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...

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans.

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We. previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1p8-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enzyme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.Proteinases of the interleukin 1(3-converting enzyme (ICE)! ced-3 family are essential for the execution of apoptotic cell death (1). These proteinases can induce apoptotic death when overexpressed in cultured mammalian (2-5) or insect (6) cells. Inhibition of ICE family enzymes by CrmA blocks apoptosis induced by factor withdrawal (7) or by the Fas and tumor necrosis factor (TNF) pathways (8). Although gene knockout experiments have revealed that ICE itself is not essential for many types of apoptotic death (9, 10), it does appear to be required for Fas-mediated cell death (10). In addition, the demonstration that apoptosis can be induced by introduction of trypsin, chymotrypsin, or proteinase K into cells (11) raises questions about the interpretation of experiments in which ICE family members are overexpressed in transfected cells. Additional studies using complementary approaches are therefore needed to clarify the role of ICE-like proteinases in apoptosis.Studies using a cell-free system in which endogenous enzymatic activities drive apoptotic events have provided independent biochemical evidence for the involvement of an ICE-like proteinase in apoptotic cell death (12). This cell-free system uses concentrated extracts from chicken DU249 cells in the condemned (committed) phase of apoptosis (13) which is similar to a sequence in pro-interleukin-1l3 that is cleaved by ICE; and it is selectively inhibited by Tyr-Val-AlaAsp-CH2Cl [YVAD "chloromethyl ketone" (YVAD-cmk)], a highly specific inhibitor of ICE family proteinases. These similarities led us to designate the PARP proteinase prICE (proteinase resembling ICE). In further experiments, treatment with YVAD-cmk at concentrations that inhibit prICE abolished all hallmark events of apoptosis ...

Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.

Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVDaminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100-and 20-fold, respectively; but there was only a 1.5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(N ␣ -benzyloxycarbonylglutamyl-N ⑀ -biotinyllysyl)-aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one-and twodimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.Recent studies (reviewed in Refs. 1-5) indicate that the cytotoxicity of virtually all chemotherapeutic agents is accompanied by apoptosis in susceptible cell lines. Likewise, experiments in animals (6 -9) and studies of circulating blasts from leukemia patients (10) have provided evidence that chemotherapy is accompanied by apoptosis in vivo. Moreover, it has been suggested that resistance to the cytotoxic effects of chemotherapeutic agents can result from resistance to chemotherapyinduced apoptosis (8,11,12). These observations highlight the potential importance of understanding the factors that control apoptosis.

Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non-small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decayaccelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrations of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well tolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundant cell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, which are required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruited natural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletion assays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC. Cancer Res; 72(10); 2609-21. Ó2012 AACR.

Molecular optical susceptibilities are calculated by deriving equations of motion for the single electron reduced density matrix, and solving them using the time dependent Hartree–Fock (TDHF) approximation. The present approach focuses directly on the dynamics of the charges in real space and completely avoids the tedious summations over molecular eigenstates. It further maps the system onto a set of coupled harmonic oscillators. The density matrix clearly shows the electronic structures induced by the external field, and how they contribute to the optical response. The method is applied to calculating the frequency-dispersed optical susceptibility χ(3) of conjugated linear polyenes, starting with the Pariser–Parr–Pople (PPP) model. Charge density wave (CDW) like fluctuations and soliton pair like local bond-order fluctuations are shown to play important roles in the optical response of these systems.

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