SummaryWe report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-l-tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala-borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by dastase-like enzymes (Ala-Ala-Pro-Valp-nitroanilide), but was inactive on the trypsin substrate, N-ol-benzyloxycarbonyl-t-lysine thiobenzyl ester, or the chymotrypsin substrate, AlaAla-Pro-Phep-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased '~10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24-kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis.

SummaryThe function of nicotinamide adenine dinucleotide (NAD) and adenosine diphosphate (ADP) ribosylation reactions in the mechanism of apoptotic cell death is controversial, although one theory postulates an essential role for NAD depletion by poly-ADP-ribose polymerase. The present study examined the role ofintracellular NAD in tumor necrosis factor (TNF) and ultraviolet (UV) light-induced activation of the 24-kD apoptotic protease (AP24) leading to internucleosomal DNA fragmentation and death. Our results demonstrate that nutritional depletion of NAD to undetectable levels in two leukemia lines (U937 and HL-60) renders them completely resistant to apoptosis. This was attributed to a block in the activation of AP24 and subsequent DNA cleavage. Normal cells show an elevation of ADP-ribosyl transferase (ADPR.T) in both the cytosol and nucleus after exposure to TNF, but before DNA fragnaentation. ADPP, T activity as well as cell death was suppressed by an inhibitor specific for mono-ADPRT. Nuclei from NAD-depleted cells were still sensitive to DNA fragmentation induced by exogenous AP24, indicating a selective function for NAD upstream of AP24 activation in the apoptotic pathway. We confirmed a requirement for intracellular NAD, activation of ADPRT, and subsequent NAD depletion during apoptosis in KGla, YAC-1, and BW1547 leukemia cell lines. However, this mechanism is not universal, since BJAB and Jurkat leukemia cells underwent apoptosis normally, even in the absence of detectable intracellular NAD. We conclude that TNF or UV light-induced apoptotic cell death is not due to NAD depletion in some leukemia cell lines. Rather, NAD=dependent reactions which may involve mono-ADPRT, function in signal transduction leading to activation of AP24, with subsequent DNA fragmentation and cell death. APoptosis is the normal physiological process of cell death essential for the maintenance of homeostasis (for reviews see references 1-3). Because of its important role in development and recent evidence implicating that abnormal regulation of this process underlies various pathological conditions (4), there has been intense interest in the biochemical mechanism of apoptosis. Work in our laboratory has analyzed the apoptotic pathway using the human monocytelike U937 leukemia as a model system. Our studies indicate that both TNF and UV light initiate similar biochemical processes culminating in mternucleosomal DNA fragmentation. A key event in this pathway is the activation of a serine apoptotic protease of 24 kD (AP24) I. This enzyme was purified from apoptotic U937 cells after exposure to UV light and was shown to initiate internucleosomal DNA fragmentation in nuclei isolated from normal U937 cells (5). Since protein synthesis is not required for apoptosis in this system (6), AP24 must be expressed in an inactive form or else is sequestered from its substrate in normal U937 cells. The upstream signaling events leading to activation of AP24 are not well understood. Since we previously have shown that inhibitors of ADP-ribosyla...

Periodontitis is a progressive inflammatory disease initiated by bacterial biofilm adhering to the tooth surface. If left untreated, periodontitis may lead to tooth loss and destruction of the alveolar bone. Regaining the lost alveolar bone is a clinical challenge because of the limited differentiation ability of osteoblasts in inflammatory environments. We have previously shown the anti-inflammatory and antiosteoclastogenic activities of parthenolide (PTL) in human periodontal ligament-derived cells by inhibiting nuclear factor kappa B (NF-κB) signaling, indicating its potential for periodontitis treatment. In this study, we further examined whether PTL could stimulate differentiation of osteoblasts from human alveolar bone in inflammatory conditions and investigated the involvement of the Wnt/β-catenin signaling pathway during this process. The results showed that PTL significantly stimulated alkaline phosphatase activity, mineralization nodule formation, and osteogenesis-related gene/protein expression of osteoblasts under the stimulation of tumor necrosis factor-α (TNF-α). In addition, PTL inhibited the NF-κB/p50 pathway and resisted the inhibition of Wnt/β-catenin signaling induced by TNF-α. Our results indicate that the stimulatory effect of PTL on the differentiation of osteoblasts in inflammatory environments may involve the activation of the Wnt/β-catenin signaling pathway, and PTL may be a promising component for bone regeneration in periodontitis treatment.