Frataxin participates to the hypoxia-induced response in tumors.

Defective expression of frataxin is responsible for the degenerative disease Friedreich's ataxia. Frataxin is a protein required for cell survival since complete knockout is lethal. Frataxin protects tumor cells against oxidative stress and apoptosis but also acts as a tumor suppressor. The molecular bases of this apparent paradox are missing. We therefore sought to investigate the pathways through which frataxin enhances stress resistance in tumor cells. We found that frataxin expression is upregulated in several tumor cell lines in response to hypoxic stress, a condition often associated with tumor progression. Moreover, frataxin upregulation in response to hypoxia is dependent on hypoxia-inducible factors expression and modulates the activation of the tumor-suppressor p53. Importantly, we show for the first time that frataxin is in fact increased in human tumors in vivo. These results show that frataxin participates to the hypoxia-induced stress response in tumors, thus implying that modulation of its expression could have a critical role in tumor cell survival and/or progression.

Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis.

Increased brain ganglioside levels are a hallmark of various neuroinflammatory pathologies. Here, we provide evidence that murine microglia can secrete disialoganglioside GD3 upon exposure to inflammatory stimuli. Comparison of different neural cell types revealed a particular and specific sensitivity of oligodendrocytes towards exogenous GD3. Oligodendrocyte death triggered by GD3 was preceded by degeneration of cellular processes, and associated with typical features of apoptosis, such as chromatin condensation, exposure of phosphatidylserine, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential, followed by the loss of plasma membrane integrity and detachment of disintegrated oligodendrocytes. Overexpression of bcl-2 partially protected oligodendrocytes from death. In contrast, treatment with the pan-caspase inhibitor zVAD-fmk did not prevent phosphatidylserine exposure, chromatin margination at the nuclear periphery, and death, although caspase-3 was blocked. Thus, GD3 produced by microglia under neuroinflammatory conditions may function as a novel mediator triggering mitochondria-mediated, but caspase-independent, apoptosis-like death of oligodendrocytes.

The human anti-bullous pemphigoid monoclonal autoantibody P22 is encoded by genes of the IGHV4 and IGLV4 families.

We identified, cloned, and biochemically characterized the full-length cDNAs encoding the heavy and light chains of a human monoclonal antibody (mAb) from the Epstein-Barr virus (EBV)-cell line P22. The cell line P22, which originated from a patient with bullous pemphigoid (an autoimmune disease causing skin blistering) expressed immunoglobulin-G (IgG) with a lambda light chain. Although the variable heavy (IGHV) chain gene family could not be assigned by IGHV repertoire analysis, the determination of its nucleotide sequence demonstrated that the heavy chain of P22 belonged to the IGHV4 family. The limited IGHV4 gene usage by memory IgG, IGA and IgE-expressing cells supports the notion of the autoreactivity-associated IGHV4 genes and stresses the strong selection pressure within germinal centres towards IGHV4 family. Alignment of P22 IGHV4 cDNA sequence to germline sequences from gene databases, revealed a remarkable divergence, suggesting that the heavy chain of the P22 mAb encodes a distinct IGHV4 gene. The variable light chain (IGLV) encodes a IGLV4 gene and is 98% similar to a previously reported IGLV gene. Furthermore, fluorescent staining with the recombinant mAb showed the same reactivity to that of the native antibody. The data reported herein, (a) reveal an autoantibody encoding a distinct IGHV4 gene, (b) confirm the notion that autoantibodies preferentially use IGHV4 genes, and (c) hypothesize that somatic hypermutation within GC may be a mechanism by which autoreactive B lymphocytes escape negative selection.

GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion.

Lipid and glycolipid diffusible mediators are involved in the intracellular progression and amplification of apoptotic signals. GD3 ganglioside is rapidly synthesized from accumulated ceramide after the clustering of death-inducing receptors and triggers apoptosis. Here we show that GD3 induces dissipation of DeltaPsim and swelling of isolated mitochondria, which results in the mitochondrial release of cytochrome c, apoptosis inducing factor, and caspase 9. Soluble factors released from GD3-treated mitochondria are sufficient to trigger DNA fragmentation in isolated nuclei. All these effects can be blocked by cyclosporin A, suggesting that GD3 is acting at the level of the permeability transition pore complex. We found that endogenous GD3 accumulates within mitochondria of cells undergoing apoptosis after ceramide exposure. Accordingly, suppression of GD3 synthase (ST8) expression in intact cells substantially prevents ceramide-induced DeltaPsim dissipation, indicating that endogenously synthesized GD3 induces mitochondrial changes in vivo. Finally, enforced expression of bcl-2 significantly prevents GD3-induced mitochondrial changes, caspase 9 activation, and apoptosis. These results show that mitochondria are a key destination for apoptogenic GD3 ganglioside along the lipid pathway to programmed cell death and indicate that relevant GD3 targets are under bcl-2 control.

