S/MAR-binding properties of Sox2 and its involvement in apoptosis of human NT2 neural precursors.

DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Regulation of DNaseY activity by actinin-alpha4 during apoptosis.

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.

Splenic T lymphocytes die preferentially during heat-induced apoptosis: NuMA reorganization as a marker.

We are investigating nuclear events during apoptosis in mouse splenic lymphocytes cultured immediately after isolation (controls) or after heat treatment (42 degreesC, 30 minutes), and have found that hyperthermia increased the level of apoptosis to double that of spontaneous apoptosis in controls within 6 hours. Immunolabelling for Nuclear Mitotic Apparatus Protein (NuMA) suggested that splenocytes were responding heterogeneously to the heat treatment. Whereas all nuclei in controls and about half of nuclei in heat-treated samples showed the usual diffuse nucleoplasmic labelling, 40-60% of nuclei in heated samples also contained numerous bright spots. We then examined whether the heterogeneity in NuMA organization might be an indication of a differential response of B and T lymphocytes to hyperthermia, and whether the presence of NuMA spots is related to the apoptotic process. NuMA labelling of heated fractionated splenocyte populations showed that 90% of nuclei in T-enriched cultures (less than or equal to 4% IgG+ cells), but only 25% of nuclei in B-enriched samples (less than or equal to 80% IgG+ cells), contained spots. As well, 2 hours after heat treatment of unfractionated cultures, greater than or equal to 90% of nuclei that were accumulating DNA strand breaks, as detected by TUNEL, exhibited NuMA spots. These data indicate that cells with NuMA spots are targetted for, or have initiated, the death program. Since most T cells, but few or no B cells, were spotty after heating, we conclude further that hyperthermia induces apoptosis preferentially in splenic T lymphocytes. The observation that the proportion of T cells was, on average, threefold greater in control than in heated samples after 24 hours in culture reinforces this conclusion.

Unique behaviour of NuMA during heat-induced apoptosis of lymphocytes.

Nuclear collapse is a key feature of apoptosis, reflecting the DNA and protein fragmentation observed biochemically. We have compared nuclear events during spontaneous and heat-induced (42 degrees C for 30 min) apoptosis at the level of individual cells, monitoring overall chromatin organization by staining with 4,6'-diamidino-2-phenylindole (DAPI), DNA cleavage by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), and the nuclear antigens NuMA, PI2, and lamin B by immunofluorescence microscopy. The data were correlated with analyses at the population level by flow cytometry and immunoblotting. We show that heat treatment doubles the level of apoptotic splenocytes with respect to the spontaneous apoptosis in controls within 6 honThe organization and disassembly of nuclear envelope antigens is identical during spontaneous and heat-induced apoptosis and proceeds in a temporally and spatially ordered manner relative to DNA fragmentation and chromatin collapse. In contrast, NuMA organization is affected by heat treatment, with about half of the cells containing many bright spots in addition to the usual diffuse labelling. The spots were still visible in many fully collapsed apoptotic nuclei. Further, this novel reorganization of NuMA and the hyperinduction of apoptosis by heat are correlated, suggesting that lymphocytes with reorganized NuMA are destined to undergo apoptosis. The data also indicate that DNA fragmentation precedes extensive remodelling of the nucleoplasm in these cells, and that chromatin begins to collapse before disassembly of nuclear envelope components.