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Clinical Cancer Research Vol. 11, 6800-6806, October 1, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Clinicopathologic and Biological Significance of Kallikrein 6 Overexpression in Human Gastric Cancer

Hisashi Nagahara1,2, Koshi Mimori1, Tohru Utsunomiya1, Graham F. Barnard3, Masaichi Ohira2, Kosei Hirakawa2 and Masaki Mori1

Authors' Affiliations: 1 Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu; 2 Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan; 3 Department of Medicine, University of Massachusetts, Worcester, Massachusetts

Requests for reprints: Masaki Mori, Department of Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu 81-874-0838, Japan. Phone: 81-977-27-1650; Fax: 81-977-27-1651; E-mail: mmori{at}beppu.kyushu-u.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Human kallikrein genes (KLK) have been reported to be involved in human malignancies and several KLKs are promising biomarkers of prostate, ovarian, testicular, and breast cancers. Herein, we investigated the clinicopathologic and biological significance of KLK6 gene expression in human gastric cancer.

Patients and Methods: Using real-time reverse transcription-PCR, we analyzed the KLK6 expression status with respect to various clinicopathologic variables in 66 patients with gastric cancer. In addition, we established a KLK6 stably suppressed gastric cancer cell line (MKN28) using small interfering RNA–mediated gene silencing, and investigated its effects on the cell proliferation rate, cell cycle, and invasiveness.

Results: The KLK6 gene expression in cancerous tissue (0.37 ± 0.53) was significantly (P < 0.000001) higher than that in noncancerous tissue (0.026 ± 0.060). Elevated KLK6 expression was significantly associated with lymphatic invasion (P = 0.03). Furthermore, patients with a high KLK6 expression had a significantly poorer survival rate than those with a low KLK6 expression (P = 0.03). Therefore, we showed that KLK6 gene silencing with KLK6 small interfering RNA effectively suppressed the cell proliferation rate (P = 0.002), cell population in the S phase (P < 0.01), and invasiveness (P < 0.01) in comparison to mock-transfected cells.

Conclusions: The KLK6 gene is markedly overexpressed in gastric cancer tissue and its expression status may be a powerful prognostic indicator for patients with gastric cancer. Our findings also suggest that KLK6 may possibly be a novel target for gastric cancer therapy by gene-silencing procedures.

The kallikrein gene family of secreted serine proteases, consisting of 15 genes, localizes tandemly on chromosome 19q13.4 and shows significant homologies at both the nucleotide and the protein levels (1–5). The human kallikrein gene 6 (KLK6), encoding human kallikrein 6 protein (hK6), has been cloned independently by three groups. Using a differential display PCR technique from breast cancer cell lines, Anisowicz et al. cloned the full-length cDNA of KLK6, named protease M (6). They showed that protease M was strongly expressed in breast cancer cell lines and in ovarian cancer tissues and cell lines. Little et al. cloned the identical cDNA, which they named ZYME, from brain tissue of a patient with Alzheimer's disease (7). Yamashiro et al. cloned the gene, which they called NEUROSIN, from a cDNA library prepared from a human colorectal cancer cell line (8).

Recent studies have suggested that human KLKs are involved in human carcinogenesis and that several KLKs are promising biomarkers of prostate, ovarian, testicular, and breast cancers (3, 5). For example, the KLK3 gene encodes prostate-specific antigen (hK3), which is a currently available cancer-specific marker, and is widely used for the screening, diagnosis, and management of prostate cancer (9, 10). KLK2 protein (hK2) can be another useful diagnostic marker for prostate cancer (11, 12). Many other KLKs have also been expected to act as tumor biomarkers (13–18). In addition, more recent evidence also implicates KLKs in many cancer-related processes, including cell-growth regulation, angiogenesis, invasion, and metastasis (4, 5). Regarding KLK6, several authors have reported that the KLK6 mRNA was highly expressed in ovarian cancer tissue and that hK6 could be a useful serum biomarker for the diagnosis and monitoring of ovarian cancer (19, 20). However, no information is available on KLK6 expression in human gastric cancer, the second most common cancer in Japan.

