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Genes to Cells (2004) 9, 661-669. doi:10.1111/j.1356-9597.2004.00754.x
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Transcriptional responses to epigallocatechin-3 gallate in HT 29 colon carcinoma spheroids

Patricia McLoughlin1, Monic Roengvoraphoj2, Cornelia Gissel2, Jürgen Hescheler2, Ulrich Certa1 and Agapios Sachinidis2,*

1 Roche Centre for Medical Genomics, F. Hoffmann-La Roche, Ltd, Postfach, 4070 Basel, Switzerland
2 Centre of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, Germany


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
Catechins have been reported to possess anti-cancer activity in vitro and in vivo. To identify target genes that may be involved in the anti-tumorigenic effect of catechins, gene expression profiles in adherent human HT 29 colon carcinoma cells, in HT 29 spheroids and in epigallocatechin-3 gallate (EGCG)-treated HT 29 cells have been analysed by high-density oligonucleotide microarrays. Treatment of HT 29 cells with EGCG (2.5–50 µM) resulted in a dose-dependent inhibition of spheroid formation of HT 29 cells. Forty transcripts were induced at least twofold in 3-day-old spheroids relative to normal adherent cells using three replicates. Oncogenes like c-fos and c-jun are significantly up-regulated in spheroids. We identified several signal transduction and proliferation genes which are down-regulated in response to EGCG treatment. Increase in the mRNA expression profile of c-Fos correlated well with protein levels in HT 29 spheroids whereas EGCG did not affect protein formation. In agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids but protein formation was totally blocked in EGCG-treated cells. Interestingly, no change in expression of cytotoxic or apoptotic related genes has been observed in EGCG-treated cells. Our findings suggest that EGCG may exert its anti-cancer activity through modulation of expression of a number of genes that are involved in cell proliferation, cell-cell contacts and cell-matrix interactions.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
Catechins belong to the flavonoid family, which is a family of polyphenolic compounds found in foods of plant origin (Hollman & Katan 1999). Flavonoids are subdivided into flavonols, flavones, catechins, flavanones, anthocyanidins, and isoflavonoids (Hollman & Katan 1999). Catechins such as 2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol (catechin), epicatechin (EC), epigallocatechin-3 gallate (EGCG), epicatechin-3 gallate (ECG), catechin-3 gallate (CG), and epigallocatechin (EGC) are the main components in green tea derived from the unfermented leaves of Camellia sinensis, and approximately 30–42% of the dry weight of green tea is composed of catechins (Graham 1992). Catechins, and in particular EGCG, have been proposed to act as chemopreventive agents against cancer as demonstrated in different animal models and tumour cell lines (for review see Mukhtar & Ahmad 2000; Yang et al. 2000). In order to explain the beneficial effects of catechins it has been postulated that they act as scavengers of reactive oxygen species (ROS) thereby impeding carcinogenesis (Hursting et al. 1999; Salah et al. 1995; Yang et al. 2000). However, there is accumulating evidence that catechins can also act at various different levels inhibiting ROS generation (Hursting et al. 1999), inhibition of growth factor receptors (Ahn et al. 1999; Sachinidis et al. 2000, 2002; Weber et al. 2003), activation of nuclear transcription factors such as NF-{kappa}B, cyclin dependent kinases (cdk), and inhibition of enzyme activities participating in malignant proliferative diseases (Chung et al. 1999; Dong et al. 1997; Dong 2000). In general, the ability of tumour cells to grow in an anchorage-independent fashion, forming spheroids, is considered to be a classic predictor of in vivo tumorigenicity (Freedman & Shim 1974; Santini & Rainaldi 1999), and this process suggests an alteration in cellular processes like proliferation and adhesion. We have previously demonstrated that EGCG, the main compound of green tea catechins, inhibits spheroid formation of human A172 glioblastoma cells and of sis-transfected NIH 3T3 colony cells (Ahn et al. 1999; Sachinidis et al. 2000). To determine whether the chemopreventive activity of EGCG might be attributed to inhibition of spheroid formation we extended our studies to examine the effect of EGCG on spheroid formation of HT 29 colon carcinoma cells. DNA microarray technology allows for multiparallel analysis of thousands of transcripts, and was applied to identify target genes that may mediate the anti-tumorigenic effects of catechins. Thus, gene expression profiles of human HT 29 colon carcinoma cells grown in monolayer, in HT 29 spheroids and in EGCG-treated cells were analysed.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Supplementary material
 References
 
