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Clinical Cancer Research Vol. 11, 2930-2936, April 15, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Phospho-Akt Expression Is Associated with a Favorable Outcome in Non–Small Cell Lung Cancer

Amit Shah1, William A. Swain1, Donna Richardson1, John Edwards2, Duncan J. Stewart1, Catherine M. Richardson1, Daniel E.B. Swinson1, Dipika Patel1, J. Louise Jones3 and Kenneth J. O'Byrne4

Authors' Affiliations: University Departments of 1 Oncology, 2 Thoracic Surgery, and 3 Pathology, University of Leicester, Leicester, United Kingdom and 4 Department of Oncology, St James's Hospital, Dublin, Ireland

Requests for reprints: Kenneth J. O'Byrne, HOPE Department, St James's Hospital, Dublin 8, Ireland. Phone: 353-1-4103546; Fax: 353-1-4103428; E-mail: obyrne{at}stjames.ie.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 DISCUSSION
 References
 
Akt, a Serine/Threonine protein kinase, mediates growth factor–associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non–small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables.

Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037).

These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC.

Key Words: Protein serine-threonine kinases • Lung cancer • Molecular diagnosis and prognosis • New targets

Lung cancer is the leading cause of cancer-related mortality worldwide with non–small cell lung cancer (NSCLC) accounting for ~80% of cases. The lack of major improvements in the 5-year survival rate of NSCLC, despite advances in surgery, chemotherapy, and radiotherapy, has driven the search for new strategies aimed at improving lung cancer management. Understanding the biology of lung cancer may lead to the identification of novel targets for the treatment and chemoprevention of this disease.

Akt, or protein kinase B, is a Serine/Threonine protein kinase, which mediates growth factor–associated cell survival (1). Akt is the cellular homologue product of the v-akt oncogene and has three isoforms, Akt-1, Akt-2, and Akt-3. All three isoforms are ubiquitously expressed in normal tissues, although the levels of expression among tissues vary. Akt-1 and Akt-2 are expressed in the brain, thymus, and lungs and Akt-3 in the brain and testis. Akt is activated downstream from phosphatidylinositol 3-kinase (PI3K), by a variety of growth factors, including insulin, insulin-like growth factor-I, and epidermal growth factor. Full activity is achieved by phosphorylation at Thr308/309 in the kinase activation loop and Ser473/474 in the COOH-terminal tail (2).

The tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) is a phospholipid phosphatase that antagonizes PI3K (3) thus regulating the activity of Akt. Specifically, loss of PTEN leads to activation of Akt, which in turn promotes antiapoptotic and pro-cell cycle entry pathways believed to be essential in tumorigenesis. Once activated, phospho-Akt (P-Akt) is a powerful promoter of cell survival as it antagonizes and inactivates various components of the apoptotic cascade such as proapoptotic Bad (2), caspase-9 (4), and forkhead transcription factor family members (5). Akt activates the transcription factor nuclear factor-{kappa}B leading to the transcription of antiapoptotic and proinflammatory genes (6). Akt has been shown to influence the cell cycle through regulation of cyclin D1 stability and inhibition of the cell cycle inhibitor p27Kip1 (7). Furthermore, Akt has been implicated in regulating angiogenesis (8) and metastasis (9), two important processes in cancer development.

The role of Akt in carcinogenesis has been well documented and Akt is overexpressed in a variety of human cancer types (2, 10). Akt has been associated with the initiation of tumorigenesis in pancreatic cancer (11) and gliomas (12) and seems to correlate with stage and tumor grade in prostate cancer (13). Furthermore, PTEN is frequently lost or inactivated by mutation in human cancers (14), whereas the positive regulator of Akt, PI3K, is commonly up-regulated (15). Finally, activated Akt has been shown to induce cellular transformation (16).