In the present study, we therefore examined the clinicopathologic and prognostic significance of KLK6 expression in gastric cancers. Furthermore, we investigated the association of such biological behaviors, as cell growth and invasiveness, with KLK6 gene expression when suppressed by gene silencing with small interfering RNA (siRNA) in a gastric cancer cell line.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tissue samples. The cell lines derived from human gastric cancer, including AZ521, KATOIII, MKN1, MKN7, MKN28, MKN45, MKN74, NUGC3, NUGC4, and human fibroblast cell line, KMST6, were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan), and maintained in RPMI 1640 containing 10% fetal bovine serum (FBS) and antibiotics at 37°C in a 5% humidified CO2 atmosphere.

The 66 tumor samples and the matched control samples taken from normal tissue located far from the tumor site of gastric cancers were frozen in liquid nitrogen immediately after a surgical resection, and were kept at –90°C until RNA extraction. The surgical samples were obtained at the Department of Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan. None of these patients received preoperative treatment, such as radiation or chemotherapy. Written informed consent was obtained from all patients according to the guidelines approved by the Institutional Research Board.

Clinicopathologic data. All data, including sex, histology, serosal invasion, lymph node metastasis, lymphatic invasion, vascular invasion, and clinical stage were obtained from the clinical and pathologic records.

RNA preparation and reverse transcription. The total RNA was isolated by the modified acid guanidinium-phenol-chloroform procedure with DNase (21). cDNA was synthesized from 2.5 µg of total RNA as described previously (22).

Oligonucleotide primers for KLK6 gene amplification by PCR. The primer sequences for KLK6 were: 5'-CATGGCGGACCCTGCGACAAGAC-3' and 5'-TGGATCACAGCCCGGACAACAGAA-3'. The forward primer was located in exon 2, whereas the reverse primer was located in exon 3. The length of the amplicon was 215 bp. The amplification was done for 28 cycles of 1 minute at 95°C, 1 minute at 57°C, 1 minute at 72°C. An 8 µL aliquot of each reaction mixture was size-fractionated in a 2% agarose gel and visualized by ethidium bromide staining. To ensure that the RNA was not degraded, a PCR assay with primers specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was carried out in each case, except that only 22 cycles were done under the following cycling conditions: 1 minute at 95°C, 1 minute at 56°C, and 1 minute at 72°C. The primer sequences for the GAPDH amplification were: 5'-TTGGTATCGTGGAAGGACTCA-3' and 5'-TGTCATCATATTTGGCAGGTT-3' (23). The length of this amplicon was 249 bp.

Real-time quantitative reverse transcription-PCR. The real-time monitoring of the PCR reactions was done using the LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Tokyo, Japan). The amplification conditions of the 40 cycles consisted of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and elongation at 72°C for 10 seconds. The products were then subjected to a temperature gradient from 68°C to 95°C at 0.1°C/s with continuous fluorescence monitoring to produce a melting curve of the products. After proportional background adjustment, the fit point method was used to determine the cycle in which the log-linear signal was distinguished from the background and that cycle number was used as a crossing-point value. The standard curve was produced by measuring the crossing point of each standard value (2-fold serially diluted cDNAs of MKN28) and plotting them against the logarithmic value of the concentration. Concentrations of each sample were then calculated by setting their crossing points to the standard curve. The expression levels were normalized by the GAPDH mRNA expression (23). We classified the 66 cases into two groups using the mean expression level of KLK6 mRNA in tumor tissue (0.37): i.e., a high-expression group (>0.37, n = 20) and a low-expression group (<0.37, n = 46).

Immunohistochemistry. Immunohistochemical studies of hK6 were done on surgical specimens from patients with gastric cancer using the avidin-biotin-peroxidase method (LSAB2 kit, Dako, Kyoto, Japan) on formalin-fixed, paraffin-embedded tissue specimens. All sections were counterstained with hematoxylin. The primary mouse monoclonal antibodies against hK6 (MCA2158, Serotec, Ltd., Oxfordshire, United Kingdom) were used at dilutions of 1:500.

