1 Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2 B5, Yamadaoka, Suita 565-0871, Japan
2 Department of Medical Biochemistry, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan
* Author for correspondence (e-mail: yokom{at}imed2.med.osaka-u.ac.jp)
Accepted 8 March 2004
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Summary |
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Key words: CD9, Tetraspanin, Apoptosis, Shc, Cancer cells
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Introduction |
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It has been reported that the expression of CD9 is closely related to the metastasis of the gastrointestinal carcinoma (Mori et al., 1998; Sho et al., 1998
; Uchida et al., 1999
; Murayama et al., 2002
). For example, reduced CD9 expression is significantly associated with venous vessel invasion and liver metastasis in colon cancer patients (Mori et al., 1998
), and is related to a poor prognosis in pancreatic cancer patients (Sho et al., 1998
). CD9 expression is inversely associated with lymph node status in gastric cancer (Murayama et al., 2002
) or esophageal squamous cell carcinoma (Uchida et al., 1999
). However, little is known about the roles of CD9 in the apoptosis of human cancer cells.
In this study, we demonstrate that antibody ligation of CD9 induced apoptosis of gastrointestinal cancer cell lines and analysed intracellular signaling during this CD9-mediated apoptosis.
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Materials and Methods |
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Construct of plasmid that produces dominant negative form of p46 Shc
HA-tagged Shc cDNA Y239/240/317F (3F) in a mammalian expression vector pKU-hyg was provided by N. Gotoh (New York University School of Medicine, New York, NY). The mutation in these three tyrosine phosphorylation sites acts as a dominant negative inhibitor of Shc signaling that is initiated by integrin and growth factors (Gotoh et al., 1997). The sequence encoding the Y239/240/317F Shc cDNA was inserted into XhoI-XbaI site of the pcDNA 3.1(+) expression vector, a geneticin-resistance plasmid, and designed pcDNA3.1(+)-HA-Y239/240/317F Shc cDNA. To produce only the p46 Shc isoform, the sequence of the dominant-negative form of p46 Shc (p46 Shc DN) was synthesized by the polymerase chain reaction (PCR) using the appropriate sets of forward and reverse primers in which the N-terminal residues from the first to second ATG of pcDNA3.1(+)-HA-Y239/240/317F Shc cDNA were deleted. The primers used in the PCR were: forward primer, 5'-GGGGGTACCCTGCATCCCAACGACAAAG-3'; reverse primer, 5'-TCGAGGCTGATCAGCGG-3'. The PCR products were confirmed by sequencing and cloned into pcDNA 3.1(+) with KpnI and XbaI sites [pcDNA3.1(+)-HA-p46 Shc DN]. We also confirmed that the pcDNA3.1(+)-HA-p46 Shc DN produces only the 46 kDa protein by transient transfection to 293T cells (data not shown).
Cell lines and transfection
Human gastric cancer cell lines (MKN-28 and MKN-45) and human lung cancer cell lines (A459 and NCI-H69) were cultured in RPMI-1640 medium (Gibco BRL, Grand Island, NY); human colon cancer cell lines (SW480, HT-29 and CaCO2), a human pancreatic cancer cell line (MIA-PaCa-2), a human hepatocellular carcinoma cell line (Hep G2) were cultured in DMEM (Gibco BRL) supplemented with 10% fetal calf serum (FCS; Whittaker Bioproducts, Walkersville, MD), 100 units ml1 penicillin and 100 µg ml1 streptomycin in a humidified atmosphere of 5% CO2 in air at 37°C. To establish MKN-28 cells stably expressing p46 Shc DN, MKN-28 cells were transfected with pcDNA3.1(+)-HA-p46 Shc DN using Lipofectamine (Gibco BRL) according to the manufacturer's instructions. The transfected cells were selected for their ability to grow in the presence of 800 µg ml1 geneticin (G418; Gibco BRL) and each G418-resistant clone was then picked up.
Flow cytometry
Cells were washed and incubated with the appropriate antibodies for 30 minutes on ice. The cells were washed with ice-cold PBS and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG1 (Cappel Organon Teknika, Durham, NC) was then added. The cells were incubated on ice for 30 minutes and then washed with ice-cold PBS. The fluorescence intensity of the cell surface was analysed using a FACScan (Becton Dickinson, San Jose, CA).