Effect of EGCG on spheroid formation of HT 29 cells

Treatment of HT 29 cells with EGCG (2.5-50 µM) resulted in a dose-dependent inhibition of spheroid formation of HT 29 cells (Fig. 1). Spheroid formation was almost completely inhibited by 20 µM EGCG (Fig. 1B). The diameter of single HT 29 cells was calculated to be 20 ± 2 µm while that of spheroids was 80 ± 4 µm (Mean ± SEM, n = 15). As shown in Fig. 1C, anchorage independent spheroid formation of HT 29 cells in bacterial petri dishes was also significantly reduced. However, spheroid formation under these conditions occurs at a much faster rate and spheroids are considerably larger (diameter: 163 ± 12 µm (Mean ± SEM, n = 21) compared to those formed in soft agar. Additionally, inhibition of spheroid formation under these conditions requires higher concentrations of EGCG: treatment of the cells with 100 µM EGCG resulted in a significant decrease of the diameter of spheroids, being reduced from 163 ± 12 µm to 44 ± 5 µm (Mean ± SEM, n = 21).



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  		Figure 1  Anchorage-independent growth of HT 29 cells in soft agar in the presence and absence of EGCG. (A) tissue culture dishes (35-mm) were underlaid with 1 mL modified Eagles medium (MEM) supplemented with 0.7% agar, 10% foetal calf serum (FCS) and EGCG. After trypsinization 5 x 104 HT 29 cells were suspended in 1.5 mL MEM supplemented with 0.35% agar, 10% FCS and 10–50 µM of each catechin compound and plated on the 0.7% agar underlay. Representative fields were photographed after 1 h and 10 days by phase-contrast light microscopy. Calibration bar represents 500 µm. (B) diagram showing spheroid diameter after treatment of the cells with EGCG for three days (Mean ± SEM, n = 15, *P < 0.05 for 10-day-old ‘spheroids’ in presence of EGCG vs. 10-day old spheroids). (C) Anchorage-independent growth of HT 29 cells in bacteriological Petri dishes in the presence and absence of EGCG. After trypsinization 5 x 104 HT 29 cells were suspended in 3 mL 10% FCS in MEM and cultured in bacteriological Petri dishes in the presence and absence of 100 µM EGCG. Representative fields were photographed after 1 h and 3 days by phase-contrast light microscopy. Calibration bar represents 1000 µm.

 
Effect of EGCG on gene expression in single HT 29 cells, in HT 29 spheroids and in EGCG-treated cells

For microarray analysis, total RNA was isolated from triplicate cultures of adherent HT 29 cells (single cells), from HT 29 spheroids generated after 3 days in bacterial Petri dishes (3-day-old spheroids) and from HT 29 cells after 3 days in the presence of 100 µM EGCG (3-day old EGCG-spheroids) (Fig. 1C). For data analysis we selected those transcripts which gave significant change factors across the three replicates as assessed by a non-paired t-test with a P-value < 0.05. Figure 2 shows 40 transcripts, clustered according to their annotated function, which were at least twofold induced (change factor = 1, P < 0.05) in 3-day-old spheroids as compared to single cells, wherein known oncogenes like c-fos and c-jun are significantly up-regulated (see Supplementary material for raw data, Table S1). Some genes (including c-fos) are represented by several probe sets on the array and where these genes were regulated a consistent response is observed for all probe pairs (Fig. 2). A comparison of the transcriptional responses in 3-day-old spheroids and spheroids formed in the presence of EGCG selects a second set of downmodulated genes (Fig. 3), with distinct functions such as proliferation, signal transduction and cell adhesion, all of which processes are modified during the course of tumour development (see Supplementary material for raw data, Table S2).



