Figure 1.
Selective inhibition of FasL and TRAIL or TRAIL-induced signaling and apoptosis.
In panel A, isogenic BJAB cells expressing GFP or GFP-FADD-DD and FADD-DDV108E were treated with increasing doses of Fas ligand, TRAIL, or agonistic anti-DR4 and anti-DR5 antibodies. The differences in responses indicate that both FADD-DD expressing cell lines were resistant to all TRAIL R targeted drugs, but that only FADD-DD expressing cells are resistant to FasL. In panel B, DISC IP experiments precipitating Fas or DR5 followed by western blotting for casp-8 or FADD demonstrate an increased recruitment of FADD-DD in place of endogenous FADD to the receptors. Panel C, illustrates that on activation of other signaling pathways leading to IkB degradation, and JNK phosphorylation, FADD-DD blocks signaling. This block leads to these events for both FasL and TRAIL, and that FADD-DD V108E blocks signaling only for TRAIL.
Figure 2.
FADD-DD and FADD-DD V108E do not inhibit killing by other apoptotic stimuli.
Isogenic BJAB cell lines were treated with increasing doses of etoposide, MS-275, oxamflatin, doxorubicin, MG132, UV, temozolomide, 5-FU, staurosporine or sorbitol as indicated followed by MTT assay to assess cell viability. All dose response curves overlap for each stimulus.
Figure 3.
FADD-DD blocks TRAIL-induced but not etoposide-induced death in long-term assays.
Isogenic control or FADD-DD expressing BJAB cells were treated with TRAIL or etoposide as indicated for 24 hours, then washed and replaced into growth media. Long term growth of surviving cells was determined by counting viable cells. Control BJAB cells died rapidly and were unable to recover any long term growth. Etoposide treated cells were completely unable to recover growth capacity whether or not FADD-DD was expressed. However FADD-DD expression protected the TRAIL-treated cells as demonstrated by overlapping growth curves with the untreated controls.
Figure 4.
FADD-DD and FADD-DD V108E do not inhibit chemotherapy-induced death in HCT-116 cells.
HCT-116 cells transfected with GFP, FADD-DD or FADD-DDV108E expression constructs were treated with increasing doses of TRAIL, FasL, etoposide, 5-FU, or doxorubicin as indicated and cell viability was assessed. FADD-DD and FADD-DDV108E inhibited FasL and TRAIL as in BJAB cells, but had no effect on tumor cell killing by the chemotherapy drugs.
Figure 5.
FADD-DD and FADD-DDV108E reduce the effectiveness of tumor eradication by etoposide in vivo.
Panel A, isogenic BJAB cells expressing GFP control, GFP-FADD-DD and GFP-FADD-DDV108E were implanted subcutaneously and tumors grown for 10 days prior to treatment with etoposide. Untreated tumors continued to grow. In control BJAB cells, etoposide caused tumor eradication; whereas, in tumors expressing either FADD-DD or FADD-DDV108E, etoposide treatment led to stabilization of tumor mass but no eradication (p<0.05 by t-test at 18 for the control versus FADD-DD and FADD-DDV108E expressing cells). Panel B, Western blot of tumor tissue from GFP control, GFP-FADD-DD and GFP-VFADD-DD V108E demonstrating similar expression of the GFP-tagged protein in all tumors.
Figure 6.
Six1-mediated TRAIL resistance reduces the effectiveness of etoposide in vivo, but not in vitro.
Panel A, parental BJAB cells and BJAB cells expressing Six1 were treated in vitro with increasing doses of TRAIL, FasL and etoposide, and cell viability was assessed by the MTT assay. Six1 caused TRAIL resistance but had little effect on FasL or etoposide-induced cell death. Panel B illustrates how etoposide treatment of subcutaneous tumors from BJAB or BJAB-Six1 cells reduces the growth of both tumors relative to untreated controls, but is less effective in the Six1-expressing cells (* p<0.001 by t-test at day 14 for the control versus Six1 expressing cells).