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DOI: 10.1530/eje.1.02213
European Journal of Endocrinology, Vol 155, Issue 2, 371-379
Copyright © 2006 by European Society of Endocrinology
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EXPERIMENTAL STUDY

Somatostatin analogues stimulate p27 expression and inhibit the MAP kinase pathway in pituitary tumours

Erika Hubina1,2, Alexandra M Nanzer1, Matthew R Hanson1, Enrica Ciccarelli3, Marco Losa5, Daniela Gaia3, Mauro Papotti4, Maria Rosaria Terreni5, Sahira Khalaf1, Suzanne Jordan1, Sándor Czirják6, Zoltán Hanzély6, György M Nagy7, Miklós I Góth2, Ashley B Grossman1 and Márta Korbonits1

1 Department of Endocrinology, William Harvey Research Institute, Barts and the London Medical School, Queen Mary College, Charterhouse Square, London EC1M 6BQ, UK, 2 Division of Endocrinology, Department of Medicine, National Medical Centre, 1135 Budapest, Hungary, 3 Departments of Internal Medicine and4 Pathology, Ospedale Molinette, University of Turin, 10100 Turin, Italy,5 Pituitary Unit of the Department of Neurosurgery, University Vita-Salute, 20132 Milan, Italy,6 National Institute of Neurosurgery, 1143 Budapest, Hungary and7 Neuroendocrine Research Laboratory, Department of Human Morphology, Semmelweis University, 1095 Budapest, Hungary

(Correspondence should be addressed to M Korbonits; Email: m.korbonits{at}qmul.ac.uk)


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Objectives: Somatostatin (SST) analogues play an important role in the medical management of somatotroph pituitary adenomas and new agonists have the potential to be effective in a wider group of pituitary and other tumours. The anti-proliferative effect of SST occurs through multiple mechanisms, one of which is cell-cycle arrest, where p27, a cyclin-dependent kinase inhibitor, is an important regulator. We hypothesised that SST may upregulate p27 protein levels and downregulate the MAP kinase pathway in these tumours.

Methods: Human pituitary adenoma cells and rat pituitary cell line (GH3) were cultured and treated in vitro with octreotide and the broad-spectrum SST agonist SOM230 (pasireotide). Immunoblotting for p27 and phospho-ERK (pERK) was performed and proliferation assessed by [3H]-thymidine incorporation. Histological samples from acromegalic patients treated with octreotide before surgery were immunostained for p27 and compared to samples from untreated patients matched for sex, age, tumour size, extension and invasiveness.

Results: We detected upregulation of p27 protein levels with SST analogue treatment in vitro in human pituitary adenoma samples. pERK1/2 was inhibited by SST analogues in both the human samples and GH3 cells. SST and its analogues inhibited the proliferation of GH3 cells. p27 immunostaining was stronger in samples from patients with longer preoperative octreotide treatment (more than 6 months) than in samples from patients with shorter treatment periods.

Conclusions: This study demonstrates that SST-mediated growth inhibition is associated with the downregulation of pERK and upregulation of p27. More potent and broader-spectrum SST analogues are likely to play an increasing role in the treatment of tumours, where the MAP kinase pathway is overactivated.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Pituitary tumours are common neoplasms, with a reported prevalence of 17% in the general population (1). Somatostatin (SST) and its stable analogues are primarily used to inhibit hormone secretion of somatotroph and thyrotroph adenomas, but there are also data suggesting that they have direct effects on tumour growth. However, while SST analogues decrease cell growth and proliferation in both normal and tumour cells in various epithelial and endocrine cell types, the exact mechanisms of cell growth arrest are poorly understood. Previous studies have suggested that the inhibitory effect of SST can be either direct inhibition of cell proliferation, or indirect via inhibition of secretion of growth-inducing hormones or inhibition of angiogenesis (2). SSTacts through five high-affinity, G protein-coupled membrane receptors, which are variably expressed in both normal tissues and tumours; the analogue octreotide activates SST receptor subtype-2 (SSTR2) and to a lesser extent SSTR5, while the new SST analogue SOM230 (pasireotide) activates SSTR1, 2, 3 and 5 receptors, with varying affinity (3,4).

