Article |
Address correspondence to Shyuichiro Matsubara, Dept. of Biochemistry, Faculty of Medicine, Kagoshima University, Kagoshima 890-8520, Japan. Tel.: 81-99-275-5246. Fax: 81-99-264-5618. E-mail: shmlmcbd{at}m.kufm.kagoshima-u.ac.jp
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Abstract |
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Key Words: -catenin; cadherin; apoptosis; p27kip1; compaction
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Introduction |
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Cadherin molecules on the cell surface transduce extracellular signals (Takeichi, 1991; Larue et al., 1996), possibly altering cell polarity (McNeill et al., 1990; Watabe et al., 1994), growth rate (Watabe et al., 1994; Bullions et al., 1997), and cellsubstratum adhesion (Miyaki et al., 1995). However, the mechanism by which cadherincatenin complexes regulate cell fate remains to be investigated.
To examine the functions of -catenin, we established DLD-1/
cell clones transfected with
-catenin (Ozawa, 1998). DLD-1/
is derived from the DLD-1 human colon cancer cell line, lacking endogenous expression of
-catenin. Cadherin-mediated cell adhesion is disrupted in this variant, despite the presence of other cadherin cell adhesion com-plex components: E-cadherin, ß-catenin, and
-catenin. We searched for the functions of
-catenin in signal transduction and found a significant reduction in death of
-cateninexpressing clones after treatment with sphingosine, an inducer (Sakakura et al., 1996; Sweeney et al., 1996; Shirahama et al., 1997) and endogenous mediator (Ohta et al., 1994, 1995) of apoptosis. The basic molecular framework for regulating and executing apoptosis comprises a functionally ordered product of expanded gene families, including the caspases and Bcl-2 family of proteins. The cadherin/catenin-derived signal may affect the functions and activities of the molecules in the apoptotic pathways. Therefore, examination of the effects of cadherin signaling on death mediators and/or regulators may uncover the molecular mechanism underlying the signal.
To determine the region of the molecule responsible for reductions in cell death induction, we examined the cellular phenotypes, resistance to cell death, and cell morphology, resulting from deletions in -catenin. We compared the effects of antiE-cadherin antibody treatment with
-catenin deficiency. We then analyzed the status of death mediator molecules in the apoptosis cascade to find possible mediators of the signals from cadherincatenin complexes. Our results indicate that increases in p27kip1, an inhibitor of cyclin-dependent kinases (cdks), correlates with resistance to cell death in the
-catenin transfectants, suggesting that expression of
-catenin effects the cell death through the anti-apoptotic function of p27kip1. Transfection-mediated upregulation of p27kip1 levels in
-catenindeficient cells decreases the levels of cell death induced by sphingosine, supporting our hypothesis.
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Results |
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Therefore, -catenin expression in DLD-1/
cells reduced sphingosine-mediated induction of cell death. At the critical concentrations of the inducer, this effect was observed in low density cultures in the presence of FCS. The effect was significant and reproducible.
To ascertain the role of -catenin in the reduction of sphingosine-induced cell death, we analyzed pairs of cells containing or lacking
-catenin. PC 3, a prostate cancer cell line, lacks
-catenin due to homozygous deletion of the gene (Morton et al., 1993). We transfected PC 3 cells with the construct used to transfect the DLD-1/
cells to establish clones expressing
-catenin (Fig. 2 A). With these cells, we got similar results (Fig. 2 B). Upon treatment with sphingosine, we observed a similar reduction of cell death dependent on
-catenin. The results indicate that the reduction in levels of sphingosine-induced cell death is a general cellular phenotype, resulting from the expression of
-catenin.
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All transfectants exhibiting resistance to cell death demonstrated compacted aggregation in multicellular suspension cultures; transfectants sensitive to cell death were loosely adherent, existing as "grape-like" aggregates in multicellular suspension cultures (Fig. 3 D). Cell compaction (Kemler et al., 1977; Takeichi, 1977; Hyafil et al., 1980) results from a cellular morphological change in which cells adhere more tightly to each other, maximizing contact areas. This effect is observed in association with cadherin-mediated cellcell adhesion (Vestweber and Kemler, 1985), involving forces generated by the actin cytoskeleton (Surani et al., 1990). Therefore, cells demonstrating the compacted morphotype suggest a link between the cadherincatenin complex and the actin cytoskeleton. These data suggest that transfectants expressing -catenin variants able to associate with the actin cytoskeleton, inducing compaction in three-dimensional cultures, are resistant to sphingosine-induced cell death.
