The apoptotic cysteine protease, caspase-3, is expressed in cells as an inactive 32-kDa precursor from which 17 kDa (p17) and 12 kDa (p12) subunits of the mature caspase-3 are proteolytically generated during apoptosis. Two amino acid sequences, ESMD2S (amino acids 25-29) and IETD2S (amino acids 172-176), in the precursor have been defined as the cleavage sites for the production of the p17 and p12 subunits. Using a cell-free assay system, we demonstrate that the caspase-3 precursor appears to be cleaved first at the IETD2S site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD2S site, generating the mature p17 subunit. The cleavage at the IETD2S site required a protease activity that was selectively inhibited by the peptide, Ac-IETD-CHO (acetyl-IETD-aldehyde), and other protease inhibitors, such as the cowpox viral serine protease inhibitor, CrmA, and N-␣-tosyl-L-phenylalanine chloromethyl ketone. The protease that catalyzed the cleavage at the ESMD/S site was selectively inhibited by another peptide, Ac-ESMD-CHO (acetyl-ESMD-aldehyde). More interestingly, the caspase-3 inhibitor, Ac-DEVD-CHO, but not the caspase-1 inhibitor, Ac-YVAD-CHO, also selectively inhibited the protease activity that cleaves at the ESMD2S site. This indicated that the cleavage at the ESMD2S site was either autocatalytic or that it required a caspase-3-like activity. In summary, we demonstrate that production of the p17:p12 form of caspase-3 is a sequential two-step process and appears to require two distinct enzymatic activities.In recent years, evidence has accumulated that the degradation of certain proteins by members of the caspase family is a general biochemical event taking place in cells undergoing apoptosis (for reviews, see Refs. 1-3). One caspase family member, caspase-3 (4 -6), has been studied extensively. In particular, several of the cellular protein targets of caspase-3 have been identifed. These include the DNA repair enzyme poly-(ADP-ribose) polymerase (PARP) 1 (5, 7), the 70-kDa protein component of the U1 small nuclear ribonucleoprotein (8), and the catalytic subunit of DNA-dependent protein kinase (DNA-PK cs ) (9 -11). Interestingly, inhibition of caspase-3 or caspase-3-like proteases in various cells has been shown to block apoptosis (6,(12)(13)(14)(15)(16)(17). In addition, the functional inactivation of the caspase-3 gene in knock-out mice results in the profound absence of apoptosis in certain tissues and lethality shortly after birth (18). Therefore, caspase-3 appears to be an extremely biologically relevant apoptotic protease.All caspase family members are initially synthesized as inactive precursors and require proteolytic processing themselves to generate the two subunits that form the active protease. This suggests that the apoptotic machinery may be regulated, in part, by a proteolytic cascade (for review, see Ref. 19). In the case of caspase-3, the mature enzyme is formed from 17 kDa (p17) and 12 kDa (p12) subunits, which are produced from a 32-kDa precurs...

Risk factors for gastric cancer are receiving renewed attention in light of the recent positive association of Helicobacter pylori infection with gastric cancer. The effect of H.pylori on the balance between oxidants and antioxidants in the stomach is not well known. In this study, we investigated if exposure of gastric cells to H. pylori increases oxidant-associated gastric epithelial cell injury. A human gastric epithelial cell line (AGS) was grown on 96-well clusters, then exposed overnight to either live H.pylori (four cagA(+) and four cagA(-)) or broth culture supernatant from an isogenic H.pylori cagA(+) strain with and without vacA activity. Incubation of AGS cells with cagA(+) and cagA(-) H.pylori strains before exposure to reactive oxygen species (ROS) reduced cell viability on average to 73.7% and 39.5% of controls, respectively. The percent viability of cells exposed to ROS after incubation with control broth, vacA(-) broth and vacA(+) broth was 97.7%, 70.5% and 63.5%, respectively. Experiments were then performed to evaluate the effects of H.pylori exposure on the activities of ROS-scavenging enzymes [catalase, glutathione peroxidase and superoxide dismutase (SOD)] and formation of 8-hydroxy-2-deoxyguanosine (8-OH-dG) adducts in AGS cells. Overnight exposure to cagA(-) strains reduced catalase activity by 42%; in contrast, exposure to cagA(+) H.pylori strains increased catalase activity by 51%. Glutathione peroxidase activity increased with exposure to both cagA(-) and cagA(+) strains by 95% and 240%, respectively. Total SOD activity increased 156% after exposure to cagA(+) strains and was marginally increased (52%) with exposure to cagA(-) strains. CuZn-SOD protein levels, assayed by enzyme-linked immunosorbent assay, were not significantly altered by exposure to H.pylori strains; however, Mn-SOD concentrations were significantly increased (P: < 0.02) after exposure to both cagA(-) and cagA(+) H.pylori strains. Exposure of AGS cells to cagA(+) and cagA(-) H.pylori was associated with, on average, 44.5 and 99.0 8-OH-dG/10(6) dG, respectively. The increase in catalase, glutathione peroxidase and SOD activity is associated with fewer 8-OH-dG DNA adducts and reduced susceptibility of AGS cells to lethal injury from ROS after exposure to cagA(+) H.pylori strains when compared with exposure to cagA(-) H.pylori strains. Alteration in the activity of ROS-scavenging enzymes by the presence of H. pylori may in part be responsible for the increased risk of gastric cancer in persons infected with H.pylori.

Summary. Resveratrol, a natural product present in wine, has recently been shown to inhibit the growth of a number of cancer cell lines in vitro. In the current study, we have demonstrated that resveratrol inhibits the growth of THP-1 human monocytic leukaemia cells in a dose-dependent manner with a median effective dose of 12 mm. It did not induce differentiation of THP-1 cells and had no toxic effect on THP-1 cells that had been induced to differentiate into monocytes/macrophages by phorbol myristate acetate. A significant fraction of resveratrol-treated cells underwent apoptosis as judged by flow cytometric analysis of DNA content, DNA fragmentation and caspase-specific cleavage of poly(ADP-ribosyl) polymerase. Resveratrol treatment had no effect on the expression of Fas receptor or Fas ligand (FasL) in THP-1 cells, nor did it induce clustering of Fas receptors. In addition, THP-1 cells were resistant to activating anti-Fas antibody, and neutralizing anti-Fas and/or anti-FasL antibodies had no protective effect against resveratrol-induced inhibition of THP-1 cell growth. The effect of resveratrol on THP-1 cells was reversible after its removal from the culture medium. These results suggest that (1) resveratrol inhibits the growth of THP-1 cells, at least in part, by inducing apoptosis, (2) resveratrol-induced apoptosis of THP-1 cells is independent of the Fas/FasL signalling pathway and (3) resveratrol does not induce differentation of THP-1 cells and has no toxic effect on differentiated THP-1 cells. Thus, resveratrol may be a potential chemotherapeutic agent for the control of acute monocytic leukaemia.