FIG. 8.

XIAP confers resistance to TRAIL-induced apoptosis in primary keratinocytes. (A) XIAP is expressed in primary keratinocytes (PK) but not in transformed keratinocytes (TK) and can be coimmunoprecipitated under native conditions with caspase 3 following TRAIL treatment. Total cellular lysates from primary keratinocytes and transformed keratinocytes were prepared, and equal amounts of protein were either analyzed by Western blotting (25 μg) with XIAP monoclonal antibodies (lysate, left and right side) or subjected to immunoprecipitation (500 μg). A 57-kDa protein representing full-length XIAP was detected in primary (right) but not transformed (left) keratinocytes. An additional band of roughly 65 kDa (asterisks) detected in total lysates was shown to be a nonspecific band (immunoprecipitation: XIAP, right lanes). Following immunoprecipitation with caspase 3 antiserum, full-length but not cleaved XIAP was detected only in immunoprecipitates of TRAIL-treated primary keratinocytes PK (immunoprecipitation: caspase 3). (B) Transfection of transformed keratinocytes with XIAP leads to decreased cleavage of full-length caspase 3 and increased detection of caspase 3 p20. Transformed keratinocytes were transiently transfected with 3 μg of pEBB-XIAP vector or pEBB control vector as indicated in Materials and Methods. Then 125 ng of TRAIL per ml was added to the six-well plates, and cellular lysates were collected 3 h later. Primary keratinocytes were treated in parallel plates. The same membrane was first incubated with monoclonal antibodies to XIAP and subsequently reprobed with caspase 3 antibodies. Rehybridization with antitubulin monoclonal antibodies confirmed comparable loading of cellular proteins. Shown is a representative experiment of a total of three independent experiments. (C) XIAP overexpression leads to relative resistance of transformed keratinocytes to TRAIL-induced apoptosis. Cells were transfected as described for B and treated with the indicated concentrations of TRAIL, and viability was determined 6 h later with annexin/propidium iodide staining. Shown are means and standard deviations for two independent experiments. (D) Primary keratinocytes were incubated for 60 min with 10 μM MG115 (right part) or diluent alone (left part) and subsequently treated with 50 ng of TRAIL per ml for the indicated time periods. TRAIL treatment resulted in cleavage of XIAP to a 29-kDa fragment (p29), with similar cleavage at early time points in the presence or absence of the proteasome inhibitor. No cleavage fragments were detected in the presence of zVAD-FMK or after incubation with MG115 alone for 4 h, at which time XIAP levels were comparable to those found under control conditions.