D,
C1D, and
C4D cells demonstrated an epithelioid morphotype when plated on solid substrate;
NM2D,
C6D, and nD cells showed a nonepithelioid morphotype with highly refractile cell borders. When plated on plastic,
NM1D cells exhibit refractile borders but not to the same extent as
NM2D,
C6D, and nD cells. It remains unclear why
NM1D cells do not exhibit an epithelial morphology on solid substrates, although these cells compact in multicellular suspension cultures. Cellsubstratum adhesion may weaken the cellcell adhesive interactions of DLD-1 cells.
AntiE-cadherin antibodies induce disruption of epithelioid morphotype but do not sensitize D cells to sphingosine-induced cell death
To examine the relationship of this change in morphology to the -catenindependent resistance to sphingosine-induced cell death, we used an antihuman E-cadherin antibody, SHE78-7, to disrupt the cellcell contacts of
D cells. The addition of SHE78-7, a function-blocking monoclonal antibody (mAb), to cultures disrupts cellcell contacts to induce decompaction (Watabe et al., 1994; Kantak and Kramer, 1998; St. Croix et al., 1998). In the presence of this antibody,
D cells demonstrated a nonepithelioid morphology on solid substrates, similar to that seen with nD cells (Fig. 4
A). Concentrations from 3 to 100 µg/ml induced a similar morphology without any observable toxicity. Treatment with sphingosine did not reduce the viability of
D cells in the presence of antiE-cadherin antibodies (Fig. 4 B). Both the MTT assay and direct counting of cells after trypsin treatment confirmed that no difference in levels of cell death was observed between antibody-treated and untreated
D cells. SHE78-7 antibodies induce disruption of homophilic cadherin binding; they are not thought to signal as a cadherin ligand. Treatment of PC-9 cells, an
-catenindeficient cell line, with this antibody overcomes the effect of
-catenin expression, resulting in cell dissociation and growth stimulation (Watabe et al., 1994). These results indicate that the inhibition of cellcell adhesion by function-blocking antibodies does not have the same effect as the inhibition caused by a deficiency of
-catenin, although the cell morphotype induced by both types of inhibition is quite similar. Therefore, close cellcell contacts are not required for resistance to cell death.
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We detected Bcl-xL protein but not Bcl-2 protein in both D cells and nD cells by Western blotting (unpublished data). Sphingosine treatment downregulated the total cellular levels of Bcl-xL protein; this decrease occurred more quickly in nD cells than in
D cells. After a 912h treatment with sphingosine, Bcl-xL levels in
D cells were greater than those observed in nD cells as observed by Western blot analysis of equivalent protein amounts (Fig. 5
A). Differences in Bcl-xL levels were also detected at 36 h, before the cells demonstrated the morphological changes that accompany apoptosis. After a 15 24h treatment with sphingosine, levels of Bcl-xL and vinculin both decreased in nD cells. Since a large fraction of nD cells floated and died in this period, these differences in protein levels could result from cell death. In contrast, no differences in the levels of vinculin were detected between
D and nD cells until 12 h after treatment. These data suggest that at least one target mediating the increased resistance of
D cells to cell death may exist upstream of Bcl-xL.
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In confluent cultures, p27kip1 protein levels were high; no differences were detectable between nD and D cells (Fig. 7)
. After trypsin/EDTA treatment, p27kip1 protein levels decreased significantly (unpublished data). After plating 2 x 105 cells per 100-mm dish, p27kip1 protein levels gradually increased during a 3-d incubation. In these low density cultures, higher levels of p27kip1 were observed in
D cells in comparison to nD cells (Fig. 7). Cell death was not efficiently induced in high density cultures, either confluent cells or cells cultured for more than 3 d after seeding as above, regardless of
-catenin expression (unpublished data). These results suggest a correlation between p27kip1 expression and resistance to cell death. DLD-1/
transfectants expressing a level of p27kip1 above a certain threshold, such as that seen in high density cultures, cannot be induced to undergo cell death by sphingosine treatment. Cells expressing lower levels of p27kip1, such as cells present in low density cultures, undergo cell death after sphingosine treatment. In this case, the upregulation of p27kip1 resulting from
-catenin expression correlates with reductions in cell death.
