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Rapid Impact Archive
Metabolism Collection
Cell Cycle, Cell Death, and Senescence
TRAIL Resistance of Breast Cancer Cells Is Associated with Constitutive Endocytosis of Death Receptors 4 and 5
Yaqin Zhang and Baolin Zhang
Yaqin Zhang
Baolin Zhang
DOI: 10.1158/1541-7786.MCR-08-0313 Published December 2008
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its agnostic antibodies, which are being evaluated clinically as anticancer therapies, selectively kill cancer cells through the death receptors DR4 and DR5. However, their therapeutic potential is limited by occurring resistance in tumor cells. Here, we compared the apoptotic response of a panel of six human breast cancer cell lines with recombinant human TRAIL and antibodies to DR4 or DR5. Despite their total mRNA and protein expression, TRAIL death receptors, with a higher frequency in DR4, are absent on cell surface in some cell lines. Loss of cell surface expression of DR4 or DR5 accounts for resistance to their corresponding antibody and, importantly, correlates with a decreased sensitivity to TRAIL. TRAIL resistance occurs when both receptors are absent on cell surface regardless of alterations in Bcl-2 family proteins or caspases. Furthermore, inhibition of endocytosis by pharmacologic inhibitors or disruption of clathrin-dependent endocytosis signaling components (adaptor protein 2 and clathrin) restores cell surface expression of the death receptors and sensitize TRAIL-resistant cells to TRAIL-induced apoptosis. DR4 endocytosis appears to be mediated by its cytoplasmic domain EAQC337LL. The results show that TRAIL death receptors undergo constitutive endocytosis in some breast cancer cells. Loss of cell surface expression of DR4 and DR5 could be evaluated as a biomarker for TRAIL resistance in breast tumors. Moreover, the clathrin-mediated endocytosis pathway could be a potential target for therapeutics to overcome tumor resistance to TRAIL receptor-targeted therapies. (Mol Cancer Res 2008;6(12):1861–71)
tumor necrosis factor-related apoptosis inducing ligand (TRAIL)
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors DR4 and DR5 expressed on the surface of target cells (1-3). Recombinant human TRAIL (rhTRAIL) preferentially induces apoptosis in cancer cells over normal cells and shows little or no overt toxicity when systemically administered to animals (4, 5). This has led to multiple clinical trials with biological agents targeting TRAIL receptors, including rhTRAIL and agonistic antibodies to DR4 and DR5. However, a significant portion of tumor cells (6-8), including breast cancer (9-11), are resistant to these agents. Nonresponsive patients will not benefit from the treatments but may still suffer from the potential side effects, highlighting the need for biomarkers that can help predict tumor sensitivity to TRAIL receptor-targeted therapies.
On ligand binding, the death receptors assemble a death-induced signaling complex (DISC) that contains the adaptor protein Fas-associated death domain (FADD) and the apoptosis-initiating protease pro-caspase-8 (or pro-caspase-10; ref. 1). In the DISC, pro-caspase-8 is processed, releasing active caspase-8 into the cytoplasm, where it cleaves and activates effector caspases, such as caspase-3, -6, and -7. Caspase-8 can also stimulate the cleavage of Bid, thus amplifying caspase activation through mitochondria. This apoptotic machinery is shared by TRAIL and antibodies against DR4 or DR5. TRAIL resistance, in some cases, is caused by the simultaneous expression of decoy receptors (DcR1, DcR2, and OPG) that lack a death domain but possess a comparable binding affinity to TRAIL. However, the presence of decoy receptors cannot explain the lack of response of many cancer cells to antibodies specifically targeting DR4 or DR5. Other defects along the pathway, including down-regulation of caspase-8 or -10 and overexpression of antiapoptotic molecules cellular FLICE-inhibitory protein (FLIP; ref. 12) and Bcl-2 and Akt (2), have been associated with TRAIL resistance in individual cell lines. However, none of these factors showed a consistent correlation with TRAIL resistance in multiple cancers (13). A better understanding of the mechanisms of TRAIL resistance may lead to the identification of predictive biomarkers for tumor responsiveness and identification of better targets for drugs to overcome the resistance.
Although the mRNA expression of both DR4 and DR5 is found in a wide variety of tumor cells including breast cancer cells (9, 10), there is no correlative link between total receptor expression levels and the sensitivity of tumors to rhTRAIL. Recent studies have suggested that the mRNA expression of TRAIL receptors does not necessarily reflect their functional protein expression due to post-translational regulation. DR4, but not DR5, was shown to be N-glycosylated in prostate cancer cells (14). Both receptors undergo O-glycosylation in various tumor cell lines, with a higher level in TRAIL-sensitive cells (13). They are also subject to regulation of endocytosis pathways (15-17). In line with these findings, the cell surface DR4 protein was down-regulated in some TRAIL-resistant cancer cells, including those of colon (18) and leukemia (19). However, little is known about the functional status of TRAIL death receptors in breast cancer cells.
Breast cancer cells are generally resistant to TRAIL, but the mechanism is unresolved (9, 10). In the present study, we found that the two TRAIL death receptors, with a higher incidence in DR4, are functionally deficient in some breast cancer cell lines due to down-regulation of cell surface expression. DR4 deficiency, which may result from defects in its protein sorting machinery, is consistent with the cellular resistance to anti-DR4 antibody and a decreased sensitivity to rhTRAIL. The results highlight the importance of determining the functionality of death receptors in predicting TRAIL sensitivity of tumor cells. In addition, the data show that the ability of a combinational therapy in restoring cell surface expression of death receptors is crucial to overcome resistance to TRAIL-related therapies.
TRAIL Requires Both DR4 and DR5 for Maximal Killing in Breast Cancer Cells
To investigate the mechanisms of TRAIL resistance in breast cancer cells, we first compared the induction of apoptosis by rhTRAIL and agonistic antibodies to DR4 and DR5 in a panel of six human breast cancer cell lines. As shown previously (9, 10), rhTRAIL was a potent inducer of apoptosis in MDA-MB-231 cells. At a concentration as low as 10 ng/mL, rhTRAIL induced substantial and rapid apoptosis, indicated by Annexin V binding (Fig. 1A and B ), and time-dependent cleavage of caspase-8 and -3 (Fig. 1C). By contrast, other cell lines were either less sensitive (MDA-MB-468 and SKBR3) or resistant (T47D, MCF7, and BT474) to rhTRAIL-induced apoptosis. The resistance of BT474 and MCF7 cells was maintained even with higher doses of rhTRAIL (up to 1 μg/mL) or longer incubation (48 h; data not shown). In TRAIL-resistant cells, no activation of caspase-8 and -3 was detected (Fig. 1C; data not shown for T47D). MCF7 cells only express minimal caspase-8 protein and are deficient in caspase-3 expression. Consistently, a general caspase inhibitor Z-VAD completely blocked TRAIL-induced apoptosis in MDA-MB-231 and two other cell lines (Fig. 1D). These results indicate that caspase activation is an essential step in rhTRAIL-induced cell killing. TRAIL-resistant cells appeared to have defective stimulation of caspase-8 processing and correspondingly less or no cleavage of caspase-3.
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).
When treated with the agonist antibodies to DR4 and DR5, these cell lines showed remarkable difference in sensitivity (Fig. 1E). Anti-DR5 induced apoptosis in cell lines, except T47D, MCF7, and BT474 cells. However, anti-DR4 showed apoptosis-inducing activity only in MDA-MB-231 cells and was inactive on the other cell lines. The sensitivities appeared to correlate with those observed for rhTRAIL (Fig. 1B; Table 1 ). For example, the hypersensitivity of MDA-MB-231 cells correlates with its susceptibility to both anti-DR4 and anti-DR5. Loss of response to either antibody correlates well with a decreased sensitivity to rhTRAIL (MDA-MB-468 and SKBR3). The cell lines (BT474, T47D, and MCF7) that are nonresponsive to both anti-DR4 and anti-DR5 are completely resistant to rhTRAIL. These results indicate that both DR4 and DR5 are required for rhTRAIL to achieve maximal killing effect of breast cancer cells. In addition, the failure of anti-DR4 or anti-DR5 to induce apoptosis raises the possibility that the death receptor may be functionally deficient in those cells.
