Molecular Human Reproduction, Vol. 8, No. 9, 789-796,
September 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Relaxin enhances in-vitro invasiveness of breast cancer cell lines by up-regulation of matrix metalloproteases
1 Department of Hematology/Oncology, Georg-August-University, Robert-Koch Str. 40, D-37075 Göttingen and 2 Department of Reproductive Biology, German Primate Center, Göttingen, Germany
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Abstract |
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Until recently, relaxin (RLX) has been known predominantly for its effects on the reproductive system, where it induces remodelling of the extracellular matrix and up-regulation of matrix metalloproteases (MMPs). In solid cancers, tissue remodelling and MMP activation are essential for invasion and metastasis. We therefore investigated the effect of RLX on invasiveness and MMP expression of human breast cancer cell lines. Upon incubation with porcine RLX, the invasiveness of SK-BR3 cells was significantly increased. Similar effects could be achieved in MCF-7 cells, especially when RLX was combined with epidermal growth factor. Enhanced invasiveness was accompanied by up-regulation of MMP production and could be almost completely blocked by the MMP inhibitor FN 439. Zymography revealed increased secretion of MMP-2, -7 and -9, associated with up-regulated mRNA concentrations of MMP-2, -9, -13 and -14. mRNA expression levels of MMP-1, -3, -7, -8, -10, -11, -12 and of tissue inhibitors of metalloproteases-1, -2, -3 and -4 were either very low or not detectably influenced by RLX. Taken together, RLX enhances in-vitro invasiveness of breast cancer cell lines by induction of MMP expression. It remains to be clarified whether RLX might play a similar role in vivo and promote tumour progression.
breast cancer/invasion/matrix metalloproteases/relaxin
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Relaxin (RLX) is a peptide hormone which is structurally related to insulin and the insulin-like growth factors (IGFs). In humans, it is produced mainly by cells and tissues of the reproductive system (Borthwick et al., 1994
). Well known effects of RLX are: inhibition of uterine contractility; induction of cervical ripening; and lengthening of the interpubic ligament prior to parturition in rodents (Kroc et al., 1959). RLX also promotes growth and development of the mammary glands and nipples as a prerequisite for successful lactation (Zhao et al., 1999, 2000). However, the broad and overlapping distribution of the two recently identified RLX receptors in reproductive and non-reproductive tissues (Hsu et al., 2002) suggests a physiological role for RLX beyond these functions.
A considerable part of the effects of RLX is based on its ability to induce modifications of stromal tissue components. RLX leads to thinning of collagen fibres and imbibition of the amorphous ground substance in murine mammary glands (Bani and Bigazzi, 1984). In human scleroderma fibroblasts, RLX decreases the synthesis of collagen (Unemori et al., 1992). The expression and catalytic activities of collagenase-1 (MMP-1), gelatinases A and B (MMP-2 and -9) and stromelysin-1 (MMP-3), members of the matrix metalloprotease (MMP) family, are increased by RLX (Unemori et al., 1996; Qin et al., 1997; Kapila and Xie, 1998; Lenhart et al., 2001).
MMPs, a family of tissue degrading enzymes, play an important role in cancer progression. They facilitate invasion and metastasis through dissolution of the basement membrane and degradation of the extracellular matrix (ECM). Their physiological antagonists are the tissue inhibitors of metalloproteases (TIMPs). Up-regulation of MMP expression and activity has been found in many tumour tissues (Okada et al., 1995; Sier et al., 1996; Zeng et al., 1996; Hagemann et al., 2001; Schmalfeldt et al., 2001) and is associated with an invasive phenotype (Ramos DeSimone et al., 1999). Expression of MMP-1, -2, -3, -9 and -14 has also been demonstrated in breast cancer (Davies et al., 1993; Brummer et al., 1999; Garbett et al., 1999; Ishigaki et al., 1999). Clinically, high levels of MMP-2 and stromelysin-3 (MMP-11) are positively correlated with disease free and overall survival of breast cancer patients (Chenard et al., 1996; Talvensaari-Mattila et al., 1998).