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-Catenin expression does not suppress the growth of DLD-1/
cells
p27kip1 inhibits cyclin E-CDK 2 activity implicated in the negative regulation of G1 progression. Therefore, we examined the effect of p27kip1 upregulation in low density cultures of D cells on cell growth.
-Catenin expression did not affect the growth of DLD-1/
cells (Fig. 10)
, in contrast to previous results with PC 9 cells (Watabe et al., 1994) and Ov2008 cells (Bullions et al., 1997). The growth of these two
-catenindeficient cell lines is retarded after the restoration of
-catenin expression.
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Discussion |
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In the present study, we could not directly measure the rate of entry into apoptosis because of the difficulties to quantify nuclear fragmentation of DLD-1/ transfectants. Cells undergoing cell death detached from dish and centrifugation to collect and to stain the cells destroyed large amount of floating cells. However, as described in Results several lines of evidence, (a) DNA fragmentation, (b) nuclear fragmentation, and (c) activation of caspase, indicate that apoptosis is taking place in our experiment and thus support the correlation between reduced viability and apoptosis.
Linkage to the cytoskeleton but not cadherin-mediated change in morphology itself is correlated with resistance to sphingosine-induced cell death
The transfection of cells with -catenin deletion mutants demonstrated that
-catenin mutants capable of association with the actin cytoskeleton can induce compaction in three-dimensional suspension cultures; these mutants also exhibit an anticell death function. These results indicate that the linkage of the cadherincatenin complex to the cytoskeleton is integral in the induction of resistance to cell death. These mutants induce increased cellcell contact when the transfectants are cultured on solid substrate, the conditions used in cell death experiments. Disruption of cell adhesion by treatment with neutralizing antiE-cadherin antibodies did not reduce the resistance to cell death, although the cadherin-mediated tight adhesion was lost from
D cells. Therefore, the increased cellcell contact does not appear to be responsible for the increased viability. This phenotype is induced at the same time as the resistance to cell death but induced independently by
-catenin expression. Further studies are needed to clarify the molecular differences between cells lacking
-catenin and cell treatment with an anticadherin antibody.
Both the middle and COOH-terminal regions may link the cadherincatenin complex and the actin cytoskeleton via different proteins. This redundancy may explain why the effect of -catenin on cell death is not specific for either domain of the molecule. The middle region of the molecule, amino acids 203381, overlaps with the vinculin-binding region reported to be contained within amino acids 326509 (Watabe-Uchida et al., 1998, Imamura et al., 1999). In addition, amino acids 325394 have been identified as an
-actininbinding site (Nieset et al., 1997). The vincu-lin- and/or
-actininmediated linkage could function in
C4D cells. The COOH-terminal 210 residues, amino acids 697906, interact directly with actin filaments in vitro (Weiss et al., 1998). In addition, amino acids 697906 of
-catenin bind to ZO-1 (Imamura et al., 1999). In
NM1D cells, linkages to the actin cytoskeleton could be established through direct binding or indirect binding via ZO-1. These two types of linkage may simultaneously influence death induction and induce close cellcell contacts.
p27kip1 as a potential mediator of the resistance to cell death dependent on -catenin
p27Kip1 may mediate the signal(s) linking the cadherincatenin complex to the apoptotic cascade. This molecule, originally identified as a cdk inhibitor involved in G1 phase arrest signaled by TGFß and cellcell contact (Polyak et al., 1994), may transduce the signal(s) from the cadherincatenin complex, resulting in growth suppression (St. Croix et al., 1998). However, these results could not be applied directly to our study, since E-cadherindependent increases in p27kip1 were reported to be observed only in three-dimensional suspension cultures not in monolayer cultures. We examined p27kip1 levels to discover that protein levels were upregulated in low density cultures of D cells compared with those observed in nD cells. The cause of the discrepancy between our results and the previous ones is not clear; differences in the cell line, cell density, or adhesive disruption method used may explain these differences. They used transfection of E-cadherin and its neutralizing antibodies, whereas we used a deficiency of
-catenin. p27kip1 protein levels were as high as the levels observed in
D cells in transfectants expressing
-catenin deletion mutants that confer resistance to cell death.
