levels, and increased apoptosis. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, mTOR, P70S6K and 4EBP1, with stable total protein levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, in association with augmented BAX:BCL2 ratio. Quantification of autophagy showed a dose-dependent increase in acidic vesicular organelles in all cells tested. CONCLUSION: In summary, our present study establishes that NVP-BKM120 presents an effective antitumour activity against T-ALL and BL cell lines.

Functional role of the nicotinic arm of the acetylcholine regulatory axis in human B-cell lines.

We studied the involvement of nicotinic acetylcholine receptors (nAChRs) in the inflammation-related activity of human B-cell lines. Activation of nAChRs in Daudi cells with epibatidine abolished the pansorbin-dependent upregulation of the pro-inflammatory marker Cox-2 both at the mRNA and protein levels, indicating that the nicotinergic signaling suppresses B-cell activation. While the anti-inflammatory action on B-cells was mediated predominantly through α7 nAChR, as could be judged from abolishing epibatidine effects with methyllycaconitine, both α7 and non-α7 nAChRs, such as α2-containing receptors, were involved in regulation of B-cell apoptosis. The net effect was antiapoptotic. To determine the role of nAChRs in regulating B-cell activation/plasmacytic differentiation, we measured changes in the CD38, CD138 and Bcl-6 gene expression. Epibatidine significantly (P < 0.05) upregulated CD38 at the transcriptional level and CD138 and Bcl-6 - at the translational levels. AR-R17779 significantly (P < 0.05) increased the protein levels of CD38 and CD138. In both cases, the effect of epibatidine was abolished with Mec, and that of AR-R17779 - by MLA, demonstrating a functional role of nAChRs in regulating Daudi cell differentiation. The obtained results revealed distinct contributions of α7 and non-α7 nAChRs to regulation of B-cell activation/differentiation, and suggested that signaling through the nicotinic arm of acetylcholine regulatory axis is important for B-cell involvement in inflammation.

Killing effect of 125I-UdR on human lymphoma Raji and Daudi cell Hoes / 中华放射医学与防护杂志

Objective To evaluate the killing effect and the uptake of 125I-UdR on human lymphoma Raji and Daudi cell lines. Methods The amount of 125I-UdR in the cells and cell nuclei were determined after incubation of different time in RPMI 1640 culturing medium containing different concentrations of 125I-UdR. The killing effects of 125I-UdR on Raji and Daudi cell lines were estimated through MTT assay and cell cycle was analyzed by propidium iodide (PI) staining. Results The amounts of 125I-UdR in Raji and Dandi cells and cell nuclei were much higher than that of Na125I(P < 0.05). The amounts of 125l-UdR in Raji and Daudi cells were 14414±95 and (6916± 53.69) Bq/106 cell when the concentration was 100 kBq/ml. The amounts of Na125I were 68± 3.8 and (324±32.8) Bq/106 cell. The uptake of 125I-UdR in Raji and Daudi cells and cell nuclei increased with the 125I-UdR concentration and incubated time. The cell surviving fractions of 125I-UdR groups was much lower than that of Na125I groups (P < 0.05). When the concentration was 500 kBq/ml and incubated time was 48 hours, the Raji and Dandi cell surviving fractions of125I-UdR groups were (19.78 ± 1.39)% and (43.17 ± 2.69) % ;those of Na125I groups were (79.10 ± 1.79) % and (80.36 ± 6.12) %. The surviving fractions of 125I-UdR groups reduced with the 125I-UdR concentration. Conclusions 125I-UdR can be specially ingested by Raji and Daudi cells and incorporated into DNA, then the cells will be killed. The uptake of 125I-UdR is dose and time dependent.

Trichostatin A Regulates hGCN5 Expression and Cell Cycle on Daudi Cells in vitro / 华中科技大学学报（医学）（英德文版）

The expression of human general control of amino acid synthesis protein 5 (hGCN5) in human Burkitt's lymphoma Daudi cells in vitro, effects of Trichostatin A (TSA) on cell proliferation and apoptosis and the molecular mechanism of TSA inhibiting proliferation of Daudi cells were investigated. The effects of TSA on the growth of Daudi cells were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. The effect of TSA on the cell cycle of Daudi cells was assayed by a propidium iodide method. Immunochemistry and Western blot were used to detect the expression of hGCN5. The proliferation of Daudi cells was decreased in TSA-treated group with a 24 h IC50 value of 415.3979 μg/L. TSA induced apoptosis of Daudi cells in a time- and dose-dependent manner. Treatment with TSA (200 and 400 μg/L) for 24 h, the apoptosis rates of Daudi cells were (14.74±2.04) % and (17.63±1.25) %, respectively. The cell cycle was arrested in G0/G1 phase (50, 100 μtg/L) and in G2/M phase (200 μg/L) by treatment with TSA for 24 h.The expression of hGCN5 protein in Daudi cells was increased in 24 h TSA-treated group by immunochemistry and Western blot (P＜0.05). It was suggested that TSA as HDACIs could increase the expression of hGCN5 in Daudi cells, and might play an important role in regulating the proliferation and apoptosis of B-NHL cell line Daudi cells.

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin / 中国病理生理杂志

AIM:This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitt's lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin.METHODS:The survivin-shRNA expression vector was constructed and transfected into Daudi cells.Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis,respectively.Apoptosis index of transfected Daudi cells was quantified by flow cytometry.The sensitivity of Daudi cells to adriamycin(ADR) before and after transfection was detected by MTT test.RESULTS:The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%,respectively,compared to those in control-shRNA treated group and PBS treated group(P

Abnormal ovarian morphogenesis in Drosophila melanogaster after injection of embryos with conditioned media from the Daudi cell line.

Drosophila melanogaster embryos were injected before the blastoderm stage with conditioned media from several male Burkitt's lymphoma human cell lines and the Daudi cell line. Such injections do not have any effect on the male genital apparatus or on the female tract. The Daudi conditioned medium modifies the ovarian morphogenesis of the flies and the rudimentary ovaries obtained look like nymphal gonads. Moreover, they have a drastically reduced number of germ cells. The ovaries that looked functional contain numerous necrotic germ cells and the mean number of ovarioles per fly is significantly smaller than that of the controls. The abnormalities observed resemble the results of experimental and genetic lack of germ cells. They disappear at very high dilution (1×10-6).

Preparation of two anti-human CD40L monoclonal antibodies and analysis of their biological functions / 中国免疫学杂志

Objective:To prepare mouse anti human CD40L antigen monoclonal antibody and study its biological characteristics and functions.Methods:Using CD40L transfected cell as antigen,cell fusion,mAb screening,immunofluorescence,Western blot and competitive test,obtain two mouse anti human CD40 mAb.Their biological functions are evaluated by the analysis of the effect of the Daudi cell proliferation and the mixed lymphocyte reaction.Results:On the basis of phenotype analysis and competition test,it was evidenced that 1B1 and 4F1 recognized different epitopes of human CD40L antigen specially,and they could reverse the growth inhibition of Daudi cell mediated by CD40L transfected cells and reduce MLR in different extents.Conclusion:Two stable hybridomas murine anti human CD40L monoclonal antibodies have been obtained and antibodies(1B1?4F1)showed a obviously blocking function for CD40/CD40L signal.