Significance of the Rac signaling pathway in HCC cell motility: implications for a new therapeutic target

Terence K. Lee1,2, Kwan Man1,2,6, Joanna W. Ho1,2, Xiang Hong Wang3, Ronnie T. Poon1,2, Chris K. Sun1,2, Kevin T. Ng1,2, Irene O. Ng1,4, Ray Xu1,5 and Sheung Tat Fan1,2

1 Centre for the Study of Liver Disease, 2 Department of Surgery, 3 Department of Anatomy, 4 Department of Pathology and 5 Institute of Molecular Biology, University of Hong Kong, Pokfulam, Hong Kong, China

6 To whom correspondence should be addressed at: Department of Surgery, The University of Hong Kong, Queen Mary Hospital, L9-55, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong, China. Tel: +86 852 2819 9646; Fax: +86 852 2819 9634; Email: kwanman{at}hkucc.hku.hk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recurrence and metastasis are commonly associated with poor prognosis of hepatocellular carcinoma (HCC). Therefore, a better understanding of molecular mechanisms involved in HCC metastasis may lead to more effective treatment for HCC patients. Rac plays important roles in cytoskeletal reorganization leading to cell motility in renal and breast carcinomas. However, the role of Rac is controversial in tumors and has not been studied in HCC. The aim of this study was to investigate the importance of the Rac signaling pathway in HCC cell motility and the anti-metastatic potential of FTY720. Recently a pair of HCC cell lines from a primary tumor (H2P) and its matched metastasis (H2M) was established. These two cell lines provide a useful tool for the study of HCC metastasis. The results show that the Rac signaling pathway is activated in the metastatic HCC cell line (H2M) compared with the primary HCC cell line (H2P). FTY720 specifically suppressed H2M cell motility by down-regulation of the Rac–GTP level through inhibition of phosphoinositide 3-kinase activity. To conclude, this study is the first to demonstrate an essential role of Rac signaling pathway activation in HCC metastasis and suppression of cell motility by FTY720 through blocking of the Rac pathway.

Abbreviations: ECM, extracellular matrix; HCC, hepatocellular carcinoma; PAK, p21 activated kinase 1; PBS, phosphate-buffered saline; PI3-K, phosphoinositide 3-kinase; Rac-DA, dominant active Rac; Rac-DN, dominant negative Rac; S1P, sphingosine 1-phosphate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hepatocellular carcinoma (HCC) is the fifth most common malignancy and is responsible for more than one million deaths annually world wide, especially in high risk regions, including Southeast Asia and sub-Saharan Africa (1,2). The 5 year survival rate is <25% in that subgroups of patients who are eligible for surgical resection with the most favorable characteristics (3). The poor prognosis of HCC is commonly associated with recurrence and intrahepatic metastasis (2). Cell motility is an important factor in the progression and metastasis of HCC. Cell motility is a complicated process that involves partial detachment from intracellular adhesions and from cell–extracellular matrix (ECM) interactions mediated by integrins, reorganization of the actin cytoskeleton and movement through the ECM (4). On the molecular level, a number of different molecules, including cadherins, integrins and growth factors, have been implicated in the regulation of cell migration (5). Although many factors have been reported in this process, the molecular mechanisms of HCC metastasis remain unclear. Recently, family members of the Rho-like GTPases, including Cdc42, Rac1 and RhoA, have been found to play important roles in distinct actin cytoskeleton changes that are required for cell adhesion, migration and invasion (6). However, their effects on migration and invasion seem to be cell type- and cell substrate-specific. Rac promotes tumor cell migration and invasion in renal and breast carcinomas (7,8). In contrast, activation of Rac inhibits migration and invasion of Madin-Darby canine kidney cells (9). Rac can activate several effectors, one of which is the PAK independent cascade the SAPK/JNK pathway (10). Up to now, a role of Rac in HCC cell motility has not been reported. Understanding the role of the Rac signaling pathway in HCC cell motility could allow the development of novel therapies targeted at the inhibition of metastasis.

