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Address correspondence to Ruth Chiquet-Ehrismann, Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Tel.: 41-61-697-24-94. Fax: 41-61-697-39-76. E-mail: chiquet{at}fmi.ch
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Abstract |
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Key Words: extracellular matrix; cancer; growth; cell adhesion; adapter protein
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Introduction |
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Therefore, it is of great interest to find out how tumor cells react to the presence of tenascin-C. In most of the published studies to date, effects of tenascin-C on cell adhesion, spreading, migration, and growth have been reported (for review see Orend and Chiquet-Ehrismann, 2000). However, the molecular mechanisms mediating these responses to tenascin-C remain largely unknown. From previous studies we know that a tenascin-C substratum supports the growth of rat primary mammary carcinoma cells more than fibronectin (Chiquet-Ehrismann et al., 1986). Furthermore, we have shown that MCF-7 human mammary carcinoma cells induce tenascin-C expression in cocultured fibroblasts surrounding the tumor cell nests (Chiquet-Ehrismann et al., 1989).
We therefore decided to screen for transcripts that are differentially expressed in MCF-7 cells grown on a tenascin-C versus a fibronectin substratum. We now report on our discovery that a tenascin-C substratum induces the expression of the phospho-serine/threoninebinding adaptor protein 14-3-3 tau in MCF-7 mammary carcinoma cells and describe its influence on cell adhesion and cell growth.
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Results and discussion |
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Possibly, this effect is related to the reported interaction of 14-3-3 tau with rap1/B-Raf (Berruti, 2000), since rap1 signaling has been implicated in the regulation of integrin-mediated cell adhesion (Bos et al., 2001). Another possibility could be an interaction of 14-3-3 with integrins themselves as has been reported for 14-3-3 beta and integrin ß1 (Han et al., 2001) or for 14-3-3 zeta with the integrin-associated docking protein p130 (Cas). Finally, an interaction of 14-3-3 zeta with the actin depolymerizing factor cofilin and its regulatory kinase LIM kinase 2 has been observed (Birkenfeld et al., 2003), and 14-3-3 zeta seems to stabilize phosphocofilin levels (Gohla and Bokoch, 2002), pointing to a further pathway for 14-3-3 proteins by affecting cell morphology through direct action on the actin cytoskeleton.
We next tested the effect of the overexpression of 14-3-3 tau on cell growth. We plated equal numbers of cells on fibronectin versus tenascin-C substrates in complete medium and cultured the parental versus the 14-3-3transfected clones 2 and 5 for 3 d. After 3 d in culture, the MCF-7 cells reached higher densities on fibronectin than on tenascin-C, whereas no distinction could be made for clone 2 or 5, respectively (unpublished data). The lower cell number of the parental MCF-7 cells on a tenascin-C substratum coincided with a reduced level of DNA replication on a tenascin-C substrate in comparison to fibronectin as measured by 3H-thymidine incorporation (Fig. 5). The 3H-thymidine incorporation by the 14-3-3 tauoverexpressing clones 2 and 5 was recovered on tenascin-C and reached values almost as high as on fibronectin (Fig. 5). A possible mechanism is that cell cycle progression is stimulated by an interaction of 14-3-3 with phosphorylated p27Kip1 and by retaining this cell cycle inhibitor in the cytoplasm (Fujita et al., 2002). Alternatively, stimulation of cell growth by 14-3-3 could be indirect by its known inhibitory action on apoptosis (Xing et al., 2000; Masters and Fu, 2001). To test whether cells on tenascin-C were protected from apoptosis by overexpression of 14-3-3 tau, we plated the parental MCF-7 cells and clones 2 and 5 on tenascin-C and fibronectin-coated wells, respectively, and analyzed the number of apoptotic cells under both conditions. However, not even the parental MCF-7 cells showed increased apoptosis on tenascin-C compared with the cells plated on fibronectin. Reduction of the serum level in the medium to 1% lead to a concomitant increase in apoptotic cells on both substrates. Therefore, the hypothesis that 14-3-3 tau could protect MCF-7 cells from apoptosis cannot be the reason for the observed increase in growth.
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Materials and methods |
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Cell growth was analyzed by measuring the incorporation of 3H-thymidine. Equal numbers of cells were plated on tenascin-C versus fibronectin in complete medium. After 22 h, the medium was removed, the cell layers were washed with medium without FCS, and medium containing 0.1% FCS was added. 22 h later, the cells were labeled with 3H-thymidine for 6 h and harvested and analyzed as described previously (Huang et al., 2001). Apoptosis was analyzed by TUNEL staining using the In Situ Cell Death Detection kit (Roche Diagnostics AG) according to the procedure of the manufacturer.
