HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/1/158 03-0420fjev1    most recent 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 SCHOENFELD, N. Articles by GRIMM, S. Search for Related Content PubMed PubMed Citation Articles by SCHOENFELD, N. Articles by GRIMM, S.

The metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates¹

Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany

²Correspondence: Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany. E-mail: sgrimm{at}biochem.mpg.de

SPECIFIC AIMS

The down-regulation of apoptosis is a hallmark of tumorigenesis. We wanted to investigate how the metastasis suppressor C33/CD82/KAI1 is able to induce cell death.

PRINCIPAL FINDINGS

1. The metastasis suppressor gene C33/CD82/KAI1 is an apoptosis inducer
Using a screen for apoptosis-inducing genes, we detected the metastasis suppressor gene C33/KAI1/CD82. CD82 is a gene that is down-regulated in many metastatic tumor cells and whose expression can attenuate the process of metastases formation in a variety of tumors. We observed cell death induction by CD82 in many different cell types. Moreover, the apoptosis inducer cycloheximide was >threefold stronger in cells with reconstituted CD82 expression compared with CD82 negative cells. This indicates that CD82 mediates a proapoptotic signal caused by specific stimuli. As CD82 is a transmembrane protein, it was speculated that it mediates its anti-metastatic effect by interacting with components of the basal membrane. When we deleted the long, glycosylated extracellular loop of CD82 implicated in mediating cellular attachment, it still caused apoptosis. These mapping experiments suggested that adherence is not obligatory for apoptosis induction by CD82.

2. C33/CD82/KAI1 induces apoptosis by the generation of reactive oxygen intermediates
CD82 seems to promote cell death by generating reactive oxygen intermediates (ROIs). After CD82 transfection, we observed a strong induction of oxygen radicals peaking after 22 h compared with control cells (Fig. 1 A). We performed apoptosis quantification by FACS analysis, which showed that CD82 apoptosis occurred significantly later than ROI production (Fig. 1B) . The superoxide scavenger Tiron and cotransfection of the ROI scavenging enzymes catalase or Cu/Zn superoxide dismutase decreased CD82-induced cell death, suggesting that ROI generation is essential for apoptosis induction by CD82 (Fig. 1C-E) . As CD82 inactivation is crucial for metastases formation; especially since micro-metastases develop before angiogenesis and concomitant oxygen supply has set in, we wanted to address the role of oxygen for apoptosis induction. CD82 was still efficient as an apoptosis inducer in an environment with reduced (1%) oxygen (hypoxia). We found that the ROIs generated by CD82 are not derived from the respiratory chain, otherwise the standard source of ROIs for cell death induction.

View larger version (28K): [in this window] [in a new window]   Figure 1. C33 leads to the production of reactive oxygen radicals during apoptosis. A) C33 induces oxidation of hydroethidine to ethidium (ET) during apoptosis. After transfecting HeLa cells with 2 µg expression plasmid for C33, p53, or control vector (Luc), ROI generation was measured by staining with hydroethidine, which detects superoxide radicals in cells. Results are expressed as % of the fluorescence intensity compared with the control transfected cells. Shown are the means and SD of 4 independent experiments. B) C33 leads to a progressive accumulation of apoptotic cells. Aliquots of the transfections described in panel A were removed when indicated and the extent of apoptosis was assessed by cells with sub-G1 DNA content using a FACS. Shown are the means and SD of 4 independent experiments. C) The antioxidant Tiron reduces C33-mediated apoptosis. 13 h after transfection of C33 or ß-gal, Tiron was added at a concentration of 300 µM. After 32 h, apoptosis was quantified by FACS analysis. D) Titration curve of Tiron. Increasing concentrations of Tiron were added to CD82-transfected HeLa cells. Shown are the means and SD of 3 independent FACS experiments. E) Cotransfection of catalase or Cu/Zn superoxide dismutase diminishes cell death by C33 expression. ß-gal (100 ng) or C33 (50 ng) and expression vectors for ß-gal or catalase (Cat), superoxide dismutase (SOD) (50 ng each) were cotransfected. Shown are the means and SD of 4 independent experiments obtained by FACS after PI staining.

3. C33/CD82/KAI1 expression renders cells sensitive to ROIs by the release of the ROI protectant glutathione (GSH)
Cotransfection experiments identified glutathione peroxidase-1 as a cell death repressor after CD82 activation. This enzyme uses glutathione (GSH), a prominent intracellular ROI scavenger, for the detoxification of ROIs. We found that the intracellular concentration of GSH dropped significantly after transfection of CD82. When the supernatant of CD82-transfected cells was tested for the presence of GSH, we observed a corresponding quantitative increase of GSH. To assess the contribution of this glutathione depletion on CD82-induced cell death, we incubated cells with increasing concentrations of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis that leads to a pronounced reduction of GSH by 10 h. BSO treatment potentiated cell death by CD82. Glutathione-monoethyl ester, a membrane-permeable GSH form, restored the CD82-mediated efflux of GSH from cells and caused pronounced reduction of ROIs and a concomitant decrease in apoptosis induction. This suggests that the efflux of GSH is necessary for cell death by CD82.

