Serine Protease Control of Lymphocyte-Mediated Cytolysis

Hudig, Dorothy; Allison, Nigel; Ewoldt, Gerald R.; Gault, Ruth A.; Netski, Dale; Pickett, Timothy M.; Redelman, Doug; Wang, Ming T.; Winkler, Ulrike; Zunino, Susan J.; Kam, Chih-Min; Odake, Shinjiro; Powers, James C.

doi:10.1007/978-1-4684-6814-4_27

Cited by 5 publications

(5 citation statements)

References 41 publications

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“…However, because it lacks free system are able to induce DNA fragmentation. 18 We an amino acid in the P4 position, Z-VAD-fmk would be therefore used DCI, a cell-permeable mechanism-based serine expected to have a broader specificity for caspases 21,23 than protease inhibitor 25 in comparison with Z-VAD-fmk. By conthe tetrapeptide inhibitors of this family such as YVAD-CHO trast to Z-VAD-fmk, DCI was active in the cell-free system and and DEVD-CHO.…”

Section: Figurementioning

confidence: 99%

Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases

1997

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The human leukemia cell line, HL60 is very sensitive to various B can activate CPP32, 13 and apoptotic activation of proteaapoptotic stimuli and p53-null. The death-related cysteine prosome 14,15 and cathepsin D 16 have been reported. A nuclear teases of the caspases family play a central role in the scaffold protease has been postulated to activate apoptotic execution phase of apoptosis, and we recently reported the nuclease(s). 17 Our previous studies in human leukemia HL60 importance of serine protease activation in camptothecincells also indicated that apoptotic DNA fragmentation can be induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CEDinduced by serine proteases in a cell-free system and sup-3-related cysteine proteases) and serine proteases in cell death pressed by serine protease inhibitors. 18,19 induced by the topoisomerase I inhibitor, camptothecin, inThe goal of the present study was to investigate the effects HL60 cells and in a cell-free system. We found that CPP32 is of two cell-permeable protease inhibitors on camptothecin- also be used in a cell-free system. 18,32 In the present report, in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32we demonstrate that CPP32 is proteolytically activated in and serine proteases are activated in camptothecin-induced response to camptothecin-induced apoptosis in HL60 cells. apoptosis. Activation of CPP32 requires both caspase and serine proteaseKeywords: camptothecin; topoisomerase I; apoptosis; caspase;activation. Furthermore, we show that in a cell-free system, serine protease; HL60only serine protease inhibitors block DNA fragmentation and lamin B degradation. These results suggest that serine proteases can function both downstream and upstream of the Introduction CPP32 activation step in a cell death pathway.Apoptosis is well defined morphologically 1 and recent studies indicate that a number of evolutionary conserved genes reguMaterials and methods late a common cell death pathway that is conserved from worms to humans. 2,3 Asparate-specific cysteine proteases Drugs and chemicals (caspases) 4 play a central role in the execution phase of apoptosis. 5,6 The interleukin-1␤-converting enzyme (ICE) has Camptothecin was a generous gift from Drs ME Wall and MC been the first described homologue of the ced-3 death gene Wani (Research Triangle Park, NC, USA). It was freshly disproduct (CED-3) of Caenorhabditis elegans. 7 Other members solved in dimethyl sulfoxide at 10 mM and further diluted in of the caspase family including caspase 3 (CPP32/Yama/ water before each experiment. The caspase inhibitor, N-benApopain) 8,9 have been identified and recent evidence suggests zyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (Zthat several caspases can be activated sequentially in the same VAD-fmk), and the serine protease inhibitor, 3,4-dichloroisoapoptotic cell. 10 Poly(ADP-ribose)polymerase (PARP) 8,9 and coumarin (...

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Section: Figurementioning

confidence: 99%

Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases

1997

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“…The presence of allogenic T lymphocytes and macrophages has been documented in the coronary vasculature (12, [18][19][20][21] and the rejecting myocardium after cardiac transplantation (22)(23)(24). These inflammatory cells contain matrix metalloproteinases (25)(26)(27)(28)(29) and an elastase indistinguishable from human neutrophil elastase (25,(30)(31)(32). Recent studies have also shown that vascular SMCs can produce an endogenous elastase (33) that is induced by serum and endothelial factors (34).…”

Section: Introductionmentioning

confidence: 99%

Elafin, a serine elastase inhibitor, attenuates post-cardiac transplant coronary arteriopathy and reduces myocardial necrosis in rabbits afer heterotopic cardiac transplantation.