[3H]-Thymidine incorporation assay
After 24 hours of serum starvation, quadruplicates of the cells were cultured in 96-well, flat-bottom microtiter plates with the indicated antibodies for 48 hours and each well was pulsed for 4 hours with 1 µCi [3H]-thymidine (Amersham Pharmacia Biotech, Little Chalfont, UK). The cells were then harvested and the [3H]-hymidine incorporation was measured using the Betaplate System (Pharmacia, Uppsala, Sweden).
Immunofluorescence microscopy
The subconfluent cells on a four-well chamber slide were fixed with 4.0% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100, 0.1% sodium citrate. The nuclei of apoptotic cells were then stained with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) using an in situ Apoptosis Detection Kit (Takara, Tokyo, Japan) according to the manufacturer's instructions. Actin filaments were stained with a 1:100 dilution of rhodamine-phalloidin for 60 minutes and nuclei were stained with a 1:5000 dilution of 4',6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Leiden, The Netherlands) for 5 minutes. The slides were washed with PBS and mounted using Parmafluor aqueous mounting medium (Immunon, Pittsburgh, PA). The stained cells were observed either in a fluorescence microscope (PROVIS Ax 80 TR; Olympus, Tokyo, Japan) or in a confocal microscope (Carl Zeiss LSM 510; Carl Zeiss, Oberkochen, Germany).
Cell cycle analysis
After stimulation, the cells were washed with PBS, resuspended in 100 µl PBS and fixed by the addition of 900 µl cold ethanol. The fixed cells were incubated with 300 µl staining buffer (100 µg ml1 RNase A, 50 µg ml1 propidium iodide, 0.1% sodium citrate and 0.3% NP-40 in PBS) at 37°C for 30 minutes. The DNA contents in the nucleus of the cells were analysed with a FACScan using the Cell Quest software.
Annexin-V staining
Cells were washed twice in PBS and resuspended in 100 µl labeling solution containing 2 µl biotin/annexin-V in PBS for 30 minutes on ice. The cells were rinsed and developed with FITC-conjugated avidin (Becton Dickinson) on ice for 30 minutes. The stained cells were then analysed with the FACScan.
Immunoprecipitation and immunoblot analysis
Cells treated with the indicated antibodies were lysed as described previously (Oritani et al., 1996). Immunoprecipitation with the indicated antibodies prebound to protein-G/Sepharose and immunoblot analysis with the appropriate primary antibody were carried out as described previously (Oritani et al., 1996
). In some experiments, the membranes were then stripped and reprobed with appropriate antibodies as an internal control for equivalent protein loadings.
Statistics
Data are expressed as the mean±s.e.m. Statistical comparisons between groups were performed with the Student's t-test. A value of P<0.01 was considered to be significant.
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Results |
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Moreover, we have examined the effect of EGFR-Fc fusion protein, which is able to neutralize all types of EGFR ligands including both soluble and membrane-anchored forms. When the interaction between EGFR and its endogenous ligands was blocked by the EGFR-Fc fusion protein, [3H]-thymidine incorporation of MKN-28 cells reduced by approximately 30% (Fig. 1F). However, MKN-28 cells treated with EGFR-Fc fusion protein showed similar cell viability to controls during our observation period (data not shown). Thus, the blocking of EGFR-EGF interactions withdrew cells from the proliferation cycle but was not sufficient to induce apoptosis. Although the growth inhibition by ALB6 was still observed even in the presence of the EGFR-Fc fusion protein, there was no difference in [3H]-thymidine incorporation of ALB6-treated MKN-28 cells with or without the EGFR-Fc fusion protein (Fig. 1F). Similar results were obtained in MKN-45, SW480, HT-29 and CaCO2 cells (data not shown). These facts might suggest that ALB6 treatment modulates not only EGFR-EGF autocrine/juxtacrine loop but also other pathways such as the pro-apoptotic signals.
Antibody ligation of CD9 induces apoptosis in MKN-28 cells
MKN-28 cells treated with isotype-matched mouse IgG1 showed cobblestone shapes with cell-cell contact preserved. ALB6 treatment induced polygonal or dendrite-like morphology within 48 hours; in addition, prolonged incubation with ALB6 resulted in shrinkage and rounding of cells for all cell lines tested here (data not shown).