On the basis of these findings, it is tempting to speculate about a putative role of RLX and its tissue remodelling ability not only for benign tissue development but also for invasiveness and metastatic potential of malignant cells. The assumption is particularly attractive for breast cancers, as RLX expression has been demonstrated in epithelial and myoepithelial cells of normal mammary ducts as well as in breast cancer cells (Tashima et al., 1994).
To evaluate the hypothesis, the influence of RLX on in-vitro invasiveness and expression of 11 MMPs and four TIMPs was investigated in two human breast cancer cell lines. Exposure to RLX was followed by significantly enhanced invasiveness of the tumour cells on the basis of increased MMP production, in particular MMP-2, -7, -9, -13 and -14.
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Materials and methods |
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Cells and reagents
Unless otherwise indicated, reagents were purchased from Sigma (Deisenhofen, Germany). The human breast cancer cell lines MCF-7 and SK-BR3 were obtained from the American Type Culture Collection (Rockville, USA). Cells were grown on RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany). Porcine RLX (courtesy of Professor O.D.Sherwood) was used at 100 ng/ml (final concentration) for all experiments, as this concentration had yielded optimal results in preparatory investigations with RLX concentrations ranging from 10 to 1000 ng/ml. Epidermal growth factor (EGF) and ß-estradiol (ßE2) were each used at 5 ng/ml (final concentration).
RNA extraction and quantitative RT–PCR
Total RNA was extracted with the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987). RT was performed from 2 µg of total RNA using oligo-dT primers and M-MLV reverse transcriptase (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Primers for MMP-3, -8, -9, -10, -13 and TIMP-4 were designed using the `Primer3 program’ (Whitehead Institute for Biomedical Research; Rozen and Skaletsky, 1998, http://www-genome.wi.mit.edu/genome_software/other/primer3.html) and synthesized by Biometra (Göttingen, Germany). The specific primers for human MMP-3 were: forward 5'-GCAGTTTGCTCAGCCTATCC, reverse 5'-GAGTGTCGG-AGTCCAGCTTC, amplifying a 214 bp fragment (55°C annealing temperature); for MMP-8: forward 5'-TCTGCAAAGTTATCCCAAGG, reverse 5'-TTGGTCCACTGAAGACATGG, amplifying a 251 bp fragment (53°C annealing temperature); for MMP-9: forward 5'-GGCGCTCATGTACCCTATGT, reverse 5'-TCAAAG-ACCGAGTCCAGCTT, amplifying a 468 bp fragment (58°C annealing temperature); for MMP-10: forward 5'-CAGAAGTTCCTTGGGTTGGA, reverse 5'-GGGGAGGTCCGTAGAGAGAC, amplifying a 598 bp fragment (58°C annealing temperature); for MMP-13: forward 5'-AGGAGATGCCCATTTTGATG, reverse 5'-GGAAGTTCTGGCCAAAATGA, amplifying a 405 bp fragment (60°C annealing temperature); for TIMP-4: forward 5'-GACCAGTGACCATCACATCC, reverse 5'-ATGACATTCGCCATTTCTCC, amplifying a 349 bp fragment (62°C annealing temperature).
Primers were synthesized according to previously described methods for MMP-1, -2, -11, -12 and -14 (Giambernardi et al., 1998), TIMP-1 (Wilhelm et al., 1989), TIMP-2 (Stetler-Stevenson et al., 1990) and TIMP-3 (Lampert et al., 1998).
Quantitative PCRs were performed on the Light Cycler PCR Analysis System (Roche). The amount of generated DNA was measured by fluorescence detection of the double strand-specific DNA-binding dye Sybr Green I (Roche) and quantified in relation to a known standard. Quantification standards were prepared by conventional PCR with the same primers. The resulting amplificate was purified, quantified by measurement of the absorbance at 260 nm and used in serial dilutions for the Light Cycler PCR. The detailed procedure for quantification has been described previously (Binder et al., 1999). The PCR reaction contained a standard PCR buffer, Sybr Green I (1:20 000), bovine serum albumin (0.05%) and 5 pmol of the specific sense and antisense primers. Forty cycles were performed with 0 s denaturation at 94°C, 5 s annealing at the respective optimal annealing temperature, 10 s extension at 72°C and 5 s fluorescence detection at 72–84°C. The melting curves were obtained at the end of amplification by cooling the sample at 20°C/s to 72°C and then increasing the temperature to 95°C at 0.1°C/s. Fluorescence was acquired every 0.1°C. To confirm comparable efficiency of RT in the samples, ß-actin was amplified and quantified according to the same protocol (primers from Clontech, Heidelberg, Germany). Experiments were performed at least in triplicate.
