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. 2008 May 9;283(19):13021–13030. doi: 10.1074/jbc.M709698200

FIGURE 2.

FIGURE 2.

Multicellular spheroids acquire resistance to apoptosis. A, mesothelioma spheroids display resistance to various apoptotic stimuli. M28, REN, and VAMT monolayers and spheroids exposed to TRAIL (1 ng/ml) plus the proteasome inhibitor MG-132 (2.5 μm) or the histone deacetylase inhibitor trichostatin A (250 nm) for 24 h were studied for apoptosis using annexin V staining and confirmed by examination of condensed nuclei following Hoechst staining. Spheroids consistently showed resistance to the different apoptotic stimuli in all cell lines tested (n = 3, *, p ≤ 0.05 monolayer versus spheroids, mean ± S.D.). B, TRAIL diffuses uniformly within spheroids. After M28 spheroids were exposed to TRAIL (1 ng/ml) for 6 h, immunohistochemistry was performed to detect TRAIL within spheroids. TRAIL diffusion into the spheroids was complete within 6 h as demonstrated by the uniform staining of spheroids exposed to TRAIL, with no staining seen in unexposed spheroids (control) (representative images of four separate experiments). C, spheroids also display resistance to non-TRAIL combinations at 24 h. Combinations of non-TRAIL apoptotic agents that were found to induce apoptosis in mesothelioma cells in monolayers were then tested on monolayers and spheroids of M28, REN, and VAMT cells. Monolayer cells underwent apoptosis following MG-132 (2.5 μm) together with either sodium butyrate (10 mm) or trichostatin A (250 nm), whereas the same cells grown as spheroids were relatively resistant. Each agent, MG-132, trichostatin A, and sodium butyrate, had little effect alone, with apoptosis being <10% (not shown). Apoptosis was measured by quantification of nuclear condensation of Hoechst-stained cells, in this and remaining apoptosis studies (n = 3, *, p ≤ 0.01 monolayer versus spheroids, mean ± S.D.). D, spheroids are also resistant to TRAIL plus the sensitizing agent, anisomycin, at 6 h. After exposure to TRAIL (1 ng/ml) plus anisomycin (25 ng/ml) for 6 h, all three mesothelioma cell lines showed apoptosis, whereas spheroids displayed resistance. The same resistance was seen at 12 and 24 h (not shown) (n = 3, *, p ≤ 0.001 monolayer versus spheroids, mean ± S.D.).