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SCCA2 inhibits TNF-mediated apoptosis in transfected HeLa cells. The reactive centre loop sequence is essential for

A F McGettrick1, R C Barnes, D M Worrall

  • 1Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.

European Journal of Biochemistry
|November 28, 2001
PubMed

Insights

Squamous cell carcinoma antigen 2 (SCCA2) inhibits tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis by preventing caspase-3 activation. Its reactive center loop is crucial for this protective function and for inhibiting cathepsin G.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Squamous cell carcinoma antigens (SCCA1 and SCCA2) are serine protease inhibitors (serpins).
  • Several serpins are known to inhibit apoptosis in mammalian cells.
  • Tumor necrosis factor-alpha (TNF-alpha) is a key inducer of apoptosis.

Purpose of the Study:

  • To investigate the role of SCCA2 in inhibiting TNF-alpha-induced apoptosis.
  • To determine if the reactive center loop (RCL) of SCCA2 is essential for its anti-apoptotic function.
  • To identify potential targets of SCCA2 involved in apoptosis.

Main Methods:

  • Stable transfection of HeLa cells with SCCA2 cDNA.
  • Induction of apoptosis using TNF-alpha.
  • Mutation of SCCA2's RCL amino acids.
  • Assessment of cell survival, DNA fragmentation, and caspase-3 activation.
  • Enzyme inhibition assays for cathepsin G.

Main Results:

  • SCCA2 overexpression increased cell survival and reduced DNA fragmentation in TNF-alpha-treated HeLa cells.
  • Mutating the SCCA2 RCL abolished its ability to inhibit TNF-alpha-induced apoptosis and cathepsin G activity.
  • SCCA2 reduced caspase-3 activation but did not directly inhibit caspases.
  • TNF-alpha treatment induced cathepsin G expression in HeLa cells.

Conclusions:

  • SCCA2 effectively inhibits TNF-alpha-induced apoptosis, likely through its RCL.
  • Cathepsin G may be a relevant target for SCCA2's anti-apoptotic activity in this system.
  • The protective mechanism of SCCA2 involves modulating caspase activation pathways.

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