Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF-kappaB/IkappaB alpha survival pathway. In this study, the consequences of altered NF-kappaB/IkappaB alpha pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular-FLICE inhibitory protein (c-FLIP), which is dependent on the NF-kappaB pathway in normal muscle cells, is down-regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF-kappaB is readily activated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF-kappaB target genes. IkappaB alpha is expressed following NF-kappaB activation independent of the CAPN3 status, whereas expression of c-FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF-kappaB-dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF-kappaB pathway could be part of the mechanism responsible for the muscle wasting resulting from CAPN3 deficiency.

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Epigenetic perturbations are increasingly described in cancer cells where they are thought to contribute to deregulated gene expression and genome instability. Here, we report the first evidence that a distinct category of chromosomal translocations observed in human tumours—those targeting 1q12 satellite DNA—can directly mediate such perturbations by promoting the formation of aberrant heterochromatic foci (aHCF). By detailed investigations of a 1q12 translocation to chromosome 2p, in a case of human B cell lymphoma, aberrant aHCF were shown to be localized to the nuclear periphery and to arise as a consequence of long range ‘pairing’ between the translocated 1q12 and chromosome 2 centromeric regions. Remarkably, adjacent 2p sequences showed increased levels of repressive histone modifications, including H4K20me3 and H3K9me3, and were bound by HP1. aHCF were associated to aberrant spatial localization and deregulated expression of a novel 2p gene (GMCL1) that was found to have prognostic impact in diffuse large B cell lymphoma. Thus constitutive heterochromatin rearrangements can contribute to tumourigenesis by perturbing gene expression via long range epigenetic mechanisms.

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Platelet !&coprotein (GP) IIb is one of the two subunits of the common platelet adhesion receptor, GPIIbIIIa. The isolation, characterization and sequencing of cDNA clones encoding for the two polypepetide chains of GPIIb are described. A number of clones were isolated from I g t l l libraries constructed with mRNA from an erythroleukemic cell line, HEL, and human megakaryocytes. Two of these clones, IIIbl, from HEL cells, and AIIb2, from megakaryocytes, cross-hybridized and were selected for detailed analysis. ythe identification of these as authentic GPIIb clones was based on immunological criteria and confirmed by the presence of nucleotide sequences in each insert encoding for known protein sequences of platelet GPIIb. These clones contained inserts of 1.54 kb and 1.39 kb, respectively, with an overlapping sequence of 801 bp. The nucleotide sequence of the overlapping region was identical indicating that HEL cells produce a protein closely related, if not identical, to platelet GPIIb. The determined nucleotide sequence of two inserts included a coding sequence for 648 amino acid residues, a TAG stop codon and 185 nucleotides of 3' non-coding sequence followed by a poly(A) tail. The coding sequence contained a portion of the heavy chain, the junction between the heavy and light chains and the entire light chain including a potential transmembrane-spanning domain and a short cytoplasmic tail. When these cDNA were used to probe for GPIIb mRNA, a single mRNA species of 3.9 kb was identified in both HEL cells and human megakaryocytes. A comparison of the deduced amino acid sequence for GPIIb with those of the c( Subunit of the vitronectin and the fibronectin receptors revealed extensive homologies. These homologies further establish that GPIIb-IIIa from platelets, together with the vitronectin and the fibronectin receptors, are members of a supergene family of adhesion receptors with a recognition specificity for Arg-Gly-Asp amino acid sequences.

This article describes the study of the functional relationship between auto‐tumor‐reactive CD4+ T cell clones (TCC) and autologous malignant B cells. Four auto‐tumor‐reactive CD4+ TCC were derived from tumor‐infiltrating T lymphocytes (TIL‐T) from a freshly isolated human follicular lymphoma by the following technique: total CD4+ TIL‐T were negatively purified by an immunomagnetic procedure, then CD4+ TCC were obtained by limiting dilution in the presence of IL‐2 and autologous non‐irradiated follicular lymphoma cells as feeders. After expansion, these CD4+ TCC were co‐cultured with non‐irradiated autologous malignant B cells. All four TCC were activated by B lymphoma cells and proliferated, as assessed by CD25 expression and cell cycle analysis. Activation and proliferation of B lymphoma cells were studied in response to activated CD4+ T cells. Although all four TCC were able to induce B lymphoma cell activation (Ki‐67 antigen induction and CD40 up‐regulation), cells were subsequently blocked in G1 phase. Activation of B‐NHL cells was mediated by TCR‐HLA class II interaction, as shown by a blocking experiment using an anti‐CD4 monoclonal antibody (mAb). Since anti‐CD40 mAb with or without IL‐4 did not induce proliferation of B lymphoma cells in contrast to normal B cells, we suggest that the blockade in G1 phase is due to the presence of abnormalities in B lymphoma cells. This is the first evidence that autologous reactive CD4+ TCC can engage follicular lymphoma B cells to enter the cell cycle and induce an aborted activation stage.

Summary Multiple mechanisms exist by which tumour cells can escape CD95‐mediated apoptosis. Previous studies by our laboratory have shown that primary B cells from non‐Hodgkin's Lymphoma (B‐NHL) were resistant to CD95‐induced cell death. In the current study, we have analysed the mechanisms underlying CD95 resistance in primary human lymphoma B cells. We report that FADD (FAS‐associated death domain protein) and caspase‐8 were constitutively expressed in lymphoma B cells and that the CD95 pathway was blocked upstream to caspase‐8 activation. However, caspase‐8 was processed and functional after treatment with staurosporine (STS). We found that the expression levels of FLICE (FADD‐like interleukin‐1 beta‐converting enzyme)‐Inhibitory Protein (c‐FLIP) and Bcl‐2‐related proteins were heterogeneous in B‐NHL cells and were not related to CD95 resistance. Finally, we report the absence of a CD95‐induced signalling complex [death‐inducing signalling complex (DISC)] in lymphoma B cells, with no FADD and caspase‐8 recruitment to CD95 receptor. In contrast, DISC formation was observed in CD95‐resistant non‐tumoural (NT) B cells. Therefore, we propose that the absence of DISC formation in primary lymphoma B cells may contribute to protect these cells from CD95‐induced apoptosis.

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