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Tenascin-C signaling through induction of 14-3-3 tau.

Martin D, Brown-Luedi M, Chiquet-Ehrismann R - J. Cell Biol. (2003)

Bottom Line: We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C.Furthermore, the growth rate on tenascin-C was increased compared with the parental cells.Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Forschungsstiftung, CH-4002 Basel, Switzerland.

ABSTRACT
We searched by a cDNA subtraction screen for differentially expressed transcripts in MCF-7 mammary carcinoma cells grown on tenascin-C versus fibronectin. On tenascin-C, cells had irregular shapes with many processes, whereas on fibronectin they were flat with a cobble stone-like appearance. We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C. To investigate the consequences of an increased level of this phospho-serine/threonine-binding adaptor protein, we transfected MCF-7 cells with a construct encoding full-length 14-3-3 tau protein and selected clones with the highest expression levels. The morphology of these cells on tenascin-C was flat, resembling that of cells on fibronectin. This was reflected by a similar pattern of F-actin staining on either substratum. Furthermore, the growth rate on tenascin-C was increased compared with the parental cells. After transient transfection of HT1080 fibrosarcoma and T98G glioblastoma cells with 14-3-3 tau, only the 14-3-3 tau-expressing cells were able to adhere and survive on tenascin-C, whereas all cells adhered well on fibronectin. Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

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Isolation of MCF-7 clones overexpressing 14-3-3 tau. Cell extracts (not normalized to protein content) of the parental MCF-7 cells (M) and of clones 1–5 isolated after transfection with Flag-tagged 14-3-3 tau were separated by 12% SDS-PAGE. Immunoblots thereof were performed using anti-Flag to identify the product from the transfected construct and anti–14-3-3 tau to detect the endogenously expressed protein. The two bands could easily be distinguished, since the Flag-tagged protein migrated slightly slower than the endogenous protein. Clones 2 and 5 (circled numbers), exhibiting the strongest band with anti-Flag relative to the presence of the endogenous protein were selected for further studies. (B) Parental MCF-7 cells and clones 2 and 5 were cultured for 24 h in complete medium on a fibronectin versus a tenascin-C substratum and photographed. The parental cells showed an altered morphology induced by the tenascin-C substratum, whereas no difference was visible in the 14-3-3–overexpressing clones 2 and 5. Bar, 20 μm.
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fig3: Isolation of MCF-7 clones overexpressing 14-3-3 tau. Cell extracts (not normalized to protein content) of the parental MCF-7 cells (M) and of clones 1–5 isolated after transfection with Flag-tagged 14-3-3 tau were separated by 12% SDS-PAGE. Immunoblots thereof were performed using anti-Flag to identify the product from the transfected construct and anti–14-3-3 tau to detect the endogenously expressed protein. The two bands could easily be distinguished, since the Flag-tagged protein migrated slightly slower than the endogenous protein. Clones 2 and 5 (circled numbers), exhibiting the strongest band with anti-Flag relative to the presence of the endogenous protein were selected for further studies. (B) Parental MCF-7 cells and clones 2 and 5 were cultured for 24 h in complete medium on a fibronectin versus a tenascin-C substratum and photographed. The parental cells showed an altered morphology induced by the tenascin-C substratum, whereas no difference was visible in the 14-3-3–overexpressing clones 2 and 5. Bar, 20 μm.

Mentions: From the literature we know that 14-3-3 proteins are implicated in the regulation of cell growth and oncogenic transformation (Takihara et al., 2000) and that they exhibit antiapoptotic activity (Xing et al., 2000; Masters and Fu, 2001). Therefore, we decided to test for an effect of elevated 14-3-3 tau levels in MCF-7 cells on their adhesion and growth behavior when cultured on tenascin-C versus fibronectin substrates. We transfected MCF-7 cells with a construct encoding 14-3-3 tau containing an NH2-terminal Flag tag. Cell clones were isolated and tested on immunoblots for expression of the transfected 14-3-3 tau in comparison to the endogenous 14-3-3 tau protein as shown in Fig. 3 A. Clones 2 and 5 exhibiting the highest amount of the transfected 14-3-3 relative to the endogenous protein were selected for further experiments. When they were grown on tenascin-C–coated plates they exhibited a cobble stone–like morphology as on fibronectin, whereas the parental cells showed an irregular morphology on tenascin-C (Fig. 3 B). This was particularly well visible in phalloidin-stained cells (Fig. 4 A).