The direct inhibition of cell proliferation by SST and its analogues may involve several signal transduction pathways including control of the MAP kinase pathway (5) and stimulation of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 (6). Cell-cycle progression in mammalian cells requires the co-ordinated action of Cdks and cyclin complexes: p27 is a widely distributed Cdk inhibitor that has a negative influence on cell-cycle progression and it can be used as a prognostic marker in human malignant tumours. Overexpression of p27 leads to cell-cycle arrest (7), while lack of p27 results in pituitary adenomas in p27 ‘knockout’ animals (8–10). We, and others, have previously shown reduced expression of p27 in a variety of pituitary adenomas (11–15), although a single study was not able to confirm this (16).

The aim of the present study was to investigate the pituitary effects of SST analogues on: (i) cell proliferation, (ii) MAP kinase activation and (iii) p27 expression using several approaches, including a pituitarycell line (for i and ii), in vitro cultured human adenomas (for ii and iii) and using histological samples from patients with acromegaly treated preoperatively with SST analogues to study the immunohistochemical expression of p27 (iii). We believe this is the first study to show these effects in the pituitary.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell culture

GH3 cells  The rat somatotroph cell line GH3 cells were cultured as previously described (17). The cells were plated at a density of 1x106 cells/well in six-well plates for western blotting for ERK, and 5x104 cells/well in 24-well plates for the [3H]-thymidine incorporation.

Human adenomas  Ten pituitary adenoma tissues (two somatotroph tumours, five non-functioning pituitary adenomas (NFPAs), one lactotroph, one corticotroph and one thyrotroph tumour) were studied for changes in p27 or phospho-ERK (pERK) expression. Patient details are shown in Table 1Go. The tumours were collected fresh during surgery and all patients gave written informed consent for the protocol, which was approved by the local Institutional Review Board. Following trans-sphenoidal surgery, human pituitary adenoma cells were dispersed in vitro (18) at a density of 1x106 cells/well in six-well plates.


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  		Table 1 Patient data and adenoma somatostatin receptor (SSTR) expression in human adenoma samples studied in vitro.
 
[3H]-Thymidine incorporation

GH3 cells were grown in media containing 2% charcoal-stripped foetal calf serum and treated with various concentrations (10–6–10–12 M) of SST (Sigma), octreotide and SOM230 (Novartis) and 12-phorbol 13-myristate acetate (PMA) 10–7 M (CN Biosciences, Nottingham, UK) for 24, 48, 72 and 96 h with the addition of 2 µCi/well of [3H]-thymidine (Amersham) for further 6 h (17); 72 h was selected for further experiments. The cells were harvested before counting in the presence of scintillation fluid using a Microbeta 1450 ß-counter (Wallac, Turku, Finland). The experiments were repeated at least three times.

Immunoblotting

Serum-starved (for 24 h) human pituitary adenoma cells were treated with octreotide and SOM230 for 72 h or 10 min for the assessment of p27 protein (patients 1–5) or pERK1/2 expression (patients 6–10) respectively. Serum-starved GH3 cells were treated for 10 min for pERK assessment. Immunoblotting and image analysis were performed as described earlier (17). Primary antibody for p27 was used at a concentration of 1:500 (Santa Cruz, CA, USA) in TBS–Tween, ß-actin at a concentration of 1:1000 (Abcam, Cambridge, UK), and anti-phosphorylated and anti-total ERK at a concentration of 1:1000 (Promega) in 5% non-fat dry milk in TBS–Tween overnight at 4 °C. After eight washes in TBS–Tween, the membranes were incubated with the secondary antibody (anti-rabbit antibody at a concentration of 1:10 000 (Dako, Glostrup, Denmark) for p27, pERK and total ERK; and anti-mouse antibody at a concentration of 1:10 000 (Dako) for ß-actin) conjugated with horseradish peroxidase. A chemiluminescent peroxidase substrate, ECL Plus (Amersham), was applied according to the manufacturer’s instructions, and the membranes were exposed briefly to X-ray film. Densito-metric readings of the resulting bands were evaluated using the Scientific Imaging System (Kodak) and the ratio to total ERK or ß-actin was calculated and compared to controls.