To rule out interference of endogenously produced RLX, expression of RLX mRNA was investigated in SK-BR3 and MCF-7 cells by conventional RT–PCR (Einspanier et al., 1997), yielding negative results for both cell lines (not shown).
Northern blot
Probes for MMP-7 were obtained by amplifying a 604 bp fragment by conventional PCR with the following primers: forward 5'-TTCAAATAGCCCAAAATGG, reverse 5'-ATGGAGTGGAGGAACAGTGC, 60°C annealing temperature. The purified product was cloned into the directional cloning vector pCR®II-TOPO using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands). After linearization of the vector with Xhol (Stratagene, Heidelberg, Germany), labelled antisense RNA probes were transcribed with Digoxigenin-11-UTP (Roche), SP6 RNA polymerase and the Stratagene RNA transcription kit according to the manufacturer’s instructions.
Total RNA (15 µg) was separated by 1% agarose gel electrophoresis and transferred to a nylon membrane (Hybond-N; Amersham, Braunschweig, Germany). Hybridization was performed under high stringency conditions (prehybridization at 68°C, hybridization at 74°C, washings at 74°C). Membrane-bound probes were detected with the Dig luminescent detection kit (Roche) as indicated by the manufacturer. Experiments were performed at least in triplicate.
Matrigel invasion assay
Invasion was measured by assessment of the breast cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber. The membrane consisted of polycarbonate (10 µm pore diameter; Nucleopore, Pleasanton, CA, USA) and was coated on ice with Matrigel (ECM gel) diluted 1:3 in serum free RPMI. RPMI (2x105 cells/ml) was seeded into the upper well of the chamber, while the lower well was filled up to the top with RPMI + 10% FCS as a chemoattractant. RLX was added at 100 ng/ml 2 h after plating the cells and renewed every 24 h during the incubation period. In the experiments, where MMP activity was inhibited, the non-selective MMP inhibitor FN 439 (MMP inhibitor I; Calbiochem-Novabiochem, Bad Soden, Germany) was added together with RLX at concentrations between 30 and 300 µmol/l. After 96 h, the content of the lower well with floating as well as adherent cells was removed and pelleted by centrifugation. The supernatant was used for zymography (see below). The cell pellet was resolved in 200 µl phosphate-buffered saline and spun down on 12 mm cover slips. After air drying, the cytospins were stained with 4',6’-diamino-2'-phenylindol (DAPI, 200 ng/ml). Cover slips were mounted in 20% mowiol 4–88 (Hoechst, Frankfurt, Germany) on glass slides. Intact nuclei were counted by UV-microscopy (Axioskop, Zeiss, Germany) and documented with a digital image editing system (Adobe PhotoShop 3.0; Adobe Systems, Tokyo, Japan). To assess the morphology of the migrated cells, control stainings with haematoxylin and eosin were performed and yielded the same results. As increased proliferation in the EGF- and ßE2-treated cells could cause a possible bias, control cells which were seeded and incubated under the same conditions as in the invasion chamber but without possibility to migrate were counted and the results corrected for variations in cell number. All experiments were performed at least in triplicate.