Tenascin-C signaling through induction of 14-3-3 tau.

Martin D, Brown-Luedi M, Chiquet-Ehrismann R - J. Cell Biol. (2003)

Isolation of MCF-7 clones overexpressing 14-3-3 tau. Cell extracts (not normalized to protein content) of the parental MCF-7 cells (M) and of clones 1–5 isolated after transfection with Flag-tagged 14-3-3 tau were separated by 12% SDS-PAGE. Immunoblots thereof were performed using anti-Flag to identify the product from the transfected construct and anti–14-3-3 tau to detect the endogenously expressed protein. The two bands could easily be distinguished, since the Flag-tagged protein migrated slightly slower than the endogenous protein. Clones 2 and 5 (circled numbers), exhibiting the strongest band with anti-Flag relative to the presence of the endogenous protein were selected for further studies. (B) Parental MCF-7 cells and clones 2 and 5 were cultured for 24 h in complete medium on a fibronectin versus a tenascin-C substratum and photographed. The parental cells showed an altered morphology induced by the tenascin-C substratum, whereas no difference was visible in the 14-3-3–overexpressing clones 2 and 5. Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172641&req=5

fig3: Isolation of MCF-7 clones overexpressing 14-3-3 tau. Cell extracts (not normalized to protein content) of the parental MCF-7 cells (M) and of clones 1–5 isolated after transfection with Flag-tagged 14-3-3 tau were separated by 12% SDS-PAGE. Immunoblots thereof were performed using anti-Flag to identify the product from the transfected construct and anti–14-3-3 tau to detect the endogenously expressed protein. The two bands could easily be distinguished, since the Flag-tagged protein migrated slightly slower than the endogenous protein. Clones 2 and 5 (circled numbers), exhibiting the strongest band with anti-Flag relative to the presence of the endogenous protein were selected for further studies. (B) Parental MCF-7 cells and clones 2 and 5 were cultured for 24 h in complete medium on a fibronectin versus a tenascin-C substratum and photographed. The parental cells showed an altered morphology induced by the tenascin-C substratum, whereas no difference was visible in the 14-3-3–overexpressing clones 2 and 5. Bar, 20 μm.
Mentions: From the literature we know that 14-3-3 proteins are implicated in the regulation of cell growth and oncogenic transformation (Takihara et al., 2000) and that they exhibit antiapoptotic activity (Xing et al., 2000; Masters and Fu, 2001). Therefore, we decided to test for an effect of elevated 14-3-3 tau levels in MCF-7 cells on their adhesion and growth behavior when cultured on tenascin-C versus fibronectin substrates. We transfected MCF-7 cells with a construct encoding 14-3-3 tau containing an NH2-terminal Flag tag. Cell clones were isolated and tested on immunoblots for expression of the transfected 14-3-3 tau in comparison to the endogenous 14-3-3 tau protein as shown in Fig. 3 A. Clones 2 and 5 exhibiting the highest amount of the transfected 14-3-3 relative to the endogenous protein were selected for further experiments. When they were grown on tenascin-C–coated plates they exhibited a cobble stone–like morphology as on fibronectin, whereas the parental cells showed an irregular morphology on tenascin-C (Fig. 3 B). This was particularly well visible in phalloidin-stained cells (Fig. 4 A).

Bottom Line: We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C.Furthermore, the growth rate on tenascin-C was increased compared with the parental cells.Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Forschungsstiftung, CH-4002 Basel, Switzerland.

ABSTRACT
We searched by a cDNA subtraction screen for differentially expressed transcripts in MCF-7 mammary carcinoma cells grown on tenascin-C versus fibronectin. On tenascin-C, cells had irregular shapes with many processes, whereas on fibronectin they were flat with a cobble stone-like appearance. We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C. To investigate the consequences of an increased level of this phospho-serine/threonine-binding adaptor protein, we transfected MCF-7 cells with a construct encoding full-length 14-3-3 tau protein and selected clones with the highest expression levels. The morphology of these cells on tenascin-C was flat, resembling that of cells on fibronectin. This was reflected by a similar pattern of F-actin staining on either substratum. Furthermore, the growth rate on tenascin-C was increased compared with the parental cells. After transient transfection of HT1080 fibrosarcoma and T98G glioblastoma cells with 14-3-3 tau, only the 14-3-3 tau-expressing cells were able to adhere and survive on tenascin-C, whereas all cells adhered well on fibronectin. Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth.

Show MeSH
Related in: MedlinePlus