Research Communication ST6GalNAcII Mediates the Invasive Properties of Breast Carcinoma Through PI3K/Akt/NF-jB Signaling Pathway

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1 Research Communication ST6GalNAcII Mediates the Invasive Properties of Breast Carcinoma Through PI3K/Akt/NF-jB Signaling Pathway Dongliang Ren 1 Li Jia 2 Yanyan Li 1 Yanxin Gong 1 Chen Liu 3 Xu Zhang 2 Ning Wang 2 Yongfu Zhao 3 * 1 Department of General Surgery, Dalian Medical University, Liaoning Province, China 2 College of Laboratory Medicine, Dalian Medical University, Liaoning Province, China 3 Department of General Surgery, The Second Affiliated Hospital of Dalian Medical University, Liaoning Province, China Abstract Metastasis of tumor cells is the most deadly attribute of breast cancer patients. Aberrant sialylation is closely associated with malignant phenotype of tumor cells, including invasiveness and metastasis. The objective of this study is to clarify the possible role and mechanism of ST6GalNAcII in the metastasis process of breast carcinoma. Real-time PCR, Western blot, and immunohistochemical were used to analyze differential expression of ST6GalNAc II in breast carcinoma cell lines and tissue specimens. PI3K/AKt signaling pathway was also analyzed. The high expression level of ST6GalNAcII was corresponding to invasive phenotype of breast cancer cells both in vitro and in vivo. Further data indicated that manipulation of ST6GalNAcII gene expression led to alter the activity of phosphoinositide-3 kinase (PI3K)/Akt signaling pathway. Blocking the PI3K/Akt pathway resulted in reduced capacity in invasion of MDA-MB-231 cells. ST6GalNAcII elucidated the unusual properties of invasion in breast cancer cell via modulating the PI3K/AKt signaling pathway. VC 2014 IUBMB Life, 66(4): , 2014 Keywords: breast carcinoma; ST6GalNAcII; PI3K/AKt signaling; metastasis Introduction Breast cancer is one of the most common cancers in women around the world. Metastasis of tumor cells, which shows a multi-step process and involves intrinsic and extrinsic factors, is the most deadly attribute of patients with breast cancer. More recently, advances in molecular biology have permitted the study of post-translational events, such as specific changes VC 2014 International Union of Biochemistry and Molecular Biology Volume 66, Number 4, April 2014, Pages *Address correspondence to: Yongfu Zhao, Department of General Surgery, the Second Affiliated Hospital of Dalian Medical University, 465 Zhongshan Road, Dalian , Liaoning Province, China. Tel: Fax: zyf0386@sina.com Received 26 February 2014; Accepted 2 April 2014 DOI /iub.1268 Published online 23 April 2014 in Wiley Online Library (wileyonlinelibrary.com) in the glycosylation pattern of cell surface glycoproteins, which have been shown to enhance the metastatic efficiency of tumor cells, and in particular that of terminal sialylation (1). In mammals, sialic acids are located at the non-reducing terminal position of the glycoproteins and glycolipids and the alteration in cell surface sialylated antigens affects many cellular properties: cell cell adhesion, cell extracellular matrix adhesion, immune defense, cell metastasis, and invasion abilities (2 5). A tendency for the addition of shorter O-glycans was founded in breast carcinomas (6), and it was associated with increased sialylation (7). Interestingly, this tendency resulted in the overexpression of sialylated antigens at the surface of cancer cells. The biosynthesis of sialylated oligosaccharide sequences is catalyzed by a family of enzymes named sialyltransferases (STs) (8). They all catalyze the transfer of a sialic acid residue from CMP-Neu5Ac to oligosaccharide chains of glycolipids and glycoproteins. STs consist of 20 members which is subjected into three subfamilies according to the linkage form: alpha-2,3- sialyltransferases (ST3GalI-VI), alpha-2,6-sialyltransferases 300 IUBMB Life

