Mine Eray

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.

Follicular Lymphoma Cell Lines, an In Vitro Model for Antigenic Selection and Cytokine‐Mediated Growth Regulation of Germinal Centre B Cells

In the periphery, B cells differentiate in germinal centres (GCs) of secondary lymphoid organs. Isolated GC cells die quickly in vitro by apoptosis. Therefore, cell lines originating from follicular lymphomas, which are the malignant counterparts of GC B cells, would provide a stable in vitro model to study the immunobiology of GC B cells. We have established three novel human follicular lymphoma cell lines that were characterized with special reference to immunophenotypic features, response to B‐cell receptor (BCR) triggering, response to cytokines and cytokine mRNA expression. One of the cell lines, HF‐1A3, has a phenotype of a centrocyte. It expresses surface immunoglobulin G (sIgG) and dies by apoptosis following BCR cross‐linking. Co‐stimulation with interleukin‐6 (IL‐6), IL‐15 or interferon‐γ (IFN‐γ) rescues HF‐1A3 cells from BCR‐induced apoptosis. The second cell line, HF‐28, also represents phenotypically an IgG+ centrocyte. Ligation of its BCR leads to the cell‐cycle arrest at G1 instead of apoptosis. HF‐28 cells express both CD45RA and RO isoforms, which is unusual in B lymphocytes apart from plasma cells, thus suggesting a transition to plasma cell phenotype. The third cell line, HF‐4.9, which phenotypically represents an sIgM+ centroblast, responds by proliferation to BCR cross‐linking. These cell lines offer a unique in vitro model to study antigenic selection and cytokine‐mediated growth regulation of human GC B cells.

The involvement of mitochondria and the caspase-9 activation pathway in rituximab-induced apoptosis in FL cells

Despite the wide use of anti-CD20 antibody rituximab in the cancer treatment of B cell malignancies, the signalling pathways of CD20-induced apoptosis are still not understood. By using dominant negative (DN)-caspase-9 overexpressing follicular lymphoma cells we demonstrated that the activation of caspase-9 was essential for rituximab-mediated apoptosis. The death receptor pathway mediated by caspase-8 activation was not involved in rituximab-mediated apoptosis since overexpression of FLIPshort or FLIPlong proteins, inhibitors of caspase-8 activation, could not inhibit rituximab-induced apoptosis. However, the death receptor pathway activation by anti-Fas antibodies showed an additive effect on rituximab-induced apoptosis. The stabilisation of the mitochondrial outer membrane by Bcl-xL overexpression inhibited cell death, showing the important role of mitochondria in rituximab-induced apoptosis. Interestingly, the rituximab-induced release of cytochrome c and collapse of mitochondrial membrane potential were regulated by caspase-9. We suggest that caspase-9 and downstream caspases may feed back to mitochondria to amplify mitochondrial disruption during intrinsic apoptosis.

Dexamethasone-induced apoptosis and up-regulation of Bim is dependent on glycogen synthase kinase-3.

Glucocorticoids are commonly used in the treatment of lymphoid malignancies. In this study, we show that apoptosis induced by dexamethasone (Dex), a synthetic glucocorticoid, was dependent on mitochondria, since overexpression of Bcl-X(L) prevented Dex-induced apoptotic changes. Dominant negative (DN) caspase-9 also prevented Dex-induced apoptotic changes including the loss of mitochondrial membrane potential indicating that caspase-9 controls mitochondrial changes. In addition, we evaluated the role of glycogen synthase kinase (GSK3) in Dex-induced apoptosis. Inhibition of GSK3 attenuated Dex-induced up-regulation of Bim, loss of mitochondrial membrane potential, release of cyt c and DNA fragmentation. These results indicate that GSK3 contributes to Dex-induced apoptosis by controlling up-regulation of Bim.

Kinetics and signaling requirements of CD40-mediated protection from B cell receptor-induced apoptosis

In the present study we used a human follicular lymphoma cell line, HF1A3, as an in vitro model for the antigen‐driven selection process in germinal centers. Apoptosis can be induced in HF1A3 cells by B cell receptor (BCR) stimulation, but the molecular mechanisms and kinetics of this process are largely unknown. We demonstrate here that there is over 12 h delay between receptoractivation and the execution phase of apoptosis, i.e. disruption of mitochondrial membrane potential, release of cytochrome c from mitochondria, caspase‐3 activation and DNA fragmentation.New protein synthesis is required for mitochondrial alterations and subsequent apoptosis to occur, as these processes are completely blocked by the protein synthesis inhibitor cycloheximide. All the apoptotic events induced by BCR triggering are completely reversed by CD40 ligation with anti‐CD40 antibody. CD40 ligation can reverse the apoptotic process in HF1A3 cells almost until the first mitochondrial events take place demonstrating that CD40‐mediated protection operates very fast and at or before mitochondrial phase of apoptosis. Using specific inhibitors of cell signaling we coulddemonstrate that Raf‐extracellular signal‐regulated kinase, phosphatidylinositol 3‐kinase, p38 or protein kinase C activation pathways are not involved in CD40‐mediated protection from BCR‐induced apoptosis in HF1A3 cells.

