1. Volume 2, Issue 4, Pages 348-358 (October 2000)
Intracellular Fas Ligand Expression Causes Fas-Mediated Apoptosis in Human Prostate Cancer Cells Resistant to Monoclonal Antibody-Induced Apoptosis 
Marc L. Hyer, Christina Voelkel-Johnson, Semyon Rubinchik, Jian-yun Dong, James S. Norris 
Molecular Therapy 
Volume 2, Issue 4, Pages (October 2000)
DOI: /mthe
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Cell surface Fas expression on different PCa cell lines as determined by FACS analysis. Cells were stained using the primary anti-Fas antibody CH-11 followed by an IgM-phycoerytherin (PE)-conjugated secondary antibody (bold line). As a control (dotted line), an irrelevant IgM isotypic primary antibody was used followed by the IgM-PE-conjugated secondary. K-562 cells (Fas–) were used as a negative control and Jurkat cells (FasR+) were used as a positive control. Percentages represent the number of Fas expressing cells minus th
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

3. FIG. 3
PCa cells transduced in vitro with AdGFPFasLTET undergo Fas-mediated apoptosis. Each PCa cell line was transduced at an m.o.i. of 100 using both AdGFPFasLTET and AdCMVGFP, separately. Fluorescent and bright-field photographs were taken at 48 h of the same field. Fluorescent photographs indicate that cells have been transduced because they are GFP and GFPFas
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

4. FIG. 4
PPC-1 and DU145 cells transduced with AdGFPFasLTET undergo apoptosis as determined by TUNEL assay. PPC-1 cells were transduced at an m.o.i. of 10 and DU145 cells at an m.o.i. of 100 with AdGFPFasLTET and AdCMVGFP, respectively. Cells were stained at 24 h using a PE conjugated anti-BrdU antibody (for control a PE conjugated isotypic antibody was used), and then examined by FACS analysis. Results are expressed as percent anti-BrdU positive over isotypic control. (A) In PPC-1 cells, 83% of the cells stained positive for BrdU while 61% of the cells had detectable GFPFasL. PPC-1 cells are very sensitive to AdGFPFasLTET treatment therefore some cells die before GFPFasL expression can be measured by fluorescence. In AdCMVGFP treatment, 93% of the cells were expressing GFP and only 3% of the cells stained positive for BrdU. (B) In DU145 cells, 35.2% of the cells stained positive for BrdU, while 98% of the cells were expressing GFPFasL. In the AdCMVGFP treatment, 91.8% of the cells were expressing GFP and only 0.3% of the cells stained positive fo
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

5. FIG. 5
Anti-Fas blocking antibody ZB-4 does not block AdGFPFasLTET-associated cytotoxicity in LNCaP and PPC-1 cells. PPC-1 cells were pretreated for 1 h using 500 ng/ml ZB-4 followed by the anti-Fas agonistic antibody CH-11 (500 ng/ml). ZB-4 treatment reduced CH-11 cytotoxicity from 39.7 to 3.1% (P < 0.005). Next we examined if ZB-4 was able to block the cytotoxic effects of AdGFPFasLTET. LNCaP and PPC-1 cells were pretreated for 1 h with ZB-4 (500 ng/ml), and then challenged with AdGFPFasLTET or AdCMVGFP (m.o.i. 100). After 24 h, MTS assay indicated minimal blockage in PPC-1 cells and no blockage in LNCa
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

6. FIG. 6
Intracellular protein localization of GFPFasL, GFP, and Fas in LNCaP cells transduced with AdGFPFasLTET and AdCMVGFP, respectively, using an m.o.i. of 10. Cells were immunostained with a Fas antibody as described under Materials and Methods 14 h after viral treatment. Images shown are a stack of 20 images captured using laser scanning confocal microscopy. In AdCMVGFP treated cells (D), diffuse GFP expression is seen both in the cell's cytoplasm and in the nucleus. In contrast, GFPFasL expression (A) has been excluded from the nucleus (arrow indicates nuclear ghost, faint staining in this area is from GFPFasL expression on the plasma membrane), and is seen in a punctated pattern within the cytoplasm. Fas expression (B and E) is seen both on the membrane and punctated within the cytoplasm (arrow in B indicates a nontransduced cell). GFPFasL and Fas (C) colocalize to the same intracellular compartment [arrow indicates an area of yellow color where GFPFasL (green) and Fas (red) are coexpressed]. This colocalization area is likely the ER, Golgi, and/or secretory vesicles. Images were captured at 400× using immersio
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

7. FIG. 2
Construction of AdGFPFasLTET and AdCMVGFP. The plasmids pLAd-T.GFsL (A) and pLAd-C.Gf (B) contain the transgene cassettes GFPFasL and GFP, respectively. Both plasmids contain the left end Ad5 ITR and packaging signals (ψ). Digestion of pLAd-T.GFsL with SwaI/AvrII releases the transgene cassette, tTA-GFPFasL-TRE (C). GFP and the FasL cDNAs have been fused using a 12-base-pair linker. GFPFasL is under the transcriptional control of the tetracycline responsive element (TRE). Within C, the tetracycline-controlled transactivator (tTA) is composed of both the tetracycline repressor (TetR) and the VP16 activation domain. tTA is constitutively expressed and binds the TRE, activating GFPFasL transcription in the absence of tetracycline. Digestion of pLAd-C.Gf with SwaI/AvrII releases the GFP cassette (D). Each transgene cassette was ligated in vitro with XbaI-digested genomic Ad5 DNA (E3, E4 deleted). Recombinant adenovirus was formed by transfecting the ligation products into the respective packaging cel
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions