The effects of CpG DNA on HMGB1 release by murine macrophage cell lines

DNA containing cytosine-guanine dinucleotide (CpG) motifs (CpG DNA) has potent immunostimulatory activities that resemble those of lipopolysaccharide (LPS) in its effects on the innate immune system. Among its activities, LPS can induce the release of high mobility group protein (HMGB1) by macrophages, a dual function molecule that can mediate the late effects of LPS. To determine whether CpG DNA can also induce HMGB1 release, the effects of a synthetic CpG oligonucleotide (ODN) on HMGB1 release from RAW 264.7 and J774A.1 cells were assessed by Western blotting of culture supernatants. Under conditions in which the CpG ODN activated the cell lines, as assessed by stimulation of tumor necrosis factor alpha and interleukin-12, it failed to cause HMGB1 release into the media. Although unable to induce HMGB1 release by itself, the CpG ODN nevertheless potentiated the action of LPS. With RAW 264.7 cells, lipoteichoic acid and polyinosinic-polycytidylic acid, like LPS, stimulated HMGB1 release as well as cytokine production. These results indicate that the effects of CpG DNA on macrophages differ from other ligands of Toll-like receptors and may lead to a distinct pattern of immune cell activation in the context of infection or its use as an immunomodulatory agent.     

The induction of HMGB1 release from RAW 264.7 cells by transfected DNA

High mobility group protein 1 (HMGB1) is a non-histone nuclear protein that can activate innate immunity when in an extracellular location. As shown in in vitro studies, while polyinosinic-polycytidylic acid [poly (I:C)] and LPS, TLR3 and TLR4 ligands, respectively, can induce HMGB1 release from macrophages, CpG DNA, a TLR 9 ligand, does not. Since DNA displays distinct immunostimulatory activity when transfected into cells, we investigated whether transfected DNA can induce HMGB1 release from macrophages. In these experiments, using RAW 264.7 cells as model, we show that DNA, either natural DNA or synthetic oligonucleotides, can induce HMGB1 release when used to stimulate cells with the transfection reagent Lipofectamine 2000; release occurred irrespective of the intrinsic activity of the DNA. The induction of HMGB1 release by transfected DNA was dependent on IFN-beta as shown by the inhibitory effects of an antibody. In addition, JNK activation mediated HMGB1 release induced by a transfected phosphorothioate oligonucleotide but not by transfected natural DNA. Together, these findings indicate that transfected DNA can stimulate macrophages to release HMGB1 under conditions in which free DNA is inactive and suggest a role of DNA in inducing inflammation when bound to molecules that influence its entry into cells.     

Isolation and identification of cancer stem-like cells from murine melanoma cell lines

In current study, cancer stem-like cells in the murine melanoma B16F10 cells were investigated. CD phenotypes of the B16F10 cells were analyzed by flow cytometry, and the specific CD phenotype cells from the B16F10 cells were isolated by MACS. Then we used colony formation assay in soft agar media, the cell growth assay in serum-free culture media as well as the tumorigenicity investigation of the specific CD phenotype cells in C57BL/6 mice, respectively, to identify cancer stem-like cells in the B16F10 cells. The results showed that the B16F10 cells could form spherical clones in serum-free culture media, and the rate of clonegenesis of CD133^＋, CD44^＋ and CD44^＋CD133^＋ cells was higher than that of CD133^-, CD44^- and CD44^＋CD133^＋ cells in soft agar media, respectively. The tumorigenic potential of CD133^＋, CD44^＋, CD44^＋CD133^＋ cells and CD44^＋CD133^＋CD24^＋ cells was stronger than that of CD133^-, CD44^-, CD44^＋CD133^- cells and CD44^＋CD133^＋CD24^- cells in mice, respectively. In conclusion, the CD44^＋CD133^＋CD24^＋ cells have some biological properties of cancer stem-like cells or are highly similar to the characteristics of cancer stem cells （CSC）. These results provide an important method for identifying cancer stem-like cells in B16F10 cells and for further cancer target therapy. Cellular ＆ Molecular Immunology.     

