Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl^– for gluconate^– shifted the reversal potential, while Cl^– channel blockers, 4,4-diisothiocyanostibene-2,2-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPS (a poorly hydrolyzable analog of ATP), 2,3-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 M suramin (a P2 receptor antagonist), and was partially blocked by 100 M pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca²⁺ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca²⁺-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.     

Lack of BCL-2 confers interferon-alpha sensitivity to B-cell lymphomas

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder with pathological manifestations usually including splenomegaly and panctopenia. Interferons (IFNs), specifically of the α subtypes have shown a significant anti-tumor effect in HCL patients, with improvement of hematological parameters within the first few months of treatment. However, the therapeutic effect of IFN-α is still rather limited. The mechanisms responsible for the beneficial action of IFN-α in HCL patients are unclear. A continuous line of cells (Eskol) from a patient diagnosed with HCL was established and shown to have several properties of HCL. Even though, Eskol cells are very resistant to anti-proliferative activity of IFN-α, Daudi cells, another human B-cell-derived cell line, are very sensitive to anti-proliferative activity of IFN-α and are commonly used as a model cell to test anti-proliferative effect of IFN-α. To understand the molecular reason(s) behind the observed obvious differences to IFN sensitivity of above cells, we have analyzed the expression levels of BCL2, caspase-1, Laminin and PARP in these cells. We found that Daudi cells do not express BCL2 at all, and probably because of that, these cells have constantly cleaved, and probably activated form of caspase-1. However, when we overexpresed BCL2 in these cells, they lost processed form of caspase-1 and became resistant to anti-proliferative activity of IFN-α. These results let us to suggest that IFN-α sensitivity of B-cell lymphomas, once again, depends on the presence or absence of BCL2.     

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

BackgroundConstitutive 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.MethodsEffects 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.ResultsNVP-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.ConclusionIn summary, our present study establishes that NVP-BKM120 presents an effective antitumour activity against T-ALL and BL cell lines.     

IL-21对CTL细胞抗daudi细胞作用及机制的研究

目的通过特异性细胞毒性T淋巴细胞(CTL)与IL-21共同作用daudi细胞,观察CTL与IL-21对daudi细胞的作用机制。方法培养肿瘤特异性CTL细胞,体外培养daudi细胞,设置对照组和实验组共6组细胞,CTL细胞与IL-21联合作用淋巴瘤细胞。MTT法检测细胞抑制率,检测细胞培养上清中IFN-γ的含量,检测CTL细胞的毒性作用,RT-PCR检测daudi细胞中Caspase-3mRNA的表达。结果各组均能抑制daudi细胞的生长,对照组培养上清中IFN-γ含量(401.32±3.54)pg/ml,CTL细胞杀伤率(82.34±2.7)%,Caspase-3 mRNA相对表达量(3.37±0.04)均明显高于对照组(P<0.05)。结论 IL-21是通过增强CTL细胞杀伤作用,上调Caspase-3蛋白表达,从而抑制daudi细胞增殖。     

EBV立刻早期蛋白Zta基因对Daudi细胞周期的影响及其机制研究

目的 探讨EBV立刻早期蛋白Zta基因对Daudi细胞周期的影响及其可能机制.方法 构建Zta基因真核表达载体,通过电穿孔将该载体转染Daudi细胞,用流式细胞术检测细胞周期的变化,用蛋白印迹试验检测细胞周期蛋白p21、Rb、E2F-1的表达.结果 成功构建了 Zta基因表达载体,转染Zta基因可抑制Daudi细胞的增殖,并促进Daudi细胞由G0/G11期[(30.0±3.4)％)]向S期[(47.7±1.1)％]的转化.同时转染Zta基因可下调Rb的表达、上调E2F-1、p21的表达.结论 转染Zta基因使Daudi细胞周期由Go/G1期向S期转变,其机制可能与Rb的表达下降、E2F-1和p21表达上调有关.     