Correlation between Cell Surface Expression of Death Receptors DR4 and DR5 and Sensitivity to rhTRAIL and Its Agnostic Antibodies in Breast Cancer Cell Lines
Deficiency in DR4 and DR5 Cell Surface Expression Correlates with Resistance to Respective Antibody and Reduced Sensitivity to TRAIL
To identify molecular determinants that control the rhTRAIL sensitivity in breast cancer cells, we first examined total protein expression of TRAIL receptors by Western blotting (Fig. 2A ). Both DR4 and DR5 were expressed across the cell lines, showing no direct relationship with the observed patterns of sensitivity. For example, DR4 protein was expressed in all the cell lines, with a higher level in MDA-MB-231 cells. DR5 was detected as two bands corresponding in sizes to the two splicing variants (20), but its overall protein level is even higher in TRAIL-resistant cells (T47D and MCF7) compared with TRAIL-sensitive cells (MDA-MB-231 and MDA-MB-468). Likewise, DcR1 and DcR2 were comparable in these cell lines irrespective of their sensitivity to rhTRAIL, suggesting that the pattern of decoy receptor expression may not be responsible for the observed resistance.
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.
Because only cell surface DR4 and DR5 are able to bind TRAIL and mediate apoptosis, we next examined the cell surface expression of TRAIL receptors by flow cytometry using phycoerythrin (PE)-conjugated antibodies. The presence of these receptors on the cell surface is indicated by a right shift of the peak after binding to a specific antibody relative to a control IgG-PE (Fig. 2B and C). DR4 was expressed on the surface of MDA-MB-231 and MCF7 cells but not those of MDA-MB-468, T47D, SKBR3, and BT474 cell lines. In contrast, DR5 was detected on the cell surface in most of the cell lines, except T47D and BT474. Only a slight shift in DR5 occurred in MDA-MB-468 and SKBR3 cells, which correlated with sensitivity to anti-DR5. The cell surface expression was confirmed by confocal microscopy analysis of endogenous proteins after immunostaining with FITC-conjugated anti-DR4 or anti-DR5 antibodies (not shown). As shown in Fig. 2D, DR4 protein was localized in the plasma membrane in MDA-MB-231 but was found predominantly in the cytosol of BT474 cells.
TRAIL-resistant cell lines (T47D, MCF7, and BT474) were no longer able to efficiently assemble a DISC after rhTRAIL treatment as shown by weak signals of the adaptor molecule FADD and pro-caspase-8 in the immunocomplexes of biotinylated rhTRAIL (Fig. 3A ). DR4 and DR5 proteins were also virtually undetectable in the coimmunoprecipitates from those cell lines, except for MCF7 cells. This was in contrast to MDA-MB-231 cells in which treatment with rhTRAIL recruited FADD into DISC and the processing of caspase-8. Taken together, the results suggest that TRAIL death receptors become functionally deficient in rhTRAIL-resistant cells as a result of down-regulation of cell surface expression. Importantly, loss of cell surface expression of the death receptors, with a higher incidence in DR4, appears to be sufficient to account for the relative sensitivity of some breast cancer cells to apoptotic induction by either rhTRAIL or its agonistic antibodies (Table 1).
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.
We also characterized expression of the regulatory proteins that are involved in the death receptor-mediated apoptosis signaling pathway. The antiapoptotic molecules FLIP, Bcl-2, and Bcl-x have been implicated in the resistance of cancer cells to apoptosis and are found to be differentially expressed in breast cancer cell lines (Fig. 3B). For instance, the long form of FLIP (FLIPL) and its p43 cleaved product were more underexpressed in MDA-MB-231 cells than other lines. In addition, MCF7 cells were deficient in caspase-3 and expressed little pro-caspase-8 protein compared with other cell lines (Fig. 1C). These factors might contribute to the observed TRAIL sensitivity in individual cell lines, but none showed a consistent correlation. Likewise, the expression levels of proapoptotic proteins Bad, Bax, and Bid were not directly related to the observed sensitivities to rhTRAIL and the agnostic antibodies.
DR4 and DR5 Undergo Constitutively Endocytosis through Clathrin-Dependent Pathway in TRAIL-Resistant Cells
Like other transmembrane proteins (21), TRAIL receptors appear to subject to membrane transport and endocytic sorting signals (15-18, 22, 23). The absence of DR4 or DR5 cell surface expression promoted us to identify defects within the translational modification processes. We first tested whether exogenous DR4 can be expressed on the plasma membrane in TRAIL-resistant cells. Cells were transfected with a plasmid expressing green fluorescent protein (GFP)-tagged wild-type DR4 (GFP-DR4-wt) and its cellular localization was detected by confocal microscopy. In MDA-MB-231 cells, DR4-GFP-wt was expressed on the cell surface but was found exclusively in the cytoplasm of BT474 cells (Fig. 4A ). These data suggest that the sorting machinery of TRAIL death receptors is defective in TRAIL-resistant cells, resulting in failure of expression on plasma membrane.
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.
We then tested whether endocytosis inhibitors could affect TRAIL receptor expression. All three inhibitors tested had no effect on the total protein levels of the receptors (Fig. 4B, top). Strikingly, phenylarsine oxide (PAO), a general inhibitor of endocytosis (24, 25), induced a dose-dependent restoration of receptor cell surface expression in BT474 cells (Fig. 4B, bottom). At threshold, the levels of surface DR4 and DR5 were comparable with those of MDA-MB-231 cells (Fig. 2B). Treatment with chlorpromazine, an inhibitor of clathrin-mediated endocytosis (26, 27), also increased the cell surface receptor expression. However, no effect was observed with the raft/caveolae endocytosis inhibitor filipin (28, 29). Consistent with these data, sequential treatment with PAO or chlorpromazine, but not filipin, followed by TRAIL induced massive apoptosis in T47D (data not shown) and BT474 (Fig. 4D) cells. Pretreatment with PAO also increased cell surface expression of the epidermal growth factor receptor, which is undetectable on the surface of wild-type BT474 cells. In contrast, Her-2/erbB-2 cell surface expression was not affected (Fig. 4D). These results suggest that clathrin-mediated endocytosis pathways may be responsible for the deficiency in DR4 and DR5 cell surface expression in TRAIL-resistant cells.
We further examined the role of clathrin pathway in TRAIL receptor endocytosis by targeted gene silencing. As shown in Fig. 5A and B , knockdown of adaptor protein 2 (AP2) or clathrin significantly increased DR4 and DR5 cell surface expression without changing their total protein levels in BT474 cells. The resultant cells became highly susceptible to TRAIL-induced apoptosis (Fig. 5A, bottom). Similar to endocytosis inhibitor (Fig. 4D), disruption of AP2 or clathrin did not alter the surface expression of Her-2/erbB-2 or transferrin receptor but remarkably increased epidermal growth factor receptor surface levels (Fig. 5C). Taken together, these results suggest that DR4 and DR5 may undergo constitutive endocytosis through clathrin-dependent pathways in TRAIL-resistant cells and experimental inhibition of endocytosis restores DR4- and DR5-mediated signaling in response to TRAIL. The results also suggest that different receptors are regulated by distinct endocytosis pathways.
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.
DR4 Endocytosis Is Mediated by Its Dileucine Sorting Signal
Sequence analysis of DR4 protein reveals two potential sorting signals within the cytoplasmic domain: EAQC337LL and 409YAML. These signals are present in many other receptors and have been implicated in mediating the rapid internalization of proteins and targeting to the endosomal-lysosomal compartments (21). To test whether these signals are responsible for DR4 sorting, we generated two DR4 mutants, LL337-338AA and Y409A, using site-directed mutagenesis. When overexpressed in BT474 cells, the DR4(Y409A) mutant, like the wild-type DR4 protein, was found in the cytoplasm (Fig. 5D, top) and remained resistant to rhTRAIL and anti-DR4 antibody (data not shown). However, the DR4(LL337-338AA) mutant showed expression almost exclusively on the cell surface. The resulting cells underwent considerable apoptosis when treated with rhTRAIL or anti-DR4 (Fig. 5D, bottom). Interestingly, restoration of DR4 cell surface expression did not alter resistance of BT474 cells to anti-DR5 antibody. These results show that the dileucine-based sorting signal (EAQC337LL) is a key structural determinant in DR4 for its sorting through the clathrin-mediated endocytosis machinery.
