ST486 Cells Research Articles

Dihydroarteannuin Ameliorates Collagen-Induced Arthritis Via Inhibiting B Cell Activation by Activating the FcγRIIb/Lyn/SHP-1 Pathway.

Background: Dihydroarteannuin (DHA), which is extracted from the traditional Chinese herb Artemisia annua L, exhibits potent immunosuppressive activity in rheumatoid arthritis (RA). Strong evidence indicates that B cells act as an essential factor in the pathogenesis of RA, but research on the immunosuppressive function of DHA in regulating B cells is limited. Objective: To investigate the modulatory effects of DHA on joint destruction, proinflammatory cytokine production, activation, apoptosis and proliferation of B cells and to explore the possible associated mechanism in RA treatment. Methods: Collagen-induced arthritis (CIA) model was established. Weight and joint oedema were record weekly, and joint damage was detected by micro-CT scan. Human Burkitt B lymphoma cells lacking endogenous Fc gamma receptor b (FcγRIIb) gene were transfected with a 232Thr loss-of-function mutant to construct a mutant cell model ST486. The proliferation of ST486 cells was assessed with Cell Counting Kit-8. Apoptosis and activation were tested by flow cytometry. The effects of DHA on the activation of FcγRIIb, protein tyrosine kinases (Lyn), and SH2-containing tyrosine phosphatase-1 (SHP-1) signaling pathways were determined by western blotting. Results: In comparison to model group, bone volume/tissue volume (BV/TV) and bone mineral density (BMD) were increased, whereas joint oedema was decreased in both of the DHA and MTX group. The mRNA and protein expression levels of Interleukin-6 (IL-6) and Tumor necrosis factor-alpha (TNF-α) were decreased after treatment with DHA. In addition, DHA treatment promoted the apoptosis, inhibited the activation and proliferation of ST486 cells. Furthermore, the protein expression levels of FcγRIIb, SHP-1, and Lyn were increased after treatment with DHA. Moreover, the expression of phosphorylated CD19 was also inhibited by DHA. Conclusion: We provide the first evidence that DHA may alleviate collagen-induced arthritis by activating the FcγRIIb/Lyn/SHP-1 signaling pathway in B cell, indicating that DHA is a novel and valuable candidate for RA therapy.

Yishen-tongbi decoction inhibits excessive activation of B cells by activating the FcγRIIb/Lyn/SHP-1 pathway and attenuates the inflammatory response in CIA rats

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Strong evidence supports that excessive activation of B cells plays a critical role in the pathogenesis of RA. Fc gamma receptor b (FcγRIIb) is the B cell inhibitory receptor and inhibits BCR (B cell receptor) signalling in part by selectively dephosphorylating CD19 which is considered a co-receptor for BCR and is essential for B cell activation. Our previous study demonstrated that a FcγRIIb I232T polymorphism presented a strong genetic link to RA and may lead to the excessive activation of B cells. Therefore, novel therapeutic strategies and drugs that can effectively inhibit the excessive activation of B cells by regulating the FcγRIIb are necessary for the treatment of RA. Therefore, we used Burkitt’s lymphoma ST486 human B cells (lacking endogenous FcγRIIb) transfected with the 232Thr loss-of-function mutant to construct a FcγRIIb mutant cell line (ST486), and we demonstrated that YSTB treatment not only reduced proliferation and promoted apoptosis in ST486 cells but also did so in a dose-dependent manner. Furthermore, the intracellular Ca2+ flux of ST486 cells was decreased after treatment with YSTB, inhibiting the excessive activation of ST486 cells, and these effects correlated with the CD19/FcγRIIb-Lyn-SHP-1 pathways. Our data showed that YSTB treatment inhibited the expression of phosphorylated CD19 and upregulated the protein expression of FcγRIIb, Lyn, and SHP-1. Additionally, the CIA model was established to explore the anti-inflammatory and inhibitory effects of YSTB on bone destruction, and we found that YSTB decreased the paw oedema and arthritis index (AI) in CIA rats. It is worth mentioning that YSTB clearly decreased the AI earlier than methotrexate (MTX) (day 10 vs 16). Moreover, synovial hyperplasia, inflammatory cell infiltration and cartilage surface erosion in CIA rats were noticeably reduced after treatment with YSTB as evidenced by histopathological examination. Finally, we found that YSTB treatment suppressed bone erosion and joint space score (JNS) in CIA rats as evidenced by radiographic assessment. In summary, these data suggest that YSTB has great therapeutic potential for RA treatment.

Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus.

BackgroundPublic health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (SARS-CoV). We have previously reported that antibodies elicited by a SARS-CoV vaccine candidate based on recombinant, full-length SARS-CoV Spike-protein trimers, trigger infection of immune cell lines. These observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages.MethodsWe have used primary human immune cells to evaluate their susceptibility to infection by SARS-CoV in the presence of anti-Spike antibodies. Fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were utilized to assess occurrence and consequences of infection. To gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (FcγR), which bind antibody-coated pathogens.ResultsWe show here that anti-Spike immune serum increased infection of human monocyte-derived macrophages by replication-competent SARS-CoV as well as Spike-pseudotyped lentiviral particles (SARS-CoVpp). Macrophages infected with SARS-CoV, however, did not support productive replication of the virus. Purified anti-viral IgGs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. Antibody-mediated infection was dependent on signaling-competent members of the human FcγRII family, which were shown to confer susceptibility to otherwise naïve ST486 cells, as binding of immune complexes to cell surface FcγRII was necessary but not sufficient to trigger antibody-dependent enhancement (ADE) of infection. Furthermore, only FcγRII with intact cytoplasmic signaling domains were competent to sustain ADE of SARS-CoVpp infection, thus providing additional information on the role of downstream signaling by FcγRII.ConclusionsThese results demonstrate that human macrophages can be infected by SARS-CoV as a result of IgG-mediated ADE and indicate that this infection route requires signaling pathways activated downstream of binding to FcγRII receptors.

Cross-linking of Surface IgM in the Burkitt's Lymphoma Cell Line ST486 Provides Protection against Arsenite- and Stress-induced Apoptosis That Is Mediated by ERK and Phosphoinositide 3-Kinase Signaling Pathways

The ST486 cell line, derived from a human Burkitt's lymphoma, is a model for antigen-induced clonal deletion in germinal center B-lymphocytes, with apoptosis induced upon cross-linking of surface IgM. Moreover, this cell line is highly sensitive to the induction of apoptosis by many chemicals, including sodium arsenite, a significant environmental contaminant with immunotoxic activity. In contrast to arsenite and other chemicals, surface IgM cross-linking induces apoptosis in ST486 cells with delayed kinetics. Moreover, the initial signaling events following IgM stimulation are associated with cell survival and proliferation and include activation of the extracellular-signal regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K) pathways. We examined the question of whether IgM-mediated activation of the ERK and PI3K pathways can influence the apoptotic response of ST486 cells following exposure to arsenite and selected drugs with different molecular targets, including cycloheximide, etoposide, and camptothecin, and a physical stress, hyperthermia. Our findings show that IgM-stimulated cells are significantly protected against arsenite and drug-induced apoptosis during a window of several hours after surface IgM cross-linking, as evidenced by an inhibition of cleavage of poly(ADP-ribose) polymerase and lack of morphological changes indicative of apoptosis. Significantly, surface IgM cross-linking also protects against arsenite-induced mitochondrial depolarization as well as caspase-9 cleavage. Furthermore, we demonstrate that this IgM-mediated protection requires the activation of the ERK and PI3K pathways, because inhibition of either pathway blocks the ability of antigen receptor activation to protect against apoptosis. Our study also provides evidence for p90(S6) ribosomal kinase as a point of convergence between the two signaling pathways resulting in the phosphorylation of the pro-apoptotic Bcl-2 family member Bad at serine 112. This investigation demonstrates, for the first time, that specific signals transduced by activation of the B-cell receptor protect cells at a common point of regulation in the apoptotic pathways for diverse stresses.

Novel genomic imbalances and chromosome translocations involving c-myc gene in Burkitt's lymphoma.