抗人CD40单克隆抗体偶联纳米胶束药物载体系统的构建及其生物学特性

目的：将抗人CD40单克隆抗体5C11与3种含有不同反应基团的聚合胶束（polymeric micelle,PM）偶联,构建相应5C11偶联的聚合胶束（5C11 coupled polymeric micelle,PM-5C11）,筛选出其中最适合与5C11偶联的PM,并观察该偶联物的生物学特性。方法：分别合成3种不同化学结构的聚合物材料,制备相应PM,与5C11偶联后制备PM-5C11,选择其中偶联率和生物安全性均较高的作为最适PM-5C11。将最适PM-5C11与人Burkitt淋巴瘤Daudi细胞株作用,观测其生物学活性及其被Daudi细胞摄取的能力。结果: 成功制备了分别含对甲苯磺酸酯基、丙烯基、羧基的3种PM,其中含对甲苯磺酸酯基和羧基的PM与5C11的偶联率均明显高于含丙烯基的PM的偶联率\[（28.08±2.24）%、（29.06±1.37）% vs （21.26±104）%,P<0.05\];由于含羧基的PM在制备过程中易残留细胞毒性物质,故选取含对甲苯磺酸酯基的PM作为最适PM。相应的最适PM成功偶联5C11形成PM-5C11,最适PM-5C11显示出5C11原有的生物学活性,并且可被Daudi细胞摄取。结论：构建的3种PM中,含对甲苯磺酸酯基的PM在生物安全性、偶联率及相应PM-5C11的生物学活性方面最优,可作为构建纳米药物载体的最佳选择。     

Sorting of cells from different cell cycle phases using surface antigen expression

Detailed procedures are presented for obtaining highly enriched human Daudi cell population in different cell cycle phases. This protocol allows harvest of enriched G₁ or S-G₂ phase cells based on the recognition of a cell surface marker antigen by anti-CD11 a antibody and sorting. Sorted cells are able to cycle and a wave of synchronisation appears. Relative advantages of the method over chemically induced cell synchronisation are discussed.Abbreviations FCS fetal calf serum - PI propidium iodide - ND neutral density - FSC forward scatter - SSC side scatter     

The effect of a mesogenic and a lentogenic Newcastle disease virus strain on Burkitt lymphoma Daudi cells

The destructive effect of Newcastle disease virus (NDV) strains on Burkitt lymphoma Daudi cells was investigated. Interaction of an active and UV-inactivated mesogenic strain (Roakin), as well as an active attenuated lentogenic strain (B1), grown in the allantoic sac of embryonated eggs, at high multiplicity, caused inhibition in cellular DNA synthesis and arrest in cell multiplication, eventually killing of the cells. The lentogenic strain cultivated in chicken fibroblasts exhibited only a moderate activity. The mechanism of the cytolytic effect is presumably linked to the increase in cell membrane permeability indicated by the elevation in⁵¹Cr release. Thus it appears that the massive adsorption and/or penetration of viral particles, active or UV-inactivated (or possibly a toxic component that resides in the virion), damages the plasma membrane and may be responsible for the killing of the cells.Abbreviations CEF chicken embryo fibroblasts - NDV Newcastle disease virus - B1 attenuated lentogenic NDV strain B1 V-188 - RO mesogenic (medium virulent) NDV strain Roakin/46 V-109 NJ - CEF-B1 B1 cultivated in CEF - RO-IRR UV-irradiated RO - m.o.i. multiplicity of infection - EID ₅₀ median egg-infective dose - PBS phosphate-buffered saline     

hGCN5在Burkkit淋巴瘤细胞株Daudi中的表达及TSA对细胞增殖凋亡的影响

目的:研究hGCN5(human general control of amino acid synthesis protein 5)在Burkkit淋巴瘤细胞株Daudi中的表达及曲古柳菌素A(TSA)对细胞增殖、凋亡的影响,探讨其抗肿瘤的分子机制.方法:采用MTT法检测细胞增殖,流式细胞仪检测细胞周期和凋亡率,采用免疫细胞化学法和Western blot观察Daudi细胞hGCN5蛋白的表达.结果:TSA可明显抑制Daudi细胞增殖;TSA(50、100μg/L)使细胞积聚于G0/G1期;TSA(200μg/L)则使细胞周期受抑于S期,而对G2/M期的作用不明显;TSA(200、400μg/L)处理24h可以诱导细胞凋亡;免疫细胞化学和Westernblot结果均显示处理组hGCN5的吸光度比未处理组明显增加(P＜0.05).结论:TSA通过上调HATs家族成员中hGCN5的表达,实现对Daudi细胞的抗增殖作用.     

人CD20跨膜区与g3pN端融合基因的克隆及其在大肠杆菌中的表达

目的 :构建g3pN1 hCD2 0跨膜及胞外区基因表达载体 ,并在大肠杆菌中进行高效融合表达。方法 :利用反转录PCR和PCR方法 ,分别从Daudi细胞和M13K0 7噬菌体中扩增hCD2 0基因和编码g3pN1蛋白N1端的基因 ,并重组到pTIG Trx表达载体中 ,在大肠杆菌中融合表达。结果 :表达产物可被抗CD2 0分子的单克隆抗体 (mAb)识别。结论 :成功地表达并鉴定了目的蛋白。