DR4 Mutations in Breast Cancer Cells
The observation on DR4 sorting signal promoted us to examine whether TRAIL resistance resulted from DR4 gene mutations. To this end, reverse transcription-PCR (RT-PCR) analyses were done using total RNA isolated from the individual cell lines and using primers specific to the different regions of DR4 mRNA. All cell lines showed a single amplification product for each portion of the gene (Fig. 6 ). DNA sequence analysis confirmed the absence of deletion or splicing variants of DR4 gene. No mutations were found in the sorting signal regions of DR4. Instead, several other mutations were identified, including A422G (His141Arg), G626C (Arg209Thr), A683C (Glu228Ala), and C853A (Leu285Ile), in different cell lines (Table 2 ). However, there is no apparent correlation between the DR4 mutational profiles and the observed cell surface expression of DR4 and DR5 and TRAIL sensitivities. These data suggest that genetic mutations in DR4 may only play a small role, if any, in TRAIL resistance of breast cancer cells.
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.
DR4 Mutations in Breast Cancer Cell Lines
Resistance of tumor cells to death receptor-induced apoptosis is a limiting factor for the clinical use of rhTRAIL and its agonistic antibodies. Predicting whether a tumor will respond to these agents is still an empirical process because no biomarker for tumor susceptibility has been identified. In this study, we show that TRAIL death receptors, particularly DR4, are functionally deficient in several breast cancer cell lines due to down-regulation of cell surface expression. Regardless of total DR4 and DR5 protein levels, their absence on cell surface is sufficient to account for the failure in forming TRAIL DISC and later events of apoptosis. Thus, loss of cell surface expression of TRAIL death receptors may be evaluated as a potential predictive biomarker for TRAIL sensitivity in breast cancer cells.
Several different mechanisms have been identified to block TRAIL-induced apoptosis in individual cancer cell lines, including the simultaneous expression of decoy receptors (30), overexpression of antiapoptotic molecules (e.g., FLIP, Bcl-2, and Bcl-XL; refs. 12, 31), and loss of caspase-8 expression (32). However, none of these factors showed a consistent correlation with TRAIL resistance in multiple cancers. A recent study showed that mRNA expression of the peptidyl O-glycosyltransferase GALNT14, which is responsible for the O-glycosylation of death receptors, correlated with TRAIL sensitivity in pancreatic carcinoma, non-small cell lung carcinoma, and melanoma cell lines. However, GALNT14 mRNA was only detected in a small portion (∼10%) of breast cancers (13).
As death receptors, DR4 and DR5 must be properly expressed on cell surface to transmit an apoptotic signal from their ligands. DR4 protein is absent on the surface in most (4 of 6) of the breast cancer cell lines studied despite its comparable levels of total mRNA (Supplementary Data) and protein. Importantly, DR4 deficiency correlated well with the cellular resistance to anti-DR4 antibody and a decreased sensitivity to rhTRAIL. As with our results, down-regulation of DR4 cell surface expression was associated with TRAIL resistance in leukemia (19) and colon cancer cells (18). The cell lines (BT474 and T47D) lacking both DR4 and DR5 cell surface expression are completely resistant to rhTRAIL and the corresponding antibody. However, the cell surface expression of DR4 and DR5 does not necessarily reflect the cellular sensitivity to TRAIL-induced apoptosis. This is the case for MCF7 cells, which express both DR4 and DR5 on cell surface but are resistant to rhTRAIL and antibodies. This may be attributed to the defects downstream of TRAIL death receptors, including lack of caspase-3 protein expression (Fig. 1B) due to gene depletion (33), lower caspase-8 protein, and higher levels of Bcl-2 relative to the proapoptotic Bad protein (Fig. 3B). The cellular FLIP, in its long form (FLIPL) and p43 cleaved form, is remarkably underexpressed in MDA-MB-231 cells compared with other cell lines, which presumably facilitate the activation of caspase-8 and execution of apoptosis by TRAIL. Nonetheless, loss of cell surface expression of death receptors appears to be sufficient to avoid apoptosis by their ligands regardless of the defects in the intracellular signaling components. Our results warrant additional studies on the functionality of TRAIL receptors in other cancer cell lines and patient specimens. If it is confirmed in clinic, the absence of cell surface expression of DR4 and/or DR5 could be a useful biomarker for TRAIL sensitivity. Furthermore, the results suggest that certain tumors may use DR4 or DR5 deficiency as a mechanism of evading death receptor-mediated immune surveillance (34).
TRAIL appears to require both DR4 and DR5 for a maximal killing in breast cancer cells (Fig. 1; Table 1). The two death receptors may act synergistically by forming heteroreceptor complexes (35). This observation is in contrast to previous studies showing that DR5 is the primary receptor leading to apoptosis in various types of cancer cells (36-39). Based on these previous results, DR5 has been the primary target for development of antibody therapy. There are currently five anti-DR5 antibody products, compared with one anti-DR4 product, in clinical trials. However, there is also evidence that TRAIL induces apoptosis exclusively through DR4 in cancer cell lines from skin (40), ovary (41), and leucocytes (19) as well as primary cells from chronic lymphocytic leukemia and mantle cell lymphoma (42). The molecular basis for this preference of TRAIL is not clear but may be due to difference in the functional status of death receptors in a specific tumor. We propose that TRAIL may have to use one receptor, whereas the other is defective. Our results suggest that DR4 may be deficient at a higher frequency than DR5 in breast cancer cells.
The molecular mechanism for TRAIL death receptor deficiency remains unclear, but it may involve defects in the protein sorting machinery. Recent studies suggest that protein transport and endocytosis pathways may play an important role in the regulation of death receptor expression (15-18, 22, 23). On ligation, TRAIL induces internalization/endocytosis of its receptors through clathrin-dependent pathways (15, 43). This process is accompanied by simultaneously activation of caspases, which in turn cleave clathrin pathway components such as AP2 or clathrin itself in cells from BJAB and colon carcinoma (15). AP2 cleavage was also observed in MDA-MB-231 but not BT474 cells (Supplementary Data). Austin et al. (15) suggested that caspase-mediated cleavage of clathrin or AP2 may provide a positive feedback loop, thereby halting the endocytic machinery and reinforcing caspase activation and apoptosis execution through TRAIL receptors. In agreement, blockade of receptor internalization rather amplified the apoptotic signaling of TRAIL (43). Moreover, Jin et al. (18) showed that a prolonged TRAIL treatment of colon cancer cells resulted in down-regulation of cell surface DR4 and TRAIL resistance. Consistent with these reports, TRAIL failed to induce apoptosis in breast cancer cell lines (T47D and BT474) lacking surface expression of DR4 and DR5 (Fig. 2; Table 1). Restoration of surface expression of TRAIL receptors, which was achieved by either endocytosis inhibitors or knockdown of clathrin or AP2, restored cellular sensitivity to TRAIL-induced apoptosis (Figs. 4 and 5). In contrast, Her-2/erbB-2 and transferrin receptor were not affected by disruption of clathrin pathway. These results suggest that DR4 and DR5 may undergo a constitutive endocytosis through clathrin-dependent pathways, resulting in failure of cell surface expression and resistance to TRAIL-induced apoptosis. However, the total protein levels of clathrin or AP2 was not directly associated with the cell surface DR4 and DR5 in the cell lines studied (Supplementary Data), suggesting that other clathrin pathway components may also contribute to the receptor deficiency. Mutational analysis reveals that the dileucine-based sorting signal (EAQC337LL) is a key structural determinant in DR4 for its endocytosis (Fig. 5D). However, no mutations were found in the sorting signal regions of DR4 gene in the cell lines studied (Table 2). Additional studies are required to identify molecular alterations in both endocytosis and transport pathways that are responsible for the deficiency of TRAIL death receptors. The acquired information may contribute to the selection of appropriate patient population for treatment and design of strategies to overcome tumor resistance to TRAIL-related therapies.
In summary, our results show that breast cancer cells may use cell surface down-regulation of DR4 and/or DR5 as a mechanism to escape apoptosis through TRAIL receptors. These data may help to predict tumor resistance likely to be found in patient samples and provide a rational approach to improve TRAIL receptor-targeted therapies.
