Involvement of caspase activation and mitochondrial stress in trichostatin A-induced apoptosis of Burkitt's lymphoma cell line, Akata

Epstein-Barr virus (EBV) infects more than 90% of the human population and has a potential oncogenic nature. Trichostatin A (TSA) has potent antitumor activity, but its exact mechanism on EBV-infected cells is unclear. This study examined the effects of TSA on proliferation and apoptosis of the Burkitt's lymphoma cell line, Akata. TSA treatment inhibited cell growth and induced cytotoxicity in both the EBV-negative and -positive Akata cells. TSA sensitively induced apoptosis in both cells, as demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to sub-G1 phase by flow cytometric analysis, and the formation of DNA ladders. This suggests that EBV has no effect on the sensitivity to TSA. Western blot analysis showed that the cleavage of PARP and Bid and the activation of caspases are closely related to the TSA-induced apoptosis of the cells. The reduction in mitochondrial transition potential and the release of apoptosis-inducing factor from mitochondria to cytosol was also observed after the TSA treatment, but was suppressed by treating the cells with a cathepsin B inhibitor. Overall, these findings suggest that besides the caspase-dependent pathway, mitochondrial events are also associated with the TSA-induced apoptosis of Akata cells.     

Role of bcl-2 in Epstein-Barr virus-induced malignant conversion of Burkitt's lymphoma cell line Akata

We have demonstrated that Epstein-Barr virus (EBV) confers enhanced growth capability in soft agarose, tumorigenesis in the SCID mouse, and resistance to apoptosis in the Burkitt's lymphoma cell line Akata. Subsequently, we have shown that EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. We constantly observed the upregulation of bcl-2 oncoprotein expression upon EBV infection and expression of EBERs. To test whether these phenotypes were due to the upregulation of bcl-2 expression, we introduced bcl-2 into EBV-negative Akata cells at various levels encompassing the range at which EBV-positive cells expressed it. As cells expressed bcl-2 at higher levels, they became more capable of growing in soft agarose and became resistant to apoptosis. However, clones expressing bcl-2 at a higher level than EBV-positive Akata cells were negative in the tumorigenesis assay in the SCID mouse. On the other hand, introduction of bax into EBV-positive Akata cells reduced the resistance to apoptosis; however, it failed to reduce the growth capability in soft agarose. These data indicate that EBV targets not only bcl-2, but also an unknown pathway(s) to enhance the oncogenic potential of Akata cells.     

MicroRNA-182-5p targets a network of genes involved in DNA repair

MicroRNAs are noncoding regulators of gene expression, which act by repressing protein translation and/or degrading mRNA. Many have been shown to drive tumorigenesis in cancer, but functional studies to understand their mode of action are typically limited to single-target genes. In this study, we use synthetic biotinylated miRNA to pull down endogenous targets of miR-182-5p. We identified more than 1000 genes as potential targets of miR-182-5p, most of which have a known function in pathways underlying tumor biology. Specifically, functional enrichment analysis identified components of both the DNA damage response pathway and cell cycle to be highly represented in this target cohort. Experimental validation confirmed that miR-182-5p-mediated disruption of the homologous recombination (HR) pathway is a consequence of its ability to target multiple components in that pathway. Although there is a strong enrichment for the cell cycle ontology, we do not see primary proliferative defects as a consequence of miR-182-5p overexpression. We highlight targets that could be responsible for miR-182-5p-mediated disruption of other biological processes attributed in the literature so far. Finally, we show that miR-182-5p is highly expressed in a panel of human breast cancer samples, highlighting its role as a potential oncomir in breast cancer.     

人B7—1和B7—2cDNA的克隆及鉴定

目的：为探索性构建全新型重组人B7－PE40绿脓杆菌外毒素融合蛋白以长期诱导免疫耐受,本研究从急性B淋巴细胞白血病细胞株Raji中隆N－末端分别缺失34和16个氨基酸的人B7－1和B7－2基因胞外区,并构建含此基因的重组质粒。方法：根据B7－1和B7－2基因序列设计合成可增B7－1和B7－2cDNA的特异性引物,用RT－PCR的方法从Raji细胞总RNA中扩增B7－1和B7－2cDNA,并克隆至pGEM－T载体中,经酶切鉴定后再进行序列分析。结果和结论：从Raji细胞中扩增出预期 的624和675bp的B7－1和B7－2cDNA,将其克隆至pGEM-T载体中,分别经EcoRI/HindⅢ和BamHI/SphI双酶切电泳和序列分析确证,为进一步构建人B7－PE40外毒素融合蛋白奠定了基础。     