Zymography
Cells, incubated as indicated, as well as the respective supernatants were used for zymography. Cells were washed and lysed in 10 mmol/l Tris–HCl pH 7.4, 1% sodium dodecyl sulphate (SDS). Protein content in the cell lysate as well as in the supernatant was determined with the bicinchoninic acid method (BCA assay; Pierce, Rockford, USA). Samples of 20 µg were mixed with sample buffer (0.03% bromophenol blue, 0.4 mol/l Tris–HCl pH 7.4, 20% glycerol, 5% SDS) and separated on 10% SDS–polyacrylamide gels containing either gelatin (1 mg/ml) or ß-casein (0.5 mg/ml). After electrophoresis, gels were washed for 1 h in renaturation buffer (2.5% Triton X-100 in aqua bidest.) and subsequently incubated for 36 h at 37°C in 50 mmol/l Tris, 200 mmol/l NaCl and 5 mmol/l CaCl2, pH 7.5. Gels were stained with 0.05% Coomassie Brilliant Blue and destained with 30% methanol and 10% acetic acid; clear zones within the blue background indicated proteinolytic activity. MMPs were identified by size and comparison with the respective recombinant protein (Calbiochem-Novabiochem) used as an additional marker. Quantification was performed by densitometry (software WinCam 2.2; Cybertech, Berlin, Germany). To rule out interferences either with the FCS or the Matrigel, which could have contained traces of MMPs themselves, both substances were submitted to zymography. At the concentrations used in the experiments, there was no evidence for gelatinolytic or caseinolytic activity. All experiments were performed at least in triplicate, and representative experiments are shown.
Statistical analysis
Calculation of means and SD was performed using Excel software (Microsoft Office for Windows 2000). Graphs were created with Sigma Plot for Windows graphic system (Sigma 2000, Jandel Corporation, USA).
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In-vitro invasiveness
Incubation of the estrogen-independent cell line SK-BR3 with 100 ng/ml porcine RLX strongly enhanced tumour cell migration through the model basement membrane (Figure 1
, Table I). In the estrogen-dependent cell line MCF-7, RLX alone was also effective, but to a lesser extent. The number of migrated MCF-7 cells could be further increased by addition of ßE2 and/or EGF (5 ng/ml each). ßE2 as well as EGF alone did not significantly influence tumour cell migration. Addition of the non-selective MMP-inhibitor FN 439 reduced enhanced invasiveness in both cell lines to almost the control levels (Table II).
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Expression of MMP proteins
Measurement of gelatinolytic activity by zymography yielded no detectable amounts of MMP-2 and -9 in untreated MCF-7 cells or in the respective supernatant. Incubation with RLX led to a strong increase in gelatinolytic activity in the supernatant which could be further augmented by addition of EGF (Figure 2
and Table III). The predominant form of MMP-9 was the smaller fragment, representing the active enzyme, while MMP-2 was detectable mostly as the latent zymogen. EGF alone enhanced MMP-2 and -9 production only weakly. ßE2 alone was ineffective and did not lead to significant additive MMP induction when given in combination with RLX, RLX + EGF or EGF. There was no evidence for caseinolytic activity in both MCF-7 control cells and their supernatant. After 24 h of incubation with RLX, MMP-7 became detectable in the supernatant, while MMP-3 remained absent (Figure 3). In contrast to the gelatinases, MMP-7 secretion was also increased by incubation with EGF, ßE2 or the combination of both agents. However, the strongest effect was exerted by the combination of all three substances, resulting in detectable up-regulation even in the cellular extracts (Figure 4 and Table IV).
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Similar to MCF-7 cells, there was no detectable expression of MMP-2, -7 and -9 in SK-BR3 control cells and only weak expression of MMP-2 and -9 in the respective supernatant. Upon incubation with RLX, secretion of all three MMPs into the supernatant was significantly up-regulated (Figures 5 and 6
). The ratio between active and latent enzymes remained unchanged. Maximal up-regulation was already detectable after 24 h. Comparing the effect of RLX on MMP induction in supernatants from cells grown on plastic and on ECM-coated membranes, no difference could be detected between the two experimental conditions, either for gelatinolytic or for caseinolytic activity.