Human breast cancer cell lines including MDA-MB-231, MDA-MB-468, T47D, MCF7, SKBR3, and BT474 were obtained from the American Type Culture Collection. MDA-MB-231 cells were grown in DMEM/F-12 (1:1 mix; Mediatech) supplemented with 5% fetal bovine serum, 4 mmol/L glutamine, 50 μmol/L β-mercaptoethanol, and 1 mmol/L sodium pyruvate at 37°C and 5% CO2 in air. Other cell lines were cultured as recommended by the American Type Culture Collection.
rhTRAIL, expressed and purified as homotrimeric protein from Escherichia coli, and monoclonal antibodies specific to the extracellular domains of human death receptors DR4 and DR5 and their PE-conjugated forms, decoy receptors DcR1 and DcR2, and PE-conjugated antibodies to transferrin receptor, epidermal growth factor receptor, and Her-2/erbB-2 were purchased from R&D Systems. Anti-DR4 conjugated with Alexa Fluor 488 was from AbD Serotec. Rabbit polyclonal antibody against a peptide corresponding to amino acids 1 to 20 of human DR4 mature protein was from Imgenex. Antibodies against human caspase-3 and -8 were from Calbiochem. Antibodies to FADD, Bcl-2, Bcl-x, Bid, Bad, Bax, and 14-3-3 were from BD Pharmingen. Anti-FLIP was from Cell Signaling Technology, and anti-actin was from Santa Cruz Biotechnology. Stealth RNA interference-negative control duplex and the validated siRNA duplexes targeting different regions of AP2 (5′-CCUGGGCCGCAUGUAUCUCUUCUAU-3′, 5′-AUAGAAGAGAUACAUGCGGCCCAGG-3′, and 5′-CCGGAAGCCGAUGAGUUGCUGAAUA-3′) and clathrin heavy chain (5′-CCGGAAAUUUGAUGUCAAUACUUCA-3′, 5′-UGAAGUAUUGACAUCAAAUUUCCGG-3′, and 5′-AAAUUCUUCUAACUCUGCAAGGCGG-3′) were purchased from Invitrogen. Inhibitors of endocytosis, PAO and chlorpromazine, were from Calbiochem; filipin III was from Cayman Chemical.
Apoptosis Assays
Cells were grown on 6-well plates to 70% to 80% confluence and treated with rhTRAIL or anti-DR4 or anti-DR5 antibodies at the indicated concentrations. As indicated, cells were pretreated with the endocytosis inhibitors. At the selected time points, cells were collected and analyzed by flow cytometry after staining with Annexin V-FITC (BD Pharmingen) and propidium iodide (44).
Flow Cytometric Analysis of Cell Surface Expression of Death Receptors
Fluorescence-activated cell sorting analysis of cell surface expression of DR4 and DR5 was done using the PE-conjugated antibodies (R&D Systems) as per the manufacturer's recommendation. In brief, cells at 70% to 80% confluence were harvested by incubation with EDTA-trypsin and washed twice in PBS. Cells (1 × 105) were incubated in 25 μL PBS containing 1% goat serum for 15 min at room temperature. Afterwards, cells were incubated with 10 μg/mL anti-DR4-PE or anti-DR5-PE (mouse IgG1-PE and IgG2b-PE as respective control) for 45 min at 4°C in the dark. Cells were then washed twice with PBS and resuspended in 0.5 mL PBS for final analysis.
Plasmids and Transfection
The DR4 cDNA was amplified by RT-PCR using total RNA from MDA-MB-231 cells and primers specific to human DR4 gene and subcloned into a pEGFP-N1 vector (BD Bioscience) for expression of DR4-GFP protein (EGFP fused to the intracellular COOH terminus of DR4). Mutations were introduced using the Quick Change Site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. All constructs were verified by DNA sequencing. Plasmids were transfected using FuGENE reagent (Roche Diagnostics). The reverse transfections of siRNA duplex (either single siRNA sequence or a mixture of the three duplexes) were done using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's instructions.
Immunofluorescence and Microscopy
Cells were cultured on glass chamber slides at 70% to 80% confluency and stained with CellTracker Red before harvest. Cells were fixed in 3% paraformaldehyde for 30 min in PBS (pH 7.4). To stain the endogenous DR4, cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min. After incubation in a blocking buffer containing 10% goat serum for 20 min, cells were incubated with FITC-conjugated anti-DR4 for 1 h and washed three times with the blocking buffer. Finally, the slides were detached from the medium chamber and mounted with Antifade reagent (Invitrogen). DR4-GFP-transfected cells were directly visualized after CellTracker Red staining. Confocal microscopy acquisitions were done on a Zeiss LAM 5 PASCAL confocal laser scanning microscope. All images are representative of three to five independent experiments.
Reverse Transcription-PCR
Total RNA was isolated from different cells using Trizol reagent (Invitrogen). RT-PCR was carried out using High-Fidelity One-Step RT-PCR kit (Invitrogen) as per the manufacturer's instructions. Primer pairs used for detection different regions of human DR4 are as follows: 1 to 500 bp (5′-ATGGCGCCACCACCAGCTA and 5′-CATGGGAGGCAAGCAAACA), 450 to 950 bp (a primer pair from R&D Systems), and 900 to 1,407 bp (5′-GAGCAACGCAGACTCGCT and 5′-TCACTCCAAGGACACGGC). β-Actin was used as an internal control. The RT-PCR products were resolved on 1% agarose gel and purified using a QIAquick Gel Extraction kit (Qiagen). DNA sequencings were done with Applied Biosystems 3100 automated sequencer (Food and Drug Administration Core Facility).
TRAIL DISC Formation
The assays were done by using biotinylated TRAIL in combination with streptavidin beads (Pierce) as described previously (16). In brief, rhTRAIL was labeled with EZ-Link Sulfo-NHS-LC-Biotin according to the manufacturer's instructions (Pierce). Cells were precooled on ice, incubated with biotinylated TRAIL (200 ng/mL) for 30 min on ice and then 30 min at 37°C, washed extensively, and lysed in a lysis buffer (Pierce) supplemented with protease inhibitors (Calbiochem). Protein concentration was determined by a BCA protein assay (Pierce). Equal amounts of extracts were incubated with streptavidin-Sepharose beads overnight at 4°C and the associated proteins were detected by immunoblotting.
Western blot analyses were done as described previously (45). In brief, cells (1 × 106) were lysed in SDS lysis buffer containing 50 mmol/L Tris-HCl (pH 7.0), 2% SDS, and 10% glycerol and incubated for 20 min at 95°C. Protein concentrations were estimated using the BCA protein assay (Pierce). Equal amounts of cell lysates (20 μg/lane) were resolved by electrophoresis using a 4% to 12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to nitrocellulose membranes (Millipore) for immunoblot analysis with an appropriate dilution of antibodies (1:1,000-1:2,000). When necessary, the membranes were stripped by Restore Western Blot Stripping Buffer (Pierce) and reprobed with appropriate antibodies. Immunocomplexes were visualized by chemiluminescence using ECL (Santa Cruz).
We thank Dr. Emily Shacter for critical reading of the article and Dr. Tatsushi Yoshida for useful discussion and comments.
Grant support: Food and Drug Administration Critical Path Fund.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).
Accepted August 28, 2008.
Received July 2, 2008.
Revision received August 8, 2008.
LeBlanc HN, Ashkenazi A. Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 2003;10:66–75.
Takeda K, Stagg J, Yagita H, et al. Targeting death-inducing receptors in cancer therapy. Oncogene 2007;26:3745–57.
Yagita H, Takeda K, Hayakawa Y, et al. TRAIL and its receptors as targets for cancer therapy. Cancer Sci 2004;95:777–83.
Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 1999;104:155–62.
Lawrence D, Shahrokh Z, Marsters S, et al. Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med 2001;7:383–5.
van Geelen CM, de Vries EG, de Jong S. Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy. Drug Resist Updat 2004;7:345–58.
Bhojani MS, Rossu BD, Rehemtulla A. TRAIL and anti-tumor responses. Cancer Biol Ther 2003;2:S71–8.
Koschny R, Walczak H, Ganten TM. The promise of TRAIL-potential and risks of a novel anticancer therapy. J Mol Med 2007;85:923–35.
Keane MM, Ettenberg SA, Nau MM, et al. Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer Res 1999;59:734–41.
Singh TR, Shankar S, Chen X, et al. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo. Cancer Res 2003;63:5390–400.
Chinnaiyan AM, Prasad U, Shankar S, et al. Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc Natl Acad Sci U S A 2000;97:1754–9.
Safa AR, Day TW, Wu CH. Cellular FLICE-like inhibitory protein (C-FLIP): a novel target for cancer therapy. Curr Cancer Drug Targets 2008;8:37–46.