Fig. 2 shows immunofluorescence microscopy observations concerning the actin cytoskeleton and the nuclear morphology of MKN-28 cells. Rhodamine-labeled phalloidin localized the actin into fine filamentous or granular patterns predominantly in the perinuclear region and adjacent plasma membrane of MKN-28 cells in the presence of isotype-matched mouse IgG1 (Fig. 2A). However, MKN-28 cells that had been treated with ALB6 showed that their actin was organized into a parallel array of filaments in the cytoplasm (Fig. 2B). In addition, fine microfilament networks or bundles of stress fibers appeared to be lost with an increased incubation time (data not shown). Morphological changes were also detected in the nuclei. Normal nuclei showed a faint blue color as a result of DAPI staining (Fig. 2C). ALB6 treatment led to the appearance of cells showing bright blue, smaller nuclei with chromatin condensation or fragmentation (Fig. 2D). To confirm the conclusion that ALB6 treatment induces apoptosis, we analysed MKN-28 cells treated with ALB6 for 96 hours using a TUNEL assay and cell-cycle analysis as well as annexin-V staining. TUNEL-positive cells were evident in the ALB6-treated cells, but only a few such cells were observed in cells treated with isotype-matched mouse IgG1 (Fig. 2E,F). In the cell-cycle analyses, the subdiploid DNA fraction in ALB6-treated cells was increased by approximately 66% compared with that in cells treated with isotype-matched mouse IgG1 (Fig. 2G,H). Annexin-V detects phosphatidylserine, which is characteristically present on the outer membrane of apoptotic cells, thus confirming the early stage of apoptosis. Under this assay condition, annexin-V-positive cells were detected at a level of approximately 60% in ALB6-treated MKN-28 cells (Fig. 2I,J). Therefore, the treatment of MKN-28 cells with ALB6 induces apoptosis.
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Caspase-3 activation and MAPK activity are involved in apoptosis induced after antibody ligation of CD9
We further investigated the issue of whether ALB6-induced apoptosis is correlated with the activation of caspase, a major pathway of apoptosis (Nagata, 1997). Caspase-3 was activated within 24 hours of incubation with ALB6 and its maximal activation was observed after 72 hours (Fig. 3A). We next determined the issue of whether a well-known caspase inhibitor (ZVAD-FMK) abrogates the apoptosis induced by ALB6 treatment. The ALB6-induced apoptosis detected by annexin-V staining was significantly abrogated by the addition of ZVAD-FMK (Fig. 3B). Therefore, caspase-dependent signaling is involved in the apoptosis of MKN-28 cells induced by ALB6 treatment.
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MAPK signaling pathways have been implicated in survival or apoptotic signals in response to many stimuli (Xia et al., 1995; Nagata and Todokoro, 1999
). As shown in Fig. 3C, the activation of JNK/SAPK and p38 MAPK reached a maximum within 5 minutes of incubation with ALB6 and was observed for 15 minutes. By contrast, the amount of constitutively phosphorylated ERK1/2 remained unaffected in the same time-course study, but was substantially suppressed within 24-48 hours of incubation with ALB6 (data not shown). Therefore, treatment of MKN-28 cells with ALB6 modulates MAPK activity, which might be related to apoptosis.
Tyrosine phosphorylation of signal-transducing molecules induced by CD9 ligation
It is known that the antibody ligation of CD9 induces tyrosine phosphorylation in some signaling molecules in platelets (Kroll et al., 1992; Yatomi et al., 1993
; Aoyama et al., 1999
). We investigated the phosphorylation of several signal-transduction molecules after treatment of MKN-28 cells with ALB6. Although ALB6 treatment failed to induce the tyrosine phosphorylation of c-cbl, Fyn, phospholipase-C
or c-Src, Shc was dramatically tyrosine phosphorylated after the stimulation (Fig. 4A). Of the Shc isoforms, only p46, not p66 or p52, was phosphorylated. In addition, we have examined the proliferation and the signaling in cells treated with the antibody ligation of CD82, another tetraspanin. The antibody ligation of CD82 did not affect either the cell proliferation or the induction of the p46 Shc tyrosine phosphorylation in MKN-28 cells (Fig. 4B), even though they expressed CD82 [mean fluorescence intensity (MFI) number: 210] on their surfaces. Moreover, we have examined whether p46 Shc tyrosine phosphorylation is mediated by the Src family kinases using Src-specific inhibitor PP2. The phosphorylation of p46 Shc after ALB6 treatment was not affected by PP2 even at a concentration that inhibited EGF-induced Stat3 phosphorylation (Fig. 4C). This result indicates that the Src-kinase family might not be involved in the tyrosine phosphorylation of the p46 Shc isoform treated with ALB6. Therefore, the antibody ligation of CD9 induces the selective tyrosine phosphorylation of the p46 Shc isoform.