2 (ST6GalI-II and ST6GalNAcI-VI), and alpha-2,8-sialyltransferases (ST8SiaI-VI) (9). ST6GalNAcI-VI catalyze the transfer of Neu5Ac to GalNAc. Some changes in ST6GalNAc expression in several tumors have been reported. ST6GalNAcI and II are capable of sialylating Neu5Aca2 6GalNAc-O-Ser/Thr (Tn antigens) which is associated with carcinoma aggressiveness and poor prognosis (9). ST6GalNAcI expression is significantly associated with better prognosis in breast cancer and is sufficient to enhance the tumourigenicity of MDA-MB-231 breast cancer cells (10,11). Upregulation of ST6GalNAcII is related to poor survival in human colorectal carcinomas (12). Down-regulated expression of ST6GalNAcII mrna is not only associated with the pathological phenotype of IgA nephropathy but also with the poor prognosis in IgA nephropathy patients (13 15). It is well known that the phosphoinositide 3 kinase (PI3K)/ Akt pathway involves in many cellular processes including proliferation, differentiation, apoptosis, cell cycle progression, cell motility and tumorigenesis, tumor growth, and angiogenesis (16,17). Dysregulation of this signal transduction pathway has been shown to be associated with more aggressive tumor behavior and poor prognosis in some subtypes of breast cancer (18 20). Platycodin D plays a substantial role in inhibiting the migration, invasion, and growth of MDA-MB-231 human breast cancer cell via suppression of epidermal growth factor receptor (EGFR)-mediated PI3K/Akt and MAPK pathways (21). Proliferation was inhibited and apoptosis was induced in MCF-7 cell by wortmannin through down-regulation of PI3K/Akt signaling and nuclear factor (NF)-kappaB protein expression (22). However, little is known about ST6GalNAcII and its signaling pathway in relation to malignant phenotype of breast cancer. Therefore, in the current study, we determine the expression levels of ST6GalNAcII in MCF-7 and MDA-MB-231 cell lines and breast cancer tissue samples. In addition, we investigated whether ST6GalNAcII participate in the regulation of tumor invasion via PI3K/Akt pathway and the possible mechanisms. Materials and Methods Cell Culture Human breast carcinoma cell lines MCF-7 and MDA-MB-231 were purchased from KeyGEN (China). Compared with MCF-7, MDA-MB-231 had a high metastasis rate. The two cell lines were cultured in 90% Dulbecco Modified Eagle Medium (DMEM) (Gibco, State of California, USA) supplemented with antibiotics (13 penicillin/streptomycin 100 U/mL, Gibco) and 10% heatinactivated fetal bovine serum (Gibco). Cells were incubated at 37 C in a humidified atmosphere containing 5% CO 2. Tissue Samples of Patients After obtaining informed consent, breast cancer and transitional tissues (3 cm from the tumor edge) were collected from the same 101 patients who underwent surgical resections from July 2010 to May 2012 at the Second Affiliated Hospital of Dalian Medical University. The investigation project and its informed consent have been certified by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University. The extracted samples were confirmed by pathological diagnosis according to the International Union against Cancer (UICC). Medical records were used to extract clinicopathological data. No patients had received chemotherapy or radiation therapy. These tissues were snap-frozen in liquid nitrogen and stored at 280 C until used. Real-time Polymerase Chain Reaction Analysis The gene expression level of ST6GalNAcII was analyzed by real-time polymerase chain reaction (PCR). The total RNA was extracted from two breast carcinoma cell lines using the RNeasy Mini Kit (QIAGEN, Valencia, CA). The cdna was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer s instruction. Real time PCR was carried out on an ABI Prism 7500 fast real time PCR system (Applied Biosystems, Foster City, CA) using QuantiTect SYBR Green PCR Kit (QIAGEN). PCR amplification was performed using 40 cycles of 95 C for 15 Sec, 60 C for 15 Sec, and 72 C for 30 Sec. The primer sequences used for amplification were as follows: F: 5 0 -CTTTGCCCTGTACTTCTCG-3 0 and R: 5 0 -CAGCACTGGAATGGAGAGA-3 0 for ST6GalNAcII; F: 5 0 -CTCC TCCACCTTTGACGCTG-3 0 and R: 5 0 -TCCTCTTGTGCTCTTGC TGG-3 0 for glyceraldehyde phosphate dehydrogenase (GAPDH). Real-time RT-PCR analysis was performed in triplicate. Expression level of target gene was normalized using the expression level of GAPDH