Figure 2.
MT-hTer expression decreases cell proliferation and viability. (A) 3H-thymidine incorporation. LNCaP clonal lines: 2.5 × 105, 1.25 × 105, and 6.25 × 104 cells were plated in triplicates and pulsed with 3H-thymidine. Columns 1–4, WT-hTER expressing lines R10, R11, R12, and R19; columns 5–11, U11-hTer expressing lines 11.1, 11.3, 11.6, 11.10, 11.12, 11.14, and 11.19; columns 12–16, AU5-hTer expressing lines 5.4, 5.5, 5.10, 5.14, and 5.15. Long-term proliferation of LNCaP lines is shown in Fig. 5. MCF-7 clonal lines: 3.0 × 105, 1.5 × 105, and 7.5 × 104 cells were plated in triplicate. Columns 1–7, WT-hTER expressing lines F4, A4, P1, K3, K2, K1, and H6; columns 8–19, 49A-hTer expressing lines C1, P1, M2, O1, K6, K5, K4, J2, G4, G3, D2, and D1. The averages (avg) for each group of clonal lines are also shown. (B) Colony-forming ability assays for LNCaP and MCF-7 clonal lines. For individual lines, the percent of the plate surface area covered by the cells is plotted. Representative plates are shown. LNCaP clonal lines: columns 1–4, WT-hTER expressing lines R10, R11, R12, and R19; columns 5–10, U11-hTer expressing lines 11.1, 11.3, 11.6, 11.10, 11.14, and 11.19; columns 11–15, AU5-hTer expressing lines 5.4, 5.5, 5.10, 5.14, and 5.15. MCF-7 clonal lines: columns 1–7, WT-hTER expressing lines F4, A4, P1, K3, K2, K1, and H6; columns 8–19, 49A-hTer expressing lines C1, P1, M2, O1, K6, K5, K4, J2, G4, G3, D2, and D1. The averages (avg) for each group of clonal lines are also shown. (C) Increased apoptosis caused by MT-hTer expression. Fraction of cells with subG1 DNA levels (determined by flow cytometry; the flow cytometry histograms are shown in Fig. 6). (Left) LNCaP: parental (−), no treatment; tam, tamoxifen treated; mif, mifepristone treated. The average percent subG1 of WT, U11, or AU5-hTer expressing LNCaP clonal lines are also shown (n = number of clonal lines). (Right) Flow cytometry analysis of parental, WT, or 49A-hTer expressing MCF-7 clonal lines.