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Roles of p46 Shc in CD9-ligation-induced apoptosis
To clarify roles of p46 Shc in ALB6-induced apoptosis, we transfected pcDNA3.1(+)-HA-p46 Shc DN into MKN-28 cells and established three clones expressing p46 Shc DN stably, designated p46 Shc DN1, p46 Shc DN2 and p46 Shc DN3 (Fig. 5A). As shown in Fig. 5B, tyrosine phosphorylation of p46 Shc was decreased in p46 Shc DN1 cells, whereas phosphorylation of p46 Shc in MKN-28V cells remained at a level similar to that of parent cells. Similar results were also observed in p46 Shc DN 2 and p46 Shc DN3 cells (data not shown), and the following experiments were performed with these three clones.
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As shown in Fig. 5C, antibody ligation of CD9 did not inhibit the proliferation of any of p46 Shc DN clones compared with the parent and MKN-28V cells. The cell viability of p46 Shc DN clones after the treatment with ALB6 was significantly higher than those of parent and MKN-28V cells (Fig. 5D). Although approximately 66% of the ALB6-treated MKN-28V cells were annexin-V-positive, annexin-V-positive cells in p46 Shc DN1 cells were detected only at 27% (Fig. 5E). Up to 72 hours after ALB6 treatment, p46 Shc DN1 cells did not show any of the typical morphological changes that were widely observed in MKN-28 and MKN-28V cells in response to ALB6 treatment (data not shown). In addition, p46 Shc DN1 cells did not show the ALB6-induced nuclear fragmentation that was observed for many MKN-28V cells treated with ALB6 (data not shown). Similar results were also observed in p46 Shc DN2 and p46 Shc DN3 cells (data not shown). Therefore, the activated p46 Shc isoform might mediate apoptotic signals after antibody ligation of CD9 in MKN-28 cells.
Effects of p46 Shc on MAPK activity and caspase-3 activation
The influence of the p46 Shc isoform on MAPK signaling pathways and the caspase cascade were then examined using the three p46 Shc DN clones. The activation of JNK/SAPK and p38 MAPK of p46 Shc DN1 cells after ALB6 treatment was markedly suppressed compared with those of the parent and MKN-28V cells (Fig. 5F). The activation of caspase-3 in p46 Shc DN1 cells after ALB6 treatment was also suppressed compared to that of the parent and MKN-28V cells (Fig. 5F). Similar observations were found in p46 Shc DN2 and p46 Shc DN3 cells (data not shown). Therefore, these results suggest that JNK/SAPK, p38 MAPK and caspase-3 are downstream of the p46 Shc isoform in MKN-28 cells treated with ALB6.
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Discussion |
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Several investigators have reported that CD9 participates in cell adhesion and migration (Maecker et al., 1997). However, only a few studies have appeared that link CD9 to apoptosis (Tai et al., 1997
; Ono et al., 1999
; Tachibana and Hemler, 1999
). In the present study, we demonstrate that the treatment of MKN-28 cells with ALB6 induces cell shrinkage, chromatin condensation and the reorganization of actin-microfilament architecture, all of which are characterized as apoptotic processes. Moreover, we confirmed the apoptotic process via the use of TUNEL staining, cell-cycle analysis and annexin-V staining. Thus, this is the first report directly to demonstrate that the antibody ligation of CD9 induces apoptosis in cancer cell lines. From the experiments using HB-EGF and EGFR-Fc fusion protein, it is possible that CD9 modulates not only the EGFR-EGF autocrine/juxtacrine loop but also other pathways, such as the pro-apoptotic signals. ALB6 treatment induced polygonal or dendrite-like morphological changes that are characteristic for apoptosis within 48 hours; in addition, much longer incubation with ALB6 was required to see cell shrinkage, round cells and detachment from the cell culture substratum. Based on the time dependency, the evidence of apoptosis through CD9 is seen much earlier than that of the cell detachment. Similarly to ALB6, the treatment of MKN-28 cells with another anti-CD9 mAb (TP82) induced apoptosis. We confirmed that TP82 completely blocked the binding of ALB6 to MKN-28 cells. Thus, these results suggest that the epitopes of these mAbs were close to each other, which might be the cause of similar inhibitory effect on the growth of MKN-28 cells (data not shown). By contrast, our experiments revealed that antibody ligation of CD82 did not affect proliferation and apoptosis of MKN-28 cells. Similar results were obtained in CD9-positive tumor cell lines in our experiments. Thus, the cell biological function that induces the growth inhibition and apoptosis in human cancer cells is not common to tetraspanins and might be specific to CD9.