In this study, CA46 and ST486, two Epstein-Barr (EBV) negative cell lines derived from sporadic BL, were analyzed by multicolor spectral karyotyping, G-banding, fluorescence in situ hybridization with single-copy gene probes, and comparative genomic hybridization (CGH). In addition to reciprocal t(8;14)(q24;q32) translocation involving c-myc and IgH loci, we identified a t(7;8;14)(q11.2;q24;q32) translocation in CA 46 cells and t(8;14;18)(q24;q32;q23) in ST486 cells. Both rearrangements were not previously described in BL and resulted in transposition of myc sequences in a new genomic configuration. Several DNA imbalances mapped by CGH at the same sites in both lines, may reflect recurrent genomic changes that are relevant to pathogenesis of BL. We tested the tumorigenicity of these lines by injecting cells intraperitoneally in SCID mice. In two separate experiments, CA46 cells produced tumors 2 weeks after cell inoculation while ST486 cells induced only one tumor after a long latency period. Partial duplication of the long arm of chromosome 1 involving variable bands but always band 1q23 is the second most common alteration in BL and is known to be associated with aggressive tumors and poor prognosis. Duplication of the bands 1q23-24 commonly observed in EBV-negative lines was identified only in highly tumorigenic CA46 cells suggesting that this region harbor gene(s) associated with tumor cell invasiveness.

Differential Induction of Apoptosis and MAP Kinase Signaling by Mitochondrial Toxicants in Drug-Sensitive Compared to Drug-Resistant B-Lineage Lymphoid Cell Lines

A panel of human B-lineage lymphoma cell lines differing in cancer drug-resistance status and Bcl-2/Bax expression were used to study the contribution of mitochondrial-based perturbations and regulation in differential induction of apoptosis. Mitochondrial dysfunction was induced in cells by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (mClCCP) and the respiratory chain inhibitor antimycin A. Cells were then assayed for early changes in MAP kinase signaling and subsequent induction of apoptosis. The cancer drug-resistant cell lines EW36 and CA46, overexpressing Bcl-2 and deficient in Bax, respectively, were both resistant to mitochondrial toxicant-induced cleavage of poly(ADP-ribose) polymerase (PARP) and morphologically detectable apoptotic cell death. In contrast, cancer drug-sensitive ST486 cell line, with low Bcl-2 expression, was sensitive to PARP cleavage and apoptosis engagement. Interestingly, mClCCP induced twofold more apoptosis than antimycin A in the ST486 cells. Exposure to the mitochondrial toxicants resulted in the early and preferential activation of the ERK and p38 MAP kinase pathways in only the drug-sensitive ST486 cell line, with mClCCP more potent than antimycin A. Specific inhibition of the p38 pathway augmented baseline and mClCCP-induced apoptosis. These results show that multi-drug-resistant and -sensitive B-lineage cells are also resistant and sensitive to compounds inducing mitochondrial dysfunction. The differential sensitivity to mitochondrial toxicant effects involved regulation by MAP kinases, since ERK and p38 were found to be preferentially activated only in the drug-sensitive B-lineage cells. Modulation of the p38 signaling pathway altered the sensitivity of cells to mitochondrial stress and may play a more general role in regulating the sensitivity of B-lineage cells to drugs and environmental toxicants.

Inhibition of gene expression and cell proliferation by triple helix-forming oligonucleotides directed to the c-myc gene.

Triple helix-forming oligonucleotides (TFOs) bind with high affinity and specificity to homopurine-homopyrimidine sequences in DNA and have been shown to inhibit transcription of target genes in various experimental systems. In the present study, we evaluated the ability of 3'-amino-modified phosphodiester TFOs directed to four sites in the c-myc gene to inhibit gene expression and proliferation of human leukemia (CEM, KG-1, and HL-60) and lymphoma (Raji and ST486) cells. GT-rich TFOs were designed to target sequences located either upstream (myc1 and -2) or downstream (myc3 and -4) of the P2 promoter, which is the major c-myc promoter. Myc2, which was directed to a site immediately upstream of this promoter, inhibited c-myc expression and proliferation of CEM cells. The effects of this TFO were sequence- and target-specific, since control oligonucleotides and TFOs directed to other sites were less or not active. Myc2 was also effective in KG-1, HL-60, and Raji cells. In contrast, ST486 cells were more sensitive to myc3, which targets a sequence in intron 1 upstream of the P3 promoter, than myc2. As result of a chromosomal translocation, P3 is the active promoter in ST486 cells. This study demonstrates the activity and specificity of TFOs designed to act as repressors of c-myc gene expression in human leukemia and lymphoma cells. Our results suggest that this is a valid approach to selectively inhibit gene expression and cancer cell growth, and encourage further investigation of its potential applications in cancer therapy.

