Article Figures & Data
Figures
- FIGURE 1.
Apoptotic response of human breast cancer cell lines to rhTRAIL and the DR4 and DR5 antibodies. A. Fluorescence-activated cell sorting analysis of apoptosis after staining with FITC-Annexin V and propidium iodide. Cells at 70% to 80% confluence were treated with 10 or 50 ng/mL rhTRAIL for 6 h and then were harvested. Bottom right quadrant, percentage of early apoptotic cells with exposed phosphatidylserine (Annexin V-FITC positive) but intact membrane (propidium iodide negative). Top right quadrant, necrotic or apoptotic cells in terminal stages with positive staining of both Annexin V-FITC and propidium iodide. B. Quantification of TRAIL-induced apoptosis (as described for A) in the indicated cell lines. Mean ± SD total percentage of the cells in the right quadrants (n = 3). C. Activation of caspase-8 and -3. Cells were treated with 10 ng/mL rhTRAIL for 1 or 4 h as indicated. Caspase activity is indicated by the decrease of pro-caspases (pro-C8 and pro-C3) and appearance of the p23 and the p20/p17 cleaved forms of caspase-8 (C-8) and caspase-3 (C-3), respectively. MCF7 cells express relatively low levels of pro-caspase-8 and are deficient in caspase-3 protein. Representative of three independent experiments. *, nonspecific bands. D. Caspase inhibitor Z-VAD blocks TRAIL-induced apoptosis. Cells were pretreated without or with a general caspase inhibitor Z-VAD at 10 μmol/L and then incubated with 50 ng/mL rhTRAIL for an additional 6 h. The extent of apoptosis was measured as described in A. E. Apoptotic responses to DR4 and DR5 antibodies. Cells were treated with 10 μg/mL anti-DR4 or anti-DR5 monoclonal antibodies for 24 h and apoptosis was analyzed as in A. Mean ± SD (n = 3).
- FIGURE 2.
DR4 and DR5 cell surface expression is down-regulated in TRAIL-resistant cells. A. Total protein expression levels of TRAIL receptors. Equal amounts of whole-cell lysates from the indicated cell lines were analyzed by Western blotting using antibodies specific to DR4 and DR5 and the decoy receptors DcR1 and DcR2, respectively. Anti-DR4 antibody recognizes a peptide corresponding to the NH2-terminal amino acids 1 to 20 of human DR4 mature protein (purchased from Imgenex). Actin was shown as a loading control. *, markers of molecular weight. B. Flow cytometry analysis of cell surface expression of DR4 and DR5. Cells were stained with PE-conjugated monoclonal anti-DR4 or anti-DR5 (open histograms). Shadowed histograms, control cells stained with isotype-matched control IgG. A single experiment representative of six independent experiments with similar results. Right shift, presence of DR4 or DR5 on cell surface. C. Quantification of results in B reveals the relative levels of cell surface DR4 and DR5. Folds of increase in fluorescence intensity compared with the corresponding IgG-PE. D. Confocal microscope images showing the subcellular localization of endogenous DR4 protein. Cells were permeabilized with 0.2% Triton X-100 and labeled with anti-DR4-FITC (green); cytoplasm was stained with CellTracker Red (red). Representative of three independent experiments.
- FIGURE 3.
TRAIL DISC does not assemble in TRAIL-resistant cell lines. A. Cells were treated with biotinylated TRAIL (200 ng/mL) at 4°C for 30 min and then at 37°C for an additional 30 min. Equal amounts of cell lysates were incubated with streptavidin agarose beads overnight at 4°C, and the coimmunoprecipitates were analyzed by immunoblotting for the presence of DR4, DR5, FADD, and caspase-8. The full-length and processed forms of caspase-8 associated with the DISC formation are seen in MDA-MB-231 and MDA-MB-468 cells. B. Comparison of protein expression of relevant TRAIL signaling components (14-3-3, Bid, Bad, Bax, Bcl-2, Bcl-x, FLIP, and FADD) between TRAIL-sensitive and TRAIL-resistant cell lines. Representative blots of three separate experiments.
- FIGURE 4.