All sections were independently examined by three investigators (H. Nagahara, K. Mimori, H. Inoue). We scored the expression as negative for hK6 when <10% of the carcinoma cells were stained in an examined area of a specimen. We examined hK6 protein expression in tumor and the corresponding normal tissues from 18 representative gastric cancer cases among 66 cases. In 18 selected cases, 6 cases showed a lower expression level of KLK6 mRNA in gastric cancer tissues than in noncancerous tissues, whereas the remaining 12 cases exhibited a higher KLK6 mRNA expression in tumor than normal tissues.

Small interfering RNA transfection. The expression vector, pSilencer 3.1-H1 hygro (Ambion Inc., Austin, TX) was used for the expression of siRNA. A hairpin siRNA designed to target the KLK6 gene (5'-GATCCCGTAAGTTGGTGCATGGCGGATTCAAGAGATCCGCCATGCACCAACTTATTTTTTGGAAA-3', sense strand) was inserted into the pSilencer according to the manufacturer's instructions, and then it was transfected into the gastric cancer cell line (MKN28) by the LipofectAMINE method (Life Technologies, Inc., Tokyo, Japan) as described previously (24). Two stably transfected clones were selected after hygromycin treatment (800 µg/mL) and used for the subsequent experiments. A mock vector-transfected clone of the cell line was used as a control.

Western blot analysis. Total protein was extracted from the samples with a radioimmunoprecipitation assay buffer. Aliquots of total protein were applied to 12% acrylamide gradient gels. After electrophoresis, the samples were electroblotted onto a polyvinylidene membrane (Immobilon; Millipore, Inc., Bedford, MA) at 0.5 Å for 50 minutes at 4°C. The hK6 protein was detected using mouse monoclonal antibody (Serotec) at dilutions of 1:1,000. The protein levels of hK6 were normalized to the level of ß-actin protein (Cytoskeleton, Inc., Denver, CO) at dilutions of 1:1,000. The blots were developed with horseradish peroxidase–linked anti-mouse immunoglobulin (Promega, Inc., Madison, WI) at dilutions of 1:5,000. Signals were detected using Supersignal (Pierce, Inc., Rockford, IL).

In vitro proliferation assay. Cells were plated at a density of 5 x 104 cells per well in three 6-cm plates and were harvested and counted on days 3, 7, and 10. The medium was changed every 72 hours. This experiment was done in triplicate.

Cell cycle analysis. Cells (2.0 x 106) were preincubated for 48 hours in serum-free medium at 37°C and then were kept in medium with serum (10% FBS) for 18 hours at 37°C. The cells were harvested and fixed in 70% ethanol at –20°C. Next, the cells were washed and resuspended in propidium iodide staining buffer (5 µg/mL propidium iodide and 0.25 mg/mL RNase) in PBS. The DNA content was evaluated using an EPICS XL flow cytometer (Beckman Coulter, Corp., Tokyo, Japan).

Measurement of bromodeoxyuridine uptake was done as described previously (25). Briefly, after siRNA-transfected cells and mock-transfected cells (2.0 x 106/plate) were incubated for 48 hours in serum-free medium at 37°C and 18 hours after addition of 10% FBS at 37°C, bromodeoxyuridine was added to the culture medium (10 µmol/L), and the cultures were incubated for 30 minutes at 37°C. The cells were fixed in 70% ethanol at –20°C. To denature the DNA, the cells were incubated for 30 minutes at room temperature in 2 N HCl with 0.5% Triton X-100. After neutralization with 0.1 mol/L sodium tetraborate (pH 8.5), the cells were incubated with anti-bromodeoxyuridine FITC (Becton Dickinson, San Jose, CA) for 30 minutes at room temperature and resuspended in 5 µg/mL propidium iodide. The cells were analyzed using an EPICS XL flow cytometer (Beckman Coulter). These procedures were also done in triplicate.