Wagner KW, Punnoose EA, Januario T, et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med 2007;13:1070–7.
Yoshida T, Shiraishi T, Horinaka M, et al. Glycosylation modulates TRAIL-R1/death receptor 4 protein: different regulations of two pro-apoptotic receptors for TRAIL by tunicamycin. Oncol Rep 2007;18:1239–42.
Austin CD, Lawrence DA, Peden AA, et al. Death-receptor activation halts clathrin-dependent endocytosis. Proc Natl Acad Sci U S A 2006;103:10283–8.
Kohlhaas SL, Craxton A, Sun XM, et al. Receptor-mediated endocytosis is not required for TRAIL-induced apoptosis. J Biol Chem 2007;282:12831–41.
Ren YG, Wagner KW, Knee DA, et al. Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle. Mol Biol Cell 2004;15:5064–74.
Jin Z, McDonald ER, III, Dicker DT, et al. Deficient tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor transport to the cell surface in human colon cancer cells selected for resistance to TRAIL-induced apoptosis. J Biol Chem 2004;279:35829–39.
Cheng J, Hylander BL, Baer MR, et al. Multiple mechanisms underlie resistance of leukemia cells to Apo2 Ligand/TRAIL. Mol Cancer Ther 2006;5:1844–53.
Screaton GR, Mongkolsapaya J, Xu XN, et al. TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL. Curr Biol 1997;7:693–6.
Bonifacino JS, Traub LM. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 2003;72:395–447.
Muppidi JR, Tschopp J, Siegel RM. Life and death decisions: secondary complexes and lipid rafts in TNF receptor family signal transduction. Immunity 2004;21:461–5.
Zhang XD, Franco AV, Nguyen T, et al. Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells. J Immunol 2000;164:3961–70.
Zeng C, Vangveravong S, Xu J, et al. Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy. Cancer Res 2007;67:6708–16.
Piper M, Salih S, Weinl C, et al. Endocytosis-dependent desensitization and protein synthesis-dependent resensitization in retinal growth cone adaptation. Nat Neurosci 2005;8:179–86.
Sun X, Yau VK, Briggs BJ, et al. Role of clathrin-mediated endocytosis during vesicular stomatitis virus entry into host cells. Virology 2005;338:53–60.
Okuyama M, Laman H, Kingsbury SR, et al. Small-molecule mimics of an alpha-helix for efficient transport of proteins into cells. Nat Methods 2007;4:153–9.
van der Luit AH, Vink SR, Klarenbeek JB, et al. A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells. Mol Cancer Ther 2007;6:2337–45.
Rejman J, Bragonzi A, Conese M. Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther 2005;12:468–74.
Pan G, Ni J, Wei YF, et al. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:815–8.
Guo BC, Xu YH. Bcl-2 over-expression and activation of protein kinase C suppress the trail-induced apoptosis in Jurkat T cells. Cell Res 2001;11:101–6.
Eggert A, Grotzer MA, Zuzak TJ, et al. Resistance to TRAIL-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression. Med Pediatr Oncol 2000;35:603–7.
Kurokawa H, Nishio K, Fukumoto H, et al. Alteration of caspase-3 (CPP32/Yama/apopain) in wild-type MCF-7, breast cancer cells. Oncol Rep 1999;6:33–7.
Finnberg N, Klein-Szanto AJ, El Deiry WS. TRAIL-R deficiency in mice promotes susceptibility to chronic inflammation and tumorigenesis. J Clin Invest 2008;118:111–23.
Schneider P, Thome M, Burns K, et al. TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-κB. Immunity 1997;7:831–6.
Ichikawa K, Liu W, Zhao L, et al. Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity. Nat Med 2001;7:954–60.
Kelley RF, Totpal K, Lindstrom SH, et al. Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling. J Biol Chem 2005;280:2205–12.
Wang Y, Engels IH, Knee DA, et al. Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway. Cancer Cell 2004;5:501–12.
Song JH, Tse MC, Bellail A, et al. Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis-inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells. Cancer Res 2007;67:6946–55.
Leverkus M, Sprick MR, Wachter T, et al. TRAIL-induced apoptosis and gene induction in HaCaT keratinocytes: differential contribution of TRAIL receptors 1 and 2. J Invest Dermatol 2003;121:149–55.
Horak P, Pils D, Haller G, et al. Contribution of epigenetic silencing of tumor necrosis factor-related apoptosis inducing ligand receptor 1 (DR4) to TRAIL resistance and ovarian cancer. Mol Cancer Res 2005;3:335–43.
MacFarlane M, Kohlhaas SL, Sutcliffe MJ, et al. TRAIL receptor-selective mutants signal to apoptosis via TRAIL-R1 in primary lymphoid malignancies. Cancer Res 2005;65:11265–70.
Kohlhaas SL, Craxton A, Sun XM, et al. Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. J Biol Chem 2007;282:12831–41.
Zhang B, Zhang Y, Shacter E. Rac1 inhibits apoptosis in human lymphoma cells by stimulating Bad phosphorylation on Ser-75. Mol Cell Biol 2004;24:6205–14.
Zhang Y, Zhang B. D4-GDI, a Rho GTPase regulator, promotes breast cancer cell invasiveness. Cancer Res 2006;66:5592–8.
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Mol Cancer Res December 1 2008 (6) (12) 1861-1871; DOI: 10.1158/1541-7786.MCR-08-0313
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text:
B2 Chapter 1
(1) The True Identity of the Beast
1 And the dragon stood on the shore of the sea. And I saw a beast coming out of the sea. He had ten horns and seven heads, with ten crowns on his horns, and on each head a blasphemous name. 2 The beast I saw resembled a leopard, but had feet like those of a bear and a mouth like that of a lion. The dragon gave the beast his power and his throne and great authority. (Revelation 13:1-2)
In order to understand this beast it is first necessary to understand what beasts represent in the Bible. For this we go to Daniel chapter 7 where Daniel saw a vision in which four beasts came up out of the sea. After Daniel describes what he saw, he asked an angel to explain it to him. The angel said:
'The four great beasts are four kingdoms that will rise from the earth.' (Dan. 7:17)
It is evident that the beasts represent great military / political kingdoms that will come upon the world, what we call empires. The first beast Daniel saw was like a lion, the second beast like a bear, the third beast like a leopard, the fourth had large iron teeth and ten horns. The beast of Revelation 13 is similar to all four beasts in that it is part lion, part bear, part leopard, and has ten horns. Therefore, the beast of Rev. 13 will be a large and powerful empire.
Empires have the power to do the most harm to the largest number of people, especially God's people, which is why they are seen as vicious beasts in the Bible. Therefore, the beast of Rev. 13 is not an individual man, called the Antichrist, the final beast is another empire that will rise to power in the Middle East. However, an individual will be leading the empire beast who I refer to as the final Antichrist, since there have been many Antichrists. The Apostle John said the spirit of Antichrist was already in the world during the first century (1 John 4:3).
(2) The Seven Heads of The Beast
Revelation 17 gives us more details about the seven heads:
9 "This calls for a mind with wisdom. The seven heads are seven hills on which the woman sits. 10 They are also seven kings. Five have fallen, one is, the other has not yet come; but when he does come, he must remain for a little while. (Rev. 17:9-10)
Even though it says wisdom is needed to figure out the meaning, people quickly jump to the most obvious conclusion, which is not the correct answer. The seven heads of the beast do not represent seven of Rome's emperors; the seven heads represent empires that have ruled over the Middle East and Europe during the course of history. One of the empires will rise again, which we will learn about in the following pages.
The text says the seven heads have a double meaning; they represent seven mountains and seven kings. The fact that five kings are fallen and one is not yet come, tells us that the kings rule in succession; one after the other. The passage does not mean that five had fallen at the time that John saw this vision. This is one of the reasons why it takes wisdom to understand the meaning.
Notice that the past tense is also used in Rev. 7:14, "these are those that came out of the Great Tribulation." Are we to believe that the Great Tribulation had already taken place when John wrote Revelation? No, likewise, this passage does not refer to five fallen emperors or even five fallen empires by the first century. Five being fallen merely brings us to the time of the 6th head, which is the head that will rise again.
