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. 1998 May 4;187(9):1477-85.
doi: 10.1084/jem.187.9.1477.

Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor

D Vercammen  1 , R Beyaert, G Denecker, V Goossens, G Van Loo, W Declercq, J Grooten, W Fiers, P Vandenabeele
Affiliations

Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor

D Vercammen et al. J Exp Med. .

Abstract

Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.

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Figures

Figure 1
Figure 1
CrmA expression enhances TNF-induced necrosis. (A) Western blot analysis of transfected L929 clones. Lane 1, control L929 cells transfected with pSV2neo alone; lanes 2–4, different clones cotransfected with pCAGGS CrmA and pSV2neo. Arrowhead, CrmA expression. (B) Sensitizing effect on TNF-mediated necrosis in L929 cells. ▿, control; •, clone 2; ○, clone 5; and ▾, clone 12.
Figure 1
Figure 1
CrmA expression enhances TNF-induced necrosis. (A) Western blot analysis of transfected L929 clones. Lane 1, control L929 cells transfected with pSV2neo alone; lanes 2–4, different clones cotransfected with pCAGGS CrmA and pSV2neo. Arrowhead, CrmA expression. (B) Sensitizing effect on TNF-mediated necrosis in L929 cells. ▿, control; •, clone 2; ○, clone 5; and ▾, clone 12.
Figure 2
Figure 2
Sensitizing effect of peptide caspase inhibitors on TNF-induced necrosis in L929 cells, added 2 h before TNF treatment. (A) Without addition of BHA. •, Ac-YVAD-cmk (100 μM); ○, zDEVD-fmk (100 μM); ▾, Ac-YVAD-cmk + zDEVD-fmk (100 μM each); ▿, zVAD-fmk (25 μM); ▪, zD-fmk (25 μM); □, zAAD-cmk (100 μM); and ♦, control. (B) With BHA (100 μM) added at the same time as TNF (same symbols as in A).
Figure 2
Figure 2
Sensitizing effect of peptide caspase inhibitors on TNF-induced necrosis in L929 cells, added 2 h before TNF treatment. (A) Without addition of BHA. •, Ac-YVAD-cmk (100 μM); ○, zDEVD-fmk (100 μM); ▾, Ac-YVAD-cmk + zDEVD-fmk (100 μM each); ▿, zVAD-fmk (25 μM); ▪, zD-fmk (25 μM); □, zAAD-cmk (100 μM); and ♦, control. (B) With BHA (100 μM) added at the same time as TNF (same symbols as in A).
Figure 3
Figure 3
Effect of zVAD-fmk on TNF-induced reactive oxygen formation and cell death. (A) Effect on TNF-induced oxygen radical production (relative DHR123 fluorescence as compared to untreated cells). •, TNF alone (500 IU/ml); ○, TNF + BHA (100 μM); ▾, TNF + zVAD-fmk (25 μM); ▿, TNF + zVAD-fmk + BHA; and ▪, zVAD-fmk alone. (B) Effect on TNF-induced cell killing determined on the basis of PI-negative cells (same experiment and symbols as in A).
Figure 3
Figure 3
Effect of zVAD-fmk on TNF-induced reactive oxygen formation and cell death. (A) Effect on TNF-induced oxygen radical production (relative DHR123 fluorescence as compared to untreated cells). •, TNF alone (500 IU/ml); ○, TNF + BHA (100 μM); ▾, TNF + zVAD-fmk (25 μM); ▿, TNF + zVAD-fmk + BHA; and ▪, zVAD-fmk alone. (B) Effect on TNF-induced cell killing determined on the basis of PI-negative cells (same experiment and symbols as in A).
Figure 4
Figure 4
Effect of zVAD-fmk on radical scavenging in L929 cells. (A) zVAD-fmk does not alter free thiol concentrations. Cells were treated with zVAD-fmk (25 μM) for 4 h [zVAD-fmk (-4)] or 2 h [zVAD-fmk (-2)] before TNF addition, or with DEM 3 h after TNF addition. Open bars, without TNF; filled bars, 1,000 IU/ml TNF. (B) Effect of zVAD-fmk on H2O2- or tBuOOH-induced oxygen radical production (relative DHR123 fluorescence as compared to untreated cells). •, H2O2 (50 μM); ○, H2O2 + zVAD-fmk (25 μM); ▾, tBuOOH (100 μM); ▿, tBuOOH + zVAD-fmk; and ▪, zVAD-fmk alone.
Figure 4
Figure 4
Effect of zVAD-fmk on radical scavenging in L929 cells. (A) zVAD-fmk does not alter free thiol concentrations. Cells were treated with zVAD-fmk (25 μM) for 4 h [zVAD-fmk (-4)] or 2 h [zVAD-fmk (-2)] before TNF addition, or with DEM 3 h after TNF addition. Open bars, without TNF; filled bars, 1,000 IU/ml TNF. (B) Effect of zVAD-fmk on H2O2- or tBuOOH-induced oxygen radical production (relative DHR123 fluorescence as compared to untreated cells). •, H2O2 (50 μM); ○, H2O2 + zVAD-fmk (25 μM); ▾, tBuOOH (100 μM); ▿, tBuOOH + zVAD-fmk; and ▪, zVAD-fmk alone.
Figure 5
Figure 5
Inhibitory effect of zVAD-fmk on TNF-mediated apoptosis in HeLa H21 cells (• and ○; 1 μg/ml actinomycin D added) and KYM cells (▾ and ▿). Open symbols, TNF only; closed symbols, 25 μM zVAD-fmk added 2 h before TNF treatment.
Figure 6
Figure 6
Possible mechanisms of action in caspase inhibitor-mediated sensitization of TNF-induced necrosis in L929 cells. A putative caspase (CASP-X ), inhibited by CrmA or zVAD-fmk, acts as a negative regulator of premitochondrial signaling (1) or mitochondrial production of reactive oxygen intermediates (ROI; 2). Alternatively, damaging of mitochondria by ROI could impair normal functioning, resulting in an even higher radical production; normally, the cell possesses a mechanism to remove these damaged mitochondria by a process involving one or more caspases (3). Interference with this clean-up process enhances necrosis.
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