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  		Figure 2  HT 29 cells were grown under standard conditions or allowed to form spheroids and thereafter total RNA was extracted for analysis. Expression levels for each gene were calculated as normalized average difference of fluorescence intensity of perfect match oligonucleotide probe sets relative to the hybridization signal of mismatch probe sets. Transcripts demonstrating a reproducible change factor of at least 1 were included in the analysis. With these criteria, 40 transcripts were shown to be up-regulated by more than twofold (change factor = 1) in the spheroids, at a probability < 0.05 (non-paired t-test). Genes were clustered according to their annotated function in public databases or as described in the literature. GenBank identification numbers are shown for each gene as are the observed change factors, represented by bars (change factor > 4 shown in red, change factor between 2 and 4 shown in yellow and change factor < 2 shown in green).

 


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  		Figure 3  HT 29 spheroids treated with 100 µM EGCG were compared to untreated HT 29 spheroids by microarray. Using the same criteria as before, 29 transcripts were found to be down-regulated by more than twofold (change factor =–1) in the EGCG-treated sample at a probability < 0.05 (non-paired t-test). Genes were clustered according to their annotated function in public databases or as described in the literature. GenBank identification numbers are shown for each gene as are the observed change factors, represented by bars (change factor > –4 shown in red, change factor between –2 and –4 shown in yellow and change factor < –2 shown in green).

 
Examination of the differentially expressed genes show a number of transcripts common to both sets; that is EGCG treatment reduces the level of up-regulation of a number of genes which are up-regulated in the spheroid state as compared to single cells. These include transcripts involved in cellular adhesion like annexin A13 and galectin-2 (Fig. 3) which supports the result shown in Fig. 1C whereby EGCG treatment produces spheroids of a smaller size (see Figs 2 and 3). EGCG did have an effect on other transcripts that were induced in spheroids but this level of change was below the cut-off used for filtering.

Effect of EGCG on IQGAP2 and c-Fos protein levels in EGCG-treated HT 29 cells

To determine if downmodulation of gene expression by EGCG is also reflected at the translational level, Western blotting analysis of selected genes was performed. Thus a gene associated with cellular proliferation (c-fos) was chosen for further analysis as was a gene involved in signal transduction (IQGAP2). As demonstrated in Fig. 4, in agreement with the DNA chip data, c-Fos protein level was increased in HT 29 spheroids and this level was not altered by treatment with EGCG under the conditions tested. Similarly in agreement with the DNA chip data, IQGAP2 protein was not increased in spheroids while treatment with 50 µM EGCG reduced the level of protein observed; increasing the dose of EGCG to 100 µM completely inhibited protein production. Although only two genes have been tested, the results obtained from the microarray analysis might correspond closely to the observed level of protein after EGCG treatment.



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  		Figure 4  Effect of EGCG on IQGAP2 and c-Fos protein levels in HT-29 single cells, spheroids and EGCG-spheroids. After trypsinization 5 x 104 HT 29 cells were suspended in 3 mL 10%FCS/MEM and cultured in bacteriological petri dishes in the presence and absence of EGCG. After 3 days protein analysis was performed by Western blotting.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Supplementary material
 References
 
In this work we have analysed the transcriptional response of a human colon carcinoma cell line, HT 29, which can form cellular aggregates known as spheroids in vitro, to treatment with a catechin found in abundance in green tea, EGCG, which is purported to be active in tumour prevention, acting through various intracellular mechanisms, as described by several experimental and epidemiological studies (Mukhtar & Ahmad 2000; Yang et al. 2000). In this context it is well established that in contrast to tumour cells grown in monolayer, spheroid formation is a suitable experimental model representing in vivo tumours (Freedman & Shin 1974; Santini & Rainaldi 1999). In the present study, we compared gene expression patterns in single HT 29 colon carcinoma cells derived from monolayer culture, in 3-day-old spheroids and in 3-day-old EGCG-treated spheroids in an effort to further delineate the chemopreventive mechanism of EGCG in a human tumour cell line. Expression studies were performed using commercial oligonucleotide microarrays with probe sets for approximately 12 000 genes from the human Unigene database (build 95). We used three biological replicates per condition so as to control for sample and chip variability using statistical analysis tools included in the RACE-A data analysis software; the importance of using multiple replicate samples is evident since this reduces the necessity to analyse each transcript by RT-PCR which is inherently prohibitive due to the number of differentially regulated genes found in a single microarray experiment.