Reverse transcriptase (RT)-PCR

SSTR expression of the human pituitary samples was studied with RT-PCR. RNA extraction and RT-PCR procedures have been previously described (18). We performed an additional second DNase treatment using RNase-free DNase (5 µl/sample; Promega) and RNA was incubated at 37 °C for 30 min followed by 68 °C for 10 min. A negative control with the omission of the RT enzyme was also included. PCR was performed at an annealing temperature of 60 °C using the following primers for SSTR1, 2, 3 and 5:

SSTR1: 5'-GCTACGTGCTCATCATTGCTA-3'; 5'-GGACTCCAGGTTCTCAGGTTG-3', product 401 bp

SSTR2: 5'-TTGGTACACAGGGTTCATCAT-3'; 5'-GTCTCCGTGGTCTCATTCAGC-3', product 459 bp

SSTR3: 5'-CTGGGTAACTCGCTGGTCAT-3'; 5'-CAGGCAGAATATGCTGGTGC-3', product 225 bp

SSTR5: 5'-TCATCTGCCTGTGCTACCTG-3'; 5'-GGAGAGGATGACCACGAAGA-3', product 233 bp

Positive controls for the PCRs were carried out using a cDNA from a somatotroph adenoma expressing SSTR1, 2, 3 and 5. SSTR PCR bands were compared to the expression of house-keeping gene gylceraldehyde-3-phosphate dehydrogenase (18).

Investigation of the effect of in vivo octreotide treatment on p27 staining in somatotrophinomas

Histological samples from 25 patients with acromegaly treated for 12 months (range, 2–62 months) with octreotide before transsphenoidal surgery and 23 untreated patients matched for sex, age, tumour size, extension and invasiveness were available for analysis (19). Clinical characteristics, proliferative (Ki-67) and apoptotic indices in this cohort have been described previously (19). Pathological sections underwent immunostaining for p27 and the details of the assessment of p27 staining have been described earlier (12). All the slides were counted by two observers blinded to the diagnosis. All patients involved in the study gave written informed consent and the study was approved by the local Institutional Review Board.

Statistical analysis

Statistical analysis was performed using the one-way ANOVA test followed by the Newman–Keuls comparison or the Kruskal–Wallis test followed by Conover–Inman comparison, the Mann–Whitney test and the Spearman correlation test (StatsDirect, Cambridge, UK); statistical significance was taken at P<0.05, data are expressed as mean±S.E.M.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Cell proliferation in GH3 cells

SST, octreotide and SOM230 treatment of GH3 cells showed a significant reduction of cell proliferation using the [3H]-thymidine incorporation assay (Fig. 1Go). Based on our time (24, 48, 72 and 96 h) and dose–response (10–6–10–12 M) curves, for further studies, we selected 72-h incubation and the concentrations for each analogue showing the highest levels of inhibition (57.7±9.9% control at 10–9 M for octreotide and 62±4.2% of control at 10–6 M for SOM230).


Figure 1
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  		Figure 1 Dose–response curve of somatostatin, octreotide and SOM230 using the [3H]-thymidine incorporation test for GH3 cells. *P<0.05, {dagger}P<0.01, {ddagger}P<0.001 vs control.

 
pERK expression in GH3 cells

Cells treated with octreotide and SOM230 showed a reduction of pERK1/2 expression in GH3 cells (octreotide, 50±1.7% control; SOM230, 61±6% control; P<0.05 compared to control), while PMA showed the expected stimulation (139±3% control; P<0.05). The co-administration of PMA and SST analogues showed a strong inhibitory effect of both analogues on pERK expression (PMA+octreotide, 54±7% control; PMA+SOM230, 76±20% control; P<0.01 when compared to PMA alone) (Fig. 2Go).


Figure 2
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  		Figure 2 Effect of 10-min octreotide (10–9 M), SOM230 (10–6 M) and 12-phorbol 13-myristate acetate (PMA) (10–7 M) treatment on ERK1/2 phosphorylation (pERK1/2) in GH3 cells. The ratio of pERK1/2 and total ERK is expressed as percentage of control. *P<0.01 vs control {dagger}P<0.01 vs PMA.

 
p27 expression in human pituitary adenomas in response to SST analogues in vitro

Both octreotide and SOM230 caused a significant increase in p27 protein levels compared to cells treated with media alone (255±43 and 213±19% control respectively; P<0.001). Figure 3AGo shows the results according to adenoma types and Fig. 3BGo is a representative immunoblot. The SSTR mRNA expression pattern of the human pituitary adenomas is shown in Table 1Go; gene transcript expression was comparable in various samples.


