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Cell and Tumor Biology |
Departments of 1 Cancer Biology and 2 Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas
Requests for reprints: Isaiah J. Fidler, Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Unit 173, P.O. Box 301429, Houston, TX 77230-1429. Phone: 713-792-8577; Fax: 713-792-8747; E-mail: ifidler{at}mdanderson.org.
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Introduction |
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One general method under consideration is the modulation of cancer progression pathways and its interaction with the organ microenvironment. The epidermal growth factor (EGF) phosphorylates EGF receptor (EGFR) by binding to the EGFR and further stimulates multiple signaling pathways that are involved in cell proliferation (e.g., Ras/mitogen-activated protein kinase), antiapoptosis (e.g., phosphatidylinositol 3-kinase/Akt, nuclear factor-
B), and others (5–8). The overexpression of EGF and EGFR by various types of malignancies has been shown to correlate with metastasis, apoptosis, resistance to chemotherapy, and poor prognosis (9–11), indicating that inhibiting EGFR signaling is a good strategy for therapeutic intervention. Cetuximab (IMC C225, Erbitux, ImClone, New York, NY) is a monoclonal antibody (mAb) to EGFR that inhibits binding of EGF to EGFR and stimulation of downstream signaling pathways (12). In locally advanced or pancreatic cancer expressing EGFR, Cetuximab in combination with gemcitabine produced a 12.2% partial response, and 63.4% of patients showed stable disease on a phase II clinical trial (13). Thus, inhibiting EGFR signaling in combination with gemcitabine for pancreatic cancer showed promising activity and has led to a phase III trial of Cetuximab plus gemcitabine.
Production of another growth modulator, vascular endothelial growth factor (VEGF), increased in most types of malignant tumors and is associated with angiogenesis and poor prognosis (14). VEGF is not only a proliferating and permeability factor but also an antiapoptotic survival factor for vascular endothelial cells (15, 16). Inhibiting VEGF receptor (VEGFR) signaling could have a therapeutic efficacy not only by preventing angiogenesis but also by causing vascular endothelial cells in the tumor microenvironment to regress. Bevacizumab (Avastin, Genentech, Inc., South San Francisco, CA) is a recombinant humanized mAb to VEGF that inhibits its binding to VEGFR and activation of downstream signaling (17). In stage IV advanced pancreatic cancer patients, Bevacizumab in combination with gemcitabine produced a median survival of 9 months and a 74% 6-month survival. The partial response rate was 21% and stable disease was achieved by 45% of patients, which are encouraging results (18). A randomized phase III trial of Bevacizumab plus gemcitabine is ongoing.
Platelet-derived growth factor (PDGF) and its receptor (PDGFR) are expressed in many types of cancer, including prostate, lung, gastric, and pancreatic (19, 20). In our previous study, 29 of 31 human pancreatic cancer specimens expressed pPDGFR (21). PDGFR signaling has been reported to increase proliferation of tumor cells in an autocrine manner (22, 23) and to stimulate angiogenesis, recruit pericytes (which stabilize the tumor vasculature; refs. 22, 24), and control the interstitial fluid pressure in stroma to influence transvascular transport of chemotherapeutic agents in a paracrine manner (25, 26). Inhibition of PDGFR activity by tyrosine kinase inhibitor STI571 (Novartis Pharma, Basel, Switzerland; ref. 27) in an orthotopic nude mouse model of pancreatic cancer decreased the growth of primary pancreatic tumors and decreased the incidence of peritoneal metastases when combined with gemcitabine (21).