Differential effect of IL-4 and IL-13 on CD44 expression in the Burkitt's lymphoma B cell line BL30/B95-8 and in Epstein-Barr virus (EBV) transformed human B cells: loss of IL-13 receptors on Burkitt's lymphoma B cells

IL-4 and IL-13, cytokines with similar biological effects may influence growth and progression of B-cell tumors through regulation of key cell surface molecules important in intercellular communications. In this study, we demonstrate that IL-4 and IL-13 exhibited differential effects on CD23 and CD44 expression and binding to hyaluronan in BL30/B95-8, a Burkitt's lymphoma (BL), and MK3.31, an Epstein-Barr virus transformed normal human B cell line (B-LCL). Studies conducted to understand the molecular mechanisms underlying this differential effect show that IL-4 induced phosphorylation of JAK1, JAK3, and STAT6 in BL30/B95-8 cells and of JAK3 and STAT6 in MK 3.31 cells. In contrast, IL-13 failed to induce the phosphorylation of JAK kinases or STAT6 proteins in these cell lines. The inability of BL30/B95-8 cells to respond to IL-13 was attributed to the loss of expression of IL-13R subunits alpha1 and alpha2, a finding confirmed for a number of other BL cell lines examined.     

Characterization of the normal lymphocyte population cytolytic to Burkitt's lymphoma cells of the EB2 cell line.

The normal human lymphocyte population which exhibits "spontaneous" cytolysis of EB2 Burkitt's lymphoma cells has been characterized. The effector cell has EA and EAC' receptors but lacks E receptors and probably surface Ig. "Spontaneous" anti-EB2 cytotoxicity was not reduced by preincubation of the effector cells with plastic or iron carbonyl or by passage through cotton wool or agarose columns but was reduced by passage through nylon wool columns. Thymocytes were not cytotoxic to EB2 cells, and chronic lymphocytic leukaemia cells (of B cell characteristics) had reduced cytotoxicity compared with normal lymphocytes. Cells from various lymphoid organs of rats and guinea-pigs were also cytotoxic to EB2 cells with reactivity in spleen greater than or equal to blood greater than lymph nodes. Spleen cells from neonatally thymectomized rats had increased cytotoxicity compared with normal rat spleen cells, suggesting that T lymphocytes are not essential. The effector cell in rat spleen did not adhere to cotton wool or agarose columns, indicating some resemblance to its counterpart in human peripheral blood.     

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate

DNA polymerase μ (Pol μ) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol μ protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol μ in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol μ to mutation of G and C residues during SHM. In vitro analysis of Pol μ misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol μ to induce transient template/primer misalignments.     

Identification of human genes involved in repair and tolerance of DNA damage

Summary An overview is presented on strategies of cloning mammalian DNA repair genes. Complementation of human and rodent repair defects and mutagen hypersensitivities by chromosome and DNA mediated gene transfer and mRNA microinjection is described, and the features of the cloned human DNA repair genes are summarized. It is shown that transfection of repair deficient cell lines with cloned bacterial and human genes may give rise to protection from the genotoxic effects of mutagens.Abbreviations MGMT O⁶-methylguanine-DNA methyltransferase - MNNG N-methyl-N-nitro-N-nitrosoguanidine - HeCNU N-hydroxyethyl-N-chloroethylnitrosourea - MMC mitomycin C - MPA mycophenolic acid - ERCC excision-repair cross-complementing rodent UV-complementation group - UV ultraviolet light - XP xeroderma pigmentosum - CHO Chinese hamster ovary Dedicated to Prof. Dr. U. Hagen on the occasion of his 65th birthdayExtended version of an oral presentation given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.90     

Gangliosides induce cell apoptosis in the cytotoxic line CTLL-2, but not in the promyelocyte leukemia cell line HL-60

Gangliosides induce apoptosis in the cells of the IL-2-dependent cytotoxic mouse line CTLL-2. Upon incubation with gangliosides for 24 h, their effect resulting in appearance of apoptotic cells, falls in a series GM2 > GM3 > GM1 > GD1a > GD1b > GT1b. In the presence of rIL-2, apoptosis induced by GM1 is suppressed, whereas that induced by GM2 is enhanced (the effect of intracellular agent C2-Cer is independent of this cytokine). The GM1-induced apoptosis is cancelled by the caspase I inhibitor. The gangliosides under study are not able to induce apoptosis in the promyelocyte leukemia cell line HL-60. Physiological aspects of the phenomenon found are discussed.