An important aspect of the present results concerns the molecular mechanisms underlying CD9-mediated apoptosis. At least two signaling pathways (the caspase cascade and the MAPK pathway) are involved in CD9-mediated apoptosis. The activation of caspases might be achieved through several molecular pathways. The best known stimuli triggering caspase cascade are the stimulation of Fas or tumor-necrosis-factor (TNF) receptors (Nagata, 1997; Baker and Reddy, 1998
). The caspase family, caspase-3 in particular, is one of the key participants in apoptotic cell death, being responsible either partially or totally for the proteolytic cleavage of several key proteins. The participation of the caspase cascade in ALB6-induced apoptosis is now clear from our results, which clearly show that the treatment of MKN-28 cells with ALB6 induced the proteolytic cleavage of caspase-3, and its chemical inhibitor significantly abrogated the ALB6-induced apoptosis. Several distinct MAPKs such as ERK1/2 (Boulton et al., 1991
), JNK/SAPK (Kyriakis et al., 1994
) and p38 MAPK (Han et al., 1994
), are also involved in the terminal stages of signaling cascades that are related not only to a cell growth and survival but also to cell death. JNK/SAPK and p38 MAPK are activated in response to a range of cellular stresses such as osmotic shock, inflammatory cytokines, lipopolysaccharides and ultraviolet irradiation (Derijard et al., 1994
). ERK1/2 inhibits apoptosis induced by a range of stimuli, such as hypoxia (Buckley et al., 1999
), growth factor withdrawal (Erhardt et al., 1999
), hydrogen peroxide (Wang et al., 1998
) and chemotherapeutic agents (Anderson and Tolkovsky, 1999
). The activation of JNK/SAPK and p38 MAPK is generally associated with the promotion of apoptosis. The downregulation of ERK1/2 activity might also accelerate caspase-3 activity, a downstream effector in the apoptosis cascade (Widmann et al., 1998
). Indeed, it has been reported that the concurrent activation of JNK/SAPK and p38 MAPK, and inhibition of ERK1/2 induce apoptosis (Xia et al., 1995
; Nagata and Todokoro, 1999
). The balance between ERK1/2, JNK/SAPK and p38-MAPK might also be a determinant of the fate of cells that are undergoing proliferation, differentiation or apoptosis. Our results, which show that treatment of MKN-28 cells with ALB6 induced the activation of JNK/SAPK and p38 MAPK along with the suppression of ERK1/2 activity, are consistent with a scenario in which the modification of MAPK signaling pathway is involved in ALB6-induced apoptosis.
The findings herein show that the antibody ligation of CD9 in MKN-28 cells selectively induces tyrosine phosphorylation of the p46 Shc isoform, but not the p66 and p52 Shc isoforms. The adaptor protein Shc exists as these three isoforms, p66, p52, and p46, which are translated from three different initiation sites (Migliaccio et al., 1997). Although Shc isoforms are activated by EGF and a range of cytokines, and contribute to the recruitment of Grb2 (Rozakis-Adcock et al., 1992
; Pronk et al., 1994
; Wary et al., 1996
), recent studies have suggested distinct physiological roles for the three isoforms (Migliaccio et al., 1997
; Okada et al., 1997
). For example, p66 Shc has been reported to act as a negative regulator of EGF-stimulated MAPK-Fos signaling pathway (Okada et al., 1997
). The p66 Shc has been shown to regulate both the oxidative stress response and lifespan in mammals (Migliaccio et al., 1999
). The p66 Shc and p52 Shc, but not the p46 Shc, have been reported to interact with and to activate c-Src in vitro and in vivo (Sato et al., 2002
). Thus, the possible existence of isoform-specific functions for each Shc isoform cannot be excluded. Our experiments using p46 Shc DN clones have clarified the crucial roles of the p46 Shc isoform in ALB6-induced apoptosis. The overexpression of p46 Shc DN in MKN-28 cells cancelled ALB6-induced apoptosis as well as the activation of JNK/SAPK, p38 MAPK and caspase-3. The mechanisms of CD9-induced p46 Shc tyrosine phosphorylation are interesting and important. At least, c-Cbl, Fyn and phospholipase-C
were not related to this event, because ALB6 treatment failed to induce their phosphorylation. Because Src inhibitor, PP2 could not inhibit CD9-induced p46 Shc tyrosine phosphorylation, Src-family kinases including c-Src that are interacted with p66 Shc and p52 Shc, did not involve in this events. Our reports suggest that the tyrosine phosphorylation of the p46 Shc isoform might be mediated by other molecules that are associated with CD9. CD9 associates with small G-proteins, and the antibody ligation of CD9 on platelets induces tyrosine phosphorylation and increases intracellular calcium concentration (Kroll et al., 1992
; Yatomi et al., 1993
). Tetraspanin proteins might contribute to integrin-mediated signaling (Shaw et al., 1995
; Yáñez-Mó et al., 1998
; Berditchdvski and Odintsova, 1999). Therefore, CD9, which acts as a key molecule in the membrane protein communication network on cell surface, might modulate the formation and/or stability of functional signaling complexes including integrins. Future studies will aim to investigate how these cellular events are triggered and what the trigger is, and experiments are in progress.