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Expression of MMP- and TIMP-mRNA
In accordance with the results obtained by zymography, measurement of mRNA expression by quantitative RT–PCR showed RLX-induced up-regulation of MMP-2 and -9 in both cell lines, as well as up-regulation of MMP-13 and -14. Up-regulation was consistently detectable, although there was a considerable variation in the absolute peak values between different experiments. The results are shown in Figure 7
. mRNA concentrations of MMP-1, -3, -8, -10, -11, -12 and TIMP-1, -2, -3 and -4 were either low (<100 fg) or undetectable and remained uninfluenced by RLX (data not shown). In contrast to the induction of MMP-7 protein, measured by casein zymography, Northern blot analysis demonstrated only low levels of MMP-7 mRNA in either cell line, showing no response to the addition of RLX alone or in combination with EGF and ßE2 (Figure 8).
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Discussion |
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The hypothesis that RLX might be involved in the up-regulation of MMPs in malignant cells is supported by the data presented. Both tumour cell lines showed only negligible expression of the investigated MMPs at the mRNA and protein levels under control conditions. Exposure to RLX was followed by strong up-regulation of MMP mRNAs, especially of MMP-2, -9, -13 and -14. At the protein level, MMP-2, -9 and -7 were induced by RLX and were mostly secreted into the supernatant.
Induction of MMP-2 and -9 in MCF-7 cells could be further enhanced by addition of EGF, which is not surprising as EGF is well known for its positive effect on MMP production in benign and malignant cells (O-Charoenrat et al., 1999; Nuttall and Kennedy, 2000). In SK-BR3 cells, maximal stimulation was achievable by RLX alone. The different responsiveness of the two cell lines to RLX may be attributed to their different biology. SK-BR3 is characterized by strong overexpression of c-erb-B2, a member of the EGF-receptor family. C-erb-B2 overexpressing tumours are often associated with high MMP production (Roetger et al., 1998). The exact pathways leading either from c-erb-B2 signalling or from RLX stimulation to MMP induction are still to be identified. However, it is tempting to speculate that some unknown cross-links may exist.
As to the role of ßE2 in this setting, the data are not conclusive. ßE2 did not influence MMP-2 and -9 protein expression when given alone. However, it led to a slight increase in MMP-7 protein concentrations, which were further augmented by the addition of either RLX or EGF. The strongest up-regulation was seen upon combination of all three substances with additional intracellular expression of MMP-7. This suggests at least an additive effect of ßE2 under these conditions and points to as yet unidentified cross-connections between the signalling pathways of RLX, EGF and ßE2. The data of other authors (Ignar-Trowbridge et al., 1992; Pillai et al., 1999) who proposed that the estrogen receptor might act as a mediator of EGF as well as of RLX signalling also lead into this direction.
Unexpectedly, up-regulation of MMP-7 protein was not accompanied by induction of the respective mRNA. While the enzyme was strongly induced by RLX after 24 h, mRNA levels were low at this time point, showing a further decline during the next 3 days. This obvious discrepancy may point to additional post-transcriptional and translational regulatory mechanisms, but may also be explained by a very early up-regulation during the first hours of exposure to RLX, which may have escaped detection.
Expression of MMP-9 has been described to be up-regulated in MDA-MB 231 breast cancer cells when grown on ECM (Balduyck et al., 2000), containing variable amounts of the protease plasminogen. Many tumour cells secrete urokinase-type plasminogen activator and are capable of activating their own MMP-9 via a protease cascade involving plasminogen and MMP-3 (Ramos DeSimone et al., 1999). In our cell lines, however, there was no evidence either for up-regulation of MMPs through contact with Matrigel or for expression of MMP-3.
The second part of our working hypothesis, that RLX might enhance invasiveness of malignant cells, is also corroborated by the results presented. RLX led to increased invasion of both SK-BR3 and MCF-7 cells through an artificial basement membrane. While migration rates in SK-BR3 cells were strongly enhanced by RLX alone, MCF-7 cells needed additional stimulation with EGF to achieve similar results.
Again, the impact of ßE2 is difficult to assess. The absolute numbers of migrated cells, as observed in our results, showed considerable variations within and between experiments, most probably due to slight differences in the thickness of the ECM layer on the hand-coated membranes. Some of these results may give the impression that the proportional effect of RLX on MCF-7 invasiveness remains the same, irrespective of any contributions of ßE2. However, numerous repetitions of the experiments showed maximal migration rates only under the combined influence of all three substances. As MMP-7 is also maximally up-regulated under these conditions, an additional effect of ßE2 seems at least imaginable.