The most recent data indicate that the biological heterogeneity of neoplasms includes expression of tyrosine kinase receptors (28). Indeed, dual immunohistochemistry of human pancreatic cancer cells growing in the pancreas of nude mice revealed that tumor cells express both EGFR and PDGFR (Fig. 1) and, thus, inhibition of the signaling of one receptor may not be sufficient to inhibit the progressive growth and spread of neoplasms. To overcome this heterogeneity and address the issue of redundancy in signaling pathways, we determined therapy of orthotopic human pancreatic cancer growing in nude mice by multiple protein tyrosine kinase inhibitors. We examined whether the simultaneous inhibition of EGFR, VEGFR, and PDGFR signaling pathway in pancreatic tumor cells, tumor-associated endothelial cells, and stroma cells would increase the therapeutic efficacy of gemcitabine against pancreatic cancer. AEE788 (Novartis Pharma) is a novel synthesized small molecule inhibitor of both EGFR and VEGFR tyrosine kinases (29), and STI571 is an inhibitor of PDGFR, BCR-ABL, and c-Kit tyrosine kinase (27). We determined whether the p.o. administrations of AEE788 and/or STI571 administered alone or combined with i.p. injections of gemcitabine inhibited the progressive growth of human pancreatic cancer cells implanted into the pancreas of nude mice and prolonged survival.
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Materials and Methods |
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Nucleotide sequence analysis of epidermal growth factor receptor in pancreatic cancer L3.6pl cell line. Mutations in exons 18, 19, and 21 of the kinase domain of EGFR have been shown to correlate with response of patients to therapy with the tyrosine kinase inhibitor Iressa (30). To exclude the possibility that the response to AEE788 was associated with mutation of the EGFR, we assayed DNA extracted from log-phase cultures of L3.6pl cells using the DNeasy Tissue kit no. 69504 (Qiagen, Inc., Valencia, CA). Mutational analysis was done by the Molecular Diagnostic Laboratory of the M. D. Anderson Cancer Center (Houston, TX). Nested PCR products of exons 18, 19, and 21 obtained using primers previously described (30) were directly sequenced in sense and antisense directions. All sequences were screened for the presence of mutations both manually and using the SeqScape software and confirmed by two independent PCR amplifications. The results indicated that the L3.6pl cells contain a wild-type EGFR.
Reagents. AEE788 (Novartis Pharma), 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a low-molecular-weight, ATP-competitive dual EGFR and VEGFR tyrosine kinase family inhibitor (29). STI571 (imatinib mesylate or Gleevec; Novartis Pharma) is a 2-phenylaminopyrimidine class protein-tyrosine kinase inhibitor of PDGFR, BCR-ABL, and c-Kit (27). For p.o. administration, AEE788 was diluted in DMSO and STI571 was diluted in sterile water. Gemcitabine (Gemzar, Eli Lilly Co., Indianapolis, IN) was maintained at room temperature and dissolved in PBS on the day of use. It was administered by i.p. injection.
Primary antibodies were purchased from the following manufacturers: rabbit anti-pVEGFR 2/3 (Flk-1; Oncogene, Boston, MA); rabbit anti-human, anti-mouse, anti-rat VEGFR (Flk-1; C1158, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-human phosphorylated EGFR (pEGFR; Tyr1173; Biosource, Camarillo, CA); rabbit anti-human EGF and rabbit anti-human EGFR for paraffin samples (Santa Cruz Biotechnology); rabbit anti-human EGFR for frozen samples (Zymed, San Francisco, CA); rabbit anti-VEGF (A20; Santa Cruz Biotechnology); polyclonal rabbit anti-PDGFR-ß, polyclonal goat anti-pPDGFR-ß, and polyclonal rabbit anti-PDGF-ß (all obtained from Santa Cruz Biotechnology); rat anti-mouse CD31 (BD PharMingen, San Diego, CA); mouse anti–proliferating cell nuclear antigen (PCNA) clone PC 10 (Dako A/S, Copenhagen, Denmark); and rabbit antidesmin (Dako; as a pericyte marker). The following secondary antibodies were used for colorimetric immunohistochemistry: peroxidase-conjugated goat anti-rabbit IgG; F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA); biotinylated goat anti-rabbit (Biocare Medical, Walnut Creek, CA); streptavidin horseradish peroxidase (Dako); rat anti-mouse IgG2a horseradish peroxidase (Serotec, Harlan Bioproducts for Science, Inc., Indianapolis, IN); and goat anti-rat horseradish peroxidase (Jackson ImmunoResearch Laboratories). The following fluorescent secondary antibodies were used: Alexa 488–conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) and Alexa 594–conjugated goat anti-rat IgG (Molecular Probes). Terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) staining was done using a commercial apoptosis detection kit (Promega, Madison, WI) with modifications.