For immunofluorescence and F-actin staining, cells were fixed with 4% PFA in PBS for 30 min and permeabilized with 0.1% Trition X-100 in PBS for 5 min. Then, they were either incubated for 1 h with 0.05 µM TRITC-phalloidin (Sigma-Aldrich) or with anti-Flag (M2; Stratagene) at a 1:500 dilution, or with both simultaneously, all in PBS containing 3% goat serum for 1 h. Secondary goat antimouse Alexa488-conjugated antibodies (Molecular Probes) diluted 1:1,000 in PBS/3% goat serum were applied for detection of the primary antibodies. Cells were washed in PBS after each incubation. Finally, the specimens were mounted in Mowiol (Calbiochem) and examined and photographed using a Axiophot microscope (Carl Zeiss MicroImaging, Inc.) connected to a 3CCD camera (Sony).
Subtractive cDNA cloning
An Oligotex Direct mRNA kit (QIAGEN) was used to isolate mRNA from two 10-cm plates of MCF-7 cells grown on either fibronectin or tenascin-C substrata for 24 h in complete medium. These mRNA samples were used for RT-PCR reactions and subtractive hybridization using the Advantage cDNA PCR kit (CLONETECH Laboratories, Inc.) and the PCR-Select cDNA Subtraction kit (CLONETECH Laboratories, Inc.) according to the manuals supplied. The PCR products were cloned into pKS and analyzed by sequencing. Primers were derived from the insert sequences to verify a differential expression of the respective transcripts in the two cDNA batches by semiquantitative PCR reactions using Taq polymerase (Roche Diagnostics AG). To amplify the 14-3-3 tau, we used the following primer pair: 5'-AGTTGCGTGTGGTGATGATCG-3' and 5'-GTATGAGTCTTCATTCAGTG-3'. Samples were taken from each reaction after completion of 15, 20, 25, and 30 cycles and analyzed on agarose gels after ethidium bromide staining. This allowed us to verify the linear range of amplification that we used for comparison.
Transfection of 14-3-3Flag and Western blots
A full-length 14-3-3 tau construct containing an NH2-terminal Flag tag was engineered by RT-PCR from mRNA of MCF-7 cells using the Advantage cDNA PCR kit (CLONETECH Laboratories, Inc.). A first set of 14-3-3-specific primers was used (5'-GCGGCCGCGGAGACGTGAAC-3' and 5'-AAGGATGACACCCTGTATGG-3') followed by a second round of PCR with (5'-ATGCGGCCGCACCATGGACTACAAGGATGACGATGACAAGATGGAGAAGACTGAGCTG-3' and 5'-ATCTCGAGTTAGTTTTCAGCCCCTTCTGC-3') to add the Flag tag and the appropriate restriction sites NotI and XhoI at respective ends needed for subsequent cloning into the expression vector pcDNA3 (Invitrogen). The resulting construct was verified by sequencing and was called 14-3-3Flag. It contained the complete coding sequence of human 14-3-3 tau available from GenBank/EMBL/DDBJ under accession no. X56468 with an NH2-terminal extension of a Flag tag. MCF-7 cells were transfected with 14-3-3Flag using the transfection reagent fugene (Roche Diagnostics AG), and clones were selected for by the addition of G418 (Life Technologies). The resistant cell clones were tested for the production of the 14-3-3Flag protein on Western blots of cell extracts using anti-Flag (M2; Stratagene), a peroxidase-labeled antimouse IgG (Cappel/ICN), and the ECL reagent (Amersham Biosciences) for chemiluminescent detection of the reactive bands. Endogenous synthesis of 14-3-3 tau was detected on the same blot using anti14-3-3 theta/tau (Research Diagnostics Inc.). The Flag-tagged 14-3-3 migrates slightly slower than the endogenous protein due to the presence of the tag.
HT1080 and T98G cells were each plated on fibronectin and tenascin-Ccoated plates. 1 d later, they were transiently transfected with the 14-3-3Flag construct using the transfection reagent fugene. On the next day, cells were fixed and stained with anti-Flag and phalloidin as described above for the MCF-7 cells.
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Acknowledgments |
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Submitted: 26 June 2002
Revised: 10 December 2002
Accepted: 10 December 2002
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References |
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