Recently, FTY720, a novel immunomodulator, has been reported to target G protein-coupled receptors for sphingosine 1-phosphate and inactivate Rac in organ transplantation models (11). Apart from its use in organ transplantation, a few reports have demonstrated that FTY720 inhibits tumor growth in breast and prostate carcinomas (12,13). In the present study we aim to elucidate the role of Rac activation in HCC cell motility and the anti-metastatic potential of FTY720 by studying the Rac signaling pathway. We demonstrate a role of the Rac signaling pathway in HCC cell motility. An elevated Rac–GTP level was found in a metastatic HCC cell line compared with the primary HCC cell line. The higher level of Rac–GTP and accompanying activated SAPK/JNK pathway correlated with HCC cell motility. We also demonstrate that FTY720 effectively and specifically inhibits Rac activity, resulting in decreased motility through down-regulation of phosphoinositide 3-kinase (PI3-K) activity. In summary, FTY720 inhibits HCC cell motility through the Rac signaling pathway.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines, drugs and cell transfection
Two human HCC cell lines, H2P and H2M (14,15) (a gift from Dr X.Y.Guan, University of Hong Kong), were maintained in RPMI medium (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL), 50 U/ml penicillin G and 50 µg/ml streptomycin (Gibco BRL) at 37°C in a humidified atmosphere containing 5% CO2. FTY720 was kindly provided by Novartis Pharmaceuticals Ltd (Basle, Switzerland). Dominant negative Rac (Rac-DN) and dominant active Rac (Rac-DA) (a gift from Dr D.Y.Jin, University of Hong Kong) and a constitutive active PI3-K plasmid (CD2-p110) (a gift from Dr D.Cantrell, University of Dundee) were transfected into H2M cells using Lipofectin (Life Technologies Inc., Carlsbad, CA) and a pool of transfectants (~50 colonies) was selected using G418. LY294002 was purchased from Cell Signal Technology (Beverly, MA).

Assay for PI3-K activity
Cultured H2M cells were treated with FTY720 at the IC10 and IC50 dosages for 3 h and then stimulated with 100 nM insulin for 10 min at 37°C. The cells were then lysed in 1 ml of NP-40 containing lysis buffer with protease inhibitor. An equal amount of protein lysate (Bio-Rad Protein Assay Kit) was incubated with 5 µl of anti-PI3 kinase antibody (Upstate Biotechnology, Lake Placid, NY) and bound with protein A–agarose beads for 1 h at 4°C. Immunoprecipitates were washed and evaluated for PI3-K activity by competitive ELISA (Echelon Biosciences, Salt Lake City, UT) (16).

Immunofluorescence and localization of F-actin
To determine the effect of the distribution of F-actin, H2P and H2M cells were fixed with 4% formaldehyde dissolved in phosphate-buffered saline (PBS) for 10 min at room temperature and permeabilized for 15 min with 0.1% Triton X-100. Cells were incubated in 1% bovine serum albumin in PBS for 30 min to block non-specific antibody binding and then incubated with 1 µg/ml FITC–phalloidin (Sigma Chemical Co., St Louis, MO) overnight at 37°C. The slides were analyzed using an Eclipse E600 image analysis system (Nikon, Japan).

Western blotting
The cells were lysed and protein extraction was performed. The samples were separated in 10% SDS–polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membrane (Amersham, Little Chalfont, UK). The membrane was blotted with 10% non-fat milk, washed and then probed for Rac-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and phospho-SEK/MKK4, phospho-SAPK/JNK and phospho-c-Jun (Cell Signal Technology) and actin (Santa Cruz, CA). After washing, the membrane was incubated with horseradish peroxidase-conjugated rabbit anti-mouse antibody (Amersham) and then visualized by enhanced chemiluminescense plus according to the manufacturer's protocol.