Contamination of human melanoma cell lines by mouse L cells.

Five of 8 cell lines from human melanomas originally established elsewhere were found to consist exclusively of mouse cells when examined in our laboratory some months after their receipt. Cytogenetic studies, including G- and C-banding, showed that the mouse cells in all cultures had originated from a single cell line, identified as the L-line. Contamination probably occurred, one year before its discovery, in a laboratory where L cells and human melanoma cells were briefly kept in the same incubator.     

Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis

Oral mucositis (OM) is a common complication in cancer patients undergoing anticancer treatment. Despite the clinical and economic consequences of OM, there are no drugs available for its fundamental control. Here we show that high-mobility group box 1 (HMGB1), a “danger signal” that acts as a potent innate immune mediator, plays a critical role in the pathogenesis of OM. In addition, we investigated treatment of OM through HMGB1 blockade using NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1Hindole-7-yl]amine). NecroX-7 ameliorated basal layer epithelial cell death and ulcer size in OM induced by chemotherapy or radiotherapy. This protective effect of NecroX-7 was mediated by inhibition of HMGB1 release and downregulation of mitochondrial oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and macrophage inflammatory protein (MIP)-1β, as well as the expression of p53-upregulated modulator of apoptosis (PUMA) and the excessive inflammatory microenvironment, including nuclear factor-kB (NF-kB) pathways. In conclusion, our findings suggest that HMGB1 plays a key role in the pathogenesis of OM; therefore, blockade of HMGB1 by NecroX-7 may be a novel therapeutic strategy for OM.     

Human malignant melanoma cell lines.

A series of 10 cell lines established from human malignant melanomas is described. The morphology varied but, in general, was epithelioid. Five produced visible melanin in culture but others produced melanin precursors. All lines were aneuploid and each had a distinctive human karyotype. One line appeared not to have the same sex as the patient but its karyotype was distinct from that of other lines.     

Characterization of NO and cytokine release by transplantable melanoma cell lines in relationship to their differentiation

Changes in the secretory activity of two transplantable melanoma lines (differing in many biological features) as regards nitric oxide (NO) release and interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and oncostatin M (OSM) secretion were the subject of the present study. The results obtained show that a spontaneous alteration of the hamster native melanoma line into an amelanotic one is accompanied by a global change of the secretory activity, but there was not a distinct correlation between the endogenous cytokine secretion and NO secretion by cells of both melanoma lines.     

Cytogenetic analysis of melanoma cell lines: subclone selection in long-term melanoma cell cultures

We employed G-banding cytogenetic analysis to follow the clonal constitution of short-term cultures of metastatic malignant melanoma compared to their long-term cultures. Eight metastatic melanoma cell lines were analyzed. No long-term culture was found to be identical to its line of origin. In all cultures there was a selection of one subclone and emergence of its own subclones. In the majority of cultured tumors (5/8), this process was associated with a decrease in the number of subclones composing the line. We suggest that subclone selection in long-term tumor cultures can be associated with a change in phenotype. Therefore, caution is required when employing long-term cultures for research and therapy.     

共表达MAGE-1与EGFP基因的小鼠黑色素瘤细胞系的建立

目的 :构建黑色素瘤抗原 1(MAGE 1)基因的真核表达载体 ,并在小鼠黑色素瘤B16细胞中进行表达。方法 :采用分子生物学的手段 ,构建了MAGE 1与增强型绿色荧光蛋白 (EGFP)基因共表达的质粒pIRES2 EGFP MAGE 1,通过脂质体以共表达质粒转染B16细胞 ,用荧光显微镜检测细胞中EGFP的表达 ,用免疫组化染色法检测细胞中MAGE 1的表达。结果 :成功地构建了真核表达载体pIRES2 EGFP MAGE 1。以其转染B16细胞后 ,经荧光显微镜及免疫组化染色法检测 ,可见细胞内有EGFP及MAGE 1的表达。结论 :成功地建立了可共表达MAGE 1与EGFP基因的B16细胞 ,为MAGE 1在肿瘤免疫治疗中的应用奠定了研究基础。     

Turnover of histamine in human melanoma cell lines

Inflammation Research -     

Spontaneous release of Fc receptor-like material from human lymphoblastoid cell lines.