In vitro invasion assay. In vitro invasion assays were done by using 24-well transwell units with polycarbonate filters (pore size, 8 µm) coated on the upper side with Matrigel (Becton Dickinson). Cells (5.0 x 104 cells/well) were placed in the upper chamber, and the lower chamber was filled with 750 µL of DMEM with 10% FBS as a chemoattractant. After 48 hours of incubation at 37°C, the membranes were labeled with Calcein, AM solutions. The invasive cells that had migrated through the membrane to the lower surface were read in a fluorescence plate reader at excitation/emission wavelengths of 485/530 nm.

Statistical analysis. The statistical analysis was done using the {chi}2 method, the Mann-Whitney U test, Student's t test, and repeated measures ANOVA analysis. Survival curves were drawn according to the Kaplan-Meier method (26), and the log-rank test (27) was applied to compare the survival curves. A probability level of 0.05 was chosen for statistical significance.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Clinical significance of KLK6 expression in gastric cancer: Expression of KLK6 mRNA in cell lines and clinical tissue specimens. Figure 1 shows the KLK6 gene expression status in the human gastric cancer cell lines. Seven of the nine cell lines (78%) substantially expressed the KLK6 gene, whereas AZ521 and NUGC3 did not. We then quantitatively determined the KLK6 mRNA expression in clinical samples by comparison with a gastric cancer cell line, MKN28, as the quantifying standard, which expresses human KLK6 well. Fifty-seven of the 63 patients (88%; P < 0.0001; Mann-Whitney U test) showed a higher expression level of KLK6 mRNA in gastric cancer tissue specimens than in noncancerous tissue specimens. The mean expression value of KLK6 mRNA in cancer tissues (0.37 ± 0.53; mean ± SD, normalized by GAPDH gene expression), was significantly higher than the value (0.026 ± 0.060) in the corresponding noncancerous tissues (P < 0.000001; Student's t test; Fig. 2A).



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  		Fig. 1. Expression of KLK6 mRNA in gastric cancer cell lines and normal fibroblast cell line. Seven of nine (78%) diverse gastric cancer cell lines are positive for KLK6 mRNA expression, whereas nonmalignant cell lines, such as fibroblast KMST6, show a reduced expression of the gene.

 


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  		Fig. 2. Expression of KLK6 mRNA in gastric cancer tissue and normal mucosa. A, the mean KLK6 expression value in the gastric cancer tissues (0.37 ± 0.53, mean ± SD) was significantly higher than that in the corresponding normal tissues (0.026 ± 0.060). B, a representative gastric cancer case exhibited the KLK6 protein expression in cancer cells (top, x40 magnification). HE staining (bottom, x40 magnification).

 
Immunohistochemistry. In 18 representative cases among the above 66 cases, we found the specific expression of hK6 protein only in cancer tissue specimens in comparison to the corresponding normal tissue specimens. In six cases with a lower expression level of KLK6 mRNA, five cases exhibited a negative or weak expression of hK6 in cancer tissue specimens. In contrast, in 12 cases with the higher KLK6 mRNA, 9 cases only exhibited a positive or strong hK6 protein expression in cancer tissue specimens.

The clinicopathologic significance of KLK6 mRNA expression in gastric cancer. The clinicopathologic factors analyzed are shown in Table 1 in relation with KLK6 mRNA expression in tumor tissue. The incidence of lymphatic invasion was significantly higher (P = 0.03) in the high-expression group (17 of 20, 85%) than in the low-expression group (27 of 46, 59%). The incidence of serosal invasion was higher (P = 0.06) in the high-expression group (16 of 20, 80%) than in the low-expression group (26 of 46, 57%). The clinical stage also correlated with the groups (P = 0.03). On the other hand, no significant difference was observed regarding sex, histology, lymph node metastasis, and vascular invasion. The 5-year actuarial overall survival rates in patients with high KLK6 mRNA levels and patients with low KLK6 mRNA levels were 26% and 55%, respectively (Fig. 3). The survival difference between these two groups was statistically significant (P = 0.03; log-rank test).


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  		Table 1. Relationships between KLK6 status and other variables

 


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  		Fig. 3. Overall survival of patients with gastric cancer according to KLK6 mRNA expression in the cancer tissue. Patients in the high KLK6 mRNA expression group (n = 20) had a significantly poorer prognosis than those in the low KLK6 mRNA expression group (n = 46).