4. The G-protein Cdc42 is activated by C33/CD82/KAI1, which is required for GSH efflux and ROI formation
A recent report suggested that CD82 activates GTPases such as Cdc42. As some of these G-proteins have been implicated in proapoptotic signaling pathways, we tested the activation of these enzymes. Figure 2 A reveals we could observe a potent stimulation of Cdc42 in an activity assay specific for this GTPase. We also detected a pronounced reduction of apoptosis by CD82 expression when a dominant-negative Cdc42 mutant was cotransfected (Fig. 2B) , suggesting a functional involvement of this GTPase in apoptosis. To investigate whether Cdc42 is necessary for the detection of ROIs induced by CD82, we cotransfected the dominant-negative Cdc42 and observed a potent reduction of ROIs (Fig. 2C) . Last, we analyzed whether Cdc42 mediates the release of cellular glutathione into the medium. Cotransfection of the dominant-negative Cdc42 prevented both the reduction of the cellular glutathione level and its detection in the supernatant of CD82-transfected cells (Fig. 2D) .

View larger version (22K): [in this window] [in a new window]   Figure 2. GTPase Cdc42 is activated by CD82, mediates apoptosis induction and glutathione efflux, and allows ROI detection. A) Cdc42 is activated by CD82. HeLa cells were transfected with plasmids for luciferase or CD82. In one transfection, zVAD, a pan-caspase inhibitor, was added. 16 h later extracts were prepared and activated (GTP-bound) Cdc42 was coimmunprecipitated from equal quantities of cell lysates with a GST-Pak1 protein that associates only with activated Cdc42, subsequently detected in an immunoblot using a specific antibody. Untransfected extracts were treated with GTP or GDP to activate or inactivate Cdc42. B) A dominant-negative Cdc42 reduces apoptosis induction by C33. An expression vector for CD82 and luciferase or the dominant-negative CDC42 T17N mutant were cotransfected at a ratio of 1:4. After 46 h apoptosis was quantified by FACS analysis. C) A dominant-negative Cdc42 reduces ROI formation. HeLa cells were transfected with expression vector for luciferase, CD82, and luciferase or CD82 and a dominant-negative Cdc42 mutant (at a ratio: 1:1). After 14 h, superoxide anions were determined by lucigenin. Shown are the means and SD of 3 independent experiments. D) A dominant-negative Cdc42 mutant inhibits the efflux of GSH from CD82-transfected cells. HeLa cells were transfected with the indicated plasmids for luciferase, CD82, and luciferase or CD82 and the dominant-negative Cdc42 at a ratio of 1:1. 25 h after transfection, cells were harvested and the glutathione level in cellular extracts and the medium was determined. Given are the percent values relative to the intracellular GSH concentration of control (ß-gal) -transfected cells.

CONCLUSIONS

A model (Fig. 3 ) might explain why it is so advantageous for tumor cells to specifically inactivate CD82 for metastases formation. After dissemination to distant tissues and before angiogenesis provides enough oxygen supply, metastases-forming cells encounter a hypoxic environment in micro-metastases. Under this condition, the respiratory chain acts as a hypoxia sensor and reduces its activity while energy generation becomes dependent on glycolysis. Many proapoptotic stimuli (e.g., TNF, ceramide, interferon-, and several cytostatic drugs) no longer function properly in such an environment since they rely on ROIs from the respiratory chain. Consequently, they cannot rescue the organism from metastases spread by inducing apoptosis. As CD82’s activity to generate ROIs relies on the efflux of GSH and is independent of the respiratory chain, it can still induce cell death under these conditions.

View larger version (5K): [in this window] [in a new window]   Figure 3. Schematic diagram.

This could be caused by an apoptosis stimulus such as cycloheximide. If CD82 is then inactivated, the survival of such tumor cells in distant tissues is made possible and metastases formation can proceed. This model links a reduced potential of cells to undergo apoptosis to the process of metastases formation. Such a correlation was indeed observed numerous times. Therefore, our findings shed light on the molecular function of the prominent metastasis-suppressor gene C33/CD82/KAI1 whose way of action was largely unknown.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0420fje

This article has been cited by other articles:

				T. Tohami, L. Drucker, H. Shapiro, J. Radnay, and M. Lishner Overexpression of tetraspanins affects multiple myeloma cell survival and invasive potential FASEB J, March 1, 2007; 21(3): 691 - 699. [Abstract] [Full Text] [PDF]

				H. Zheng, K. Tsuneyama, C. Cheng, H. Takahashi, Z. Cui, K. Nomoto, Y. Murai, and Y. Takano Expression of KAI1 and tenascin, and microvessel density are closely correlated with liver metastasis of gastrointestinal adenocarcinoma J. Clin. Pathol., January 1, 2007; 60(1): 50 - 56. [Abstract] [Full Text] [PDF]

				B. He, L. Liu, G. A. Cook, S. Grgurevich, L. K. Jennings, and X. A. Zhang Tetraspanin CD82 Attenuates Cellular Morphogenesis through Down-regulating Integrin {alpha}6-Mediated Cell Adhesion J. Biol. Chem., February 4, 2005; 280(5): 3346 - 3354. [Abstract] [Full Text] [PDF]

				B. Zhou, L. Liu, M. Reddivari, and X. A. Zhang The Palmitoylation of Metastasis Suppressor KAI1/CD82 Is Important for Its Motility- and Invasiveness-Inhibitory Activity Cancer Res., October 15, 2004; 64(20): 7455 - 7463. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/1/158 03-0420fjev1    most recent 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 SCHOENFELD, N. Articles by GRIMM, S. Search for Related Content PubMed PubMed Citation Articles by SCHOENFELD, N. Articles by GRIMM, S.