Inhibitors of endocytosis restore DR4 and DR5 cell surface expression and sensitize TRAIL-resistant cells to TRAIL-induced apoptosis. A. Wild-type DR4 ectopically expressed in BT474 cells fail to be translocated to the cell surface. MDA-MB-231 and BT474 cells were transfected with a plasmid expressing wild-type DR4 tagged with GFP (DR4-GFP), counterstained with CellTracker Red, and analyzed by confocal microscopy. B. BT474 cells were treated for 20 h with filipin III (FLN; an inhibitor of raft/caveolae endocytosis; left to right, 0, 0.5, and 5 μg/mL), chlorpromazine (CPZ; an inhibitor of clathrin-mediated endocytosis; left to right, 0, 1, and 5 μg/mL), or PAO (a general inhibitor of endocytosis; left to right, 0, 0.1, 0.5, and 5 μg/mL). The protein expressions of DR4 and DR5 were analyzed by Western blotting (top) and flow cytometry using PE-conjugated antibodies (bottom). C. Cells were sequentially treated with the indicated inhibitors (5 μg/mL) for 20 h and rhTRAIL (20 ng/mL) for an additional 6 h. Apoptosis was measured as in Fig. 1A. D. Effects of PAO on epidermal growth factor receptor and Her-2/erbB-2 cell surface expression. Cells were treated with 5 μg/mL PAO for 20 h and analyzed for surface expression of the indicated receptors by fluorescence-activated cell sorting using PE-conjugated antibodies.
- FIGURE 5.
Clathrin-dependent, constitutive endocytosis of DR4 and DR5 in TRAIL-resistant cells. A. Knockdown of AP2 or clathrin sensitizes BT474 cells to TRAIL-induced apoptosis. Cells were transfected with a RNA interference-negative control duplex (siCtrl) or siRNA against AP2 (siAP2) and clathrin heavy chain (siCLT). Top, Western blots of the indicated proteins after 48 h post-transfection. As determined by densitometry, AP2 and clathrin proteins were reduced by 60% and 80%, respectively, in cells transfected with the corresponding siRNA. The levels of DR4 and DR5 proteins were not affected. Representatives of three independent transfections with siRNA sequence 5′-CCUGGGCCGCAUGUAUCUCUUCUAU-3′ (AP2) or 5′-CCGGAAAUUUGAUGUCAAUACUUCA-3′ (clathrin). Similar results were obtained with other two siRNA sequences (see Materials and Methods). Bottom, apoptosis of cells after treatments with 20 ng/mL rhTRAIL for an additional 6 h. B and C. Effects of AP2 or clathrin knockdown on cell surface expression of the indicated receptors. Transfection of siRNA was as in A. Fluorescence-activated cell sorting analyses were done using PE-conjugated antibodies specific to DR4, DR5, Her-2/erbB-2, transferrin receptor, or epidermal growth factor receptor. D. DR4 endocytosis is mediated by its cytoplasmic domain EAQC337LL. Top, mutation in the sorting sequence of DR4 restores its cell surface expression in BT474 cells. BT474 cells were transiently transfected with plasmids for GFP fusion proteins of wild-type DR4 or mutants containing the 409Y(A) or 337LL(AA) mutations. Representative of three independent experiments. Bottom, expression of DR4-337LL(AA) but not DR4- 409Y(A) restores BT474 cell sensitivity to apoptotic induction by rhTRAIL or anti-DR4. Mean ± SD (n = 3). After transfection, EGFP-positive cells were isolated by fluorescence-activated cell sorting and treated with TRAIL (10 ng/mL) or the indicated antibodies (10 μg/mL) for 24 h.
- FIGURE 6.
DR4 gene expression and mutations. Semiquantitative RT-PCR analyses were done using equal amount of total RNA (0.4 μg) from the indicated cell lines using primers for the different regions of DR4 mRNA (1-1407 bp). Amplified products corresponding to regions 1 to 500, 450 to 950, and 900 to 1,407 bp of the DR4 gene. Right, schematic view of the locations of the mutated residues in DR4 protein (1-468 amino acids). TMD, transplasma membrane domain (240-268 amino acids). DD, death domain (379-445 amino acids). DR4 mutations are summarized in Table 2.
Tables
- Table 1.
Correlation between Cell Surface Expression of Death Receptors DR4 and DR5 and Sensitivity to rhTRAIL and Its Agnostic Antibodies in Breast Cancer Cell Lines
Cell lines DR4 surface* DR5 surface* Relative sensitivity† Anti-DR4 Anti-DR5 rhTRAIL MDA-MB-231 ✓ ✓ +++++ +++++ +++++ MDA-MB-468 x ✓ − +++ ++++ T47D x x − − − BT549 x ✓ − +++ +++ MCF7 ✓ ✓ − − − SKBR3 x ✓ − +++ + BT474 x x − − − ↵* Presence (✓) or absence (x) on cell surface.
↵† Apoptosis relative to MDA-MB-231 cells after treatment with the indicated agent (+, sensitive; −, resistant; data from Fig. 1A and B and data not shown for BT549).
- Table 2.
DR4 Mutations in Breast Cancer Cell Lines
Mutation Cell lines A422G (His141Arg) MDA-MB231 MCF7 SKBR3 G626C (Arg209Thr) MDA-MB-231 MCF7 SKBR3 A683C (Glu228Ala) BT474 C853A (Leu285Ile) T47D
Supplementary Data, Zhang, et al.
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Volume 6, Issue 12