Ki67 immunofluoresence staining
Cells were plated onto chamber slides in RPMI medium at ~70% confluence for 24 h. The cells were then treated with FTY720 at dosages of 2 and 5 µM for 48 h. They were fixed in ice-cold acetone and methanol (1:1), washed with PBS and then stained with mouse anti-human Ki67 (Oncogene Research Products, San Diego, CA) overnight at 4°C. After washing, the cells were probed with goat anti-mouse FITC-conjugated secondary antibody for 30 min at room temperature and then counterstained with propidium iodide (50 µg/ml) for 30 min at room temperature. The cells were examined under a fluorescence microscope. A total of 500 cells were counted in five fields per sample. The percentage of proliferating cells was calculated as the number of proliferating cells over the total number of cells counted x 100.

Rac and Rho activation assay
Cells were seeded onto a 14 cm diameter tissue culture plate to 70% confluence. For the Rac and Rho activation assay the cells were serum starved for 24 h, stimulated with 100 ng/ml platelet-derived growth factor BB or lysophosphatidic acid for 30 min and then lysed with 0.5 ml of ice-cold lysis buffer. The protein lysate was incubated with PAK-PBD beads or Rhotekin-RBD, respectively, for 1 h at 4°C and washed three times with 1x wash buffer (25 mM Tris–HCl, pH 7.5, 1 mM dithiothreitol, 30 mM MgCl2, 40 mM NaCl, 1% Nonidet P-40) and twice with the same buffer without Nonidet P-40. The bead pellet was finally suspended in 20 µl of Laemmli sample buffer. Proteins were separated by 12% SDS–PAGE, transferred to nitrocellulose membrane and blotted with Rac or RhoA antibodies (Cytoskeleton, CO).

Wound healing assay and three-dimensional growth of cells in ECM
Cell migration was assessed by measuring the movement of cells into an area scraped free with a 200 µl pipette tube (time 0) and three-dimensional growth of cells in the ECM. Cell concentration was first adjusted to 4–6 x 104/ml and 100 µl of cell suspension was then transferred to an Eppendorf tube to which 100 µl of collagen type I solution (Sigma Chemicals) was added. These were gently mixed and dropped onto a 60 mm Petri dish and left for 30 min until solidification. Aliquots of 3–5 ml of RPMI medium (Gibco BRL) was added slowly to the gel. The development of organoids in this three-dimensional culture model is dependent on migration of single cells into cell aggregates. Organoid development was monitored on day 7 using an inverted light microscope (Zeiss Axioscope 25) and photographed with a Polaroid camera at x100.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Rac signaling pathway is significantly elevated in a metastatic HCC cell line and is correlated with cell motility
Recently, a pair of cell lines have been established from a primary HCC (H2P) and a matched metastatic thrombosis in the portal vein (H2M) (14,15). G-banding analysis and fluorescence in situ hybridization with chromosome arm painting probes have demonstrated that these two cell lines (all at passage 4) were of the same origin. These two cell lines provide a very useful tool to investigate genes associated with HCC metastasis. A significant increase in Rac–GTP level was found in the H2M cells when compared with H2P cells by Rac–GTP pull-down assay, but no difference in Rho–GTP level was determined by Rho-GTP pull-down assay (Figure 1A). Activated Rac was found to play some role in cytoskeletal reorganization (17). With phallodin staining rapid and pronounced membrane ruffling and lamellipodia formation with significant cortical actin polymerization (Figure 1B) was found in H2M cells, which presented a higher level of Rac–GTP. To further confirm the role of Rac in HCC cell motility, Rac-DN was transfected into H2M cells. A Rac–GTP pull-down assay showed decreased Rac–GTP in Rac-DN-transfected H2M cells when compared with the empty vector control (Figure 1C). Closure of wounds of ~0.75 mm in monolayers of H2M but not H2P cells occurred at 48 h in a wound healing assay (Figure 1D). Increased motility of H2M cells was further confirmed by the formation of three-dimensional elongated colonies in matrix gel, when compared with the round and packed H2P colonies (Figure 1E). Although the degrees of cell motility were different between H2M and H2P cells, their proliferation rates were similar (Figure 1F). The role of Rac in HCC cell motility was further confirmed by transfection of Rac-DN into H2M cells followed by a wound healing assay. Compared with the empty vector control, delayed wound closure was found in the H2M cells with a lower level of Rac–GTP, which was down-regulated by Rac-DN (Figure 1D). An effector of Rac, the SAPK/JNK pathway, was found to be significantly elevated in H2M cells when compared with H2P cells, but not in Rac-DN-transfected H2M cells (Figure 1G).