Taken together, RLX leads to enhanced invasiveness as well as to induction of MMPs, suggesting a causal connection between these findings. Consistently, the enhanced invasiveness was almost completely blocked by the broad spectrum MMP-inhibitor FN 439. An essential role of MMP up-regulation for tumour cell invasion and metastasis has been demonstrated in various tumour types. MMP-2 and -9 are known to convey an invasive phenotype and lead to an unfavourable clinical outcome (Tokuraku et al., 1995; Walther et al., 1997). Concomitant up-regulation of MMP-14 and -2—as demonstrated in our cell lines—has been shown to increase activation of proMMP-2 and to be associated with lymph node and distant metastases (Ueno et al., 1997). MMP-7 up-regulation and activation is involved in the formation of liver metastases (Zeng et al., 2002).
A special role in the development of breast cancer has been attributed to MMP-3. It has been demonstrated not only to induce invasive behaviour in benign mammary epithelia, but also to achieve this effect by only a transitory presence during a short `vulnerable’ period of the whole process (Lochter et al., 1997). Also, RLX is present in significant concentrations only during certain stages of the reproductive cycle. As rat mammary stroma has been shown to dramatically modify breast cancer cell migration depending on its MMP content according to the endocrine status (Bemis and Schedin, 2000), an effect of RLX on MMP production, stroma remodelling and tumour cell dissemination seems imaginable. In our cell lines, MMP-3 was undetectable, which is not surprising, as the main source for MMP-3 in breast cancers is the stromal compartment.
To our knowledge, the presented findings are the first to suggest a potential role of RLX in tumour cell invasion. Bani and co-workers investigated the effect of RLX on the growth of MCF-7-derived mammary tumours in athymic nude mice (Bani et al., 1999). RLX did not foster tumour enlargement, but rather promoted differentiation. Their results may be considered contradictory to ours; however, the experimental conditions are not comparable. The implanted xenografts, whether exposed to RLX or not, were described as encapsulated by stromal tissue. This may be interpreted as wound healing as well as immunological host reactions in the not completely immunodeficient nude mice and may render local invasion difficult, all the more as invasiveness of MCF-7 in animal models is known to be low. The development of disseminated micrometastases and MMP expression were not investigated as this was not the aim of the study.
Other members of the insulin/IGF-family have also been implicated in malignant progression. Enhanced protein expression of RLX-like factor has been found in malignant breast epithelia as compared with their benign counterparts (Hombach-Klonisch et al., 2000). Up-regulation of MMP-9 in MCF-7 cells and enhanced invasiveness similar to our results has also been demonstrated for IGF-1 (Mira et al., 1999). Expression of IGF-I increases during prostate cancer progression (Kaplan et al., 1999), and elevated serum IGF-I levels are found in patients with prostate, colorectal and lung cancer (Grimberg and Cohen, 2000). Recently, a positive correlation between elevated serum concentrations of RLX itself and active metastatic disease in breast cancer patients has been demonstrated by our own group (Binder et al., 2001).
The data presented in this report strongly support the hypothesis that RLX enhances the in-vitro invasiveness of the investigated breast cancer cell lines by up-regulation of MMP-2, -7, -9, -13 and -14. As the experimental conditions were artificial and the applied RLX concentrations unphysiologically high, further investigations will show whether these effects may play a role in vivo and may contribute to the progression and dissemination of breast cancers.
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We are grateful to Professor O.D.Sherwood for generous provision of porcine RLX. This work was supported by grants no. 10/1270 Bi I from the Dr Mildred Scheel-Foundation (Deutsche Krebshilfe) and no. Ei 333/8–1 from the Deutsche Forschungsgemeinschaft.
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3 To whom correspondence should be addressed. E-mail: cbinder{at}med.uni-goettingen.de
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References |
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Submitted on September 6, 2001; resubmitted on February 18, 2002; accepted on May 15, 2002.
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