Transfection-mediated overexpression of p27kip1 decreases sensitivity to apoptosis induced by DNA-damaging drugs (Dimanche-Boitrel et al., 1998; Eymin et al., 1999a,b). In leukemic cells, p27kip1 overexpression inhibits cytochrome c release from mitochondria and procaspase 3 activation (Eymin et al., 1999a,b). Release of cytochrome c from mitochondria is controlled by proteins of the Bcl family; proapoptotic members of the family, Bax and Bak, accelerate the opening of mitochondrial permeability transition pore, whereas antiapoptotic members of the family, such as Bcl-xL, inhibit (Shimizu et al., 1999). Increases in permeability result in the relocation of cytochrome c from mitochondria to the cytosol, aiding the activation of caspases (Li et al., 1997). These results suggest that p27kip1 may prevent cell death upstream of mitochondrial events, possibly through Bcl family proteins. In this study, analysis of the antiapoptotic Bcl family and the death mediator caspases demonstrated that the target(s) facilitating the resistance of D cells to death may function upstream of Bcl-xL and/or caspase 3 (or caspase 3like proteases). This is consistent with the idea that p27kip1 may link the signal(s) mediated by the cadherincatenin complex to death mediator molecules involved in sphingosine-induced cell death. Finally, increased expression of p27kip1 in
-catenindeficient (DLD-1/
) cells decreases the induction of cell death by sphingosine.
The precise mechanism whereby p27kip1 affects the cell death cascade remains unknown; two possible mechanisms may mediate this effect dependent or not dependent on cell cycle progression. In the present study, -catenin expression did not suppress cell growth; therefore, a cell cycledependent mechanism does not appear likely. In addition, caspase-mediated cleavage of p27kip1 into a 15-kD NH2-terminal fragment is required for antiapoptotic activity (Eymin et al., 1999b). A point mutation of amino acid 108 (D to E), the putative cleavage site for the caspase, resulted in both resistance to cleavage and the loss of the antiapoptotic function, preventing both the release of cytochrome c from the mitochondria and the activation of caspase 3 (like proteases). These findings suggest that p27kip1 possesses an unknown function, independent of the inhibition of cell cycle progression. The molecular mechanism(s) underlying the anticell death function remains to be investigated.
Recently, Vasioukhin et al. (2001) showed that -catenin deletion induced increased cell proliferation and downregulation of p27kip1 but not elevated apoptotic rate. We do not know why downregulation of p27kip1 does not lead an elevated apoptosis in their system. One possible explanation could be the difference in the cell types used in the experiments, that is, normal keratinocytes in their experiments versus cancer cells in ours.
We demonstrated that -catenin expression increases resistance to cell death. The deletion mutants of
-catenin, capable of increasing cellcell adhesion, can also decrease the susceptibility to cell death after sphingosine treatment. However, the anticell death function does not require cellcell contact itself as indicated by treatment with antiE-cadherin antibodies. The
-catenininduced increases in p27kip1, a cdk inhibitor, may mediate the anticell death function.
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Materials and methods |
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Cells and transfection
DLD-1/ cells, a DLD-1 human colon cancer cell line variant, were provided by Dr. S.T. Suzuki (Aichi Human Service Center, Kasugai City, Aichi, Japan). Cells were grown and transfected as described previously (Ozawa, 1998). G418-resistant clones were examined by immunoblotting for the expression of either
-catenin or the various deletion mutants. Positive cells were subcloned; a representative for each construct from multiple transfectant clones was chosen to be used for further studies.
For multicellular suspension cultures, cells were plated in 35-mm dishes coated previously with polyhydroxyethylmethacrylate (Frisch and Francis, 1994).
Sphingosine treatment
Cells were seeded at 5 x 104 cells in 35-mm dishes containing 2 ml culture medium and incubated for 24 h. To induce apoptosis, culture medium containing either sphingosine or the drug vehicle (DMSO) alone was dispensed into the dishes. The culture dishes were then incubated for 324 h before analysis. Cells were seeded at 2,500 cells per well in 96-well plates in 100 µl of culture medium containing either SHE78-7 or control IgG, then treated as above for the experiments using an antiE-cadherinblocking antibody.
