Effect of Protease Inhibitors on Early Events of Apoptosis

Hara, Shuta; Halicka, H. Dorota; Bruno, Silvia; Gong, Jianping; Traganos, Frank; Darzynkiewicz, Zbigniew

doi:10.1006/excr.1996.0092

Cited by 49 publications

(20 citation statements)

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“…Low concentrations of TPCK inhibit internucleosomal cleavage and differentially modulate apoptosis dependent on the stimulus Low concentrations of TPCK (5 ± 10 mM) inhibited the internucleosomal cleavage of DNA induced by different apoptotic stimuli (Figure 6) in agreement with its effects in several different systems (Bruno et al, 1992;Chow et al, 1995;Fearnhead et al, 1995a;Hara et al, 1996;Neamati et al, 1995). This may be related to the ability of TPCK to inhibit a 24 kDa protease, which can initiate internucleosomal cleavage of DNA (Wright et al, 1994).…”

Section: High Concentrations Of Tpck Induce Apoptosissupporting

confidence: 58%

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

et al. 1997

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N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK), a chymotrypsin-like serine protease inhibitor, affected apoptosis in human monocytic THP.1 cells differently dependent on both the concentration used and the apoptotic stimulus. TPCK (50 ± 75 mM) induced both biochemical and ultrastructural changes characteristic of apoptosis, including proteolysis of poly (ADP-ribose) polymerase (PARP) and lamins together with formation of large kilobase pair fragments of DNA, particularly of 30 ± 50 and 200 ± 300 kilobase pairs in length but without internucleosomal cleavage of DNA. The induction of apoptosis by TPCK also involved the processing of CPP32 and Mch 3 to their catalytically active subunits. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), an ICE-like protease inhibitor, completely prevented all the biochemical and morphological changes induced by TPCK demonstrating the involvement of ICE-like proteases in the execution phase of apoptosis. Lower concentrations of TPCK (5 ± 20 mM) prevented internucleosomal cleavage of DNA induced by other apoptotic stimuli. TPCK (10 mM) inhibited cell death induced by etoposide but potentiated that induced by cycloheximide demonstrating that it differentially affected apoptosis in THP.1 cells dependent on the stimulus used. These results are consistent with at least three distinct TPCK targets, one being important for cell survival, the second in facilitating internucleosomal cleavage of DNA and the third in the modulation of apoptosis induced by different apoptotic stimuli.

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Section: High Concentrations Of Tpck Induce Apoptosissupporting

confidence: 58%

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

et al. 1997

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“…Chromatin condensation, associated with the initial step of DNA cleavage generating high molecular weight DNA fragments, is considered a characteristic feature of apoptosis, and it occurs independently from the subsequent and dispensable internucleosomal DNA fragmentation (24). Apoptotic cells therefore were identified by the altered nuclear morphology showing chromatin condensation (25,26) as detected by the staining with the DNA dye propidium iodide (PI).…”

Section: Methodsmentioning

confidence: 99%

Prenatal exposure to high levels of glucocorticoids increases the susceptibility of cerebellar granule cells to oxidative stress-induced cell death

Ahlbom

Gogvadze

Chen

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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There is growing concern that prenatal exposure to excessive glucocorticoids may have deleterious effects on the development of various organs, including the nervous system. This study aimed at evaluating whether prenatal exposure to high levels of glucocorticoids might produce long-term effects on neuronal cell survival. Pregnant rats were injected i.p. with 0.1 mg͞kg dexamethasone (DEX) from day 14 postconception, and cerebellar granule cells (CGC) were prepared from 1-week-old rats from DEX-treated and control dams. After 7 days in culture, cells were exposed to H 2O2, methylmercury, or colchicine at concentrations known to induce apoptotic cell death. After exposure to H 2O2 or methylmercury, both inducing oxidative stress, the number of apoptotic cells was significantly higher in DEX-than in control-CGC. Because mitochondria play a key role in apoptosis, mitochondrial function was investigated, and a decrease in the threshold level of Ca 2؉ necessary for induction of mitochondrial permeability transition, in Ca 2؉ accumulation rate, and in oxygen consumption was detected in DEX-CGC. Moreover, the activity of the antioxidant enzyme catalase was significantly decreased in DEX-CGC. A similar decrease in catalase activity was observed in cerebellar homogenate from newborn and 40-day-old DEX-rats. In conclusion, these results indicate that prenatal exposure to high levels of glucocorticoids induces long-lasting changes in CGC rendering them more sensitive to oxidative stress. With the increasing use of multiple doses of glucocorticoids in preterm infants, the possibility that prenatal exposure to excess glucocorticoids may lead to long-term neurological consequences becomes a relevant issue.

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“…The inhibitory effect of TPCK on apoptosis has often been ascribed to inhibition of chymotrypsin-like proteases [35] but because TPCK can both induce caspases and cell-death [6] while at the same time inhibiting the DNA laddering associated with apoptosis [8] it is predicted that there are at least two distinct apoptosis-relevant TPCK targets [10]. TPCK can also interact and label other cellular proteins at residues other than histidine [36,37] and the apoptosisrelevant targets for TPCK are not known.…”

Section: Discussionmentioning

confidence: 98%

TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells

Fábián

O’Brien²,

Pajęcka

et al. 2009

Apoptosis

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N-alpha-Tosyl-L: -phenylalanyl chloromethyl ketone (TPCK) is an affinity label for chymotrypsin-like proteases and has been extensively used as an experimental tool in apoptosis research to probe the role of proteases in cell death. While TPCK blocks some apoptotic changes and induces others, the cellular target or targets for TPCK have not been identified. Here we investigated for the first time the cellular targets for TPCK using a polyclonal anti-tosyl antibody. We have found that TPCK rapidly and irreversibly labels numerous intracellular proteins and have identified one as RPB1, the largest subunit of RNA polymerase II. We show that TPCK inhibits DNA binding by RNA polymerase and that TPCK inhibits transcription. Inhibition of transcription is known to induce apoptosis and while TPCK may trigger death through interaction with multiple targets, our data suggests that the pro-apoptotic effects of TPCK may be explained in part by the inhibition of RNA polymerase II activity.

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Effect of Protease Inhibitors on Early Events of Apoptosis

Cited by 49 publications

References 0 publications

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

Prenatal exposure to high levels of glucocorticoids increases the susceptibility of cerebellar granule cells to oxidative stress-induced cell death

TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells

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