QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01201

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Murayama, Y. Articles by Shinomura, Y. Search for Related Content PubMed PubMed Citation Articles by Murayama, Y. Articles by Shinomura, Y. Social Bookmarking                         What's this?

CD9-mediated activation of the p46 Shc isoform leads to apoptosis in cancer cells

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

View larger version (32K): [in a new window]   Fig. 1. Antibody ligation of CD9 inhibits the cell proliferation of human cancer cell lines. (A) The indicated cell lines were serum-starved for 24 hours and subsequently cultured for 48 hours in the presence of 50 µg ml–1 ALB6, isotype-matched mouse IgG1 or medium alone, and their proliferation was measured by a [3H]-thymidine incorporation assay. Data are represented as the means±s.e.m. from six replications and are expressed as a percentage of the values found for cells cultured in medium alone. *P<0.01 vs control. (B) MKN-28 cells were serum-starved for 24 hours and subsequently incubated for 48 hours with the indicated concentrations of ALB6, and their proliferation was measured by a [3H]-thymidine incorporation assay. Data are represented as the means±s.e.m. from six replications and are expressed as a percentage of the values found for cells treated with isotype-matched mouse IgG1 (50 µg ml–1). *P<0.01 vs control. (C) After MKN-28 cells (2x104 cells per well) were cultured on six-well plates for 24 hours, the medium was replaced with fresh serum-deprived medium containing 50 µg ml–1 ALB6 or isotype-matched mouse IgG1. Their cell viability after ALB6 or isotype-matched mouse IgG1 treatment for the indicated periods was estimated by the trypan-blue dye-exclusion method. Data are represented as the mean cell viability (%) ±s.e.m. (n=4). *P<0.01 vs control. (D) After MKN-28 cells (1x104 cells per well) were cultured on six-well plates for 24 hours, the medium was replaced with fresh serum-deprived medium containing different concentrations of ALB6. Their viable cell numbers were estimated by the trypan-blue dye-exclusion method after 96 hours of culture. Data are represented as mean cell viability (%) ±s.e.m. (n=4). *P<0.01 vs control. Similar results were observed in five independent experiments. (E) Effect of HB-EGF on ALB6-induced growth suppression in MKN-28 cells. The indicated cell lines were serum-starved for 24 hours and subsequently cultured in the presence of 50 µg ml–1 ALB6 or isotype-matched mouse IgG1 together with or without 20 ng ml–1 HB-EGF for 48 hours and their proliferation was measured by a [3H]-thymidine incorporation assay. Data are presented as means±s.e.m. from six repeats of the experiment and are expressed as a percentage of the values found for isotype-matched mouse IgG1 (50 µg ml–1) treatment cells. (F) Effect of EGFR-Fc on ALB6-induced growth suppression in MKN-28 cells. The indicated cell lines were serum-starved for 24 hours and subsequently cultured in the presence of 50 µg ml–1 ALB6 or isotype-matched mouse IgG1 together with or without 20 µg ml–1 EGFR-Fc for 48 hours, and their proliferation was measured by a [3H]-thymidine incorporation assay. Data are presented as means±s.e.m. from six repeats of the experiment and are expressed as a percentage of the values found for isotype-matched mouse IgG1 (50 µg ml–1) treatment cells.

View larger version (44K): [in a new window]   Fig. 2. Antibody ligation of CD9 induces apoptosis in MKN-28 cells. MKN-28 cells were cultured on chamber slides for 96 hours in the presence of 50 µg ml–1 IgG1 (A,C,E,G,I) or ALB6 (B,D,F,H,J). The cultured cells were then fixed with 4.0% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 at 4°C for 2 minutes and subjected to staining with rhodamine-phalloidin (A,B), DAPI (C,D) or TUNEL (E,F). Scale bars, 50 µm (A-D), 200 µm (E,F). The cultured cells were harvested, stained with propidium iodide and their nuclear contents analysed by flow cytometry (G,H). The cultured cells were harvested and stained with annexin-V (shaded bars). Control staining is also shown (open bars) (I,J). Similar results were obtained in three independent experiments.

