Recent studies suggest that proteases of the interleukin 1-beta-converting enzyme (ICE)/ced-3 family are involved in initiating the active phase of apoptosis. Here we identify a novel protease resembling ICE (prICE) that is active in a cell-free system that reproduces the morphological and biochemical events of apoptosis. prICE cleaves the nuclear enzyme poly(ADP-ribose) polymerase (PARP) at a tetrapeptide sequence identical to one of two ICE sites in pro-interleukin-1-beta. However, prICE does not cleave purified pro-interleukin-1-beta, and purified ICE does not cleave PARP, indicating that the two activities are distinct. Inhibition of prICE abolishes all manifestations of apoptosis in the extracts including morphological changes, cleavage of PARP and production of an oligonucleosomal ladder. These studies suggest that prICE might be pivotal in initiating the active phase of apoptosis in vitro and in intact cells.

Although proteases related to the interleukin 1,8-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cellfree model system for apoptosis (S/M extracts A key question in cell death research is whether the apoptotic cascade is driven by the action of a single interleukin 1l3-converting enzyme (ICE)-related protease (IRP) (1-7) or by multiple IRPs acting in concert (8). In the nematode Caenorhabditis elegans, a single IRP is required for all developmental cell deaths (1, 9). In contrast, cDNA cloning experiments show that at least seven IRP mRNAs are expressed in a single human cell type (4, 7, 10, 11), raising the possibility that multiple IRPs might be required for completion of apoptosis in vertebrates. The individual roles of these multiple IRPs during apoptosis are currently unclear.To begin to address this question, we have compared proteolytic cleavage of two apoptotic substrates by cloned IRPs expressed in Escherichia coli and by cell-free extracts (named S/M extracts, prepared from chicken DU249 hepatoma cells committed to apoptosis by an S-phase aphidocolin block and subsequently collected in M phase) (12, 13). Exogenous nuclei incubated in S/M extracts recapitulate nuclear apoptotic events, including endonucleolytic cleavage of DNA, chromatin condensation, and fragmentation of the nucleus (12). Incubation of nuclei or purified poly(ADP-ribose) polymerase (PARP) in S/M extracts results in rapid, quantitative cleavage of the PARP to a 85-kDa fragment indistinguishable from that observed in a wide variety of apoptotic cells (13)(14)(15). This cleavage occurs at a conserved DEVDUG sequence and is mediated by an enzyme with substrate recognition properties and inhibitor sensitivity similar to ICE. We termed this proteolytic activity prICE [protease(s) resembling ICE (13)]. Subsequent investigations have shown that the cloned human IRPs CPP32, Mch2a, and Mch3a as well as the C. elegans IRP CED-3 all cleave PARP (5,7,11,16,17). ICE itself can also cleave a PARP subfragment when added in considerable excess (18); however, at near physiological levels, it does not cleave full-length native PARP (5, 13).Although PARP was the first apoptosis-specific IRP substrate to be identified, the physiological significance of PARP cleavage in apoptosis is presently unknown (for review, see ref.15). In contrast, cleavage of the nuclear lamins is a proteolytic event that appears to be required for completion of nuclear reorganization during apoptosis. Lamin A is cleaved in S/M extracts (8) to fragments that are indistinguishable from those produced in cells undergoing apoptosis (8,(19)(20)(21). The inhibitor profile of the lamin protease suggests that lamin cleavage depends upon the activity of an IRP distinct from the PARPcle...

Abstract.We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn 2÷, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.

Abstract. We describe a novel set of polypeptide antigens that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, we have provisionally termed them the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100 % of the detectable INCENPs remain scaffold associated. We were therefore unprepared for the fate of the INCENPs at anaphase. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAbl77) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, -250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAbl77 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAbl77 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.