Recently, West et al. (17) showed the rapid activation of Akt by nicotine and other tobacco carcinogens. When taken together with the data showing constitutively activated Akt in 90% of experimented NSCLC cell lines and that Akt confers chemoresistance in NSCLC cell lines (18, 19), the use of Akt as a prognostic and predictive marker may contribute to the assessment of its relevance as a therapeutic target for the future management of NSCLC.

In this study, we investigated the patterns of Akt expression and P-Akt levels in NSCLC and their correlation with clinicopathologic variables. Survival analysis was done to define the prognostic relevance of P-Akt expression.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 DISCUSSION
 References
 
Tissues. Archival snap-frozen and paraffin-embedded tissue specimens from 82 sequential patients, with stage I to IIIA NSCLC, treated with surgery alone over a 24-month period were studied. The final staging and histologic diagnosis was based on the histopathology report. Fifty-three patients were male and 29 female with a median age at time of surgery of 70.6 years (range, 34.2-84.9). Squamous cell carcinoma was the most frequent cancer subtype with 46 cases. Twenty-two patients had adenocarcinoma and 14 cases were classified as "other", which included large cell carcinoma and those that were pathologically unclassified. Survival data were available for all 82 patients. Only patients surviving >60 days after operation were included in the study to avoid bias from perioperative death.

Western blotting. Snap-frozen samples were homogenized mechanically in a buffer [0.16 mol/L NaCl, 0.11 mol/L Tris-HCl (pH 8.0), 0.01% Triton, 9.04 mg/mL diethylpyrocarbonate, and 2.1 mol/L EDTA (pH 8.0); Sigma, St. Louis, MO] containing protease inhibitors, before sonification and centrifugation at 4°C for 15 minutes. Samples were loaded sequentially onto a vertical, 10% SDS-PAGE, at a concentration of 150 µg protein per well. Proteins were resolved by the application of 150 V to the gel, following which they were transferred onto a nitrocellulose membrane (Hyperbond-Enhanced Chemiluminescence, Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Membranes were blocked for 1 hour with 5% nonfat dry milk in Tris-HCl buffered saline containing 0.1% Tween 20 (TBST; for Akt and {alpha}-actin), or 7% nonfat dry milk in TBST (for P-Akt). The membranes were incubated overnight at 4°C with primary antibodies raised against Akt1 [polyclonal anti-Akt1 goat IgG; Santa Cruz Biotechnology, Santa Cruz, CA; 1:2,000 in TBST with 5% nonfat dry milk], {alpha}-actin (polyclonal anti-{alpha}-actin goat IgG, Santa Cruz Biotechnology, 1:500 in TBST with 5% nonfat dry milk) and P-Akt [polyclonal anti-phospho-Akt (Ser473) rabbit IgG; New England Biolabs, Beverly, MA; 1:1,000 in TBST]. Membranes were incubated with an anti-goat horseradish peroxidase-conjugated secondary antibody for Akt1 and {alpha}-actin (Santa Cruz Biotechnology) or anti-rabbit for P-Akt (Amersham Pharmacia Biotech) for 1 hour at room temperature. Membranes were developed using enhanced chemiluminescence and then exposed to Hyperfilm, following the manufacturer's protocol (Amersham Pharmacia Biotech). The membranes were chemically stripped of antibodies using stripping buffer [2% SDS, 20 mmol/L Tris (pH 6.5), and 7 mmol/L ß-mercaptoethanol], and reblocked before reprobing. P-Akt was probed first, followed by Akt and {alpha}-actin. Developed films were scanned and band densitometry calculated on a densitometer (ChemiGenius 2 Bio Imaging System and Gene Tools software; Syngene, Cambridge, United Kingdom). Background activity was automatically deducted from each sample by the software. An epidermal growth factor–stimulated BEAS2B cell line was used as a positive control and was run on each gel. Akt and P-Akt densitometry results were also normalized to {alpha}-actin. All experiments were done initially in triplicate (19 cases) and subsequently in duplicate when reproducibility of the technique had been demonstrated (see Results). The mean score was used as the final result in each case. Although with our experience of semiquantitative Western blotting protocols, we expected the densitometry values to be in the linear part of the dose-response curve, we did not assess this specifically in this study. Therefore, the values were analyzed as categorical variables with the median value as the cut point (20).