In this study, we demonstrated that CD9 delivers specialized signals to induce apoptosis in human cancer cell lines and selectively induced tyrosine phosphorylation of p46 Shc isoform that mediated pro-apoptotic signals through the activation of JNK/SAPK and p38 MAPK as well as caspase-3. These results suggest that CD9 plays an important role in intracellular signals that control not only cell growth, migration and adhesion but also apoptosis. Thus, it is important that we provide a specific biochemical link between a particular member of the tetraspanin family and a signaling pathway leading to apoptosis. Further analysis of the signaling pathways via CD9 might provide the insights for new therapeutic strategies for patients with cancers.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, C. N. G. and Tolkovsky, A. M. (1999). A role for MAPK/ERK in sympathetic neuron survival: protection against a p53-dependent, JNK-independent induction of apoptosis by cytosine arabinoside. J. Neurosci. 19, 664-673.
Aoyama, K., Oritani, K., Yokota, T., Ishikawa, J., Nishiura, T., Miyake, K., Kanakura, Y., Tomiyama, Y., Kincade, P. W. and Matsuzawa, Y. (1999). Stromal cell CD9 regulates differentiation of hematopoietic stem/progenitor cells. Blood 93, 2586-2594.
Baker, S. J. and Reddy, E. P. (1998). Modulation of life and death by the TNF receptor superfamily. Oncogene 17, 3261-3270.[Medline]
Baudoux, B., Castanares-Zapatero, D., Leclercq-Smekens, M., Berna, N. and Poumay, Y. (2000). The tetraspanin CD9 associates with the integrin alpha6beta4 in cultured human epidermal keratinocytes and is involved in cell motility. Eur. J. Cell Biol. 79, 41-51.[Medline]
Berditchevski, F. (2001). Complexes of tetraspanins with integrins: more than meets the eye. J. Cell Sci. 114, 4143-4151.
Berditchevski, F. and Odintsova, E. (1999). Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling. J. Cell Biol. 146, 477-492.
Berditchevski, F., Zutter, M. M, and Hemler, M. E. (1996). Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins). Mol. Biol. Cell 7, 193-207.[Abstract]
Buckley, S., Driscoll, B., Barsky, L., Weinberg, K., Anderson, K. and Warburton, D. (1999). ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am. J. Physiol. 277, L159-L166.[Medline]
Boucheix, C., Soria, C., Mirshahi, M., Soria, J., Perrot, J. Y., Fournier, N., Billard, M. and Rosenfeld, C. (1983). Characterization of platelet aggregation induced by the monoclonal antibody ALB6 (acute lymphoblastic leukemia antigen p24). Inhibition of aggregation by ALB6 Fab. FEBS Lett. 16, 289-295.
Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., Dephino, R. A., Panayotatos, N., Cobb, M. H. and Yancopoulos, G. D. (1991). ERK's: a family of protein serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65, 663-675.[Medline]
Cajot, J. F., Sordat, I., Silvestre, T. and Sordat, B. (1997). Differential display cloning identifies motility-related protein (MRP1/CD9) as highly expressed in primary compared to metastatic human colon carcinoma cells. Cancer Res. 57, 2593-2597.[Abstract]
Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M. and Davis, R. J. (1994). JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76, 1025-1037.[Medline]
Erhardt, P., Schremser, E. J. and Cooper, G. M. (1999). B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. Mol. Cell. Biol. 19, 5308-5315.