Amplification of the translocated c-myc genes in three Burkitt lymphoma cell lines.

Translocations of the coding exons of the human c-myc gene are consistent features of human Burkitt lymphomas (BL). In the BL cell lines CA46, JD40, and ST486, the second and third c-myc exons have been translocated into the immunoglobulin heavy chain locus. In addition to this rearrangement, in all three cell lines, we have found that the translocated c-myc exons show low-level amplification relative to restriction fragments from the germ-line c-myc gene. The patterns of hybridization of an IgM switch region probe suggest that immunoglobulin heavy chain sequences have been co-amplified with the translocated c-myc sequences. Differential sedimentation was used to determine whether the amplified sequences reside in high-molecular-weight chromosomes or low-molecular-weight extrachromosomal DNA. In JD40 and ST486 cells, the amplified c-myc sequences were found on high-molecular-weight chromosomes ST486 cells also contained translocated C-myc sequences in low-molecular-weight, extrachromosomal DNA, as did CA46 cells. These conclusions were corroborated by fluorescence in-situ hybridization (FISH) of HeLa, CA46, ST486 and JD40 metaphase chromosomes. These results suggest that there is ongoing selection for cells containing amplified copies of the expressed c-myc sequences. and that there is continuous generation of extrachromosomal copies of the translocated c-myc sequences in ST486 and CA46 cells.

Anti-IgM-mediated Regulation of c-myc and Its Possible Relationship to Apoptosis

Anti-IgM treatment of Burkitt's lymphoma cells is followed by either growth arrest or induction of apoptosis. In this study we have explored the role of c-myc in these events. Our results in Ramos cells indicate the following. (a) The decline in c-myc mRNA occurs at about 4 h; inhibition of about 80% being observed. (b) The stability of c-myc message is involved since the half-life of c-myc mRNA is decreased from about 30 min in untreated cells to about 15 min following treatment with anti-IgM. In the presence of cycloheximide, a protein synthesis inhibitor, the half-life is increased to about 50 min and was unaltered by treatment with anti-IgM. (c) By contrast, nuclear run-on experiments indicated no change in transcription rates for c-myc message due to treatment with anti-IgM. (d) A decrease in c-myc causes apoptosis since specific repression of c-myc with antisense oligonucleotides decreases the levels of c-Myc, inhibits growth rate, decreases viability, and induces apoptosis. (e) Anti-CD40 inhibition of apoptosis occurs without alteration in anti-IgM-induced down-regulation of c-myc mRNA, suggesting that it acts distally to c-myc down-regulation. Other cell lines were also investigated. In Epstein-Barr virus (EBV)-positive cell lines (Daudi, Raji, and Namalwa), anti-IgM treatment for 24 h results in growth inhibition without induction of apoptosis. In EBV-negative cell lines (ST486 and CA46, as well as Ramos), a more heterogeneous pattern of responses to anti-IgM are observed. Ramos and ST486 cells both show growth inhibition and apoptosis upon anti-IgM treatment; CA46 cells shown only growth inhibition but not apoptosis. Anti-IgM causes a decline in c-myc mRNA levels in all of these lines, as well as in c-Myc protein level in the two lines investigated, Daudi and Ramos, regardless of apoptosis. Addition of antisense c-myc oligonucleotides to the cells reduced growth in both Daudi and Ramos cells lines, however it resulted in substantial apoptosis only in Ramos cells. These results suggest that anti-IgM destabilizes c-myc mRNA by a process that involves mRNA turnover, rather than transcription rates. However anti-IgM exerts differential effects in EBV-positive and EBV-negative cell lines. EBV-positive cells are uniformly resistant to apoptosis, while EBV-negative cell lines show a tendency to apoptosis but with exceptions. Growth inhibition can be uncoupled from apoptosis in EBV-positive cell lines, but not in those EBV-negative cell lines prone to apoptosis. Furthermore, down-regulation of c-myc message correlates with growth inhibition in these cells, but is an insufficient link to apoptosis. By contrast inhibition of apoptosis by anti-CD40 occurs even though c-myc mRNA is decreased.