Immunohistochemistry. For the detection of P-Akt, the streptavidin-biotin complex (StreptABC) method was used employing the polyclonal anti-phospho-Akt (Ser473) immunohistochemistry-specific rabbit IgG antibody (New England Biolabs). Four-micrometer-thick sections were cut from routinely processed, formalin-fixed, paraffin-embedded tissue blocks. Sections were dewaxed and rehydrated through graded alcohols. For antigen retrieval, the sections were boiled in 10 mmol/L citric acid buffer (pH 6.0) in a pressure cooker, for 2 minutes, followed by washes in distilled water and TBST. Following each incubation step, three 5-minute washes in TBST were done. Endogenous peroxidase activity was quenched by incubation in TBST containing 3% H2O2 for 10 minutes. To block nonspecific background staining the sections were incubated in TBST containing 5% normal goat serum for 1 hour. The primary antibody was applied (dilution 1:100) in TBST containing 5% normal goat serum and incubated overnight at 4°C. Sections were incubated in biotinylated goat anti-rabbit IgG antibody (1:300) for 30 minutes at room temperature. The streptavidin-biotinylated peroxidase solution was prepared as recommended by the manufacturer and applied for 15 minutes at room temperature. Sections were stained with liquid Diaminobenzidine Plus (Dakocytomation, Cambridge, United Kingdom) for 2 minutes and counterstained in Mayers hematoxylin. Sections were dehydrated and mounted in DPX (BDH Chemicals Ltd., Poole, England) and analyzed by standard light microscopy. High Gleason grade prostate cancer sections with high levels of P-Akt (13) served as positive controls. Sections were immunostained with omission of the primary antibody as a negative control.

Assessment of P-Akt. The extent and pattern of P-Akt specific immunostaining within a tissue section was determined by the percentage of cells with staining of the nucleus, cytoplasm or cell membrane. Tumor sections were inspected at x100 and x400 magnification and the overall percentage of cells on the section immunostaining was determined according to the pattern of intracellular localization. Sections were assessed in a blinded fashion by two independent observers (A.S. and D.R.). Where a disagreement was recorded a consensus was determined using a dual-headed microscope. J.L.J., a consultant histopathologist, adjudicated unresolved differences.

Statistical analysis. Statistical analysis was done using the SPSS software system (SPSS for Windows, version 9.0; SPSS, Inc., Chicago, IL). The {chi}2 test was used to analyze associations with categorical clinicopathologic variables. P-Akt Western blot densitometry values from the first two of the three runs were compared with linear regression analysis. Survival curves were plotted using the Kaplan-Meier method and the log-rank test was used to assess any differences. Multivariate analysis was done using a forward, stepwise, Cox proportional hazard model, with P < 0.1 on univariate analysis required for entry into the model. The effect of P-Akt levels, in terms of Western blot densitometry and immunohistochemical staining, was incorporated into these models as categorical variables using the median value as the cut point to define two groups of low and high levels. All Ps are two sided and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
DISCUSSION
 References
 
Western blotting analysis. Immunoreactive bands were seen, corresponding with the P-Akt positive control at Mr 57,000. Figure 1 shows a representative blot of eight NSCLC tumor samples. The median values of P-Akt and Akt taken as a percentage of the positive control and then corrected for {alpha}-actin were 12% and 139%, respectively. There was a close correlation between the P-Akt densitometry values of the first two runs, expressed as a percentage of the value for the positive control sample on each gel (r = 0.978, Fig. 2).



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  		Fig. 1. P-Akt (Ser473) band densities for eight NSCLC tumor samples (top). Corresponding Akt band densities (middle). Bands obtained after probing for {alpha}-actin (bottom).

 


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  		Fig. 2. A close correlation was observed between the P-Akt densitometry values of the first two runs (r = 0.978). Each sample is expressed as % value for the positive control sample on each gel.