Gotoh, N., Toyoda, M. and Shibuya, M. (1997). Tyrosine phosphorylation sites at amino acid 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation. Mol. Cell. Biol. 17, 1824-1831.[Abstract]
Han, J., Lee, J. D., Bibbs, L. and Ulevitch, R. J. (1994). A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265, 808-811.[Medline]
Hemler, M. E. (2003). Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu. Rev. Cell Dev. Biol. 19, 397-422.[CrossRef][Medline]
Higashihara, M., Maeda, H., Shibata, Y., Kume, S. and Ohashi, T. (1985). A monoclonal anti-human platelet antibody: a new platelet aggregating substance. Blood 65, 382-391.[Abstract]
Higashiyama, S., Iwamoto, R., Goishi, K., Raab, G., Taniguchi, N., Klagsbrun, M. and Mekada, E. (1995). The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-like growth factor. J. Cell Biol. 128, 929-938.[Abstract]
Inui, S., Higashiyama, S., Hashimoto, K., Higashiyama, M., Yoshikawa, K. and Taniguchi, N. (1997). Possible role of coexpression of CD9 with membrane-anchored heparin-binding EGF-like growth factor and amphiregulin in cultured human keratinocyte growth. J. Cell Physiol. 171, 291-298.[CrossRef][Medline]
Jones, P. H., Bishop, L. A. and Watt, F. M. (1996). Functional significance of CD9 association with beta 1 integrins in human epidermal keratinocytes. Cell Adhes. Commun. 4, 297-305.[Medline]
Kroll, M. H., Mendelsohn, M. E., Miller, J. L., Ballen, K. K., Hrbolich, J. K. and Schafer, A. I. (1992). Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium. Biochim. Biophys. Acta 1137, 248-256.[CrossRef][Medline]
Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J. and Woodgett, J. R. (1994). The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369, 156-160.[CrossRef][Medline]
Maecker, H. T., Todd, S. C. and Levy, S. (1997). The tetraspanin superfamily: molecular facilitators. FASEB J. 11, 428-442.
Mori, M., Mimori, K., Shiraishi, T., Haraguchi, M., Ueo, H., Barnard, G. F. and Akiyoshi, T. (1998). Motility related protein 1 (MRP1/CD9) expression in colon cancer. Clin. Cancer Res. 4, 1507-1510.[Abstract]
Murayama, Y., Miyagawa, J., Shinomura, Y., Kanayama, S., Isozaki, K., Yamamori, K., Mizuno, H., Ishiguro, S., Kiyohara, T., Miyazaki, Y. et al. (2002). Significance of the association between heparin-binding epidermal growth factor-like growth factor and CD9 in human gastric cancer. Int. J. Cancer 98, 505-513.[CrossRef][Medline]
Migliaccio, E., Mele, S., Salcini, A. E., Pelicci, G., Lai, K. M., Superti-Furga, G., Pawson, T., di Fiore, P. P., Lanfrancone, L. and Pelicci, P. G. (1997). Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase Fos signaling pathway. EMBO J. 16, 706-716.
Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Reboldi, P., Pandolfi, P. P., Lanfrancone, L. and Pelicci, P. G. (1999). The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402, 309-313.[CrossRef][Medline]
Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355-365.[Medline]
Nagata, Y. and Todokoro, K. (1999). Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis. Blood 94, 853-863.
Ohto, H., Shibata, Y., Takeuchi, A., Chen, R. F. and Maeda, H. (1985). Expression of the platelet-common acute lymphoblastic leukaemia associated antigen on normal eosinophils. Scand. J. Haematol. 34, 281-287.[Medline]
Okada, S., Kao, A. W., Ceresa, B. P., Blaikie, P., Margolis, B. and Pessin, J. E. (1997). The 66-kDa Shc isoform is a negative regulator of the epidermal growth factor-stimulated mitogen-activated protein kinase pathway. J. Biol. Chem. 272, 28042-28049.
Ono, M., Hanada, K., Witheres, D. A. and Hakomori, S. (1999). Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation. Cancer Res. 59, 2335-2339.
Oritani, K., Wu, X., Medina, K., Hudson, J., Miyake, K., Gimble, J. M., Burstein, S. A. and Kincade, P. W. (1996). Antibody ligation of CD9 modifies production of myeloid cells in long-term cultures. Blood 87, 2252-2261.
Ozaki, Y., Satoh, K., Kuroda, K., Qi, R., Yatomi, Y., Yanagi, S., Sada, K., Yamamura, H., Yanabu, M., Nomura, S. et al. (1995). Anti-CD9 monoclonal antibody activates p72syk in human platelets. J. Biol. Chem. 270, 15119-15124.