mrna and compared between MC F-7 and MDA-MB-231 cell lines. Western Blot Analysis Whole cell proteins were electrophoresed under reducing conditions in 10% polyacrylamide gels. The separate proteins were transferred to a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk in phosphate buffer solution (PBS) containing 0.1% Tween 20 (PBST), the membrane was incubated with antibody (anti-st6galnacii, 1/500 diluted, Thermo, Rockford, USA; anti-pi3k, 1/200 diluted, Thermo; anti-p-akt T308, 1/200 diluted, Thermo; anti-p-akt S473, 1/200 diluted, Abgent; anti-akt 1/500 diluted, Abgent; anti-nfkb, 1/500 diluted, Abgent, San Diego, USA) and then with peroxidase-conjugated anti-rabbit IgG (1/5,000 diluted; GE Healthcare UK Ltd., Little Chalf-ont, UK). A GAPDH antibody (1/200 diluted; Santa Cruz Biotechnology, Santa Cruz, CA) was used as a control. All bands were detected using ECL Western blot kit (Amersham Biosciences, UK), and the bands were analyzed with LabWorks (TM ver4.6, UVP, BioImaging Systems, State of California, USA). RNAi Assay MDA-MB-231 cells were incubated in appropriate antibioticfree medium with 10% fetal bovine serum (Gibco), transferred to a 6-well tissue culture and incubated at 37 C, in a CO 2 incubator to obtain 60 80% confluens. According to the manufacturer s instruction, the cell cultures were transfected with ST6GalNAcII specific sirna Transfection Reagent Complex Ren et al. 301

3 IUBMB LIFE Fig 1 Expression levels of ST6GalNAcII are different in MDA-MB-231 and MCF-7 cell lines revealed by real-time PCR and Western blot. A: ST6GalNAcII gene expression was significantly higher in MDA-MB-231 cells than that in MCF-7 cells. B: ST6GalNAc II protein was abundant in MDA-MB-231 cells consistent with mrna level. Asterisk indicates significant difference from the groups than without an asterisk (*P < 0.05). (Santa Cruz Biotechnology). Transfer cells were cultured and incubated at 37 C for 6 H, followed by incubation with complete medium for additional 24 H. The cell transfection efficiency was 85% and the survival rate 92%, respectively. In Vitro Extracellular Matrix Invasion Assay The cell invasion in vitro was demonstrated by using 24-well transwell units (Corning, NY) with 8 lm pore size polycarbonate filter coated with extracellular matrix (ECMatrix gel; Chemicon, Amersfoort, The Netherlands) to form a continuous thin layer. Cells ( ) were harvested in serum-free medium containing 0.1% bovine serum albumin (BSA) and added to the upper chamber. The lower chamber contained 500 ll DMEM. Cells were incubated for 24 H at 37 C, 5% CO 2 incubator. At the end of incubation, the cells on the upper surface of the filter were completely removed by wiping with a cotton swab. Then the filters were fixed in methanol and were stained with Wright-Giemsa. Cells that had invaded the Matrigel and reached the lower surface of the filter were counted under a light microscope at a magnification of In Vivo Tumorigenicity Assay The tumorigenicity of ST6GalNAcII in vivo was investigated using a xenograft tumor model in the nude mice. Forty-five female athymic nude mice (5-week-old) were obtained from Animal Facility of Dalian Medical University, and were provided with sterilized food and water. They were divided into three groups and MDA-MB-231 cells (with or without ST6GalNAcII shrna interference and control shrna) were injected subcutaneously into the mammary fat pads area of each nude mouse, respectively. Once bearing palpable tumors (about 4 weeks after tumor cell inoculation), mice were sacrificed and their tumors were isolated, weighed, and photographed. Immunohistochemical Staining Analysis Immunohistochemistry assay was performed on paraffin embedded tumor sections. The slides were dried, deparaffinized, rehydrated, and then were immersed in 3% hydrogen peroxide for 10 min to quench the endogenous peroxidase. After consecutive washing with PBS, the slides were labeled overnight at 4 Cwith antibodies (Abcam, Cambridge, UK) at a dilution of 1:200 or isotype immunoglobulins (Santa Cruz Biotechnology) as negative control. The following staining was performed at room temperature for 60 min with secondary streptavidin-horse radish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology). Finally, the sections were counterstained with hematoxylin and cover-slipped. The Image-Pro Plus 4.5 Software (Media Cybernetics, Rockville, Maryland, USA) was used to analyze the expression of proteins. 302 ST6GalNAcII in the Invasive Properties of Breast Carcinoma

