Tumor necrosis factor-related apoptosis-inducing ligand can induce apoptosis in subsets of premalignant cells

Abstract

During the transformation from a normal to a malignant cell, several mutations are required to bypass the pathways responsible for controlling proliferation. Premalignant cells have acquired some, but not all of these mutations and consequently have not yet attained a malignant phenotype characterized by tumor formation in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in malignant cells while sparing normal ones and is currently being considered as adjuvant therapy for various human malignancies. Whether TRAIL is effective in inducing apoptosis in premalignant cells is unclear, however. We studied the effect of TRAIL on two human premalignant cell lines the SV7tert and HA1E cells. Both cell lines had been immortalized by the addition of simian virus 40 large T antigen and the telomerase subunit hTERT, but had not been transformed into malignant cells. TRAIL initiated apoptosis by activating both the mitochondrial-independent and -dependent apoptotic pathways in both cell lines at relatively low doses whereas it had no effect on normal human pulmonary artery smooth muscle cells even at high doses. These results suggest that TRAIL can induce apoptosis in premalignant cells and suggests a novel therapy for the treatment of premalignant lesions in vivo.

Figure 1

TRAIL induces apoptosis in SV7tert cells but not in human PA SMCs. A: Light microscopy of SV7tert cells exposed to TRAIL (0 or 100 ng/ml) for 6 hours. Human PA SMCs showed no changes (not shown). B: Effect of increasing doses of TRAIL at 6 hours on apoptosis in human PA SMCs and SV7tert cells as determined by flow cytometry. Data are mean ± SEM, n = 3 separate experiments; *, P < 0.05 versus human PA SMCs. C: Ethidium bromide-stained agarose gel of DNA harvested from human PA SMCs and SV7tert cells 24 hours after exposure to 100 ng/ml of TRAIL. DNA laddering was seen only in the SV7tert cells.

Figure 2

TRAIL activates caspase 8 in angiomyolipoma cells. A: Western blot of SV7tert cell lysates harvested 30 minutes after exposure to increasing doses of TRAIL. B: Western blot showing the time course of caspase 8 activation in SV7tert cells treated with 100 ng/ml of TRAIL. Representative blot from three separate experiments.

Figure 3

TRAIL activates both the mitochondrial-independent and -dependent apoptotic pathways in angiomyolipoma cells. A: Western blot of SV7tert cell lysates harvested 6 hours after exposure to 100 ng/ml of TRAIL shows cleavage (and activation) of caspase 3. Cleavage of the nuclear protein PARP confirmed complete activation of the caspase pathway. B: TRAIL cleaves BID to its truncated form indicating activation of the mitochondrial-dependent apoptotic pathway. The presence of Smac/Diablo and cleaved caspase 9 confirm this observation. Representative blot from three separate experiments. Truncated BID shown at 2.5 hours; Smac/DIABLO and cleaved caspase 9 are shown at 6 hours.

Figure 4

Inhibiting protein synthesis with cycloheximide (CHX) amplifies TRAIL-induced apoptosis in angiomyolipoma cells. A: Effect of TRAIL alone and in combination with CHX (25 μmol/L) on apoptosis of SV7tert cells as determined by flow cytometry. Data are mean ± SEM, n = 3 separate experiments; *, P < 0.05 versus TRAIL (100 ng/ml) alone. B: Western blot of SV7tert cell lysates harvested 6 hours after TRAIL exposure demonstrating increased activation of caspases 8 and 9 along with an increase in cleaved PARP in the presence of CHX. Representative blot from three separate experiments.

Figure 5

TRAIL induces apoptosis in HA1E cells. Effect of increasing doses of TRAIL at 6 hours on apoptosis in human PA SMCs, SV7tert, and HA1E cells as determined by flow cytometry. Data are mean ± SEM, n = 4 separate experiments; *, P < 0.05 for both SV7tert and HA1E cells versus human PA SMCs. These apoptosis experiments were done simultaneously in all three cell lines and represent different experiments from those shown in Figure 1B.

Figure 6

TRAIL activates caspase 3 and cleaves PARP in HA1E and SV7tert cells, but not human PA SMCs. Three bead populations with distinct fluorescence intensities have been coated with capture antibodies specific for cleaved PARP (C-PARP), Bcl-2, and activated caspase 3 (human apoptosis kit from Becton-Dickinson, see Materials and Methods). Six hours after exposure to control or TRAIL (100 ng/ml) whole cell lysate protein from human PA SMCs (A), HA1E cells (B), and SV7tert cells (C) was harvested, normalized, and then incubated with these beads. The beads were then analyzed by flow cytometry. The y axis indicates the distinct bead identification number based on increasing red fluorescence intensity (bead ID FI). The top band (most intense) represents C-PARP, the middle band represents Bcl-2, and the bottom band (least intense) represents activated caspase 3. The x axis indicates the fluorescence intensity of the phycoerythrin-conjugated detection reagent (FI of PE). The figures on the left demonstrate data obtained from a single experiment; the bar graphs on the right indicate the statistical analysis of four separate experiments. *, P value < 0.05. Each experiment was compared to its (untreated) control and changes denoted by fold-change over baseline. Note that after incubation with TRAIL the fluorescence intensity of activated caspase 3 and cleaved PARP increases (ie, shifts to the right) in HA1E and SV7tert cells, but not in human PA SMCs. Bcl-2 is unchanged in any of the three cell types after TRAIL exposure.