Figure 3
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  		Figure 3 (A) Human pituitary adenomas (somatotroph adenoma, GH, non-functioning adenoma, NFPA and prolactinoma, PRL) were cultured in vitro. The SSTR subtype expression of individual samples (patient (Pt) 1–5) is shown. The effect of octreotide and SOM230 treatment on p27 expression is shown as the ratio of band density of p27 to ß-actin (percentage of control; S.E.M. is shown for the middle panel). (B) Representative blot from the sample of patient 2.

 
pERK expression in human pituitary adenomas in response to SST analogues in vitro

pERK1/2 levels decreased significantly after both octreotide (60±8% control; P<0.01) and SOM230 (59±7% vs control; P<0.01) treatment. PMA (10–7 M; known to activate the PKC–ERK pathway) caused the expected increase in ERK1/2 phosphorylation (324±37% control; P<0.001), while co-treatment with PMA and SST analogues caused a significant inhibition of pERK activation (PMA+octreotide, 87±19% control and PMA+SOM230, 88±22% control; P<0.01 for both). Figure 4Go shows the results according to adenoma types, while Fig. 4BGo shows a representative immunoblot.


Figure 4
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  		Figure 4 (A) The effect of 10-min in vitro octreotide, SOM230 and PMA treatment is presented in cultured human pituitary adenoma cells (TSH, thyrotrophinoma; GH, somatotroph adenoma; ACTH, corticotrophinoma; NFPA, non-functioning pituitary adenoma). Immunoblotting for phosphorylated ERK1/2 is shown as the ratio to total ERK (percentage of control, S.E.M. is shown for the last panel). (B) Representative blot from patient 7.

 
The effect of preoperative octreotide treatment on p27 staining in somatotrophinomas

We studied nuclear p27 immunostaining of the previously carefully described (19) cohort of adenoma samples obtained from 25 acromegalic patients treated for a median of 12 months (range, 2–62) with octreotide before transsphenoidal surgery and from 23 untreated patients matched for sex, age, tumour size, extension and invasiveness. Octreotide-treated patients showed a significant decrease in their growth hormone (GH)/insulin-like growth factor-I levels (see detailed data in (19)). No significant difference in p27 staining was observed between the octreotide-treated and untreated groups overall (percentage of cells showing p27-positive staining, median 81% (range, 9–96%) vs 58% (range, 6–100%; P>0.05), although octreotide-treated patients showed a lower Ki-67 staining (1.8±0.3%) compared to untreated patients (3.8±0.7%; P< 0.05)) (19). The p27 staining was predominantly nuclear, as previously reported, and the occasional diffuse cytoplasmic staining did not reveal any further differences between the groups (20,21). We also studied the correlation between Ki-67 and p27 staining: in the octreotide-treated group this showed a negative trend with R=–0.4, P=0.07. However, when we divided the octreotide-treated patient group into those who had been treated for more or less than 6 months (n=13 and 11 respectively), patients with longer than 6 months’ octreotide treatment showed significantly higher p27 staining than patients with shorter treatment periods (Fig. 5Go, P<0.05). We had 25 pre- and post-treatment MRIs available for the treatment group, with four cases showing more than 20% tumour shrinkage. These four patients had been treated for more than 6 months with octreotide (range, 8–27 months) and their tumours showed a relatively high p27 (73±11%) and a low Ki-67 staining (0.6±0.2%), as compared to those patients showing less shrinkage.


Figure 5
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  		Figure 5 Analysis of p27 staining in patients treated for less than or more than 6 months with octreotide before transsphenoidal surgery for pituitary adenoma. Data are expressed as lower and upper quartiles, and minima and maxima. P<0.05 between groups.