4 Fig 2 Effects of ST6GalNAcII on the invasive and migration ability of MDA-MB-231 cells in vitro. Silencing of ST6GalNAcII in MDA-MB-231 cells was analyzed by RNAi approach. A: Using real-time PCR analyzed, ST6GalNAcII gene expression was significantly reduced (*P < 0.05). B: After shrna transfection, distinct reduction of ST6GalNAcII was observed at protein levels by Western blot analysis (*P < 0.05). C: The average number of cells that invaded through the filter was counted by In vitro ECMatrix gel analysis. MDAMB-231-ST6GalNAcII shrna cells were significantly less invasive (*P < 0.05) than the MDA-MB-231 cells and MDA-MB-231-control shrna cells. Data are the average 6 SD of triplicate determinants. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Ren et al. 303

5 IUBMB LIFE Fig 3 ST6GalNAcII gene knockdown inhibits the tumorigenicity of MDA-MB-231 cells in vivo. A: Significant reduction of mean tumor weight in mice group with MDA-MB-231-ST6GalNAcII shrna1 tumors was observed, as compared with MDA-MB-231 and control groups (*P < 0.05). B: Reduced regulation of ST6GalNAcII was also shown by IHC staining in xenograft tumors derived from MDA-MB-231-ST6GalNAcII shrna1 cells (4003) (*P < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Inhibition of the PI3K/Akt Signaling LY (Sigma, Missouri, USA) were used to suppress the activity of the PI3K/Akt signaling in MDA-MB-231 cells. Briefly, MDA-MB-231 cells ( cells per well) were incubated with dimethyl sulfoxide (DMSO) or the PI3K inhibitor LY (20 lmol/l) dissolved in DMSO, and collected after 24 H. In addition, Akt expression was also silenced by RNAi. Changes in protein expression were measured by Western blot analysis. The tumorigenicity was also analyzed when PI3K/Akt signaling was blocked in xenograft tumor model. Sixty female athymic nude mice (5-week-old) were divided into four groups and MDA-MB-231 cells (with DMSO, LY294002, control shrna, and AKt sirna, respectively) were injected subcutaneously into the mammary fat pads area of each nude mouse, respectively. Once bearing palpable tumors (about 4 weeks after tumor cell inoculation), mice were sacrificed and their tumors were isolated, weighed, and photographed. Statistical Analysis SPSS 13.0 (SPSS Inc., Chicago, IL) was used. Each assay was performed at least in triplicate. Data are expressed as means 6 standard deviation (SD). Student s t-test and Chi-square test were used to determine the significance of differences in multiple comparisons. P < 0.05 was considered statistically significant. Results Differential Expression of ST6GalNAcII in Breast Carcinoma Cell Lines Compared to MCF-7 cells, MDA-MB-231 cells showed a remarkable increase in mrna level of ST6GalNAcII gene (5.88-folds). In protein level, Western blot analysis also confirmed this tendency (Fig. 1). These data indicated that ST6GalNAcII might be associated with metastasis of human breast carcinoma cells. 304 ST6GalNAcII in the Invasive Properties of Breast Carcinoma

6 Fig 4 ST6GalNAcII gene knockdown inhibits the activity of PI3K/Akt-PAK1 signal pathway. With Western blot analysis, the main signal molecules of PI3K/Akt/NF-jB signaling pathway were found to be downregulated at protein level in MDA-MB-231 cells with ST6GalNAcII shrna, as compared with those in MDA-MB-231 cells (*P < 0.05 vs. MDA-MB-231 cells and MDA-MB-231 cells with control shrna). The data are means 6 SD of three independent assays. Altered Expression of ST6GalNAcII Effects on the Invasive Ability of Breast Cancer Cells In Vitro and In Vivo ST6GalNAcII was silenced by shrna in order to elucidate its effect on the invasion and metastasis of breast cancer cells. As shown in Figs. 2A and 2B, compared to the control, the expression level of ST6GalNAcII was significantly reduced in MDA-MB-231 