Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

Identification of ETS-like transcription factor 4 as a novel androgen receptor target in prostate cancer cells.

Transcriptional control by androgens via androgen receptor (AR) is strongly involved in prostate cancer development, but the critical target genes have remained elusive. We have characterized E twenty-six-like transcription factor 4 (ELK4) (also known as serum response factor accessory protein 1) as a novel AR target in human prostate cancer cells. In-silico screening identified three putative AR response elements (AREs) within -10 kb from the transcription start site of ELK4. Both ARE1 at -167/-153 and ARE2 at -481/-467 bound AR in vitro and mediated androgen induction as isolated elements in transcription assays in non-prostate cells. However, merely the ARE2 that cooperates with a proximal forkhead box A1-binding site was critical for the AR-dependent activation of ELK4 promoter in prostate cancer cells. Preferential loading of holo-AR onto the ARE2 and concomitant recruitment of RNA polymerase II onto the ELK4 promoter was confirmed in prostate cancer cells by chromatin immunoprecipitation. Database searches indicated that the expression of ELK4 is markedly increased in prostate cancers relative to normal prostates. Moreover, prostate cancer tissue immunostainings showed that nuclear ELK4 levels are significantly increased in androgen-refractory prostate cancers compared to untreated tumours. Reduction of the amount of ELK4 in LNCaP cells by RNAi retarded cell growth. In conclusion, ELK4 is a direct AR target in prostate cancer cells. Androgens may thus contribute to the growth of prostate cancer via influencing ELK4 levels.

Human B cells and macrophages cooperate in T-cell-independent type 2 response.

We have analysed separately the role of B-cell receptor (BCR) stimulation and the soluble second signal in the T-cell-independent type 2 (TI-2) B-cell response. We were able to show that human B cells and macrophages (Mphi) could function together in TI-type microbial response. Interestingly, BCR cross-linking of peripheral blood (PB) B cells enhanced IgG production induced by Mphi-derived growth factors whereas interleukin (IL)-12 + IL-18 had milder effect on IgG production. We demonstrated that B-cell-derived soluble mediators primed lipopolysaccharide (LPS)-stimulated Mphi for tumour necrosis factor-alpha (TNF-alpha) and IL-6 production significantly better than IFN-gamma, confirming the role of B cells in the activation of Mphi. We could show that human PB B cells were active cytokine producers and could be induced to produce interferon (IFN)-gamma mRNA in the presence of known Mphi cytokines, like IL-12 and IL-18. BCR stimulation also stabilized and enhanced the IFN-gamma mRNA production induced by IL-12 and IL-18. In addition, our novel finding was that a known Mphi cytokine, IL-10, induced the expression of IFN-gamma mRNA from human B-cell line (HF28R0) cells. In summary, we propose a model for the active role of B cells in the induction of the inflammatory response during TI antigen challenge in close collaboration with Mphi.

CD45RA and RO isoforms have distinct effects on cytokine- and B-cell-receptor-mediated signalling in human B cells.

The common leucocyte antigen, CD45, is widely expressed on the surface of lymphocytes. In T and B cells, CD45 has an important role in the early events of receptor signalling. However, the role of various CD45 isoforms in B-cell receptor (BCR)- and cytokine-induced signalling and proliferation is still unclear. In the present study, we establish two follicular lymphoma cell lines expressing either CD45RA (HF28RA) or CD45R0 (HF28R0) isoforms. It was observed that the two isoforms had distinct effects on BCR- or cytokine-induced cellular proliferation. BCR stimulation significantly increased the proliferation of HF28R0 cells, in contrast to a decreased proliferation of HF28RA cells. Moreover, proliferation of HF28R0 cells significantly increased after the addition of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, interferon-gamma and tumour necrosis factor-alpha cytokines, whereas most of these cytokines significantly inhibited the proliferation of HF28RA cells. In addition, the cell lines had their individual cytokine mRNA expression profiles after BCR stimulation. We also analysed the effect of CD45 isoforms on intracellular signalling after BCR stimulation. It was found out that the kinetics of ERK1/2 MAP kinase phosphorylation was clearly faster in HF28R0 than in HF28RA cells. The phosphorylation of other analysed MAP kinases or PTKs was very similar in the cell lines.

Flow cytometric analysis of apoptotic subpopulations with a combination of annexin V-FITC, propidium iodide, and SYTO 17.

BACKGROUND: We have previously characterized apoptotic cell death induced in a follicular lymphoma cell line, HF-1, after triggering via the B-cell receptor (BCR) or treatment with Ca(2+) Ionophore A23187. We analyzed the kinetics of apoptosis induced by these two treatments, as two alternative models of classical apoptosis, by flow cytometry using a novel combination of cytofluorometric stains. METHODS: Cells were stained with a combination of Annexin V-FITC, propidium iodide (PI), and SYTO 17 and analyzed by a two-laser flow cytometry system using 488-nm argon and 633-nm HeNe air-cooled lasers. RESULTS: In both apoptotic models, the first apoptotic cells were detected by SYTO 17 staining. The alteration in SYTO 17 staining intensity was followed by an increased uptake of PI. Finally, the apoptotic cells were labeled with Annexin V in BCR-induced apoptosis. On the contrary, on treatment with Ca(2+) Ionophore A23187, cells became positive for Annexin V earlier than for PI. CONCLUSIONS: The novel cytofluorometric dye, SYTO 17, discriminates apoptotic alterations before Annexin V and PI. PI also discriminates apoptotic alterations before the loss of plasma membrane asymmetry by BCR but not by Ca(2+) Ionophore A23187-induced apoptosis. Finally, the combination of these three cytofluorometric dyes allows effective detection of apoptotic subpopulations and ordering of apoptotic events by flow cytometry.