Animals and orthotopic implantation of tumor cells. Male athymic nude mice (NCI-nu) were purchased from the Animal Production Area of the National Cancer Institute Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed and maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and NIH. The mice were used in accordance with institutional guidelines when they were 8 to 12 weeks old.
To produce pancreatic tumors, L3.6pl cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with medium containing 10% FBS and the cells were washed once in serum-free medium and resuspended in HBSS. Only suspensions consisting of single cells with >90% viability were used for injection into the pancreas of nude mice as described previously (21).
Treatment of established human pancreatic carcinoma tumors growing in the pancreas of athymic nude mice. Twenty-one days after the intrapancreatic injection of 0.5 x 106 viable L3.6pl cells in 50 µL HBSS, the pancreatic tumors reached the size of 5 to 6 mm. At that time, the mice were randomized to the following eight treatments (n = 10): (a) Control mice: administration of water diluted at 1:20 with DMSO-0.5% Tween 80 (diluent) by p.o. gavage thrice weekly, daily p.o. gavage with sterile water, and i.p. injections of PBS twice a week; (b) administration of diluent by p.o. gavage thrice weekly, daily p.o. gavage with sterile water, and twice weekly i.p. injections of gemcitabine (50 mg/kg); (c) p.o. gavage of AEE788 (50 mg/kg), thrice weekly, daily p.o. gavage with sterile water, and twice weekly i.p. injections of PBS; (d) p.o. gavage of AEE788 (50 mg/kg) thrice weekly, daily p.o. gavage with sterile water, and twice weekly i.p. injection of gemcitabine (50 mg/kg); (e) daily p.o. gavage of STI571 (50 mg/kg), diluent of AEE788 by p.o. gavage thrice weekly, and i.p. injections of PBS twice weekly; (f) daily p.o. STI571 (50 mg/kg), p.o. gavage of diluent for AEE788 thrice weekly, and i.p. injections of gemcitabine (50 mg/kg) twice weekly; (g) combination of p.o. AEE788 (50 mg/kg) thrice weekly, daily STI571 (50 mg/kg), and twice weekly i.p. injections of PBS; and (h) combination of p.o. AEE788 (50 mg/kg) thrice weekly, STI571 (50 mg/kg) seven times weekly, and twice weekly i.p. injections of gemcitabine (50 mg/kg). All mice were treated for 4 weeks and killed on day 49 of the experiment.
For survival studies, 21 days after the intrapancreatic injection of 1.0 x 106 tumor cells in 50 µL HBSS, at which time the tumors in the pancreas exceeded 6 to 8 mm in diameter, the mice were randomized (n = 10) to one of the eight treatment groups as described above. The mice were killed and necropsied when they became moribund. Survival was evaluated by the Kaplan-Meier method. The study was repeated.
Necropsy procedures and histologic studies. In the first treatment study, the mice were killed on day 49 after tumor cell injection, weighed, and necropsied. Tumors growing in the pancreas were excised and weighed. For immunohistochemical staining procedures, one part of the tumor tissue was fixed in formalin and embedded in paraffin and the other was embedded in optimum cutting temperature compound (Miles, Inc., Elkhart, IN), rapidly frozen in liquid nitrogen, and stored at –70°C.
Immunohistochemical analysis to detect EGF, VEGF, PDGF-BB, EGFR, VEGFR, PDGFRß, pEGFR, pVEGFR, and pPDGFRß in pancreatic tumors. Paraffin-embedded pancreatic tumors of mice from all treatment groups were immunostained to evaluate the expression of EGF, VEGF, PDGF-BB, EGFR, VEGFR, PDGFRß, pEGFR, pVEGFR, and pPDGFRß. The sections were deparaffinized in xylene, dehydrated with alcohol, and rehydrated in PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide in PBS. Samples were exposed to protein block (5% normal horse serum and 1% normal goat serum in PBS) and incubated overnight at 4°C with each primary antibody at the appropriate dilution. After 1-hour incubation at room temperature with peroxidase-conjugated secondary antibody, positive reaction was detected by exposure to stable 3,3'-diaminobenzidine (Phoenix Biotechnologies, Huntsville, AL). Slides were counterstained with Gill's no. 3 hematoxylin. Sections stained for immunoperoxidase or H&E were examined in a Nikon Microphot-FX microscope equipped with a three-chip charged coupled device color video camera (Model DXC990, Sony Corp., Tokyo, Japan). Digital images were captured using Optimas Image Analysis software (Media Cybernetics, Silver Spring, MD).