 
There was a significant correlation between Akt/{alpha}-actin expression and P-Akt/{alpha}-actin levels (P < 0.001). Akt/{alpha}-actin (data not shown) had no significant correlations with any clinicopathologic variables. P-Akt/{alpha}-actin correlated significantly with poorly differentiated tumors (P = 0.02) and with the histologic subtype (P = 0.039), as shown in Table 1. No other correlations were seen.


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  		Table 1. Correlations between P-Akt and clinicopathologic characteristics using the {chi}2 test

 
Immunohistochemistry analysis. Of the 78 cases evaluated, 96% (75 of 78) were immunoreactive for P-Akt. P-Akt expression was confined to tumor cells only. On each individual section, the percentage of tumor cells staining positive ranged from 1% to 90% (median, 15%); however, cytoplasmic P-Akt (CP-Akt) staining was observed in all tumor cells staining positive (Fig. 3A). In addition, nuclear staining (NP-Akt) was observed in 42% (32 of 78) of cases (Fig. 3B). Nuclear staining was analyzed as either present or absent. Membranous staining was seen in only 5% (4 of 78) of cases with a range of 1% to 5% of tumor cells staining positive. Both CP-Akt and NP-Akt correlated strongly with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt correlated with the presence of nodal involvement (P = 0.013) and with squamous versus nonsquamous histologic subtype (P = 0.037). No further correlations were seen between CP-Akt and NP-Akt and other clinicopathologic factors (Table 2).



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  		Fig. 3. A, There is positive cytoplasmic immunostaining in the NSCLC tumor cells (large arrow) but negative staining in the nuclei (small arrow; bar, 40 µm) and surrounding stroma. B, There is strong nuclear immunostaining in the NSCLC tumor cells (arrow; bar, 40 µm) with concurrent cytoplasmic staining.

 

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  		Table 2. Correlations between cytoplasmic and nuclear P-Akt and clinicopathologic characteristics using the {chi}2 test

 
Prognostic effect of P-Akt. In univariate analysis, high P-Akt/{alpha}-actin ratio correlated with a favorable prognosis when assessed as a categorical variable (greater or less than median value; P = 0.007; Fig. 4). In multivariate analysis with the clinicopathologic factors (identified by univariate analysis, P < 0.1), P-Akt/{alpha}-actin and tumor-node-metastasis stage were the only independent prognostic factors (P = 0.04 and 0.009, respectively; Table 3). Pathologic tumor stage was not an independent prognostic factor (P = 0.059). Gender, histologic subtype, tumor grade, and nodal status were not prognostic variables in univariate analysis (P > 0.1). No prognostic significance was seen with either CP-Akt or NP-Akt expression (P = 0.368 and 0.291, respectively).



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  		Fig. 4. The median densitometry units were used as a cut point (P = 0.007, log rank).

 

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  		Table 3. Cox multivariate regression analysis

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 DISCUSSION
References
 
This study further supports the growing evidence that Akt is expressed and activated in NSCLC, regardless of the histologic subtype, and can be detected using immunohistochemistry and Western blotting techniques. A considerable proportion of the samples tested (96%) showed P-Akt immunoreactivity in tumor cells. Moreover, P-Akt was only detected in tumor cells and the surrounding normal alveolar epithelium and stroma remained negative. Similar to previous reports (21, 22), we observed P-Akt staining localized to the cytoplasm and the nucleus. P-Akt staining in prostate cancer, and to some extent breast cancer, seems to be largely localized at the cell membrane (13, 23), whereas a study in primary epithelial ovarian carcinomas showed a predominant nuclear localization (24). It has been documented that Akt, once activated, translocates to the cytoplasm and the nucleus (25). Unfortunately, unlike the cytoplasmic function of Akt, the role of nuclear translocation is yet to be completely defined. It is possible that it may be simply a regulatory mechanism or may in actual fact be targeting nuclear substrates. Elucidating a nuclear function for Akt will provide critical information that will enable us to further understand its physiologic role.