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We report that cell proliferation in the rat pituitary GH3 cell line is inhibited by the SST analogues, octreotide and SOM230, and this occurs in parallel with inhibition of ERK1/2 phosphorylation. In GH3 cells, the p27 gene is not expressed due to the methylation of the promoter (22), so we were unable to explore its modulation in this model system. In human pituitary adenomas, we have shown that despite their variable expression of SSTRs, they all show a response to SST agonist stimuli, with both inhibition of pERK expression and increased expression of p27. p27 expression increased in all the adenomas to at least 150% of control, while basal unstimulated pERK decreased to at least 80% or less of control and to 50% or less of PMA-stimulated samples by octreotide and/or SOM230. This indicates that these parameters significantly change in response to SST analogue treatment in a range of pituitary adenomas. In the clinical study, while octreotide treatment was not obviously associated with an increase in p27 expression overall, long-term treatment (>6 months) positively correlated with an increase in p27 expression. The trend for a negative correlation between p27 and Ki-67 in SST analogue-treated samples supports this hypothesis.

In vitro treatment of human NFPAs with octreotide or lanreotide inhibits cell proliferation (23,24) and octreotide has been shown to induce G0/G1 cell-cycle arrest and thus prevent DNA synthesis in rat GH3 cells (25), but changes in the p27 and the MAP kinase pathways in response to SST analogues have not been previously investigated in pituitary adenomas, despite the fact that pituitary adenomas are one of the primary treatment targets of SST analogues. We have shown previously that normal somatotroph cells contain more p27 protein than somatotroph adenoma cells: for example, 75% of somatotroph cells in the normal pituitary show positive immunostaining for p27, while somatotroph cells in adenomas show only 40% p27 staining (26). This staining is predominantly nuclear, where it is considered that p27 acts as a Cdk inhibitor to modulate cell proliferation. The effect of SST on p27 has previously been studied in other cell types (6,27–29). In our study, both octreotide and SOM230 significantly increased p27 expression in human adenomas, and, while the sample size is small, the expression of the distribution of SSTRs in pituitary adenomas appeared to influence the response to the analogues (Fig. 3Go and 4Go). Previous studies have indicated that all SSTRs (other than SSTR4) may be involved in the inhibition of cell proliferation (30). However, octreotide only demonstrates specific binding with high affinity to the SSTR2 and, to a lesser extent, the SSTR3 and SSTR5 subtypes (31,32). The recently described new SST analogue, SOM230, now known as pasireotide, has a wider spectrum of activity, with significant binding to SSTR1, 2, 3 and 5 subtypes; affinity to SSTR1 is 30 times that of octreotide, to SSTR3 5 times and for SSTR5 50 times (3,4). It also has a much longer half-life and causes less desensitisation (3), both accounting for the differences seen in the shape of the dose–response curve. It may be that the differential responsiveness to SOM230 and octreotide is related to different and selective receptor expression, but further studies in this area, possibly using techniques such as RNA interference, will be helpful.

The effect of SST on cell proliferation occurs through multiple direct and indirect mechanisms. SST limits cell growth by inhibiting the release of growth factors such as hormones and cytokines, via inhibition of angiogenesis, and several studies describe a strong effect on apoptosis. Apoptosis plays a crucial role in the proliferation and turnover of cells in various malignant tumours and downregulation of p27 may be the main cause of cell differentiation dysfunction and an attenuation of apoptosis. p27 can induce apoptosis of human gastric cancer cells in vitro (33) and p27 can modulate Akt stability, cell survival and tumourigenicity in human breast carcinomas (33). A new molecular pathway for p27 action has been described recently: octreotide induces expression of the tumour suppressor gene Zac1 (34); Zac1 also induces cell-cycle arrest and apoptosis and is highly expressed in normal pituitary, mammary and ovarian glands, but is downregulated in pituitary, breast and ovarian tumours. Zac1 is a target of the phosphatidylinositol 3-kinase (PI3K) survival pathway. Besides the involvement of p27 and MAP kinase pathways, stimulation of tyrosine phosphatase activity may also mediate part of the anti-proliferative effect of SST. In one study, octreotide treatment decreased the tyrosine phosphorylation levels of the PI3K regulatory subunit p85, induced dephosphorylation of phosphoinositide-dependent kinase 1 and Akt, and activated glycogen synthase kinase 3ß (34).