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Fig. 1. The Rac signaling pathway plays an important role in HCC cell motility. (A) Rac–GTP pull-down showed that there was a significant increase in Rac–GTP level but not Rho–GTP level in metastatic HCC cell line H2M. Quantitation of specific bands was by densitometric analysis and is shown as fold change at the bottom of each band as compared with H2P. (B) Consistent with the increased Rac activity, increased membrane ruffling and lamellipodia formation (indicated by arrows) with significant cortical actin polymerization were observed in H2M. (C) To confirm the role of Rac in HCC cell motility, Rac-DN was transfected into H2M cells. A decreased Rac–GTP level was observed and confirmed by Rac–GTP pull-down assay. (D) Cell motility was examined by wound healing assay and 3-dimensional growth of cells in a matrix gel. In the wound healing assay closure of the wound was observed in H2M cells but not in H2P cells at 48 h. Rac-DN transfection resulted in failure to close the wound when compared with the empty vector control at 48 h. (E) Elongated 3-dimensional colonies (indicated by arrows) were also observed in H2M but not H2P cells. (F) To examine whether the difference in cell motility related to proliferation, Ki67 immunostaining was performed. No significant difference in proliferation rate between H2P and H2M cells was observed. (G) There was a significant increase in the phosphorylation level of SEK/MMK4, SAPK/JNK and c-jun protein in H2M cells when compared with H2P cells. However, there was no significant change in Rac-DN transfected H2M cells, suggesting that the Rac/JNK pathway was activated mainly in the metastatic HCC cell line.

 
FTY720 suppresses Rac-induced cell motility
To determine the effect of FTY720 on Rac–GTP level in H2M cells, Rac activity was evaluated by Rac–GTP pull-down assay at dosages of 2 and 5 µM (IC10 and IC50 for H2M cells are 9 and 17 µM, respectively). FTY720 inhibited Rac activity in a dose-dependent manner (Figure 2A), accompanied by decreased membrane ruffling, lamellipodia formation and a disorganized actin structure (Figure 2B). On decreased Rac activity using FTY720, significantly decreased cell motility was observed in the wound healing assay, as was three-dimensional growth of cells in matrix gel (Figure 2C and D). However, FTY720 did not decrease H2M cell proliferation significantly at 2 and 5 µM (Figure 2E). To exclude the possibility that other proteins could be involved in this malignant phenotype and non-specificity of Rac inactivation by FTY720, we introduced constitutively active Rac1 to inhibit the effect of FTY720 and using F-actin staining showed that Rac-DA transfection reversed the morphological effects of FTY720 in H2M cells (Figure 2B). In the wound healing assay we found that closure of the wound still occurred at 2 and 5 µM FTY720 (Figure 2C). Expression of Rac–GTP and Rac1 protein in Rac-DA-transfected H2M cells was examined by western blot (Figure 2F).




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Fig. 2. FTY720 suppresses Rac-induced HCC cell motility. (A) FTY720 significantly inhibited Rac activity in a dose-dependent manner. (B) Along with decreased Rac activity, decreased membrane ruffling and lamellipodia formation were observed in FTY720-treated H2M cells. (C) FTY720 inhibited closure of the wound in a wound healing assay. The effect of FTY720 on cell motility was inhibited by introduction of constitutively active Rac-1 into H2M cells. (D) With FTY720 there was a decreased number of 3-dimensional colonies at dosages of 2 and 5 µM. (E) The above dosages did not significantly affect the proliferation rate of H2M cells. (F) The specificity of inhibition of Rac activity by FTY720 was examined by transfection of Rac-DA into H2M cells. Rac-DA-transfected H2M cells also exhibited membrane ruffling and lamellipodia formation upon administration of FTY720 at dosages of 2 and 5 µM. In (A) and (F) quantitation of specific bands was by densitometric analysis and is shown as fold change at the bottom of each band as compared with the untreated control and empty vector, respectively.

 
FTY720 down-regulates the Rac–GTP level through inhibition of PI3-K activity
The role of PI3-K in Rac activity was investigated by Rac–GTP pull-down assay after adding a pharmacological inhibitor of PI3-K, LY294002. The Rac–GTP level was down-regulated by LY294002 in a dose-dependent manner (Figure 3A). FTY720 inhibited PI3-K activity by 32 ± 3.1 and 63 ± 2.1%, respectively (Figure 3B). To further determine that inhibition of PI3-K is critical to the suppression of cell motility by FTY720, a constitutively active PI3-K plasmid (CD2-p110) was transiently transfected into H2M cells. Western blotting showed increased expression of PI3-K (Figure 3C). Transfection of PI3-K opposed the effect of FTY720 on cell motility, as evidenced by phallodin staining (Figure 3D) and a wound healing assay (Figure 3E).



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Fig. 3. FTY720 inhibits Rac activity mediated by PI3-K. (A) PI3-K was found upstream of Rac by performing a Rac–GTP pull-down at dosages of 25 and 50 µM of the PI3-K inhibitor LY294002. Rac–GTP level significantly decreased in a dose-dependent manner. Quantitation of specific bands was by densitometric analysis and is shown as fold change at the bottom of each band as compared with untreated H2M cells. (B) FTY720 inhibited PI3-K activity by 32 ± 3.1 and 63 ± 2.1%, respectively, at dosages of 2 and 5 µM. (C) A western blot showed increased expression of PI3-K after transfection of plasmid CD2-p110 (expressing a constitutively active PI3-K enzyme) in H2M cells. (D) CD2-p110-transfected H2M cells also exhibited membrane ruffling and lamellipodia formation upon administration of FTY720 at a dosage of 5 µM. (E) The effect of FTY720 on cell motility was inhibited by the introduction of CD2-p110 into H2M cells, as demonstrated by a wound healing assay. Values are mean percentages ± SD from at least three independent experiments.

 
FTY720 inhibits the Rac-induced SAPK/JNK pathway
Previous studies demonstrated that Rac activates JNK (18) and AKT through activation of PI3-K (19). In addition, from the above results we can surmise that the Rac-mediated SAPK/JNK pathway is activated in a metastatic HCC cell line when compared with a primary HCC cell line. We then further investigated the effect of FTY720 on the JNK/SAPK pathway. Firstly, we found that FTY720 dephosphorylated SEK/MKK4, SAPK/JNK and c-Jun in H2M cells in a dose-dependent manner (Figure 4A). To further confirm the specificity of FTY720 on the Rac-mediated SAPK/JNK pathway, the same dosages of FTY720 were administered to Rac-DA-transfected H2M cells. From the western blot we found that there were no significant changes in the phosphorylation levels of SEK/MKK4, SAPK/JNK and c-Jun proteins in Rac-DA-transfected H2M cells (Figure 4B). The results show that FTY720 specifically inhibits the Rac-induced SAPK/JNK pathway.



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Fig. 4. Assessment of the SAPK/JNK cascade in FTY720-treated H2M cells. (A) There was a significant dose-dependent decrease in phosphorylation level of SEK/MKK4, SAPK/JNK and c-Jun protein. (B) There was no significant decrease in phosphorylation level of the SAPK/JNK cascade in Rac-DA transfected H2M cells. Quantitation of specific bands was by densitometric analysis and is shown as fold change at the bottom of each band as compared with untreated H2M cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rac regulates continuous turnover of cytoskeletal elements to form lamellipodia and filopodia resulting in cell movement (20), thus this GTPase is thought to play a critical role in cell motility and invasiveness. Cell motility, as in many cancers, is an important factor in the progression and metastasis of HCC. However, the role of Rac in cell motility remains controversial. Active Rac can induce cell motility in most cell types (7,8), but it can also suppress cell motility in certain cell types (9). In the present study an increase in the activated form of Rac was found in a metastatic HCC cell line. Ectopic Rac-DN decreased HCC cell motility and cell invasiveness. Following the activation of Rac in H2M cells, one of its downstream effectors, the SAPK/JNK cascade, was correspondingly activated. This result suggests that the Rac signaling pathway might play an important role in HCC cell motility.