A significant association between P-Akt levels and histologic subtype was seen. Using Western blotting, low P-Akt levels correlated with adenocarcinoma (P = 0.039). On immunohistochemistry, NP-Akt correlated with squamous histologic subtype (P = 0.037). This is in contrast to the study by Lee et al. (21) that found no difference with histologic subtype, but this was in only 43 cases. Whereas there was a significant inverse relationship between P-Akt and histologic grade using immunohistochemistry (P = 0.008 and 0.017, CP-Akt and NP-Akt, respectively), a contradictory significant association was seen between P-Akt and poorly differentiated tumors with Western blotting (P = 0.02). The reason for this is unknown. A {chi}2 analysis was done to evaluate the relationship between the two methodologies and no correlation was observed between P-Akt analyzed by Western blotting and either CP-Akt or NP-Akt (P = 1.000 and 0.650, respectively). The lack of correlation seen between the two methodologies may not be all that surprising as both methods characterized P-Akt in different ways. Immunohistochemistry can only truly describe the localization of P-Akt and quantification is more subjective. In contrast, whereas Western blotting can quantify total P-Akt, the method cannot distinguish its source in whole tumor homogenates. This may explain why there was no association between P-Akt and nodal stage on Western blotting (P = 0.647), as seen in the NP-Akt immunohistochemistry (P = 0.022). Furthermore, in this study, the staining in the cytoplasmic and nuclear compartments was scored separately.

The correlation between P-Akt and tumor grade has been seen in previous studies with other tumor types (12, 13). Our study showed a strong correlation between both CP-Akt and NP-Akt and well-differentiated NSCLC tumors (P = 0.008 and 0.017, respectively), which is in keeping with the findings of Mukohara et al. (26). These findings suggest that those tumors with relatively high P-Akt levels may remain well-differentiated and exhibit a slower growth rate whereas those that survive and grow independent of Akt activity may form poorly differentiated tumors with increased proliferation rate. Several studies have implicated Akt in promoting the progression of the cell cycle (7, 27, 28); however, there has been a lack of correlation between Akt and the proposed substrates of the cell cycle in studies analyzing human tumor samples (12, 23). Furthermore, dominant-negative Akt has less of an effect on induction of the cell cycle than on cell death (29). Finally, overexpression and stabilization of cyclin D1 does not always lead to cell cycle progression and in some cases may be associated with decreased cell proliferation (30). Therefore, the possibility exists that increased Akt activity may lead to reduced cellular growth. In keeping with this proposal, Perez-Tenorio et al. showed P-Akt to strongly associate with a lower S-phase fraction (23).

The association of P-Akt and metastasis has been previously reported in other tumor types (26, 31). Despite this, evidence suggests that P-Akt may play a role in the development of NSCLC rather than the progression of the disease. Lee et al. saw no difference in P-Akt levels between the primary NSCLC tumor and the corresponding metastatic node (21). Our study showed a similar finding when the percentage of tumors staining positive for P-Akt, regardless of subcellular localization was analyzed (data not shown); however, it was interesting to find a correlation between NP-Akt and lymph node metastasis (P = 0.022). This may suggest a possible role of NP-Akt in NSCLC, although the mechanism by which P-Akt may regulate metastatic progression in the nucleus is unknown. Arboleda et al. found that Akt2, but not Akt1 or Akt3 was able to increase cell invasiveness (32) and it may be possible that the different isoforms, although thought to have similar physiologic actions, may in fact differ in their subcellular localization, have different substrate specificities and thus differ in their physiologic actions in various tissue types. The relevance of the association of NP-Akt with nodal metastases found in this study needs further investigation.