Differential effect of heat shock on RNA metabolism in human Burkitt's lymphoma B-cell lines

Thermal stress induces expression of a family of heat shock proteins which may regulate the synthesis of various cellular genes. We investigated the effect of heat shock on polyadenylation in Epstein-Barr Virus (EBV) negative and EBV transformed human Burkitt's lymphoma (BL) B-cell lines. Incubation of the BL B-cell line P3HR-1, carrying the defective EBV genome [EBV nuclear antigen-2 gene deletion] at 46 °C for 15 min increased nuclear poly(A) polymerase (PAP) activity. Thereafter, enzymatic activity declined and at 60 min it was reduced to about 50% of that observed in cells incubated at 37 °C. In contrast, no significant increase in PAP activity was observed at 15 min or thereafter in an EBV − BL cell line, ST-486, in response to elevated temperature. Furthermore, no heat shock mediated change in nuclear poly(A)-specific endonuclease activity was observed in either P3HR-1 or ST-486 cells suggesting a specific effect on PAP activity. However, thermal stress dependent increase in c-myc expression was detected only in P3HR-I cells. These results suggest an association between EBV transformation and enhanced expression of c-myc and PAP activity. To further determine the role of EBV, an EBV − BL cell line, BL-30, and BL-30 cells infected in vitro with a wild type strain of EBV, BL-30/B95-8, were investigated. BL-30/B-95-8, unlike the parental BL-30 cells, exhibited c-myc and PAP gene upregulation at 15 min but were downregulated at 60 min following exposure of cells to elevated temperatures. These results suggest that infection of human B-cells with EBV is associated with their ability to respond to thermal stress by increased PAP activity which may stabilize mRNA through enhanced polyadenylation.

Opposite replication polarity of the germ line c-myc gene in HeLa cells compared with that of two Burkitt lymphoma cell lines.

To study the cell type specificity of the direction of replication of the human c-myc genes and the relationship of replication polarity to transcriptional activity, we analyzed the directions of replication of the c-myc genes in two Burkitt lymphoma cell lines, CA46 and ST486, and in HeLa cells. On the basis of in vitro runoff replication of forks initiated in intact cells, we found that transcribed c-myc genes in the germ line configuration in HeLa cells were replicated in the direction of transcription from origins in the 5'-flanking DNA, while the repressed, unrearranged c-myc genes of CA46 and ST486 cells were replicated in the antitranscriptional direction. In contrast, the transcribed c-myc genes of CA46 cells were replicated in the transcriptional direction, while the translocated, amplified c-myc genes of ST486 cells showed no preferred polarity of replication. The data also provided evidence for the existence of an endogenous barrier to DNA polymerases in the flanking DNA immediately 5' to the HeLa c-myc genes.

Translocated c-myc oncogene of Burkitt lymphoma is transcribed in plasma cells and repressed in lymphoblastoid cells.

We examined somatic cell hybrids between Burkitt lymphoma cells and either human lymphoblastoid cells or mouse plasmacytoma cells for the expression of the translocated c-myc oncogene. The results of this study indicate that the translocated c-myc oncogene is transcribed in plasma cells but is repressed in lymphoblastoid cells. Thus, the factors necessary for translocated c-myc transcription are present in plasma cells and Burkitt lymphoma cells but are absent or inactive in lymphoblastoid cells. Since the distance between the rearranged immunoglobulin loci and the c-myc oncogene can even exceed 30-50 kilobases, we speculate that the translocated c-myc oncogene is under the transcriptional control of enhancer-like elements capable of acting over long distances. The activity of this long-range enhancer may depend on the interaction with transacting factors that are active in plasma cells and in Burkitt lymphoma cells but are not active in lymphoblastoid cells. We also examined the transcription of the first exon of the c-myc oncogene, which becomes separated from the second and third exon because of the chromosomal break involving the first intron. This exon is transcribed at high levels in ST486 Burkitt lymphoma cells with the t(8;14) chromosome translocation. Hybrids between lymphoblastoid and ST486 cells expressed high levels of transcripts of the first exon, whereas hybrids between plasma cells and ST486 cells did not. Thus, transcription of the separated first exon can be enhanced in lymphoblastoid and Burkitt lymphoma cells because of its close proximity to the heavy chain enhancer that is normally located between the joining and the switch region of the C mu gene. Such enhancement, however, does not occur in plasma cells, possibly because these cells are able to suppress completely the c-myc oncogene, unless it has been placed in the proximity of a rearranged immunoglobulin constant region gene.