View larger version (32K): [in a new window]   Fig. 3. Effects of antibody ligation of CD9 on the activation of caspase-3 and MAPK activity. (A) MKN-28 cells were incubated with 50 µg ml–1 ALB6 or isotype-matched mouse IgG1 for the indicated periods. The adherent cells were harvested and subjected to immunoblots with anti-caspase-3 antibody. The arrow indicates the procaspase-3. The arrowhead indicates the cleaved caspase-3. (B) Annexin-V staining of the ALB6-treated MKN-28 cells is shown. MKN-28 cells were incubated with 50 µg ml–1 ALB6 alone (left, shaded bars) or with ALB6 plus 50 µM ZVAD-FMK for 24 hours (right, shaded bars). Open bars show the staining of control cells. (C) MKN-28 cells were serum-starved for 24 hours, and then incubated with 50 µg ml–1 ALB6 or isotype-matched mouse IgG1 for the indicated periods. The total cell lysates were analysed by immunoblotting with the indicated antibodies. Each figure shows representative data from four similar experiments.

View larger version (27K): [in a new window]   Fig. 4. CD9 ligation induces tyrosine phosphorylation of p46 Shc. (A) MKN-28 cells were serum starved for 24 hours and then stimulated with 50 µg ml–1 ALB6 or isotype-matched mouse IgG1 for 10 minutes. The total cell lysates were immunoprecipitated with the indicated antibodies and the blots were analysed with PY20 (top). The same filters were then stripped and reprobed with the indicated antibodies to confirm equal loadings (bottom). Each figure shows one of four similar experiments. (B) MKN-28 cells were serum starved for 24 hours and subsequently cultured for 48 hours in the presence of 50 µg ml–1 isotype-matched mouse IgG1, 50F11 or ALB6, and their proliferation was measured by a [3H]-thymidine incorporation assay. Data are represented as the means±SEM from six replications and are expressed as a percentage of the values found for isotype-matched mouse IgG1 (50 µg ml–1) treatment cells (top). *P<0.01 vs control. MKN-28 cells were serum starved for 24 hours, and then stimulated with 50 µg ml–1 isotype-matched mouse IgG1, 50F11 or ALB6 for 10 minutes. The total cell lysates were immunoprecipitated with anti-Shc antibody and the blots were analysed with PY20 (bottom). (C) MKN-28 cells were serum starved for 24 hours and were subsequently pretreated either 10 µM PP2 or 10 µM PP3 for 30 minutes, and then stimulated with or without 50 µg ml–1 ALB6 for 10 minutes. The total cell lysates were immunoprecipitated with anti-Shc antibody and the blots were analysed with PY20.

View larger version (43K): [in a new window]   Fig. 5. Expression of p46 Shc DN cancels growth inhibition and apoptosis induced by ALB6 treatment. (A) The total cell lysates of each p46 Shc DN clone were analysed by immunoblotting using anti-HA antibody. (B) MKN-28 cells, MKN-28V cells and p46 Shc DN1 cells at subconfluent density were serum starved for 24 hours and then stimulated with 50 µg ml–1 ALB6 or isotype-matched mouse IgG1 for 10 minutes. The total cell lysates were immunoprecipitated with anti-Shc antibody and the blots were analysed for PY20 (top). The same filters were then stripped and reprobed with anti-Shc antibody (bottom). (C) The indicated cells were serum starved for 24 hours, and subsequently cultured for 48 hours in the presence of 50 µg ml–1 ALB6, and their proliferation was measured using a [3H]-thymidine incorporation assay. Data are represented as the means±s.e.m. from four replications and are expressed as a percentage of the values found for cells cultured in medium alone. *P<0.01 vs control. (D) After the indicated cells (2x104 cells per well) were cultured on six-well plates for 24 hours, the medium was replaced with fresh serum-deprived medium containing 50 µg ml–1 ALB6. Their cell viability after ALB6 treatment for the indicated period was estimated by the trypan-blue dye-exclusion method. Data are represented as the mean cell viability (%) ±s. e.m. (n=4). *P<0.01 vs control. (E) MKN-28 cells, MKN-28V cells and p46 Shc DN1 cells were incubated with 50 µg ml–1 ALB6, stained with annexin-V and analysed with a FACScan. Broken lines show the staining of untreated cells, solid lines show the staining of MKN-28 cells and shaded bars show the staining of p46 Shc DN1 cells. (F) The indicated cells were serum starved for 24 hours and then incubated with 50 µg ml–1 ALB6 for 5 minutes. The total cell lysates were analysed by immunoblotting with the indicated antibodies The indicated cells were incubated with 50 µg ml–1 ALB6 for 48 hours. The adherent cells were harvested and subjected to immunoblots with anti-caspase-3 antibody. The arrow indicates procaspase-3. The arrowhead indicates cleaved caspase-3. Each figure shows one of four similar experiments.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter