Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation.

Abstract

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.

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions

“…Previous reports have suggested the implication of serine proteases in apoptosis induced by TNF and UV, 43,44 dexamethasone, 45 hypoxia reoxygenation, 46 DNA damage, 47 NGF deprivation 48 and the overexpression of s-Myc and cMyc. 49 Moreover, serine proteases were shown to play roles in the cleavage of chromatin into 50-300 kb pieces, 45 the activation of an endonuclease, 50 the processing of caspase-2 48 and caspase-3 44 and the cytochrome c-mediated activation of caspase-9.…”

Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant

“…The results are the average of three independent experiments several caspases and vigorously induces apoptosis (Stennicke and Salvesen, 1999). Several other proteinases including the cysteine proteinase, calpain, apoptotic serine proteinase p24 and cathepsin D have also been shown to be involved in apoptosis (Squier et al, 1994;Wright et al, 1994;Deiss et al, 1996;Hirsch et al, 1998;Nakagawa and Yuan, 2000). Our results demonstrated that at 0.1 mM DOX, PR3-mediated DOX-induced apoptosis in HL-60 cells was independent of the caspase-8 and -9 pathways, but was associated with the cleavage of caspase-3 in HL-60 cells.…”

Ligation of CD40 rescues Ramos‐Burkitt lymphoma B cells from calcium ionophore‐ and antigen receptor‐triggered apoptosis by inhibiting activation of the cysteine protease CPP32/Yama and cleavage of its substrate PARP

“…It is known that ICE-like proteases degrade lamins during apoptosis and may thereby trigger collapse of the chromatin [23,24]. A partially characterised 24-kDa protease has been shown to induce DNA degradation characteristic of apoptosis when added to purified nuclei possibly by activating an endonuclease as it does not itself exhibit any nuclease activity [25]. Activated CPP32/Yama or apopain can cleave poly-(ADP-ribose) polymerase (PARP) and thereby inhibit most of its DNA repair activity [11,12,18,19,26].…”