Immunohistochemical determination of proliferating cell nuclear antigen, CD31/platelet endothelial cell adhesion molecule 1 (endothelial cells), and terminal deoxynucleotidyl transferase-mediated nick end labeling (apoptosis). Paraffin-embedded tissues were used for immunohistochemical identification of PCNA. Frozen tissues used for identification of CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1) were sectioned (8-10 µm), mounted on positively charged slides, and air-dried for 30 minutes. Frozen sections were fixed in cold acetone (5 minutes), in acetone/chloroform (v/v; 5 minutes), and again in acetone (5 minutes), and washed with PBS. Immunohistochemical procedures were done as described previously (21). Control samples exposed to a secondary antibody alone showed no specific staining. For the quantification of mean vessel density in sections stained for CD31, 10 random 0.159 mm2 fields at x100 magnification were captured for each tumor and microvessels were quantified. For quantification of PCNA expression, the number of positive cells was counted in 10 random 0.159 mm2 fields at x100 magnification.
Analysis of apoptotic cells was done by using a commercially available TUNEL kit (Promega) with the following modifications: Samples were fixed and incubated with an equilibration buffer followed by a reaction buffer (containing nucleotide mix and terminal deoxynucleotidyl transferase enzyme). Immunofluorescence microscopy was done in a Zeiss Axioplan microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with an HBO 100 mercury lamp, narrow bandpass filters to individually select for green, red, and blue fluorescence (Chroma Technology Corp., Brattleboro, VT). Images were captured using a cooled charged coupled device Hamamatsu Orca camera (Hamamatsu Corp., Bridgewater, NJ) and Image Pro Analysis software (Media Cybernetics). Photomontages were prepared using Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA). The number of TUNEL-positive cells in 10 random 0.159 mm2 fields at x100 magnification was used to quantify apoptosis.
Double immunofluorescence staining for CD31/platelet endothelial cell adhesion molecule 1 and EGFR, pEGFR, VEGFR, pVEGFR, PDGFRß, pPDGFRß, pericytes (desmin-positive cells), and terminal deoxynucleotidyl transferase–mediated nick end labeling. Frozen sections of pancreatic tumors were mounted on slides and fixed. Immunofluorescence for CD31 was done using Alexa 594–conjugated secondary antibody and samples were again blocked briefly in a blocking solution (5% normal horse serum and 1% normal goat serum in PBS) as described above and incubated with antibody against human EGFR, pEGFR, VEGFR, pVEGFR, PDGFRß, pPDGFRß, or desmin at 4°C overnight. After washes and blocking with blocking solution, samples were incubated with Alexa 488–conjugated secondary antibody. Endothelial cells were identified by red fluorescence and EGFR, pEGFR, VEGFR, pVEGFR, PDGFRß, pPDGFRß, and desmin-positive cells (pericytes) were identified by green fluorescence. The presence of growth factor receptors and phosphorylated receptors on endothelial cells were detected by colocalization of red and green fluorescence, which appeared yellow.
The coverage of pericytes on endothelial cells was determined by counting CD31-positive cells in direct contact with desmin-positive cells and CD31-positive cells without direct association with desmin-positive cells in five randomly selected microscopic fields (at x200 magnification; refs. 31–33).
TUNEL-positive apoptotic cells were detected by localized green fluorescence within cell nuclei and endothelial cells were identified by red fluorescence. Apoptotic endothelial cells were identified by yellow fluorescence within the nuclei. Quantification of apoptotic endothelial cells was expressed as the ratio of apoptotic endothelial cells to the total number of endothelial cells in 10 random 0.159 mm2 fields at x100 magnification.