Regarding the pathology of human cancer, six characteristic properties can be attributed to the cancer cell: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, ability to evade apoptosis, limitless replicative potential, ability to sustain angiogenesis, and ability to invade tissues and metastasize (Hanahan & Weinberg 2000). The sequence in which these capabilities are acquired vary within and between tumours. Thus in this study where we have exploited HT 29 cells as a model for an early developmental stage of colon cancer, spheroid formation is accompanied by growth promotion given that there is an increase in cell number; additionally spheroids are not subject to the same restrictions as normal somatic cells in terms of cell adhesion and contact inhibition. It is well known that catechins inactivate several proteins that are associated with proliferative diseases probably via physicochemical interactions (Ahn et al. 1999; Chen et al. 1998; Kitano et al. 1997; Nam et al. 2001; Okabe et al. 1999; Sachinidis et al. 2000, 2002; Sazuka et al. 1996, 1998; Wang et al. 1988). More recently, we showed that plasma membrane incorporated EGCG or soluble EGCG directly interacts with growth factors such as PDGF, thereby preventing specific receptor binding (Weber et al. 2003). However, less is known about the transcriptional changes resulting as a consequence of such interactions.

Based on our gene expression analysis, a comparison between normal single cells and cells that underwent spheroid formation, there are a clear number of candidates suggestive of a tumorigenic pathway in effect (Fig. 2). Among the 40 induced transcripts, several belong to classes of genes that are typically associated with the process of tumorigenesis, such as proliferation and differentiation, signal transduction, cellular adhesion and metabolic processes (Fig. 2). Both c-fos and c-jun are known to function in proliferation and differentiation processes and their involvement in the epidemiology of human cancers is well-documented (Jochum et al. 2001; Shaulian & Karin 2001), and in this work both molecules were found to be up-regulated in HT 29 spheroids (Figs 2 and 4) while being refractory to EGCG treatment at both the mRNA and protein levels (Figs 3 and 4). Several transcription factors have also demonstrated significant changes over the two conditions in support of the theory of subverted cellular function in tumorigenesis. A number of hypothetical proteins and ESTs were also induced in spheroids and these are involved in oncogenesis, as in the case of the gene product for kiaa0429 (AB007889 [GenBank] ), which corresponds to a gene called metastasis suppressor 1, which is involved in regulating the actin cytoskeleton (Mattila et al. 2003).

Examining the difference between spheroids treated with EGCG and those which were not treated reveals another set of genes, expression of which is repressed by EGCG (Fig. 3), with similar ontology classifications as for the set of genes induced in spheroids, including proliferation and differentiation, signalling, cell adhesion, and metabolism. For example expression of the signalling molecule IQGAP2 is affected by catechin treatment (Fig. 3) as is the production of protein from the gene (Fig. 4). IQGAP1 and IQGAP2 are RasGTPase-activating-protein (RasGAP)-related proteins that interact with calmodulin, F-Actin and with the small RhoGTPases, Cdc42 and Rac1 and Rho, thereby regulating cadherin-mediated cell-cell adhesion (Brill et al. 1996; Li et al. 2000; Zhang et al. 1997).

In terms of the global effect of EGCG on gene expression, there are two different modes visible. Certain transcripts that are induced in spheroids as compared to single HT 29 cells, are relatively unaffected by EGCG treatment, at the concentration of 100 µM used here. Conversely, EGCG exposure has an appreciable effect on other genes that are induced in spheroids, resulting in down-regulation of their expression to levels close to that found in single HT 29 cells. Genes that are most responsive to EGCG include molecules that are involved in adhesion, like galectin-2 and annexin A13, which is in support of the theory that EGCG functions to reduce the loss of contact inhibition that allows spheroids to form. The observed effect of EGCG on spheroid formation in soft agar where spheroids are significantly smaller than those formed from untreated cells also indicates such an effect (Fig. 1C). Galectins modulate cell-cell and cell-matrix interactions and have a demonstrated role in colon cancer metastasis (Hittelet et al. 2003; Rabinovich et al. 2002). These findings suggest a mechanism whereby physicochemical interactions of catechins with certain cellular proteins results in a suppression of the expression of genes that are involved in the development of proliferative diseases. This concept is supported by several experimental studies (Weber et al. 2003).