In vitro studies on human cells studying pituitary hormone secretion used 10–8 M concentrations and showed inhibition of GH, thyrotrophin (TSH) and {alpha}-subunit release, with octreotide having a stronger effect than SOM230 (35–37). However, the maximal effective dose of SST analogues could be different for hormone release and anti-proliferative effects. In cases where the SSTR2 is the most important of the receptor subtypes mediating an effect, SOM230 is an order of magnitude less effective than octreotide. For example, inhibition of ghrelin secretion by octreotide and SOM230 in rats is mainly via SSTR2, so SOM230 requires a higher dose to have the same effect as octreotide (30 vs 3 µg/kg) (38). In another study, SOM230 generally showed a lower potency for the inhibition of GH release, compared with octreotide (36). In humans, 250 µg SOM230 induced a significantly greater suppressive effect on circulating GH secretion than the 100 µg dosage of SOM230, while the inhibitory effect of octreotide (100 µg) on GH levels was equivalent to SOM230 (250 µg) (39). This may also explain the lower potency of SOM230 compared to octreotide in patients with acromegaly. The test drug concentration levels, at which half of the maximum drug effect is observed (EC50), are 46 and 553 pg/ml for octreotide and SOM230 with considerable interpatient variability, mainly attributable to the heterogeneous responses among acromegalic patients (40). Although the overall effects of octreotide and SOM230 on the stimulation of p27 and inhibition of pERK1/2 were not significantly different in our human samples, the variable effects of octreotide and SOM230 on the individual human samples can be explained by several mechanisms. The level of receptor expression in each tumour for each receptor subtype is different and, in addition, Jaquet and colleagues observed that SSTR mRNA levels do not directly correlate with SSTR protein levels (41). SST and its analogues could produce homo-or heterodimerisation of the SSTR2 and 5 subtypes resulting in an increased binding affinity and effects in tissue where both are expressed (42,43). There are considerable differences in receptor expression pattern and the activity of octreotide in rat and human SSTR5 (160-fold greater affinity for the rat SSTR5 (44)), and therefore it may be incorrect to simply extrapolate potency estimates in rodents to human tissue. In addition, there are definite differences in SSTR subtype expression in different types of pituitary adenoma (Table 1Go). The high expression levels of SSTR2 in GH3 cells may explain the higher efficacy of the SSTR2 preferring agonist octreotide in our rat cells. SOM230 inhibits cell proliferation at concentrations of 10–7 and 10–6 M, but not at concentrations 10–8 and 10–9 M in human umbilical vein endothelial cells expressing SSTR1, -2 and -5 (45). Octreotide was only effective at a 10–6 M dose in a recent in vitro study (34), again suggesting that the pharmacological data do not always translate into efficacy in cell systems under the same conditions.

Independent of the effects of SST analogues on hormone secretion, tumour shrinkage has been observed after long-term SST analogue treatment, particularly in somatotroph tumours, usually of the order of ~50% (46–48). Morphological changes, especially perivascular fibrosis, have been noted in histological studies. Tumour shrinkage has also been reported in 50% of cases during octreotide treatment in patients with TSH-secreting pituitary adenomas (49), as well as in a very limited number of NFPAs during long-term SST analogue treatment (50). The mechanism of these effects is poorly understood, but regulation of apoptosis, as mentioned above, may be involved. In our samples, as a group, octreotide did not significantly increase p27 staining in somatotroph tumours, although it was associated with a reduction in Ki-67 staining.

In clinical studies, shrinkage is usually evident within 3 months of SST analogue therapy, although further responsiveness over a longer duration is possible (48). Indeed, there are long-term data, which suggest that the anti-proliferative effects of SST analogues may be mediated, at least in part, by p27-induced inhibition of the cell cycle (51).

In conclusion, this study demonstrates that SST-mediated growth inhibition is associated with the downregulation of pERK and upregulation of p27. More potent and broader-spectrum SST analogues are likely to play an increasing role in the treatment of tumours where the MAP kinase pathway is overactivated.


   Acknowledgements
 
E H was supported by the Royal Society and the Hungarian Endocrine Society. M K was supported by the MRC. MIG was supported by a Hungarian National Research grant (OTKA/2007). We are grateful for Novartis for supplying the octreotide and SOM230.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received 3 January 2006
Accepted 24 May 2006




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