Cowan¹,

Bar-On²,

Crack³

et al. 1996

J. Clin. Invest.

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We have related experimentally induced post-cardiac transplant coronary arteriopathy to increased elastolytic activity, IL-1 ␤ , fibronectin-mediated inflammatory and smooth muscle cell (SMC) migration, and SMC proliferation. Since our in vitro studies show that a serine elastase releases SMC mitogens and facilitates IL-1 ␤ induction of fibronectin, we hypothesized that administration in vivo of the specific serine elastase inhibitor, elafin, would decrease the post-cardiac transplant coronary arteriopathy. Cholesterol-fed rabbits underwent a heterotopic cardiac transplant without immunosuppression and received elafin (1.79 mg/kg per d continuous infusion after a 9 mg bolus, n ϭ 6) or vehicle ( n ϭ 6). 1 wk later, hearts were harvested for morphometric, immunohistochemical, and biochemical analyses. A Ͼ 70% decrease in the total number of coronary arteries with intimal thickening in elafin-treated compared to control donor hearts ( P Ͻ 0.002) was associated with reduced vascular elastolytic activity judged by fewer breaks in the internal elastic lamina ( P Ͻ 0.03), less accumulation of immunoreactive fibronectin ( P Ͻ 0.02), and reduced cell proliferation quantified by proliferating cell nuclear antigen ( P Ͻ 0.0001). Despite myocardial lymphocytic infiltration, wet weight of elafin-treated donor hearts was reduced by 50% compared to untreated controls ( P Ͻ 0.002) and associated with relative preservation of myocyte integrity, instead of extensive myocardial necrosis ( P Ͻ 0.004). This protective effect correlated with decreased myocardial elastolytic activity ( P Ͻ 0.0001) and inflammatory cell proliferation ( P Ͻ 0.0001) and with an elafin-inhibitable elastase in lymphocytes. Serine elastase activity thus appears an important therapeutic target for post-cardiac transplant coronary arteriopathy and myocardial necrosis induced by rejection. ( J. Clin. Invest. 1996. 97:2452-2468.)

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“…As a serine protease, granzyme B is an "Asp-ase," cleaving at aspartic acid residues. The involvement of serine proteases in cytolysis was first suspected on the grounds that serine proteases inhibitors could block cytotoxic T-lymphocyte activity as measured by chromium release (9,10). Later experiments showed that granzyme B can specifically induce rapid DNA fragmentation, but only when cells are simultaneously exposed to sublytic quantities of perforin (11).…”

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confidence: 99%

Nuclear Transport of Granzyme B (Fragmentin-2)

Jans¹,

Jans²,

Briggs³

et al. 1996

Journal of Biological Chemistry

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Cytotoxic T and natural killer cells are able to kill their target cells through synergistic action of the poreforming protein perforin and the serine protease granzyme B, resulting in very distinctive nuclear changes typical of apoptosis. Whereas perforin acts at the membrane, granzyme B appears to be both capable of entering the cytoplasm of target cells and accumulating in isolated nuclei. In this study we examine nuclear transport of fluoresceinated granzyme B both in vivo in intact cells in the presence of perforin and in vitro in semipermeabilized cells using confocal laser scanning microscopy. Granzyme B alone was observed to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of sublytic concentrations of perforin, however, it accumulated strongly in intact cell nuclei to levels maximally about 1.5 times those in the cytoplasm after about 2.5 h. In vitro nuclear transport assays showed maximal levels of nuclear and nucleolar accumulation of granzyme B of about 2.5-and 3-fold those in the cytoplasm. In contrast to signal-dependent nuclear accumulation of SV40 large tumor antigen (TAg) fusion proteins in vitro, nuclear/nucleolar import of granzyme B was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTP␥S (guanosine 5-O-(3-thiotriphosphate)). Similar to T-Ag fusion proteins, however, granzyme B nuclear and nucleolar accumulation was dependent on exogenously added cytosol. Specific inhibitors of granzyme B protease activity had no effect on nuclear/nucleolar accumulation, implying that proteolytic activity was not essential for nuclear targeting. The results imply that granzyme B (32 kDa) may be transported from the cytoplasm to the nucleus through passive diffusion and accumulate by binding to nuclear/nucleolar factors in a cytosolic factor-mediated process. Active and passive nuclear transport properties were normal in the presence of unlabeled granzyme B, implying that the nuclear envelope and pore complex are not granzyme B substrates.The serine protease granzyme B (fragmentin-2) (1-3) is integrally involved in apoptotic cell death induced in target cells upon their exposure to the contents of lysosome-like cytoplasmic granules (or cytolytic granules) found in cytotoxic T-lymphocytes and natural killer cells (4 -6). Granzyme B acts in conjunction with a second cytolytic granule component, perforin, which can induce complement-like pores within target cell membranes (7,8). As a serine protease, granzyme B is an "Asp-ase," cleaving at aspartic acid residues. The involvement of serine proteases in cytolysis was first suspected on the grounds that serine proteases inhibitors could block cytotoxic T-lymphocyte activity as measured by chromium release (9, 10). Later experiments showed that granzyme B can specifically induce rapid DNA fragmentation, but only when cells are simultaneously exposed to sublytic quantities of perforin (11). This implies that granzyme B is the causative agent triggering apoptosis, but that perforin is required in order for...

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Serine Protease Control of Lymphocyte-Mediated Cytolysis

Cited by 5 publications

References 41 publications

Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases

Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases

Elafin, a serine elastase inhibitor, attenuates post-cardiac transplant coronary arteriopathy and reduces myocardial necrosis in rabbits afer heterotopic cardiac transplantation.

Nuclear Transport of Granzyme B (Fragmentin-2)

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