If the seven heads referred to Roman Emperors of the first century, it would require that Revelation be written in 68 A.D. The five fallen emperors are supposed to be Augustus, Tiberius, Caligula, Claudius, and Nero who committed suicide in 68. The next emperor only ruled a few months, as did the next, both in 68. John wrote Revelation in about 96 A.D., during the rule of Domitian, who was the fourteenth emperor. Those who say it refers to Roman emperors ignore the ones who ruled only a few months, because if they count those that ruled a short time, then it messes everything up. Even though the Bible clearly states that one of the heads will rule only a short time, but the short-reigning rulers are skipped over.
All the previous heads of the beast ruled near or bordered the Mediterranean Sea, especially over Asia Minor and Israel. Even though Israel was not an official nation for almost 1900 years, there were Jews still living in the land, and it was still God's land and his holy city Jerusalem. As far as the Bible is concerned, Jerusalem is the center of the world, which is why the majority of Bible prophecy deals with that part of the world.
If we count all the individual ruling bodies that have existed in that part of the world, there are way more than seven: Egypt, Assyria, Babylon, Media-Persia, Greece, Rome, several Arabic caliphates, Seljuk Turk, Crusader kingdoms, Kingdom of Saladin, Mongol, Ottoman Turk, British, and even more. Often more than one ruled at the same time, side by side, some with many ruling dynasties, and others that were short -lived kingdoms. So what is the answer? First, I believe to be counted as one of the heads they must have ruled over the land of Israel because Israel is the main focus of the Bible.
The Mongols did not rule Palestine or even Syria, so they did not conquer far enough into the region to be counted; likewise the U.S.S.R. did not rule within the Middle East. The Crusader kingdoms were small, mostly encompassing Palestine. But even if you don't count these kingdoms, there are too many.
We can examine it any number of ways, to exclude one empire or another, like counting all the Islamic-based kingdoms as one, but it is still hard to come up with a definitive list that most people can feel are the right kingdoms without raising many objections. In the end I believe the number 7 gives us the answer.
In the Bible, the number 7 appears more than any other number, from Genesis to Revelation, and it carries much symbolic meaning, which boils down to fullness, completeness. Therefore, I believe the 7 heads of the beast are not to be taken as an exact number, but merely represent the total number of empires that have or will rule over the Mediterranean / Middle Eastern world. Perhaps the seven heads represent the last seven empires. The main heads of the beast are: Babylon, Media-Persia, Greece, Rome, Arabic Islamic kingdoms, Ottoman Empire, and the British Empire.
(3) Seventh Head of the Beast
Because there are 7 heads on the beast, many people believe the 7th head of the beast will be the final head, but that is not the case, because Rev. 17 clearly describes an 8th head that will be explained shortly. About the 7th head, Rev. 17:10 says, "but when he does come, he must remain for a little while."
Since the British Empire was the last empire to rule in that part of the world, and it only ruled there a short time, it must be the 7th head of the beast. The British Empire conquered the Ottoman Empire in WWI and ruled Egypt and the Middle Eastern nations after the war. It remained only a short time because it gradually gave independence to most of its subject nations during the 20th century, thereby ceasing to be a beastly empire (see BK1). At this time there are no empires in any part of the world, in the classical sense. So the beast is currently dead, but will soon come to life again.
Some people may want to argue that the British Empire existed long before it ruled over Egypt, the Middle-East, and Palestine; that is true, but not only did it rule Israel after the Ottoman Empire, it also came into being after the Ottoman Empire came into being and did not last as long as many of the previous heads of the beast.
And it ruled the nations around the Mediterranean and Israel the shortest length of time of all the previous empires. So, it stands to reason that the British Empire is the seventh head that we are told will rule only a short time. Especially since no other empire took its place. The final eighth head of the beast will be one of the former heads, which is why we only see seven heads.
What about the USSR? It came into being in 1917 and only lasted about 74 years; wouldn't it be the seventh head of the beast? It cannot be one of the heads of the beast for the simple reason that it never ruled over the nation of Israel and never ruled over any Middle Eastern /Mediterranean or Western European nations. Though Satan has had many empires in history, they are not shown to us in Revelation, because the main focus of Rev. is the Middle Eastern / Mediterranean / European region.
Even though the beast will be among the nations that burn America with fire during WW3, the attackers will not be able to conquer and rule over the U.S.A. for centuries the way they conquered and ruled the Christians of the Eastern Roman Empire, from Egypt to the Balkans. Which is why many Christians have historically been, "out of the serpent's reach" (Revelation 12:14, see Bk1 for a full discussion of this passage).
(4) The Final Eighth Head of the Beast
The next question becomes, what about the next head of the beast? Rev. 17, which will be discussed shortly, says the beast actually has eight heads, not seven, but it only shows seven because the eighth will be one of the previous seven, revived. All the nations that Assyria, Babylon, and Media-Persia ruled over are today Islamic. The same is true of the Ottoman Empire, which was the 6th head.
The nation of Greece is not Islamic, but Greece is today very weak, and barely able to survive as a nation because of extreme economic problems. Even though the four generals who ruled Alexander's empire were Greek, if you look at a map of all four kingdoms, today those kingdoms are 90% Islamic.
The Roman Empire converted to Christianity in the fourth century and was so large it became divided with two capitals; the city of Rome was the western capital, and Constantinople the eastern. When the Roman Empire supposedly fell, it was actually only part of the western half that fell; the eastern capital continued to rule most of the empire but is referred to by historians as the Byzantine Empire even though it was officially known as the Roman Empire. I refer to it in this book as the Eastern Roman Empire.
("Byzantine" was a term sometimes used to refer to the Eastern Romans because of Constantinople's heritage, like the original British and first Americans are known as Anglo-Saxons because of their heritage. And even in the Bible, the Babylonians are called Chaldeans, which is what they were in past centuries.)
The Eastern Roman Empire successfully held back the barbarians, even the Vikings were held back. It ruled the regions of Egypt, Palestine, Iraq, Syria, Greece, Bulgaria, Albania, Bosnia, Croatia, many islands of the Mediterranean, Asia Minor (Turkey), and part of North Africa. For a time it even ruled most of Italy after the fall of the city of Rome.
The Eastern Roman Empire maintained the Justinian laws which became the basis for the laws of most European nations.
[The Eastern Roman Empire] supervised education and maintained hospitals and asylums for the old, the orphaned, the sick, and the destitute, and imperial legislation as a whole reflected the constant concern of the emperor for the welfare of his subjects. (Funk & Wagnalls New Encyclo., V.4, p.421)
With up to a million inhabitants, Constantinople was the largest and richest city during the Middle Ages and was a major center of trade. But the Eastern Roman Empire was gradually conquered by the Muslims over a period of more than 800 years.
The final Islamic empire was the Ottoman Empire that was defeated in WW I, so if one of the former heads of the beast is going to rise again, it is a virtual certainty that it will be Islamic. The chances of it rising out of Iraq are slim because Iraq is just a boiling caldron of chaos with many different tribal and Islamic factions fighting for control. In Syria, the fighters are trying to topple the ruler, Bashar al-Assad.
Many of the fighters have stated that they want to establish the caliphate. The caliphate was originally in Mecca, then moved to other cities such as Damascus, and Baghdad. So Damascus and Baghdad have long histories as the capitals of Islam. However, the most powerful country in the region is Turkey, and it was the last country to have the caliphate, so the chances are very high that it will be the principle country of the next Islamic empire. Later I will present a lot of evidence that points to Turkey.
As we learned in Book One, Revelation chapter 9 tells us that 4 angels bound at the Euphrates River will be released to kill 1/3rd of mankind. The Euphrates River begins in Turkey and flows through Syria, then through Iraq. So this tells us for a fact, that WW 3 will begin in this region, or at least be started by the people that live in this region.
So if we are talking about a revived empire that is going to wage war with a desire to destroy Jews and Christians, then the final head of the beast can only be a revived Islamic empire, most likely Ottoman. The reason it cannot be a revived British Empire is because if Britain had any interest in having an empire it would not have willingly given independence to most of its subject nations (except Northern Ireland). It is also not trying to reconstitute its empire. But most importantly, it was not killed in warfare like the Ottoman and Babylonian beast-empires were killed, Britain gave up its empire willingly.
B1 Introduction 2023
B1: Chapter 1: Keys To Understanding Bible Prophecy
B1: Chapter 8: Daniel's 10 Toes
B1: Chapter 13: Daniel's 70 Weeks
The Little Horn and the Taliban
Is Afghanistan the First Horn of the Beast?
