SCCA2 inhibits TNF‐mediated apoptosis in transfected HeLa cells.

Abstract

Abbreviations: SCCA1, squamous cell carcinoma antigen 1; SCCA2, squamous cell carcinoma antigen 2; TNF, tumour necrosis factor-alpha; RCL, reactive centre loop; PI-9, proteinase inhibitor 9; PAI 2, plasminogen activator inhibitor 2; crmA, cytokine response modifier A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; succ-AAPF-pNA, succinyl-Ala-Ala-Pro-Phep-nitroanilide; Ac-DEVD-amc, acetyl-Asp-Glu-Val-Asp7-amido-4-methylcoumarin.Eur. J. Biochem. 268, 5868-5875 (2001) q FEBS 2001 protects the cells against apoptosis by inhibiting certain caspases [14]. Two ov-serpins, PI-9 and PAI-2, have been found to protect cells against cytotoxic T-cell and TNFamediated apoptosis, respectively [15,16]. In the case of the ov-serpin PAI-2, mutating the P 1 -Arg to an Ala caused PAI-2 to lose its ability to protect cells against TNFainduced apoptosis [16] and the C -D interhelical region has also been found to be essential for this function [17]. We have previously reported preliminary findings that SCCA2 can protect cells from TNF-mediated apoptosis [18] and protection from radiation-induced apoptosis has also been reported [19].In this study we demonstrate that HeLa cells, stably transfected with SCCA2, are more resistant to TNFmediated apoptosis than untranfected or antisense controls, and that caspase-3 activity is decreased. Furthermore, SCCA2 mutated in the RCL cleavage site failed to protect, suggesting that the serpin inhibitory function is required for the anti-apoptotic mechanism. M A T E R I A L S A N D M E T H O D S MaterialsThe Escherichia coli strain BL21 (DE3) was obtained from Stratagene (La Jolla, CA, USA). The bacterial cloning vector PCR2.1 and expression vector pRSETC were supplied by Invitrogen. Q-Sepharose, iminodiacetic acid (metal affinity matrix), cathepsin G, caspase-3 and their substrates succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (succ-AAPF-pNA) and acetyl-Asp-Glu-Val-Asp7-amido-4-methylcoumarin (Ac-DEVD-amc), respectively, geneticin sulfate (G418), cycloheximide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium and the horseradish peroxidase-conjugated anti-(rabbit IgG) Ig were purchased from Sigma. All oligonucleotide primers were obtained from Genosys (Cambridge, UK). Taq polymerase and MMLV reverse transcriptase were purchased from Promega. All restriction enzymes were purchased from New England Biolabs. Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum were supplied by Shaw Scientific Ltd. Glutamine, penicillin and streptomycin were purchased from GibcoBRL. The IMx tumour marker kit was purchased from Abbotts Laboratories. Cytobuster TM protein extraction reagent was purchased from Novagen. BM chemiluminescence blotting substrate (POD) was purchased from Roche Molecular Biochemicals. The rabbit anti-(cathepsin G) Ig was purchased from Calbiochem. Construction of the mutantsMutants were generated using site directed mutagenesis. The plasmid template used was pRSETC/SCCA2, a His 6 -tagged fusi...

Serine protease inhibitor 2A inhibits caspase‐independent cell death

“…For example, the inhibition of cathepsins has been described by the human serpins SCCA 1 and 2 and the mouse serpin SQN-5 [23,24]. Furthermore, overexpression of SCCA2 protected HeLa cells from TNF-a-mediated PCD [25].…”

Targeted expression of maspin in tumor vasculatures induces endothelial cell apoptosis

“…With regard to the location of action, some serpins act on the cell surface. For example, PAI-2 and SCCA-2 are involved in the inhibition of TNF-mediated apoptosis through cell surface interaction (Dickinson et al, 1998;McGettrick et al, 2001). Other serpins act to inhibit intracellular proteases such as granzyme B and caspases Bird et al, 1998).…”

Electrophoretic analysis of the cleaved form of serpin, squamous cell carcinoma antigen‐1 in normal and malignant squamous epithelial tissues

“…Namely, SCC antigen-1 inactivated cysteine proteinases, e.g., papain, cathepsins, L, S, K, and L2, as well as chymotrypsin, a serine proteinase [11][12][13][14], while SCC antigen-2 inactivates serine proteinases, e.g., cathepsin G and mast cell chymase [15]. As for the biological significance of the proteinase inhibitor activity of SCC antigens, more recently, McGettrick et al [16] reported that SCC antigen-2 attenuated tumor necrosis factor a (TNFa)-mediated apoptosis in HeLa cells by inhibiting TNFa-induced cathepsin G. However, there is no evidence of the interaction of native SCC antigen-1 with specific endogenous proteinases in vivo. When a native serpin such as plasminogen activator inhibitor-2 (PAI-2) interacts with an endogenous target proteinase, the specific cleavage of the serpin within its RCL leads to the formation of the cleaved and thermodynamically more stable conformation [17].…”