transfectants. Transwell assay evaluated that knockdown of ST6GalNAcII gene significantly inhibited invasive activity in MDA-MB-231-ST6GalNAcII shrna cells (Fig. 2C). In xenograft tumor model, the mean tumor weight in nude mice bearing MDA-MB-231-ST6GalNAcII shrna was significantly reduced compared to control (Fig. 3A). Then we performed immunohistochemical assay to study the expression of ST6GalNAcII in mice tumor tissues. It also revealed that ST6GalNAcII was lowly expressed in tumors derived from MDA-MB-231-ST6GalNAcII shrna cells (Fig. 3B). All of the results further indicated that ST6GalNAc II might play important roles in metastasis of human breast carcinoma cells. ST6GalNAcII Gene Mediates the Activity of PI3K/Akt Signaling Pathway in Breast Carcinoma Cells Given the critical role of PI3K/Akt pathway in tumor cells, we investigated whether ST6GalNAcII activated the PI3K/Akt pathway and whether this pathway played a pivotal role in ST6GalNAcII-mediated cell invasion. Western blot analysis showed that the levels of PI3 kinase p110a (the catalytic subunit of PI3K) and phosphorylation Akt were decreased in MDA- MB-231 cells with ST6GalNAcII shrna1 transfection (Fig. 4). Concomitantly, the degrees of phosphorylation of Akt at Ser473 and Thr308 and NF-jB were decreased markedly. By contrast, there was no variation in the total amount of Akt protein, which indicated a true decrease in phosphorylation status. These data might indicate a possible mechanism of ST6GalNAcII involved in PI3K/Akt pathway which regulated the invasion of breast carcinoma cell lines. Blocking PI3K/Akt Modulates the Invasive Ability of MDA-MB-231 Cells In Vitro and In Vivo To further determine the role of the PI3K/Akt signaling pathway, specific inhibitor of PI3K/Akt or Akt shrna to silence Akt were selected to treat MDA-MB-231 cells. The protein levels of PI3K110a, Akt Ser473, Akt Thr308, Akt, and NF-jB were measured. As shown in Fig. 5A, MDA-MB-231 cells with the inhibitor LY294002, and Akt sirna treatment exhibited significantly decreased protein levels of the main signal molecules of PI3K/Akt pathway. The inhibition of PI3K/Akt pathway made the MDA-MB-231 cells less invasive (Fig. 5B). The similar results were also observed in vivo tumorigenicity analysis. Reduced tumor weight were measured in mice group bearing MDA-MB-231 tumors with impaired PI3K/Akt signaling (Fig. 5C). All of the data implicated a role of PI3K/Akt signaling in modulating the invasive properties of MDA-MB-231 cells. Differential Expression of ST6GalNAcII in Breast Cancer and Transitional Tissues Altered expression of ST6GalNAcII was found in breast cancer and transitional tissues by immunohistochemistry staining (Table 1). ST6GalNAcII was expressed at a high level in breast cancer tissues compared with transitional tissues (P ). Interestingly, we also observed that differentiation, lymph node metastasis, and clinical stage were correlated Ren et al. 305

7 IUBMB LIFE Fig 5 PI3K/Akt inhibition modulates the invasive ability of MDA-MB-231 cells both in vitro and in vivo. A: The MDA-MB-231 cells were pretreated LY or Akt sirna. Expression of PI3K/Akt/ NF-jB signaling molecules were then examined by Western blot analysis. B: LY or Akt sirna treatment decreased the invasive ability of MDA-MB-231 cells in vitro. C: In xenograft tumor model, mean tumor weight was significantly reduced in LY or Akt shrna treatment group. *P < 0.05 vs. DMSO treatment cells; #P < 0.05 vs. control sirna treatment cells. The data are means 6 SD of three independent assays. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 306 ST6GalNAcII in the Invasive Properties of Breast Carcinoma