The prognostic significance of P-Akt tumor cell expression has been examined in other cancers, although evidence is still limited. In renal cell carcinoma, P-Akt was associated with poor prognosis on univariate but not multivariate analysis (33). Ermoian et al. found no association between P-Akt and patient survival in gliomas; however, the expression of PTEN significantly predicted prognosis in this patient group (12). P-Akt does not seem to predict survival in breast cancer; however, it does predict patient relapse (23). Evidence thus far would suggest that P-Akt in other tumor types might be associated with factors that predict poor prognosis, such as tumor stage, grade, histology, metastatic disease, and importantly, loss or mutation of PTEN. In NSCLC, the prognostic value of P-Akt has not been well described.

The expression of P-Akt assessed by immunohistochemical techniques did not correlate with patient prognosis, a similar finding to that of Tsao et al. (34). However, P-Akt protein levels, as assessed by Western blotting and semiquantitative densitometry, correlated with a good prognosis in univariate and multivariate analysis. This is in keeping with the recent findings from the study by Cappuzzo et al. in which tumors positive for P-Akt expression was associated with better response rate, time to progression, and disease control rate when treated with gefitinib. In multivariate analysis, P-Akt positivity was associated with a reduced risk of disease progression (35). Possible explanations for this unexpected association may be due to a reduced growth rate and well-differentiated tumor phenotype as a result of increased Akt activity. A recent study showed insulin-like growth factor-I, a potent mitogenic factor, was in fact growth inhibitory to a human lung adenocarcinoma cell line mediated by sustained Akt activation (36). A number of previous experimental studies support a role for mitogenic and antiapoptotic factors as possessing growth inhibitory actions. Lawlor et al. showed induction of the cell cycle kinase inhibitor p21Cip1/WAF1 by constitutively activated Akt (37). Another study reported that epidermal growth factor was growth inhibitory to lung tumor cells (38). That antiapoptotic modulators may confer a favorable patient outcome in NSCLC has previously been reported. Overexpression of Bcl-2, an antiapoptotic protein, has been shown in some studies as being a favorable independent prognostic indicator of survival in early-stage NSCLC (39, 40).

In breast cancer, estrogen receptor status is well recognized as a good prognostic factor and yet is an effective target for anti-hormone therapies such as tamoxifen (41, 42). Likewise, P-Akt deserves investigation as a potential novel target in NSCLC. Studies have shown Akt playing a pivotal role in survival of cells when exposed to different apoptotic stimuli such as growth factor withdrawal and DNA damage, but more importantly also promotes chemoresistance (18). In vitro and in vivo studies that have combined small molecule inhibitors of the PI3K/Akt pathway with standard chemotherapy have been successful in attenuating chemotherapeutic resistance. Thus, specific small molecules (43–45) and natural compounds, such as deguelin (46, 47), targeting Akt and other components of the pathway are now being developed for clinical use to overcome therapeutic resistance to conventional treatment, such as cytotoxic chemotherapy and radiotherapy. Specifically inhibiting Akt activity may be a valid approach to treat cancer and increase the efficacy of chemotherapy.

In conclusion, high levels of P-Akt protein, as assessed by Western blot analysis, are a favorable prognostic factor in NSCLC independent of stage; however, evidence to date suggests that Akt has a key role in chemotherapy and radiotherapy resistance (18, 48). Restoration of PTEN or inhibition of Akt may enable patients to respond better to cytotoxic drugs that initiate the apoptotic process (18, 46, 49, 50). The high percentage of P-Akt positive patients seen in this study, support an important role for P-Akt in the pathogenesis of this malignancy. The physiologic role of nuclear localization of P-Akt along with the role of P-Akt on the cell cycle and tumor differentiation requires further investigation. The inhibition of Akt is a potential novel therapeutic target for the management of NSCLC. Furthermore, Akt inhibitors may have a role to play in the chemoprevention of the disease.


   Footnotes
 
Grant support: The Health Foundation, London, United Kingdom (A. Shah).

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 7/14/04; revised 11/ 9/04; accepted 11/29/04.


   References
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 Abstract
 Materials and Methods
 Results
 DISCUSSION
 References
 

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