Pronk, G. J., de Vries-Smits, A. M., Buday, L., Downward, J., Maassen, J. A., Medema, R. H. and Bos, J. L. (1994). Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras. Mol. Cell. Biol. 14, 1575-1581.[Abstract]
Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G. et al. (1992). Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinase. Nature 360, 689-692.[CrossRef][Medline]
Rubinstein, E., le Naour, F., Lagaudriere-Gesbert, C., Billard, M., Conjeaud, H. and Boucheix, C. (1996). CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. Eur. J. Immunol. 26, 2657-2665.[Medline]
Seehafer, J. G. and Shaw, A. R. (1991). Evidence that the signal-initiating membrane protein CD9 is associated with small GTP-binding proteins. Biochem. Biophys. Res. Commun. 179, 401-406.[Medline]
Shaw, A. R., Domanska, A., Mak, A., Ginlchrist, A., Dobler, K., Visser, L., Poppema, S., Fliegel, L., Letarte, M. and Willett, B. J. (1995). Ectopic expression of human and feline CD9 in a human B cell line confers ß1 integrin-dependent motility on fibronectin and laminin substrates and enhanced tyrosine phosphorylation. J. Biol. Chem. 270, 24092-24099.
Shi, W., Fan, H., Shum, L. and Derynck, R. (2000). The tetraspanin CD9 associates with transmembrane TGF-alpha and regulates TGF-alpha-induced EGF receptor activation and cell proliferation. J. Cell Biol. 148, 591-602.
Sho, M., Adachi, M., Taki, T., Hashida, H., Konishi, T., Huang, C. L., Ikeda, N., Nakajima, Y., Kanehiro, H., Hisanaga, M. et al. (1998). Transmembrane 4 superfamily as a prognostic factor in pancreatic cancer. Int. J. Cancer 79, 509-516.[CrossRef][Medline]
Sato, K., Nagao, T., Kakumoto, M., Kimoto, M., Otsuki, T., Iwasaki, T., Tokmakov, A. A., Owada, K. and Fukami, Y. (2002). Adaptor protein Shc is an isoform-specific direct activator of the tyrosine kinase c-Src. J. Biol. Chem. 277, 29568-29576.
Tachibana, I. and Hemler, M. E. (1999). Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance. J. Cell Biol. 146, 893-904.
Tai, X. G., Toyooka, K., Yashiro, Y., Abe, R., Park, C. S., Hamaoka, T., Kobayashi, M., Neben, S. and Fujiwara, H. (1997). CD9-mediated costimulation of TCR-triggered native T cells leads to activation followed by apoptosis. J. Immunol. 159, 3799-3807.[Abstract]
Uchida, S., Shimada, Y., Watanabe, G., Li, Z. G., Hong, T., Miyake, M. and Imamura, M. (1999). Motility-related protein (MRP-1/CD9) and KAI1/CD82 expression inversely correlate with lymph node metastasis in oesophageal squamous cell carcinoma. Br. J. Cancer 79, 1168-1173.[CrossRef][Medline]
Wang, X., Martindale, J. L., Liu, Y. and Holbrook, N. J. (1998). The cellular response to oxidative stress: influences of mitogen-activated protein kinase signaling pathways on cell survival. Biochem. J. 333, 291-300.[Medline]
Wary, K. K., Mainiero, F., Isakoff, S. J., Marcantonio, E. E. and Giancotti, F. G. (1996). The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell 87, 733-743.[Medline]
Widmann, C., Gibson, S. and Johnson, G. L. (1998). Caspase-dependent cleavage of signaling proteins during apoptosis. A turn-off mechanism for anti-apoptotic signals. J. Biol. Chem. 273, 7141-7147.
Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J. and Greenberg, M. E. (1995). Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326-1331.[Abstract]
Yáñez-Mó, M., Alfranca, A., Cabañas, C., Marazuela, M., Tejedor, R., Ursa, A., Ashman, L. K., de Landázuri, M. O. and Sanchez-Madrid, F. (1998). Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TATP-1 and CD151/PETA-3 with 3ß1 integrin localized at endothelial lateral junctions. J. Cell Biol. 141, 791-804.
Yatomi, Y., Ozaki, Y., Satoh, K. and Kume, S. (1993). Anti-CD9 monoclonal antibody elicits staurosporine inhibitable phosphatidylinositol 4, 5-bisphosphate hydrolysis, phosphatidylinositol 3,4-bisphosphate synthesis, and protein-tyrosine phosphorylation in human platelets. FEBS Lett. 322, 285-290.[CrossRef][Medline]