Statistical analysis. Body weight, tumor weight, PCNA-positive cells, mean vessel density (CD31/PECAM-1), and TUNEL-positive cells were compared using the Mann-Whitney U test. Survival analysis was computed by the Kaplan-Meier method and compared by the log rank test.
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Results |
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Cell proliferation (proliferating cell nuclear antigen), apoptosis (terminal deoxynucleotidyl transferase–mediated nick end labeling), and mean vessel density. Cell proliferation was evaluated by staining for PCNA (Fig. 5). In tumors from control mice, the median number of PCNA-positive cells was 371 ± 88. As shown in Table 2, treatment with gemcitabine alone or STI571 alone decreased the number of dividing PCNA-positive cells. A significant decrease of PCNA-positive cells was found in tumors from all other treatment groups, with the highest inhibition produced in tumors from mice treated with AEE788, STI571, and gemcitabine (155 ± 54, P < 0.001).
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Mean vessel density in the tumors was determined by immunohistochemical staining with antibodies against CD31 (Table 2). The median number of CD31-positive tumor cells from control mice was 46 ± 11. Treatment with gemcitabine alone or STI571 alone did not decrease mean vessel density. The number of CD31-positive cells was significantly decreased in tumors from all other treatment groups, with the largest decrease in mean vessel density in tumors from mice treated with AEE788, STI571, and gemcitabine (16 ± 6; P < 0.001).
Immunofluorescence double staining for CD31/platelet endothelial cell adhesion molecule 1 and terminal deoxynucleotidyl transferase–mediated nick end labeling. Next, we determined whether therapy was associated with apoptosis of endothelial cells by using the CD31/TUNEL fluorescent double-labeling technique (Fig. 5B). Tumors from control mice had no apoptosis in tumor-associated endothelial cells. Treatment of mice with AEE788, STI571, and gemcitabine produced a median of 8 ± 5% apoptosis in tumor-associated endothelial cells (Table 2).
Pericyte coverage on tumor-associated endothelial cells. The effect of the different treatments on pericyte coverage on tumor-associated endothelial cells was evaluated using the double immunofluorescence staining technique with anti-CD31 antibody and antidesmin antibody (Fig. 6A). Pericyte coverage rate in tumors from control-treated mice was 35.4 ± 9.8% (median ± SD). Treatment with STI571 alone or STI571 and gemcitabine produced a significant decrease in pericyte coverage (P < 0.05, 18.8 ± 14.7%, 18.1 ± 10.3%, respectively; Fig. 6B). In contrast, treatment with gemcitabine alone, AEE788 alone, or treatment including AEE788 did not produce a measurable decrease in pericyte coverage. Thus, in this study, we did not find a correlation between inhibition of pericyte coverage of endothelial cells and a decrease in mean vessel density.
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Discussion |
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In the present study, human pancreatic cancer cells growing in the pancreas of nude mice expressed high levels of EGF, VEGF, PDGF-BB, and their receptors, and the receptors were phosphorylated. In addition to the tumor cells, tumor-associated endothelial cells also expressed these receptors, probably in response to specific ligands produced by tumor cells (19). Oral treatment with AEE788 inhibited the phosphorylation of EGFR and VEGFR (but not the expression of EGF, VEGF, EGFR, and VEGFR) on pancreatic tumor cells and tumor-associated endothelial cells. Oral treatment with STI571 inhibited phosphorylation of PDGFR but did not alter PDGF-BB and PDGFR expression levels. When AEE788 and STI571 were combined, phosphorylation of the EGFR, VEGFR, and PDGFR was inhibited on both the implanted human pancreatic cancer cells and the tumor-associated endothelial cells of the recipient mice.
