SCCA2 inhibits TNF-mediated apoptosis in transfected HeLa cells. The reactive centre loop sequence is essential for this function and TNF-induced cathepsin G is a candidate target.
The squamous cell carcinoma antigens, SCCA1 and SCCA2, are members of the serine protease inhibitors (serpin) superfamily and are transcribed by two tandomly arrayed genes. A number of serpins are known to inhibit apoptosis in mammalian cells. In this study we demonstrate the ability of SCCA2 to inhibit tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. HeLa cells stably transfected with SCCA2 cDNA had increased percentage cell survival and reduced DNA fragmentation. We investigated if the reactive centre loop (RCL) was necessary to allow SCCA2 to inhibit TNF alpha-mediated apoptosis. The RCL amino acids (E353Q, L354G, S355A), flanking the predicted cleavage site, were mutated and the resulting SCCA2 lost both the ability to inhibit cathepsin G and to protect stably transfected cells from TNF alpha-induced apoptosis. The presence of SCCA2 caused a decrease in the activation of caspase-3 upon induction with TNF alpha but no direct inhibition of caspases by SCCA2 has been found. Expression of cathepsin G was found to be induced in HeLa cells following treatment with TNF alpha. This protease has recently been shown to have a role in apoptosis through cleavage of substrates, so maybe the relevant target for SCCA2 in this system.
McGettrick
A F
AF
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
Barnes
R C
RC
Worrall
D M
DM
D000951:Antigens, Neoplasm / Q000235:genetics / Q000502:physiology*; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D056649:Cathepsin G; D002403:Cathepsins / Q000096:biosynthesis; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D015843:Serpins; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D000951:Antigens, Neoplasm; D011994:Recombinant Proteins; D015843:Serpins; D014409:Tumor Necrosis Factor-alpha; C051072:squamous cell carcinoma-related antigen; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases