Evidence that Notch signaling is active in Ewing tumors. RT-PCR data showing expression of NOTCH-1, NOTCH-2, DLL-1, DLL-2, DLL-3, JAG-1, JAG-2, MFNG, and the Notch target gene HES-1 in five individual primary Ewing tumors. All of the lesions expressed the Notch-regulated target gene HES-1. The two cell lines examined in this study also expressed all of the ligands, receptors, and the target gene HES-1.

Notch signaling regulation in Ewing sarcoma cell lines. A: Immunofluorescence detection of GFP, expressed by the bidirectional tetracycline-regulated expression construct, showing that doxycycline is able to tightly regulate expression in the cell lines. Expression driven by the tetracycline-off construct was suppressed in the presence of doxycycline (DOX). B: Western analysis using an antibody to FLAG showing regulated expression of the three Notch-1 constructs by doxycycline. “+” lanes are loaded with protein extracts from cells treated with doxycycline, and lanes labeled “−” are loaded with protein extracts from cells treated without doxycycline. C: Activation of the HES-1 luciferase reporter showing increased Notch transcriptional activation with expression of the constitutively active Notch signaling activation to more than twice the level of cells in which the construct was not active, whereas expression of the dominant-negative construct resulted in activation of the construct at roughly half of the level observed in control cells. The control cell data are pooled data from cells in which the constructs are not expressed. CAN1 is the constitutively active construct, DNN1 is the dominant-negative construct, and WTN1 is the wild-type construct. Data are given as relative luminescent units and 95% confidence intervals and are provided for each cell line. Statistically significant differences (P < 0.05) are present when the 95% error bar does not cross the mean of the comparison data. Data for both cell lines are presented.

Notch signaling regulates cell proliferation and apoptosis. A: BrdU incorporation in cell lines expressing the constitutively active, dominant-negative, and wild-type Notch-1 constructs. Percentage of cells over 10 high-powered fields showing positive staining and 95% confidence intervals are shown. Expression of the dominant-negative construct reduced cell proliferation in the RD-ES cell line. Treatment with the γ-secretase inhibitor also reduced cell proliferation but not to as great a degree as with expression of the dominant-negative construct. B: Apoptosis rate as percentage of cells undergoing apoptosis and 95% confidence intervals. Expression of the dominant-negative Notch constructs decreased cell apoptosis rate in both cell lines, whereas expression of the constitutive active construct in the SK-ES-1 cell line also resulted in a decrease in cell apoptosis. Treatment with the γ-secretase inhibitor did not alter the apoptosis rate.

Notch signaling has an effect on the size of tumors that formed when the cell lines are implanted into nude mice. The cell lines expressing Notch constructs were implanted into nude mice, and half of the mice were treated with doxycycline to suppress expression. Data are given as mean volume and 95% confidence intervals. A: Data from the RD-ES cell line, and B shows data from the SK-ES-1 cell line. Statistically significant differences (P < 0.05) are present when the 95% error bar does not cross the mean of the comparison data. As such, there is a significant difference for SK-ES-1 cells expressing the dominant-negative construct compared with the control cells. The wide variability on grafted tumor size in apparent in the large 95% confidence intervals.

Histology reveals an altered cytology in cells with Notch inhibition. Cells from xenograft tumors expressing the dominant-negative Notch construct were larger in size and had more of a neuroblastoma-like appearance than control cell lines or cells expressing the constitutively active construct.

Notch inhibition causes expression of markers of a neural phenotype. Immunohistochemistry of cells from xenograft tumors expressing the dominant-negative Notch construct shows intense staining for markers of neural differentiation.