PDTC enables type I TRAIL signaling in type II follicular lymphoma cells.

Based on Bcl-X(L) overexpression studies we identified type I and type II follicular lymphoma cell lines in response to TRAIL. We demonstrate here that either amount of caspase-8 activation or Bid cleavage could not define the dependence on mitochondria. Furthermore, an inhibitor of NF-kappaB, PDTC, enabled TRAIL to activate type I apoptotic pathway in type II cells. However, an inhibitor of IKK did not switch apoptosis to type I pathway in type II cells, indicating that NF-kappaB might not be responsible for the switch.

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‐xL, c‐FLIPlong or dominant negative (DN) caspase‐9. Fas‐induced apoptosis was dependent on the caspase‐8 activation, since the overexpression of c‐FLIPlong, 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‐xL 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 (ΔΨm) 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.

Somatic hypermutations in the immunoglobulin genes of two new human lymphoma lines of lymphatic follicle origin.

Variable immunoglobulin heavy‐chain regions (VDJ) of two newly established human lymphoma cell lines (HF‐1 and HF‐4) were sequenced. The most homologous germline VH gene found for both the HF‐1 and HF‐4 sequences was VH26 of the VH3a (V gene) family (82% and 91 % homologies, respectively). The JH region of the HF‐4 heavy‐chain sequence contained two nucleotide differences compared to the published germline JH3 gene. The DHJH region of the HF‐1 gene had a record high number (20%) of somatic mutations.

The Effect of Microenvironmental CD40 Signals on TRAIL- and Drug-induced Apoptosis in Follicular Lymphoma Cells

Follicular lymphoma (FL) cells are malignant counterparts of germinal centre (GC) B cells. Microenvironment of FL B cells has an important role in the progression of FL and might also have an impact on the treatment of FL. CD40 is an important mediator of microenvironmental survival signals in GCs. Here we studied responses of CD40 signalling on TRAIL‐, dexamethasone‐ and doxorubicin‐induced apoptosis in three human FL cell lines. In two of the FL cell lines, CD40 protected cells from apoptosis which was entirely dependent on the activation of NF‐κB. In one of the FL cell lines, CD40 induced apoptosis itself. However, inhibition of NF‐κB induced apoptosis in all three FL cell lines. Therefore, our results indicate that inhibitors of NF‐κB or critical downstream anti‐apoptotic targets of NF‐κB instead of blocking CD40 antibodies in combination with TRAIL or other cytotoxic agents should be considered in the treatment of FL in order to prevent the protective effect of the microenvironment.

Differential Expression of Bcl-2 Family Proteins Determines the Sensitivity of Human Follicular Lymphoma Cells to Dexamethasone-mediated and Anti-BCR-mediated Apoptosis.

Bcl-2 family comprises proapoptotic and antiapoptotic proteins. The balance between these proteins is critical for the survival of the cells. Overexpression of the antiapoptotic protein, Bcl-2, is the hallmark of follicular lymphoma (FL). High expression of Bcl-2 provides survival advantage and may facilitate chemotherapeutic resistance in FL. In the present study, we examined expression profile of Bcl-2 family proteins such as Bcl-2, Bcl-xL, and Bim in human FL cell lines, HF1A3 and HF28. We assessed the correlation between the expression levels of these proteins and cells’ sensitivity to dexamethasone (Dex)-mediated and B-cell receptor (BCR)-mediated apoptosis. Here, we show that Dex and anti-BCR-induced synergistic apoptosis which correlated with significant downregulation of Bcl-xL, inhibition of ERK1/2 phosphorylation and accumulation of nonphosphorylated Bim. However, HF28 cells were less sensitive than HF1A3 cells to Dex-induced and anti-BCR-induced apoptosis due to high Bcl-2 protein level. It is interesting to note that, a Bcl-2-specific inhibitor, ABT-199, sensitized HF28 cells to Dex-induced or anti-BCR-induced apoptosis. In addition, overexpression of Bcl-xL prevented Dex-mediated, anti-BCR-mediated, and ABT-199-mediated apoptosis, indicating that mitochondria were involved. In conclusion, these data show that the expression levels of Bcl-2 family proteins may serve to predict tumor response to BH3 mimetics and the sensitivity of FL cells to Dex-induced and anti-BCR-induced apoptosis. Moreover, our results show that BCR-targeted apoptosis might have therapeutic benefit against FL and B-cell lymphomas.