Lipid signaling in CD95-mediated apoptosis.

Ceramides play an important role mediating different cell responses such as proliferation, differentiation, growth arrest and apoptosis. They are released upon sphingomyelin hydrolysis which occurs after triggering of a number of cell surface receptors including CD95. Ceramide generation also regulates glycosphingolipid and ganglioside metabolism. In particular, ganglioside GD3 biosynthesis represents an important event for the progression of apoptotic signals generated by CD95 and mediated by ceramide in hematopoietic cells.

Requirement for GD3 ganglioside in CD95- and ceramide-induced apoptosis.

Gangliosides participate in development and tissue differentiation. Cross-linking of the apoptosis-inducing CD95 protein (also called Fas or APO-1) in lymphoid and myeloid tumor cells triggered GD3 ganglioside synthesis and transient accumulation. CD95-induced GD3 accumulation depended on integral receptor "death domains" and on activation of a family of cysteine proteases called caspases. Cell-permeating ceramides, which are potent inducers of apoptosis, also triggered GD3 synthesis. GD3 disrupted mitochondrial transmembrane potential (DeltaPsim), and induced apoptosis, in a caspase-independent fashion. Transient overexpression of the GD3 synthase gene directly triggered apoptosis. Pharmacological inhibition of GD3 synthesis and exposure to GD3 synthase antisense oligodeoxynucleotides prevented CD95-induced apoptosis. Thus, GD3 ganglioside mediates the propagation of CD95-generated apoptotic signals in hematopoietic cells.

Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation.

Human tonsillar B cells were separated into naive IgD+CD38-CD23- (Bm1) and IgD+CD38-CD23 (Bm2), germinal center IgD-CD38+CD23- centroblasts (Bm3) and IgD-CD38+CD77- centrocytes (Bm4) and memory IgD-CD38- (Bm5) subsets. Previous IgVH sequence analysis concluded that the triggering of somatic mutations occurs during the transition from Bm2 subset into the Bm3 subset. To determine the initiation of isotype switching, sterile transcript expression was analyzed by amplification, cloning, and sequencing. A selective sterile I gamma, I alpha, and I epsilon expression was observed at centrocyte (Bm4) stage, suggesting that isotype switch is triggered within germinal centers, after somatic mutation is initiated with centroblasts (Bm3). Finally, the high level of 5'S gamma-S mu 3' DNA switching circles observed in germinal center B cells indicates that within human tonsils, germinal center is a major location for isotype switching.

Interleukin-10 induces immunoglobulin G isotype switch recombination in human CD40-activated naive B lymphocytes.

Upon activation, B lymphocytes can change the isotype of the antibody they express by immunoglobulin (Ig) isotype switch recombination. In previous studies on the regulation of human IgG expression, we demonstrated that interleukin 10 (IL-10) could stimulate IgG1 and IgG3 secretion by human CD40-activated naive (sIgD+) tonsillar B cells. To assess whether IL-10 actually promotes the DNA recombination underlying switching to these isotypes, we examined the effect of IL-10 on the generation of reciprocal products that form DNA circles as by-products of switch recombination. The content of reciprocal products characteristic of mu-gamma recombination was elevated after culture of CD40-activated tonsillar sIgD+ B cells with either IL-4 or IL-10, although high levels of IgG secretion were observed only with IL-10. Unlike IL-4, IL-10 did not induce reciprocal products of mu-epsilon and gamma-epsilon switch recombination. These results demonstrate that IL-10 promotes both switching to gamma and IgG secretion.

B-chronic lymphocytic leukemias can undergo isotype switching in vivo and can be induced to differentiate and switch in vitro.

B-chronic lymphocytic leukemias (B-CLL) represent expanded clones of resting B lymphocytes that mostly express surface IgM (sIgM). The present study shows that B-CLL cells, freshly isolated from two patients, were sIgM+, sIgG-, and sIgA- but expressed IgG and IgA transcripts. cDNA cloning and sequencing showed that the VDJ segments associated to gamma and alpha heavy chain transcripts are identical to those from mu transcripts, thus showing that B lymphocytes giving rise to CLL cells have undergone isotype switching in vivo. Stimulation of these B-CLL cells through surface CD40 in the presence of interleukin-10 induced them to secrete IgG and IgA, proving that they can also differentiate into Ig-secreting cells. Finally, CD40-stimulated B-CLL cells were induced to switch towards IgE in response to interleukin-4, as shown by the presence of specific VDJ-C epsilon transcripts and the secretion of IgE. Therefore, B-CLL cells tested herein can undergo isotype switching in vivo and can be induced to undergo further isotype switching and differentiation in vitro.