 
Biological significance of KLK6 expression in gastric carcinoma: The KLK6 small interfering RNA–transfected gastric cancer cell lines stably suppress both KLK6 mRNA and hK6 protein. Although the highest KLK6 expression was found in MKN74 (Fig. 1), this cell line showed an inherently low proliferation rate. Therefore, we used the MKN28 line for subsequent experiments. Among 45 stable KLK6-suppressed clones established using the RNA interference (RNAi) method (with KLK6 mRNA expression <0.3 compared with MKN28 parental cells), we selected two clones (RNAi-1 and RNAi-2) and determined their expression levels of hK6 protein by Western blot analysis. These two stable KLK6-suppressed clones were confirmed to express markedly lower levels (about one fifth) of hK6 protein than the MKN28 parental cells (Fig. 4).



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  		Fig. 4. Real-time reverse transcription-PCR and Western blot analysis of two KLK6-suppressed clones. The KLK6 mRNA expression is about 80% decreased in both treated clones (RNAi-1 and RNAi-2). The protein levels are measured by the NIH imager with ß-actin protein normalization. We also observed a reduced expression of hK6 protein in both clones.

 
Lower proliferation activity of KLK6-suppressed cancer cells. We analyzed whether suppression of KLK6 expression would alter the growth rate of MKN28 gastric cancer cells. As shown in Fig. 5A, there was a significant difference in growth rate between the KLK6-suppressed cells and the mock-transfected cells (P = 0.002). The KLK6-suppressed clones did not reach confluency by 10 days. To investigate whether the KLK6-suppressed cells showed low proliferation activity, we analyzed the cell cycle after serum starvation and after re-feeding with serum (Fig. 5B). The percentage of bromodeoxyuridine-positive cells (cells in S phase) in KLK6-suppressed cells was significantly lower than that in the mock-transfected cells after serum starvation for 48 hours and after addition of serum for 18 hours (P < 0.01). These results suggested that both KLK6 mRNA and hK6 protein expression were closely associated with cell proliferation.



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  		Fig. 5. Proliferation activity and cell cycling of KLK6-suppressed cells. The RNAi-1 clone in Fig. 4 was used for the following experiments. A, in vitro growth rate of KLK6-suppressed cells in the presence of 10% FBS. The cell number was counted on days 3, 7, and 10 in triplicate; bar, SD; P < 0.01; repeated measures ANOVA analysis. B, cell cycle of KLK6-suppressed cells after 48 hours of serum starvation followed by 18 hours of re-feeding with 10% FBS. (a) mock cells after 48 hours of serum starvation followed by 18 hours of re-feeding. (b) KLK6-suppressed cells after 48 hours of serum starvation followed by 18 hours of re-feeding. (c) mock and KLK6-suppressed cells after 48 hours of serum starvation. (d) mock and KLK6-suppressed cells 18 hours after re-feeding. C, the percentage of bromodeoxyuridine-positive cells in KLK6-suppressed cells after 48 hours of serum starvation. Each experiment was done in triplicate.

 
Low invasive potential of KLK6-suppressed cancer cells. In a clinicopathologic study, we found that the incidence of lymphatic invasion was significantly higher in the high-expression group than in the low-expression group. To verify these findings in an in vitro assay, we examined the invasive potential of the KLK6-suppressed cells using an in vitro Matrigel invasion assay (Fig. 6). The KLK6-suppressed cells exhibited significantly less invasive potential than the mock-transfected cells (P < 0.01), suggesting that high expression of KLK6 enhanced tumor invasiveness.