L3.6pl cells growing in the pancreas of nude mice were resistant to treatment with gemcitabine (Fig. 1; Table 1). When combined with AEE788, however, gemcitabine reduced tumor growth by nearly 75% and significantly prolonged survival (P < 0.0001). This therapeutic effect was significantly better than that from treatment with AEE788 alone (P < 0.05). Indeed, the combination treatment using AEE788 and gemcitabine induced a significantly higher level of apoptosis in tumor and tumor-associated endothelial cells, decreased the number of proliferating cells, and a decreased mean vessel density compared with control. These data indicate that inhibition of both the EGFR and VEGFR signaling pathways on tumor cells and tumor-associated endothelial cells combined with a chemotherapeutic reagent is superior to either treatment administered alone.
STI571 as a single treatment had a limited effect on the inhibition of tumor growth and prolongation of survival. However, the combination of STI571 with AEE788 significantly lowered the number of PCNA-positive cells and the mean vessel density and increased the number of apoptotic tumor cells and apoptotic endothelial cells; all these were associated with prolongation of survival. Similar data were produced by combining AEE788 with gemcitabine. The best therapy, however, was produced by combining AEE788 with STI571 and gemcitabine. This combination led to a decrease in tumor size, prolonged survival (P < 0.0001), the fewest PCNA-positive tumor cells, the lowest mean vessel density, and the highest number of apoptotic cells.
In our study, tumor-associated endothelial cells expressed not only EGFR and VEGFR but also PDGFR, which would provide another target for inhibition of its signaling by STI571. PDGFR as well as EGFR and VEGFR signaling, which activates the antiapoptotic protein Akt and bcl-2, acts like a survival factor for endothelial cells (36–38). With the inhibition of survival mechanisms by AEE788 and STI571, tumor-associated endothelial cells, whose proliferating frequency is 20 to 2,000 times higher than that of endothelial cells in normal organs (39, 40), would be more sensitive to anticycling chemotherapeutic treatment. Indeed, we found the largest number of apoptotic cells on tumor-associated endothelial cells (Table 2).
Until now, antiangiogenic therapy has focused mainly on endothelial cells. Recent studies, however, imply that pericyte can also play an important role in angiogenesis (22–24). Because pericyte recruitment and covering of endothelial cells for stabilization and maturation of vessel structure is dependent on PDGFRß signaling (22), the inhibition of PDGFR signaling by a protein tyrosine kinase inhibitor should inhibit pericyte recruitment and attachment to endothelial cells that would in turn confer resistance to VEGFR antagonists on endothelial cells (41, 42). In agreement with other reports, we found that treatment with STI571 decreased pericyte coverage on tumor-associated endothelial cells, whereas AEE788 did not. However, administration of AEE788 seemed to reverse the effect of STI571, suggesting that AEE788 may target endothelial cells or targeted endothelial cells with relatively poor pericyte coverage.
The increased interstitial hyperpressure found in tumor stroma can decrease delivery of drugs. A number of studies reported that inhibition of PDGFR signaling can decrease this pressure and, hence, enhance the effects of chemotherapeutic reagents (25, 26). Increased vascular permeability is a major reason for increased interstitial high pressure (43, 44). Anti-VEGF mAb treatment can lower vascular permeability by normalization of vascular architecture and function (43). Taken together, these reports suggest that treatment with AEE788 and STI571 may decrease interstitial pressure as well as vascular permeability and, hence, increase delivery of gemcitabine to cancer cells.
In conclusion, pancreatic cancer cells produce EGF, VEGF, and PDGF. These ligands can activate their receptors on tumor cells by an autocrine manner and on tumor-associated endothelial cells by a paracrine manner. As a consequence, both tumor cells and tumor-associated endothelial cells have increased survival and resistance to chemotherapeutic agents (36). Inhibiting these signaling pathways by tyrosine kinase inhibitors combined with conventional chemotherapy induced a significant apoptosis in tumor-associated endothelial cells and tumor cells, resulting in decreased tumor size and significant prolongation of survival. The success of this multimodality therapy can be attributed to the heterogeneous nature of cancer. Targeting both tumor cells and tumor-associated endothelial cells can, therefore, be of great therapeutic benefit.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Walter Pagel for critical editorial review and Lola López for expert assistance with the preparation of the manuscript.
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Footnotes |
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Received 5/26/05. Revised 9/ 6/05. Accepted 9/ 8/05.
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