05 considered statistically significant. All statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL). Results Reporter Cell Numbers were Linearly http://www.selleckchem.com/products/kpt-330.html Associated with Luciferase Activity When Imaging In order to confirm the correlation of luciferase activity in images with reporter cell numbers, we did a series of dilution for Fluc labeled tumor cells (termed ��reporter cells��). 100, 250, 500, 750, 1000, 2500, 5000, 7500 and 10000 Panc1Fluc or HT29Fluc tumor cells were seed into 96 well plates in 6 replicates the day before imaging. The imaging was performed 5 minutes after adding D-luciferin using the NC100 instrument. The photons from each well were collected and subsequently analyzed by two-tailed ANOVA. The results indicated that photons/sec were linearly associated with cell numbers seeded in wells (Fig.

1A, 1B, R2=0.9967 in Panc1Fluc cells and R2=0.9973 in HT29Fluc cells respectively). Figure 1 Growth-Stimulating properties of dying tumor cells irradiated at various doses. Irradiated Dying Tumor Cell Stimulated Living Tumor Cell Growth We carried out a series of experiments to examine the effects of dying, irradiated tumor cells at various doses on living tumor cells. To simulate in vivo scenarios where the vast majority of tumor cells are killed by radiation or chemotherapy, we seeded a small number (103) of Fluc labeled human pancreatic cancer Panc1 cells or human colonic cancer HT29 cells onto a bed of a much larger number (2.5��105) of unlabeled homologus cancer cells. The latter cancer cells termed ��feeder cells�� were irradiated at 2 Gy, 6 Gy, 10 Gy, 14 Gy and 20 Gy, or untreated (0 Gy) respectively.

Growth of the small number of living ��reporter cells�� was monitored by epi-fluorescent microscopy at 3 day intervals and by bioluminescence imaging on day14 (Fig. 1C, 1D). Luciferase activities were used as surrogates for the number of ��reporter cells�� which was verified by our linear association experiment (Fig. 1A, 1B). Our results indicated that reporter cells grew significantly faster when seeded onto dying cells than when seeded alone. In addition, feeder cells irradiated with 6 Gy showed the highest growth enhancing ability than other doses did, with non-irradiated feeder cells showing no supportive role. In tumor cells irradiated with doses higher than 6 Gy, growth stimulating ability was reduced with increasing irradiation dose (Fig.

1C, 1D). These observations were true for both HT29 cells and Panc1 cells. Activation of SHH Signaling Pathway Correlated Positively with Dying Cell Stimulated Living Tumor Cell Growth To examine whether SHH signaling pathway activation was associated with stimulation of tumor cell growth by dying cells, we carried out Cilengitide Western blot experiments with two cancer cell lines, Panc1 (Fig. 2A) and HT29 (Fig. 2B). Activated SHH signaling was confirmed by the protein levels of Shh and Gli1 which were quantified by measuring the signal of the 19-kD and 160-kD bands, respectively.