Cell viability studies
Cell viability after sphingosine treatment was assessed using the MTT assay described by Alley et al. (1988). Samples were incubated with MTT for 1 h; detached cells were then collected by centrifugation and combined with attached cells. Cell viability was expressed as a percentage of the control absorbance after subtracting the background absorbance of culture medium without cells.
DNA fragmentation assay
Nucleosomal DNA degradation was assayed as described by Sinha et al. (1995) with the following modifications. In brief, 2 x 105 cells were seeded in a 100-mm culture dish and treated with 17.5 µM sphingosine for 18 h. After drug treatment, suspension cells were collected and centrifuged. The remaining cells were collected by trypsinization and combined with the suspension cells. After a wash with ice-cold PBS, cells were lysed for 10 min at 4°C in 100 µl of lysis buffer comprised of 10 mM Tris (pH 7.4), 10 mM EDTA, and 0.5% Triton X-100. The lysate was centrifuged at 27,000 g for 20 min; the resulting supernatant was treated with RNase A (400 µg/ml) for 30 min at 37°C, followed by treatment with proteinase K (400 µg/ml) for 1 h at 37°C. DNA was precipitated with an equal volume of iso-propanol in the presence of 0.5 M NaCl overnight at -20°C. Total DNA from each sample was visualized on a 2% agarose gel.
DAPI staining
Cells were grown on coverslips and stained according to the method of the manufacturer (Rosche Diagnostics GmbH). In brief, the sphingosine-treated cells were washed once with DAPI-methanol (1 µg/ml) and then incubated in the same solution for 15 min at 37°C. After washing once with methanol, cells were examined under a fluorescence microscope with a 330385-nm excitation filter.
Immunoblotting
Before protein analysis, cells were seeded at 2 x 105 per 100-mm dish in 8 ml culture medium. Culture medium containing sphingosine was dispensed after a 48-h incubation to induce apoptosis. Preliminary experiments demonstrated that cell viability after sphingosine treatment was similar to that of cells treated in 35-mm dishes as described above. After drug treatment, floating cells were centrifuged. After two washes with ice-cold PBS, we added SDS sample buffer (Laemmli, 1970) to both the cells collected by centrifugation and those attached to the dish. Cells were lysed, combined, and boiled for 5 min. After SDS-PAGE (12.5% polyacrylamide), proteins were transferred to nitrocellulose membranes. Proteins were detected as described previously (Ozawa, 1998) with the following antibodies: antiBcl-2 (0.2 µg/ml), antiBcl-xL (0.5 µg/ml), anti-catenin (1.25 µg/ml), anti-HA (0.2 µg/ml), anti-p27kip1 (0.5 µg/ml), anti-p21cip1 (0.25 µg/ml), and antivinculin (8.4 ng/ml). Antivinculin antibodies were used to monitor fluctuations in the total protein amount. Blots were quantified with a scanner (Scan Jet 4c/T; Hewlett Packard) and NIH Image 1.62 f software.
Measurement of caspase 3 (like) activity
Cells were treated with sphingosine in 100-mm dishes as described above. After harvesting and washing, cells were resuspended in a hypotonic cell lysis buffer (20 mM Hepes, pH 7.5, 10 mM KCl, 2mM MgCl2, 2 mM DTT, 2mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml pepstatin) and lysed by four cycles of freeze-thaw. Cell lysates were centrifuged for 20 min at 16,000 g; the resulting supernatant was used as the source of enzyme. Caspase 3 (like) activity was measured with a colorimetric tetrapeptide, Ac-DEVD-pNA, in the presence (negative control) or absence (assay) of the caspase 3 inhibitor Ac-DEVD-cmk as described by Datta et al. (1997). The activity was calculated to be the difference between the 405-nm absorbance of the assay mixture and the negative control.
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Footnotes |
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Acknowledgments |
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This work was supported by a grant from the Ministry of Education, Science and Culture of Japan, a Grant-in-Aid for Science Research on Priority Areas (B), and a grant from the Kodama Memorial Foundation.
Submitted: 21 March 2001
Revised: 22 June 2001
Accepted: 26 June 2001
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