8 TABLE 1 Correlation between the clinicopathologic characteristics and expression of ST6GalNAcII protein in breast carcinoma ST6GaINAcII (%) Characteristics n ST6GaINAcII (high) ST6GaINAcII (low) P value Group Cancer tissue (71.3%) 29 (28.7%) Transitional tissue (52.5%) 48 (47.5%) Age (years) (57.4%) 29 (42.6%) < (54.3%) 16 (45.7%) Differentiation E1, E2, or well (41%) 46 (59%) E3, E4, or poorly (78.3%) 5 (21.7%) Lymph node metastasis Absent (47.8%) 24 (52.2%) Present (74.5%) 14 (25.5%) Distant metastasis Yes (63.2%) 7 (36.8%) No (53.7%) 38 (46.3%) Clinical stage I II (45.5%) 24 (54.5%) III IV (73.7%) 15 (26.3%) significantly with ST6GalNAcII expression (P , 0.006, 0.004, respectively). Discussion Intensive research is currently underway to identify the signaling pathways and molecular mechanisms that underlie the multistage process of metastasis of breast cancer (23 26). The ultimate goal of the present study was to intensively investigate the possible mechanism of ST6GalNAcII on the tumor invasion in human breast carcinoma cell lines MDA-MB-231 and MCF-7. We further analyzed the differential expression of ST6GalNAcII in breast cancer tissues. Alteration in the expression pattern of glycogens encoding glycosyltransferases has been shown to have enormous impact on cell behavior and functions (27). STs are essential for the biosynthetic pathway of sialylated glycans. The data shown here indicated that the expression profile of ST6GalNAcII gene was remodeled between MCF-7 and MDA-MB-231 using a real-time PCR analysis. Compared to MCF-7 cells, MDA-MB- 231 cells showed up-regulating expression of ST6GalNAcII mrna (Fig. 1A). Our previous report indicated that high metastatic potential cells were active in a2,3- and a2,6-linked sialylation (core sialylation) in human hepatocarcinoma cell lines (28). These results suggested that high expression of ST6GalNAcII might be associated with tumor metastasis, and be a tumor metastasis-associated gene in breast cancer. With this result, we further analyze the effects of ST6GalNAcII on the invasive and migration ability of MDA-MB-231 cells in vitro and in vivo. All these results suggested that knockdown of ST6GalNAcII gene inhibited the invasive and migration ability of MDA-MB- 231 cells in vitro and in vivo (Figs. 2 and 3). Taken together, these results supported and emphasized that the altering expression of ST6GalNAcII could be more important as indicators and functional regulators of tumor metastasis. In addition, we also investigated the molecular mechanism by which ST6GalNAcII-mediated PI3K/Akt signaling pathway regulates cell invasiveness. We have explored that ST6GalI and ST8SIAII are related to high activation of PI3K/Akt Ren et al. 307

9 IUBMB LIFE signaling pathway in human hepatocarcinoma cell lines (28). In this study, MDA-MB-231 cells exhibited higher PI3K/Akt activity than MCF-7 cells. Furthermore, the invasive properties of MDA-MB-231 cells were reversed, when the PI3K/Akt pathway was inhibited. These results confirmed that ST6GalNAcIImodulated invasion was, at least in part, PI3K/Akt-dependent in breast cancer. It was reported that STs with altered mrna expression in carcinoma tissues are regarded as prognostic factors and potential targets for therapeutic approaches (29,30). In the present study, we demonstrated that ST6GalNAcII was overexpressed in human breast carcinoma tissues and was associated with differentiation, lymph node metastasis, and clinical stage. Once again the findings from the clinical samples confirm that the altered level of ST6GalNAcII is probably associated with metastasis and of breast cancer. On basis of the above results, it might be possible to utilize ST6GalNAcII as a useful biomarker for clinical diagnosis of breast cancer metastasis and as potential targets in the future therapeutic approaches. In conclusion, our results indicated that ST6GalNAcII elucidated the unusual properties of invasion in breast cancer cell via modulating the PI3K/AKt signaling pathway. We also found that the elevated expression of ST6GalNAcII was associated with differentiation, lymph node metastasis, and clinical stage of breast cancer. Our findings were of potential pathophysiological importance for understanding the integration of migration-related signaling and provided a basis for designing future therapeutic strategy for blocking metastasis in patients with breast carcinoma. References [1] Dall Olio, F. and Chiricolo, M. (2001) Sialyltransferases in cancer. Glycoconj J. 18, [2] Harvey, B. E., Toth, C. A., Wagner, H. E., Steele, G. D. Jr., and Thomas, P. 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