FTY720, a new immunomodulator, has been shown to be effective in Rac inactivation in organ transplantation models by targeting G protein-coupled receptors for sphingosine 1-phosphate (11). Since we have found in the present study that elevated Rac is related to HCC cell motility, we further investigated the effectiveness of FTY720 in inhibition of HCC cell motility. We found that FTY720 effectively inhibited HCC cell motility, accompanied by a dose-dependent decrease in the Rac–GTP level. From the above data it might be postulated that FTY720 inhibits Rac-induced HCC cell motility. However, FTY720 may not be a specific inhibitor of Rac, although it targeted G protein-coupled receptors during Rac inactivation in organ transplants. Thus other proteins or effectors, such as CDC42 and Rho, might be involved in this metastatic phenotype. To test the specificity of FTY720 in the inhibition of Rac-mediated cell motility, we transfected constitutively active Rac1 into H2M cells. In a wound healing assay, Rac-DA-transfected H2M cells exhibited wound closure in various dosages of FTY720. In addition, phosphorylation of a downstream effector of Rac, SAPK/JNK, was unchanged, in contrast to untransfected H2M cells. These data suggest that FTY720 specifically inhibits Rac-mediated HCC cell motility. FTY720 not only decreased membrane ruffling and lamellipodia formation, but also stress fiber formation, and led to a disorganized actin structure (Figure 2). Apart from Rac, Rho also plays an important role in cell motility, by increasing the formation of actin stress fibers (21). There is cross-talk between Rac and Rho and a report has indicated that Rac activates Rho activity (22). The precise function of Rho in HCC cell motility will be investigated in further studies.

PI3-K plays an important role in cell migration through effector molecules such as Rac. Although there is a close correlation between PI3-K and Rac, a critical question has been raised regarding whether PI3-K acts upstream or downstream of Rac (23,24). Inhibition of PI3-K activity with LY294002 resulted in a decrease in Rac–GTP level in H2M cells in a dose-dependent manner. This result suggests that PI3-K may act upstream of Rac in the HCC cell line. Upon administration of FTY720 significantly decreased PI3-K activity was observed in H2M cells, accompanied by a decrease in Rac activity. Therefore, FTY720 inhibits the Rac–GTP level by inhibition of PI3-K activity. FTY720 was found to target sphingosine 1-phosphate (S1P) receptors, a subset of G protein-coupled receptors of the endothelial differentiation gene family (10). There is cross-talk between PI3-K and S1P1. Therefore, FTY720 might inhibit PI3-K activity through binding to the S1P1 receptor. Rac can regulate several different pathways through different effectors. One of them is the PAK-independent cascade SAPK/JNK (25). The present study has demonstrated for the first time that FTY720 might inhibit cell motility through the PI3-K-mediated Rac signaling pathway by dephosphorylation of SEK/MKK4, SAPK/JNK and c-Jun in a dose-dependent manner. The results confirm a role of the SAPK/JNK cascade in tumor cell invasion (26), and its inhibition suppressed tumor cell motility.

In summary, the present study has demonstrated for the first time an essential role of Rac in HCC cell motility. FTY720 significantly inhibited HCC cell motility through the PI3-K-mediated Rac signaling pathway. It is a potential novel drug for clinical application, targeting inhibition of tumor motility and metastasis of HCC.


    Acknowledgments
 
We thank Dr D.Y.Jin of the University of Hong Kong for providing the Rac-DN and Rac-DA plasmids. Financial support was provided by the Sun Chieh Yeh Research Foundation of Hepatobiliary and Pancreatic Surgery of the University of Hong Kong.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 7, 2004; revised December 6, 2004; accepted December 7, 2004.