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  		Fig. 6. Invasion potential of KLK6-suppressed cells. The invasive cells migrating through the membrane to the lower surface were counted by a fluorescence plate reader at excitation/emission wavelength of 485/530 nm; bar, SD; P < 0.01; Student's t test.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we clearly showed a sharp contrast between gastric cancer tissue and the corresponding normal counterpart with respect to KLK6 mRNA expression, as illustrated in Fig. 2. To date, several authors have reported that KLK6 mRNA is highly expressed in human cancer tissues (28–30). Among such gastrointestinal malignancies as colorectal cancer, Schuster et al. investigated the gene expression levels of KLK6 by real-time reverse transcription-PCR of colorectal cancer and normal colon mucosa tissues (31). They found the mean KLK6 expression value in normal samples to be approximately two logs lower than that of the cancer samples. Yousef et al. also reported that KLK6 was overexpressed in colorectal cancer tissues compared with normal colon tissues (30). Moreover, our study showed a drastic change in the KLK6 mRNA expression between the gastric cancer tissue and the normal mucosa. However, none of the previous studies investigated the clinicopathologic significance of the KLK6 expression in cancer tissues. Thus, in this study, we examined the clinicopathologic correlation to the KLK6 expression status in gastric cancer. The findings indicate that the overexpression of KLK6 was significantly associated with both an increased incidence of lymphatic invasion and a poor prognosis for patients with gastric cancer. These findings suggest that an enhanced expression of KLK6 might play an important role in various pathologic processes of gastric cancer.

To ascertain the contribution of KLK6 to cell proliferation and invasiveness, we used MKN28 cells, which inherently expressed high levels of both KLK6 mRNA and hK6 protein, and established the relevant suppressed clones by gene silencing using RNAi techniques. RNAi is mediated by siRNAs that are produced from the long dsRNAs of exogenous or endogenous origin by an endonuclease of the RNase-III type, called Dicer (32, 33), and has emerged as a powerful tool for understanding the gene function. With the aid of RNAi techniques, we showed that the KLK6-suppressed clones had markedly reduced cell growth, proliferation, and invasiveness. Regarding other KLKs, such as KLK2 or KLK3, several reports have indicated that hK2 and hK3 might stimulate the growth and survival of tumor cells by degrading insulin-like growth factor binding proteins (IGFBP2, 3, 4, and 5), thereby liberating the mitogenic growth factor insulin-like growth factor I (IGF-I), which binds to its cell-surface receptor (IGF-IR) and induces cell proliferation and prevents apoptosis (34, 35). Furthermore, Magklara et al. reported that hK6 can degrade in vitro fibrinogen and collagen type I, basic constituents of the extracellular matrix, as well as collagen type IV, a major component of the basement membrane (36). Another group has also shown that hK6 can digest laminin and fibronectin in the tumor parenchyma (37). The lysis of certain components of the extracellular matrix disrupts their dynamic interactions with cells and is linked with an altered regulation of cell proliferation that can lead to tumor cell growth and malignant transformation. The results of our studies suggest that the increased hK6 expression may be associated with pericellular proteolysis and tumor invasion. Because our in vitro experiments showed that KLK6 gene silencing successfully reduced tumor cell proliferation and invasiveness, siRNA-mediated gene silencing of KLK6 might conceivably be a suitable candidate in therapy for patients with gastric cancer.

Finally, the current study indicates that KLK6 mRNA was remarkably overexpressed in gastric cancer tissues and high KLK6 expression levels were associated with lymphatic invasion and poor patient prognosis. hK3 has been well-documented to be an excellent tumor marker for prostate cancer. Moreover, hK6 is a promising serum biomarker for ovarian cancer. Therefore, studies are now under way to investigate whether hK6 may also be a useful biomarker for gastric cancer using serum samples from patients at our institute.


   Acknowledgments
 
We thank Drs. Mitsuhiko Ohta, Tomoya Sudo, Kazuhiko Ogawa, Fumiaki Tanaka, and Hiroshi Inoue for important discussion. We thank M. Kasagi, M. Oda, K. Ogata, T. Shimooka, and M. Nagahara for their technical valuable assistance.


   Footnotes
 
Grant support: Grant-in-aid for Scientific Research (B) (15390398, 15390379, 16390381); grant-in-aid for Scientific Research (C) (15591412, 15591411) from Japan Society for the Promotion of Science; grant-in-aid for Exploratory Research (16659337) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Public Trust Haraguchi Memorial Cancer Research Fund and the Sankyo Foundation of Life Science also supported this study.

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.

Received 4/30/05; revised 7/ 5/05; accepted 7/12/05.


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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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