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 s erine protease inhibitors (serpin) superfamily and are transcribed by two t andomly arrayed genes. A number of serpins are known to inhibit apoptosis i n mammalian cells. In this study we demonstrate the ability of SCCA2 to inh ibit tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. HeLa cells stably transfected with SCCA2 cDNA had increased percentage cell survival a nd 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 inhibi t cathepsin G and to protect stably transfected cells from TNF alpha -induc ed apoptosis. The presence of SCCA2 caused a decrease in the activation of caspase-3 upon induction with TNF alpha but no direct inhibition of caspase s by SCCA2 has been found. Expression of cathepsin G was found to be induce d in HeLa cells following treatment with TNF alpha This protease has recent ly been shown to have a role in apoptosis through cleavage of substrates, s o maybe the relevant target for SCCA2 in this system.