Recently in a study of the effect of EGCG on a defined set of 250 genes associated with kinases and phosphatases that are involved in signalling in cell cycle regulation, apoptosis and metabolic pathways, a total of 25 genes were found to be modulated in an adherent prostate carcinoma cell line (16 induced and 9 repressed); these 25 genes were associated with different pathways implying multiple combinatorial downstream effects of EGCG (Wang & Mukhtar 2002).

In general, inhibition of genes by EGCG on a transcriptional level might reflect inhibition at the protein level as demonstrated for IQGAP2 and c-Fos (Fig. 4). However, although only two genes have been tested, we may speculate that the results obtained from the microarray analysis might correspond closely to the level of proteins. Work performed to date would imply that there is not a clear mutually dependent relationship between transcript and protein levels, due to post-translational modifications, post-transcriptional control of gene expression as well as changes in expression levels, protein synthesis and degradation rates (Anderson & Seilhamer 1997; Gygi et al. 1999; Lee 2001).

Overall, we have shown that exposure of HT 29 cells to EGCG has a direct effect on the size and quantity of spheroids produced in suspension culture (Fig. 1C), and that such an outcome may be directly related to the altered transcriptional response observed. Rather than focusing on one specific pathway within the cell it would appear that the effect of EGCG is rather more heterogeneous in nature, producing pleiotropic effects in diverse pathways such as proliferation, differentiation, adhesion and metabolism. Remarkably, no expression of cytotoxic or apoptotic related genes has been observed in EGCG-treated cells suggesting a non-toxic and non-apoptotic inhibitory intracellular mechanism.

We have presented a novel experimental strategy exploiting an in vitro model of tumorigenesis representative of in vivo tumours that not only provides insight into the molecular mechanisms of different chemopreventive agents but also allows discovery of new target genes for development of drugs for cancer therapy and prophylaxis.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Supplementary material
 References
 
Materials

Modified Eagles medium (MEM), and McCoy's 5 A medium with L-glutamine were obtained from Gibco BRL, Eggenstein, Germany. (-)-EGCG was obtained from Sigma Chemical Company, Deisenhofen, Germany. Monoclonal mouse antibodies to IQGAP2 and rabbit polyclonal antibodies to c-FOS (sc-52) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Human Genome GeneChip® U95Av2 arrays were obtained from Affymetrix (Santa Clara, California, USA). Plastic tissue culture dishes were obtained from Falcon (BD Biosciences, Heidelberg, Germany) and bacteriological culture dishes were obtained from Greiner (Kremsmünster, Austria).

Cell culture

Human HT-29 colon carcinoma cells were provided by the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) cell bank (established from the primary tumour of a 44-year old Caucasian woman with colon carcinoma in 1964). Cells were cultured in McCoy's 5A medium supplemented with 10% foetal calf serum (FCS).

Soft agar assay

The soft agar assay was performed as previously described (Sachinidis et al. 2000). Briefly, 35-mm tissue culture dishes were underlaid with 1 mL MEM supplemented with 0.7% agar, 10% FCS and varying concentrations of EGCG. After trypsinization, 5 x 104 single HT 29 cells were suspended in 1.5 mL MEM supplemented with 0.35% agar, 10% FCS and either 20 or 50 µM EGCG and plated on the 0.7% agar underlay. Cells were fed once per week with 2 mL MEM supplemented with 10% FCS (10%FCS/MEM) and either 50 or 100 µM of EGCG. Cells were imaged by phase-contrast light microscopy after 1 h and 10 days. Data are expressed as the arithmetic mean ± SEM. Statistical analysis was performed using the Mann-Whitney U-test and by a non-paired t-test. P < 0.05 was considered statistically significant.

Bacteriological petri dish assay

After trypsinization single cells were cultured for different periods of time in 10% FCS/MEM on petri dishes (Greiner, 664102, 10 cm). After 1 h and 3 days cells were photographed by phase-contrast light microscopy. Data are expressed as the arithmetic mean ± SEM. Statistical analysis was performed using the Mann-Whitney U-test and by a non-paired t-test. P < 0.05 was considered statistically significant. This assay has been used in order to isolate total RNA from the spheroids using RNA-Bee from ams biotechnology, Europe.

Target preparation and microarray hybridization

Triplicate samples of 10 µg total RNA, prepared from single HT 29 cells, from spheroids that were formed after three days (3-day-old spheroids) and from small spheroids that were formed after 3 days in the presence of 100 µM EGCG (EGCG spheroids) were used to generate biotinylated cRNA following the Affymetrix standard protocol. 15 µg cRNA was hybridized for 16 h at 45 °C to Human Genome U95Av2 GeneChip® oligonucleotide arrays, which carry probes representing ~12 000 sequences previously characterized in terms of disease association or function, from the Human Unigene database (Build 95). Following hybridization arrays were washed and stained with streptavidin-phycoerythrin and thereafter scanned using an Affymetrix scanner according to the manufacturer's protocols.

Data analysis

Raw data was collected using Microarray Suite software, version 5.0 from Affymetrix®. Differential expression analysis was carried out using RACE-A (Roche Affymetrix Chip Experiment–Analysis) as previously described (Certa et al. 2003). Expression data were calculated using mean-based normalization, non-paired t-test calculation and Nalimov test for outlier removal at 95% confidence interval. Changes in mRNA expression levels are referred to as ‘change factors’ which is defined as ‘(cond2/cond1) –1’ in the case of an increase, ‘(cond1/cond2) +1’ for a decrease and ‘0’ if no change is exemplified. A change factor of at least ±1 and a positive non-paired t-test at the 95% confidence interval were used to identify and restrict the number of differentially expressed genes. Hierarchical clustering was performed using a Euclidean distance algorithm with centred weighting.

Electrophoresis, and Western blot analysis

Cells or spheroids were lysed with 1 mL RIPA buffer (50 mM NaCl, 20 mM Tris-HCl, 50 mM NaF, 10 mM EDTA, 20 mM Na4P2O7 10H2O, 1% Triton X-100, pH 7.4) containing 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 µg/mL leupeptin, 10 µg/mL anti-pain, and 0.023 TIU/mL aprotinin. Proteins were separated on a 7.5% SDS polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulose membranes for Western blotting. Enhanced chemiluminescence Western blotting analysis using primary monoclonal anti-IQGAP2 (1 : 200) and anti-Fos (1 : 200) was carried out as previously described (Sachinidis et al. 2002). Equal amounts of protein (15 µg per lane) were analysed after stripping and reblotting of the membranes with monoclonal anti-ß-tubulin (1 : 500) and a secondary horseradish peroxidase-labelled anti-mouse IgG (1 : 5000).


   Supplementary material
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
References
 
The following material is available from: http://www.blackwellpublishing.com/products/suppmat/GTC/GTC754/GTC754sm.htm

Table S1  Mean values (n= 3) of the normalized raw data after filtering for significant changes are shown for the comparison of Control HT 29 cells vs. HT 29 spheroids. Affymetrix IDs for probes sets are shown as are descriptions of each transcript. Change factors for the conditions are shown together with the calculated P-value. For more details see legend to Fig. 2. Table S2  Mean values (n= 3) of the normalized raw data after filtering for significant changes are shown for the comparison of HT 29 spheroids virus HT 29 spheroids grown in the presence of EGCG. Affymetrix IDs for probes sets are shown as are descriptions of each transcript. Change factors for the conditions are shown together with the calculated P-value.


   Acknowledgements
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Sa 568/5-3). We thank Senait Mesghenna for excellent technical assistance.


   Footnotes
 
Communicated by: Carl-Henrik Heldin

* Correspondence: E-mail: a.sachinidis{at}uni-koeln.de


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
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Received: 27 February 2004
Accepted: 30 April 2004




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