In the culture medium of some human lymphoblastoid cell lines material is released with the following properties: (a) hemagglutination reaction of IgG-sensitized erythrocytes; (b) enhancement of precipitation of DNA-anti-DNA complexes; (c) inhibition of binding of C1q to immune complexes; (d) inhibition of immune complex binding to lymphocytes; (e) inhibition of antibody-dependent lymphocytotoxicity. The material is not identical with C1q or rheumatoid factor, it is heat resistant (30 min at 56 degrees C); the molecular weight is about 100 000 daltons and it is capable of inhibiting antibody production in vitro. It is suggested that this material consists of Fc receptors spontaneously shed from lymphocyte membranes.     

下调人高迁移率族蛋白1对人前列腺癌细胞PC-3的影响

目的研究小干扰RNA(siRNA)抑制高迁移率族蛋白1(HMGB1)基因对PC-3侵袭和转移的影响。方法构建真核表达载体Pgenesil-1/HMGB1siRNA,转染PC-3细胞系,通过RT-PCR和Western blot检测HMGB1的mRNA及蛋白;通过细胞划痕、transwell和明胶酶谱分析检测PC-3体外侵袭能力。结果成功构建siRNA表达载体Pgene-sil-1/HMGB1siRNA,可使PC-3细胞HMGB1mRNA和蛋白表达水平显著降低(P<0.05),并能有效抑制PC-3体外侵袭和转移活性(P<0.05)。结论应用siRNA技术能有效的抑制基因的表达,同时也能有效抑制PC-3细胞的体外侵袭和转移,为肿瘤的生物学治疗提供了新思路。     

Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1

High mobility group box 1 (HMGB1) can be actively secreted by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli (such as bacterial endotoxin, TNF-alpha, IL-1, and IFN-gamma) or passively released by necrotic cells and mediates innate and adaptive inflammatory responses to infection and injury. Here, we demonstrated that a reactive oxygen species, hydrogen peroxide (H(2)O(2)), induces active and passive HMGB1 release from macrophage and monocyte cultures in a time- and dose-dependent manner. At nontoxic doses (e.g., 0.0125-0.125 mM), H(2)O(2) induced HMGB1 cytoplasmic translocation and active release within 3-24 h. At higher concentrations (e.g., 0.25 mM), however, H(2)O(2) exhibited cytotoxicity to macrophage and monocyte cell cultures and consequently, triggered active and passive HMGB1 release. In addition, H(2)O(2) stimulated potential interaction of HMGB1 with a nuclear export factor, chromosome region maintenance (CRM1), in macrophage/monocyte cultures. Inhibitors specific for the JNK (SP600125) and MEK (PD98059), but not p38 MAPK (SB203580), abrogated H(2)O(2)-induced, active HMGB1 release. Together, these data establish an important role for oxidative stress in inducing active HMGB1 release, potentially through a MAPK- and CRM1-dependent mechanism.     

Natural human polyreactive IgM induce apoptosis of lymphoid cell lines and human peripheral blood mononuclear cells

Natural polyreactive IgM autoantibodies, encoded by unmutated germline Ig V genes, represent a major fraction of the normal circulating IgM repertoire. We have previously shown that therapeutic preparation of pooled IgM exerts immunomodulatory effects as assessed by in vitro and in vivo studies. Here, we show that the IgM preparation induces cell death in lymphoblastoid cell lines and in human peripheral blood mononuclear cells. The IgM-induced cell death involved classical features of apoptosis such as nuclear fragmentation and activation of caspases. Treatment of leukemic cells with IgM resulted in the cleavage of poly-(A)DP ribose polymerase, a substrate of caspase, and in a reduction in mitochondrial transmembrane potential during the early period of apoptosis induction. Natural IgM-induced apoptosis was inhibited by soluble Fas molecules and affinity-purified Fas antibodies from pooled IgM preparation induced apoptosis in lymphoblastoid cells, suggesting the involvement of the Fas receptor. Our results suggest a role for normal IgM in controlling cell death and proliferation, and imply a possible therapeutic role for IgM in autoimmune and lymphoproliferative disorders.     