B2 Table of Contents
B2 Introduction
B2 Chapter 2: The Quran and Hadith
B2 Chapter 3: A Brief History of Islam
B2 Walid Shoebat's Wrong Interpretation of 666
New Insights into the Mark of the Beast
In Memory of the 50 Million Victims of The Orthodox Christian Holocaust
Book 3:
B3 Chapter 1: Who is Babylon the Great?
B3 Chapter 8: Prophecy of the Statue of Liberty
B3: Chapter 17 Prophecies of Civil War
Book 3 Update
More Prophecies About the Future of the USA
B4 Sibylline Oracles
Vision of Pastor John J. Hinkle
Updates on Books
Additional Historical info on Daniel 11
End Time UPDATES
USA water supply in danger
Iran escalates enrichment
The Causes of the Great Tribulation
Another step toward the invasion of the USA
Sri Lanka Economy has Collapsed
More nations in economic trouble
Russia Making Deals with Iran
Famine is Coming
Russia in Central America
Iran says it's starting up new centrifuges
David Wilkerson predicts nations will default
General Update: 2020, 2021, 2022
Early Pentecostals on the Great Tribulation
Proof that the USA is Mystery Babylon
The Prophets are Wrong
Replacement Theology or NOT
What Could Happen in the Near Future
Vision of Tsunami hitting East coast of USA, and economic collapse
Updated Bible Prophecy Graphics
Are We In the Great Tribulation?
Quotes from David Wilkerson
The Day and Hour Unknown?
Nostradamus Predicts the Rise of An Islamic Empire and End Days
Prophecy of Roman Catholicism in the Old Testament (but not a good one)
Michael D. Fortner
My other website:
MichaelFortner.com
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Copyright Michael D. Fortner

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On Episode 2 of Just Run With It, Tara Wommack of Greensboro sheds some light on running with young children and how she opened a Fit4Mom franchise to encourage and support moms and their kids through fun group exercises.
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redpajama_set_name: RedPajamaGithub


It's been exactly 130 years since France gave the US one of its most memorable gifts - the Statue of Liberty. Here are a few other things the French have given the US over the years.
With the Americans sick of yo-yo diets and quick tips for losing weight, Frenchwoman Mireille Guiliano rocked America's socks off with her book French Women Don't Get Fat in 2004, which raced to number one on The New York Times Bestseller list.
Of course, she was criticized for presenting idealized versions of women who live in a few chic neighbourhoods in Paris, but she certainly contributed to a revolution that allowed her to pen two more best-sellers "French Women for all Seasons" and "French Women Don't Get Facelifts".
The layout of the capital of the United States of America was basically all thanks to the hand of Frenchman Pierre Charles L'Enfant, who fought in the War of Independence then hung around to become a city planner for George Washington.
He turned what was then a hilly, forest-covered sprawling area into a the city that it is today, known for its wide avenues and public squares.
Indeed, this city could be the biggest physical gift to come from the French. Read a lot more about it here.
Yeah, yeah, yeah, French fries might have originated in Belgium - but we'll never know for sure and they're called "French" fries anyway. Did you know that Thomas Jefferson apparently ate "potatoes served in the French manner" at a White House dinner in 1802?
"What? That's not French!" you say. "That was designed by three American architects!" You're partly right. The building was indeed an American design, but did you know it was inspired by the Palais Garnier, better known as the Paris Opera House? Compare the two pictures and see for yourself.
A few years ago, the concept of French parenting went viral in the United States. The Wall Street Journal went as far as to blast the headline "Why French Parents Are Superior" across its website.
The basic idea is that being a bit of "tough love" can raise better-behaved children, and the whole world seemed curious. Whether it's the right way to raise your children or not is for you to decide, but if you raise perfectly well-behaved children thanks to a few of those tips, then you can be sure to thank the French for that too.
We wouldn't go as far as saying a French general gave the US its independence... but it wouldn't be a stretch. Gilbert du Motier, Marquis de Lafayette - or General Lafayette in short - was a key figure in the War of Independence and scored a decisive victory over the British in 1781.
He crossed the Atlantic in just 38 days - a voyage that confirmed his renown as a hero of the American Revolution. They've even remade his frigate, Hermione, 235 years on for another memorial crossing.
At least 42 US counties and cities and hundreds of streets and squares -- including the famed Lafayette Square opposite the White House -- are named after him or after his ancestral home in France, La Grange.
Where do Americans go on holiday (if they want to leave their own country)? France of course.
L'Hexagon, as France is also known, is among the most popular holiday destinations for Americans.
The New York Times reported that an estimated 2.49 million Americans will visit France in 2015, which makes it just a fraction away from being the most popular travel destination in Europe. In fact, it's only the UK that will be more popular in Europe among Americans with 2.54 million expected to visit this year. That means France is the fourth most popular international destination for Americans in general, after Mexico, Canada, and the UK.
Tony Parker, a basketball star for the San Antonio Spurs in the NBA, is arguably the best French athlete in American sports. He's loyal (has played with the Spurs since his rookie season in 2001), he's a winner (he steered the team to four championships), and he moves like an assassin on the court. If you're not into basketball, you might remember him as the ex-husband of Desperate Housewives star Eva Longoria.
New Orleans, at the mouth of the Mississippi river, was once owned by the French. In fact, Napoleon sold the whole state of Louisiana to the US for $15 million back in 1803 to keep it out of the hands of the British. Now, the city has a thriving French quarter (known as Vieux Carrée) where you can see everything from classic French architecture to cafes and restaurants named after characters from France's history pages.
Piketty seemed to be nothing more than an ordinary economist until his book "Capital in the 21st Century" shot him to fame worldwide, but particularly in the US, where his work topped the New York Times bestseller list.
It was in the US, a country with one of the greatest levels of inequality, that his work to explain how the rich were getting richer and the poor were getting poorer really struck a chord. It earned him a trip to the White House to meet the president's advisors and Barack Obama made tackling the issue a top priority.
Whether he has or not remains to be seen, but Frenchman Piketty did his bit.
Yes, 130 years ago to this day, French steamer Isère made its grand entrance into the New York port with what would become a 93-metre-high statue in its cargo. The statue, which weighs 204 tonnes and is visited by almost 100 million people a year, was designed by Frédéric Auguste Bartholdi, with the help of Gustave Eiffel.


More than a dozen families are being evicted from a trailer park in Gypsum as the school district works on its Eagle Valley High School expansion.
GYPSUM — More than a dozen families are being evicted from a Gypsum mobile home park so Eagle County Schools can build athletic facilities and a parking lot at Eagle Valley High School.
It's part of the school district's $31.2 million renovation of the high school, and part of the school district's $144 million voter-approved plan ($233 million with interest) to rebuild and renovate schools up and down the Vail Valley.
About half of the trailer park property will be parking spaces to accommodate Eagle Valley High School's projected growth to 1,300 students. Some of that land will be a bus loop and secondary access to the school.
The other half will be tied to expansion of the school's athletic complex, which will be tied to Gypsum's expansion plans of its recreation and athletic complex, said Sandy Mutchler, the school district's chief operating officer.
The school's expansion will cover some of the existing parking.
The school district paid Virginia resident Steven L. Peters $1.95 million for his trailer park. The parties went under contract on Feb. 3, and the property changed hands on May 15.
The residents learned on Feb. 16 about the pending sale when they received a notice from the school district.
They learned on May 17 that the deal had been closed two days before. May 17 was also the day they learned they had until Nov. 15 to leave and take their mobile homes with them.
The families told their stories, gathered around a picnic table in a Gypsum park on a stunning summer afternoon. Some have children in neighborhood schools; some attended local schools themselves.
They said that this past winter, a property manager from Dalco Realty walked through the park, knocking on their doors and telling them that the school district was buying the park from Peters, and that they would have to move.
Because they've known each other for years, they met in the middle of the park. They asked each other what they would do.
No one had an answer, they said. Many still don't.
"We've cried a lot," said Patricia Bryant.
Eva Meraz is studying nursing at Colorado Mountain College and works at Colorado Mountain Medical in Avon.
She lives with her parents and younger brothers. Her brothers walk to their neighborhood school. Eva and her family are on three waiting lists for an apartment. No one has called back yet, she said.
Eva said if she can't live with her parents, she's not sure she can continue college.
"It's not an expensive college, but it costs more than I have," she said.
Elio Fernandez' son is working and also studying at CMC. Elio said he has a brother in Texas, and he may head that way. He doesn't know if his son will come with him.
