Enhancement of cisplatin-induced apoptosis by expression of survivin Thr³⁴→Ala, and histology of YUSAC2 tumors in vivo. (A) YUSAC2/T34A-C4 cells were cultured for 3 days in the presence or absence of tet and 3 μM cisplatin, as indicated, and stained for DNA content as described in Fig. 1. Percentages shown are for the sub-G₁ fraction (left marker), corresponding to apoptotic cells. (B) Sections of (untransfected) YUSAC2 tumor (×400) were stained with antibodies to survivin or HMB-45 (Zymed) as indicated.

Characterization of tet-regulated induction of survivin Thr³⁴→Ala in YUSAC2/T34A-C4 cells. (A) YUSAC2/T34A-C4 cells were cultured in the presence or absence of tet (0.5 μg/ml) and harvested at the indicated time intervals, and lysates (100 μg/lane) were subjected to Western blotting with antibodies to survivin (Upper) or β-actin (Lower). (B) YUSAC2/WT or YUSAC2/T34A-C4 cells were cultured for 24 h in the presence or absence of tet, and lysates (50 μg/lane) were analyzed by Western blotting with an antibody to phosphorylated T34* (α-survivinT34*) (Upper) or β-actin (Lower). For A and B, molecular mass markers in kDa are indicated at the left of each image. (C) YUSAC2/T34A-C4 cells were cultured in the presence or absence of tet for the indicated time intervals, and nonadherent and adherent cells were pooled and analyzed for DNA content by propidium iodide staining and flow cytometry. Percentages shown are for the sub-G₁ fraction (left marker), corresponding to apoptotic cells, and the G₂/M fraction (right marker), corresponding to mitotic cells. (D) YUSAC2/T34A-C4 cells were cultured for 48 h in the presence or absence of tet. Arrows indicate TUNEL-reactive cells with apoptotic morphology.

Effect of survivin Thr³⁴→Ala mutant in melanoma tumors in vivo and tet-regulated apoptosis of tumor lines reestablished in vitro. (A) Untransfected YUSAC2 cells (Left) or YUSAC2/T34A-C4 cells (Right) were injected s.c. into CB.17 mice, and tet was added (left side of images) or withheld (right side of images) from the drinking water as indicated. Photographs were taken of representative mice 8 weeks after injection. (B) YUSAC2/T34A-C4 tumors were established in 30 animals maintained on tet. When tumors became apparent (day 0), tet was maintained in the drinking water of 10 animals (tet+, ●) and withheld from 20 animals (tet−, ○), and tumors were monitored for 3 weeks. Bars = SD, P < 0.0001 for days 12–21 by two-tailed t test. (C) Histology of established YUSAC2/T34A-C4 tumors from tet+ (Upper) and tet− (Lower) animals after 8 weeks by hematoxylin/eosin staining (×100). (D) YUSAC2/T34A-C4 tumors were established in animals on tet, and when tumors became apparent (day 0), tet was maintained in the drinking water of half the animals (tet+, ●) and withheld from the others (tet−, ○). Tumors were excised on days 4, 7, 10, 13, and 21 and subjected to TUNEL and BrdUrd staining for respective determination of apoptotic (upper graph) and proliferative (lower graph) indices. Bars = SEM from 2 animals for days 4 and 13, 4 animals for days 7 and 10, and 10 animals for day 21. Adjacent images are representative fields (×400) from day 7 tumors ± tet as indicated. (E) Cell lines from several YUSAC2/T34A-C4 tumors slowly growing in vivo after withdrawal of tet were reestablished in vitro. The DNA content analysis for a representative line after culturing for 72 h in the presence (Upper) or absence (Lower) of tet is shown. The marker and percentages indicate the sub-G₁ fraction, corresponding to apoptotic cells.