Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation

Abstract

Programmed cell death or apoptosis provides an irreversible mechanism for the elimination of excess or damaged cells. Several recent studies have implicated the activation of the interleukin 1beta-converting enzyme/Ced-3 (ICE/Ced-3) family of proteases as the "point of no return" in apoptotic cell death, while others have suggested that loss of mitochondrial membrane potential (delta psi(m)) is the ultimate determinant of cell death. The temporal relationship of these two events during apoptosis and the role of Bcl-2 proteins in inhibiting these steps has not been defined. To examine these issues, control and Bcl-x(L)-transfected Jurkat T cells were treated with Fas antibodies in the presence and absence of the ICE protease inhibitor zVAD-FMK. ICE/Ced-3 protease activity was monitored by following the cleavage of poly(ADP-ribose) polymerase (PARP) and delta psi(m) was followed by rhodamine 123 fluorescence. Although Bcl-x(L) expression did not block Fas-induced protease activation, it substantially inhibited the subsequent loss of delta psi(m) and cell death in Fas-treated cells. In contrast, zVAD-FMK blocked PARP cleavage as well as loss of delta psi(m) and cell death. Together these data demonstrate that Bcl-x(L) can maintain cell viability by preventing the loss of mitochondrial membrane potential that occurs as a consequence of ICE/Ced-3 protease activation.

Figure 1

Bcl-x_L protects against anti-Fas-induced cell death, but not PARP cleavage. (A) Jurkat cells transfected with either a bcl-x_L (Bcl-x_L, ▪) expression vector or the vector control (Neo, •) were treated with anti-Fas for a 96-hr time course. Viability was determined at indicated time points by propidium iodide exclusion. The data presented are the mean and standard deviation of three independent determinations and are representative of four independent experiments. (B) Jurkat cells transfected with a bcl-x_L expression vector (Bcl-x_L) or vector control (Neo) were treated for 24 hr with nothing (Medium), anti-Fas (αFas), or anti-Fas and zVAD (αFas/zVAD). At 6 and 24 hr, cells were removed and lysates were produced for Western blot analysis. Western blot analysis of PARP was performed with the PARP antibody 2C-10, which recognizes the 116-kDa intact PARP as well as an 85-kDa cleaved product observed during apoptotic death (14). The data are representative of four independent experiments and have been reproduced using two additional clones of each transfectant.

Figure 2

Bcl-x_L and protease inhibition can block loss of mitochondrial membrane potential. Jurkat T cells were transfected with a bcl-x_L expression vector (Bcl-x_L) or control vector (Neo) and treated with anti-Fas in the presence and absence of zVAD as described. At the indicated time points, cells were removed for FACS analysis of viability by propidium iodide staining (y axis; PI) and changes in mitochondrial membrane potential by rh123 staining (x axis; Rh123). The data are presented as one of three determinations from a representative experiment. Similar data were obtained in at least five independent experiments performed with three independent clones of each transfectant.

Figure 3

Bcl-x_L-transfected cells can proliferate following removal of anti-Fas. (A) Jurkat T cells transfected with a bcl-x_L expression vector (Bcl-x_L, □) or a control vector (Neo, ○) were incubated with 100 ng/ml of anti-Fas for a 96-hr time course. At indicated time points, cells were removed for determination of viability by propidium iodide exclusion and for cell counts. The number of viable cells was determined by multiplying the cell count by the fraction of cells that exclude propidium iodide. (B) Jurkat T cells transfected with a bcl-x_L expression vector (Bcl-x_L, ▪) or a control vector (Neo, •) were treated with 100 ng/ml of anti-Fas for 6 hr. Cells were isolated by centrifugation, washed three times, and resuspended in fresh medium. Cell counts were performed after washing to determine cell loss due to washing and counts adjusted accordingly. Viable cell number was determined as described in A. The data are presented as the mean ± standard deviation of three separate determinations from a representative of two individual experiments.

Figure 4

Commitment to Fas-induced cell death occurs at an early time point relative to loss of viability as measured by ΔΨ_m or dye exclusion. (A) Jurkat T cells were treated with anti-Fas for 6 hr at which point one-half of the cells were removed, washed three times in fresh medium, and resuspended in fresh medium. The number of total viable cells was determined, as described in Fig. 3, at indicated time points. The cells which remained continually in anti-Fas are represented by open symbols and solid lines while the washed cells are represented by filled circles and dashed lines. The data are presented as the mean ± standard deviation of six independent determinations and are representative of two independent experiments. (B) Jurkat T cells were treated as described in A, and viability and ΔΨ_m were monitored by staining cells with propidium iodide (y axis; PI) and rh123 (x axis; Rh123) at the indicated time points. The data represent one of three replicate samples from a representative experiment. The data are representative of four independent experiments.