Molecular cytogenetic evaluation of 10 uveal melanoma cell lines

Uveal melanoma is the most common intraocular tumor in adults and often results in unilateral blindness and/or death. Previous cytogenetic characterizations of this tumor consistently revealed chromosomal abnormalities involving chromosomes 3, 6, and 8; reports of other abnormalities vary in frequency. We defined cytogenetic abnormalities of this tumor using complementary in situ hybridization techniques on 10 uveal melanoma cell lines. Synthesis of comparative genomic hybridization (CGH) and spectral karyotyping (SKY) results revealed that chromosomal rearrangement is involved in DNA sequence copy number abnormalities throughout the genome, but monosomy 3 was not found. Monosomy 3 is thought to be a significant prognostic indicator, so its absence was investigated further. Fluorescence in situ hybridization (FISH) for chromosome 3 revealed approximately 1 centromere signal per cell, but probes for 3p and 3q revealed multiple telomere signals per cell, suggesting chromosomal rearrangement without whole-chromosome loss. Based on combined CGH, SKY, and FISH data, we propose that chromosome 3 is more frequently involved in chromosomal rearrangements than whole-chromosome loss in uveal melanoma. Future approaches should be designed to confirm and enhance the resolution of regions of imbalance in primary tumors. Once identified, conserved chromosomal alterations that contribute to uveal melanoma may reveal the underlying aspects of uveal melanoma onset, metastasis and resistance to current treatment modalities.     

晚期糖基化终末产物诱导人卵巢颗粒细胞株COV434凋亡并上调促炎因子HMGB1自分泌

目的:探讨晚期糖基化终末产物(advanced glycation end products,AGEs)是否诱导人卵巢颗粒细胞株COV434凋亡,并探究其可能的机制。方法:采用不同浓度AGEs牛血清白蛋白与人卵巢颗粒细胞株COV434共同孵育,流式细胞术观察细胞凋亡率,Western blot观察caspase-3和cleaved caspase-3的蛋白水平,ELISA检测细胞培养液上清中高迁移率族蛋白B1(high mobility group box 1 protein,HMGB1)的含量。结果:与对照组比较,100 mg/L AGEs组和200 mg/L AGEs组早、晚期凋亡率显著增加,各组caspase-3蛋白水平的差异无统计学显著性,但与对照组比较,100 mg/L AGEs组和200 mg/L AGEs组cleaved caspase-3的蛋白水平显著增加(P0.05)。此外,与对照组比较,100 mg/L AGEs组和200 mg/L AGEs组细胞培养液上清中HMGB1促炎介质的水平显著上升(P0.05)。结论:晚期糖基化终末产物诱导人卵巢颗粒细胞株COV434凋亡可能与促炎反应有关。     

HMGB1: guiding immunity from within

Two of the main challenges that eukaryotic multicellular organisms faced during evolution were to eliminate and replace dying cells and to cope with invading microorganisms. The innate immune system evolved to handle both tasks: to scavenge cellular debris and to form the first line of defence against microbes. In this review, we focus on high mobility group box 1 (HMGB1) protein as a common signal that alerts the innate immune system to excessive or deregulated cell death and to microbial invasion. HMGB1, which is well known nuclear protein, has revealed unexpected facets as an extracellular mediator. The role of HMGB1 as an endogenous molecule that facilitates immune responses and has an important role in tissue homeostasis and disease will be highlighted here.