The humor gets blacker as the evening sun begins to sink, and the longer the discussion goes on.
"Maybe when Halloween rolls around we can trick-or-treat for money," they say.
They all say they're working, some multiple jobs and all said they are in the country legally.
Patricia Bryant says she has made her home in the Gypsum mobile home park for more than 40 years. She will not make 50.
The school district is not being completely heartless, they said.
• $4,000 for rent reimbursement.
• $4,000 for mobile home moving expenses.
• $2,000 for personal relocation expenses.
The school district also offered to help them prequalify for one of the new Red Table Apartments in Gypsum.
Rents for a two-bedroom apartment are expected to range from $1,050 to $1,500, plus $150 for a garage.
"I don't make that on Social Security," said Bryant, who added that her husband died in 2013.
The Red Table apartments are scheduled to be ready by November, but delays are always possible, said a letter from the school district to the residents.
So far, some are managing to move.
The school district said one family has moved its mobile home to another local park. Another purchased a home and vacated the property. Two other families have been hired by Eagle County Schools and applied for district housing.
One resident sold his trailer to the construction company to house its workers.
Another recently moved to Glenwood Springs and one is planning to purchase a recreational vehicle.
Under the Colorado Manufactured Home Community Act, the school district does not have to give residents any money, but must give residents six months to vacate.
The school district offered to try to sell residents' mobile homes as public property, but doesn't actually own the trailers, so it cannot, the school district said.
"We are trying to help make the transition as smooth as possible for displaced families," said Kate Cocchiarella, school board president.
The school district has been talking to Peters about the property for years, well before voters approved the bond this past November, Cocchiarella said.
"The trailer park land is beneficial for the evolution of the (school) campus as a whole, and will enable us to provide the best facilities and experiences for our students," Cocchiarella said.
Of those 15 mobile homes, Peters owns four and rents them. The rest of the families own theirs.
The school district acquired all four of Peters' trailers when it bought the property, and will dispose of them.


text:
BarryDouglasposition Defender
NationalityScottish
Date of birth04/09/1989
Barry Douglas signed for Leeds United from Wolverhampton Wanderers in July 2018 for an undisclosed fee.
Starting his career at Queen's Park, Douglas also had stints with Dundee United, Lech Poznan and Konyaspor.
He joined Leeds on the back of helping Wolves to win promotion from the Sky Bet Championship in the 2017/18 season.
In Douglas's final season with Wolves, he provided the most assists in the Championship.
His first season at Leeds was marred by injury as he only started 21 times. Douglas will be hoping to bounce back from injury ahead of the new season and recapture his form from the 2017/18 season.
Join the Leeds United Mailing List and receive the latest news from inside Elland Road! Just enter your email address here.


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text:
Report considers abolition of prison sentences under a year
A Commons report has called for the abolition of prison sentences under a year to be considered this week.
Cross-party bill could force Theresa May to request deadline extension
Organisations respond to Labour pledge to "ban" privatising services
How does the parliamentary Whipping System work?
News | Published April 1st 2019
With the role of the whips and the extent to which MPs defy them playing a crucial role in Brexit, The Parliamentary Review examines the the Whipping system as a whole, how it works and notable examples of its use.
Labour to support Customs Union and Common Market 2.0
Labour have announced they will whip their MPs to support a "Common Market 2.0", a Brexit proposal that would see the UK remaining in the single market. They will also support a Customs Union and a confirmatory vote.
Report shows investment in UK healthcare continues to grow
Knight Frank reported an increase of more than ten per cent in UK healthcare investment during 2018 this week.
News | Published March 31st 2019
Dominic Grieve loses confidence vote over Brexit debate
Conservative MP and prominent remainer Dominic Grieve has suffered a vote of no confidence after a "rowdy" Brexit discussion in his Beaconsfield constituency on Friday. Grieve lost the vote 182 to 131 at Beaconsfield's Conservative association's annual general meeting.
Frank Field: "Rampant injustice" in the gig economy
Labour MP Frank Field proposed six recommendations this week that aim to reduce the injustice faced by gig economy workers in the UK.
'Gender pay gap' found in 96% of government departments and 90% of universities
Figures submitted to the Government Equalities Office have shown that 90 per cent of universities and 96 per cent of government departments pay the average male employee more than the average female employee.
May likely to call fourth vote on her deal
Theresa May will again bid to get her Brexit deal through parliament this week after it was rejected MPs on Friday.
Withdrawal Agreement defeated for third time
The Withdrawal Agreement has been defeated for a third time in the House of Commons, losing by 344 votes to 286. Unlike the two previous votes, this vote was not on the deal as a whole but only the Withdrawal Agreement, leaving out the political declaration. This means the result is not classified as a "meaningful vote."
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Employability Solutions, a Liverpool-based social enterprise, believe that society is too quick to give up on its most vulnerable. Since their registr... Read more
Mab's Cross Primary School
At the heart of Mab's Cross's ethos is their motto: "Bringing out the best in everyone". Forming a culture of responsibility and leadership has been o... Read more
Bricknells Letting Agents
Operating across South Yorkshire, Bricknells Rentals are an independent residential letting agency. As members of the Association of Residential Letti... Read more


Home » Paragliders » EN B » Gin Carrera Plus M (85-105Kg) Less than 25 hours of flight !
For sale my Gin Carrera Plus, color sundance (yellow) with 25-30 flights and less than 25 hours (total flight time).
Reason: stepping up to EN C.
It's in mint condition, like new.
It's a genuine Plus wing, not upgraded.
No rips tears or patches.
Never been SIV or acroed.
Excellent wing for a B pilot.
You can receive the wing at your home, anywhere in UK or any other country (like France, Germany, Italy..) in 4-5 days. Shipping included in the price (If the shipping exceeds 35£ you have to pay the extra).


Hi. I'm Sara, and I'm a cycle-a-holic.
I didn't think it had come this far, but here I am. My knee hurts badly; I know that I shouldn't bike tomorrow morning, and that makes me feel bereft. I don't bike every single morning but I don't take kindly to knowing that it's off limits for now. The cravings are beginning. Driving the car is, at best, a necessary evil. There's no joy in it; it just gets me from A to B and back. But cycling… aside from the obvious fitness benefits, it does wonders for my seasonal depression. Also my soul, my wallet, and my general sense of well-being.
When I'm cycling, I'm close to the ground and there's nothing between me and the world around me. I sing out "good morning" whenever we pass a pedestrian. We stop to examine the fire hydrants that haven't been installed yet. We greet the same construction workers and crossing guards every time. Cycling gives me this sense of being part of the city instead of separated from it in a metal-and-glass bubble.
Nature is right beside me all along the road. I see the trees in various stages of autumnal undress, the places where there are more weeds than grass, the wetness of the road after a night of rain. The air feels clean in the morning, especially on the residential streets that make up most of my ride. I can see the sky – not a piece of it through a windshield, but the whole expanse – and I often marvel that even on cloudy days there is usually a clear patch of blue peeking through somewhere.
Throughout my ride, I have many opportunities to make it easier or harder for myself. I can relax and ride slowly if I feel like it, or I can push myself to the limit and set a new time record. I can lean into the turns more, challenging my skill and balance. I'm up against my own limits, and I often astonish myself.
I can chat with my children, point out landmarks, and ruffle their hair at stop signs. I get to watch them wiggle in time to the music from my iPod. When I greet pedestrians with a smile the children and I get to watch wizened old faces and jaded young faces break into a tentative smile or a grin and a surprised laugh. Even the grumpy-looking old man with the tiny dog (practically a fixture in our parking lot at drop-off and pick-up times) looks handsome when he smiles at us.
Even at my fastest, my speed probably tops out at 18 km/h. Fast enough to get where we're going, and slow enough to not feel harried and rushed. Traffic means very little to me, gliding along residential streets and through pathways where cars can't travel.
It's possible to drive a car on autopilot. We've all done it, I'm sure, getting somewhere and realizing we have no particular memory about the journey. I've never been able to bike on autopilot. Cycling makes me feel connected – to nature, to the city, to the people, to myself and my limits. To God.
To paraphrase the milk commercials of my youth, cycling does a body good… but it does a soul great.
I must admit, when I read the post heading, I immediately thought of something else.
I love the image of you and your bakfiets. Hope your knees cooperate!


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