ACLS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brown, R. E.
Right arrow Articles by Law, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brown, R. E.
Right arrow Articles by Law, A.
Annals of Clinical & Laboratory Science 36:421-426 (2006)
© 2006 Association of Clinical Scientists

Communication

Morphoproteomic Demonstration of Constitutive Nuclear Factor-kappaB Activation in Glioblastoma Multiforme with Genomic Correlates and Therapeutic Implications

Robert E. Brown1 and Amy Law2
1 Division of Laboratory Medicine and 2 Department of Adult Hematology and Oncology, Geisinger Medical Center, Danville, Pennsylvania

Address correspondence to Robert E. Brown, M.D., at his new address: Department of Pathology, University of Texas Medical School, 6431 Fannin Street, MSB 2.286, Houston, TX 77030, USA; tel 713 500 5332; fax 713 500 0733; e-mail robert.brown{at}uth.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Glioblastoma multiforme (GBM) presents a major challenge to neurosurgeons, neuro-oncologists, and radiation therapists by virtue of its location with a blood-brain barrier, chemoradioresistance, highly malignant phenotype, and angiogenic potential. Because nuclear factor-kappaB (NF-{kappa}B) can transcriptionally activate genes leading to the synthesis of anti-apoptotic, chemoresistant, growth promoting, and angiogenic proteins; we assessed the state of activation of NF-{kappa}B in 6 GBM cases at diagnosis. Morphoproteomic analysis confirmed the constitutive activation of NF-{kappa}B by demonstrating the phosphorylation (p) and nuclear translocation of p-NF-{kappa}Bp65 (Ser 536) in these cases. This observation coincides with (a) previous immunohistochemical findings showing nuclear translocation of total p65, (b) demonstration of NF-{kappa}B DNA binding activity, (c) the results of electrophoretic mobility shift assays, and (d) existing genomic data in GBM. Furthermore, such constitutive activation of the NF-{kappa}B pathway helps to explain some of the tumor biology and supports the incorporation of NF-{kappa}B pathway inhibitors into the treatment of GBM.

Keywords: glioblastoma multiforme, nuclear factor-kappaB, tumor morphoproteomics


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Glioblastoma multiforme (GBM) is a high grade, malignant glioma of astrocytic lineage, characterized by (a) variable histologic and cytologic features that include anaplastic and giant gemistocytic components, with pleomorphic karyomegaly and hyperchromaticity in the latter, and occasional sarcomatous transformation; (b) mitotic activity; (c) foci of necrosis, some rimmed by palisading tumor cells; and (d) endothelial/microvascular proliferation [1]. Its clinical course is generally aggressive and marked by chemoradioresistance and postoperative recurrence with a median duration of survival of 9 to 15 mo [2]. This study was designed to determine whether or not NF-{kappa}B is constitutively activated in GBM when assessed by morphoproteomic analysis and, if so, how this correlates with previous reports using complementary methods of assessment and also with the collective genomic data on GBM.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
With Institutional Review Board approval, archival, paraffin-embedded blocks from 6 representative cases of GBM at diagnosis were studied. The patients were 4 men and 2 women, age 41–74 yr. The immunohistochemical probe was an antibody for detection of phosphorylated (p)-NF-{kappa}Bp65 (phosphorylated at serine 536 [Cell Signaling Technology, Beverly, MA]). The immunohistochemical method was previously described [3]. Positive and negative controls stained appropriately in the immunohistochemical reaction.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Brightfield microscopy revealed moderate to strong expression (up to 3+ intensity on a scale of 0 to 3+) of p-NF-{kappa}Bp65 (Ser 536) in tumoral nuclei from all cases (Fig. 1Go). Such nuclear translocation of phosphorylated NF-{kappa}Bp65 represents an endpoint in the NF-{kappa}B pathway that creates an opportunity for the tumor cell to form p-NF-{kappa}Bp65·DNA complexes, thus allowing transcriptional activation. Furthermore, this morphoproteomic finding indicates that certain preceding steps in the NF-{kappa}B pathway have been completed, which include (a) translational synthesis of cytoplasmic NF-{kappa}Bp65, (b) phosphorylation and proteasomal degradation of IkappaB·NF-{kappa}Bp65 complexes, and (c) phosphorylation and release of p-NF-{kappa}Bp65 for nuclear translocation (see Fig. 2Go).


Figure 1
View larger version (137K):
[in this window]
[in a new window]

  		Fig. 1. Hematoxylin-eosin (H & E) stained sections containing portions of glioblastoma multiforme (GBM; left-hand frames) with contiguous non-neoplastic brain (lower left). Immunohistochemical phosphospecific probe for NF-{kappa}Bp65 (phosphorylated on serine 536) reveals nuclear translocation of this activated protein analyte with variable moderate to strong brown (DAB chromogen) signal intensity in the tumor cells (right-hand frames). Compare and contrast with immunonegativity in nuclei of glial cell component in contiguous non-neoplastic brain parenchyma (lower right-hand frame). (Original magnification x600.)

 

Figure 2
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 2. Diagramatic depiction of essential components of the NF-{kappa}Bp65 pathway, which include transcription of the NF-{kappa}Bp65 gene in the nucleus leading to the release of NF-{kappa}Bp65-mRNA (shown in green); translational synthesis of NF-{kappa}Bp65; complexing of NF-{kappa}Bp65 with its inhibitor, I-{kappa}B; phosphorylation of the latter by I kappa Kinase (IKK) leading to proteasomal degradation and release of activated (phosphorylated) NF-{kappa}Bp65; translocation of p-NF-{kappa}Bp65 to the nucleus for complexing with DNA and activation of transcription, leading to tumorigenicity (red pathway). Opportunities for therapeutic intervention (shown in blue) include siRNA to NF-{kappa}Bp65 to block the downstream translational synthesis of NF-{kappa}B, IKK inhibitors, Velcade to block cytoplasmic ptoteasomal degtadation with release of p-NF-{kappa}Bp65, oligonucleotide decoys to complex with cytoplasmic p-NF-{kappa}Bp65 in an effort to block nuclear translocation, and Velcade or rofecoxib to interfere in p-NF-{kappa}Bp65 DNA binding. Additionally, Velcade could increase nuclear I-{kappa}B and/or block nuclear proteasomal degradation of p-NF-{kappa}Bp65, I-{kappa}B complexes, and promote p21 WAF1, resulting in the inhibition of Gl cell cycle progression [6,26–38]. Genomic correlates of a constitutively activated NF-{kappa}B pathway in glioblastoma multiforme (GBM) are depicted in green [ 8–25].

 
Morphoproteomics utilizes phosphospecific probes and cellular compartmentalization to assess directly the state of activation of protein analytes in lesional tissue [4] and, as noted above, has confirmed the constitutive activation of NF-{kappa}Bp65 and the NF-{kappa}B pathway in GBM. The present observations are consistent with and complemented by the findings of other investigators. Specifically, Guo et al [5] reported that the NF-{kappa}B DNA binding activity correlates with the degree of malignancy in human astrocytomas. Robe and colleagues [6] showed that there is constitutive NF-{kappa}B activity in nuclear extracts from primary cultures of 3 human malignant gliomas using electrophoretic mobility shift assays. Additionally, they detected total p65 (NF-{kappa}Bp65) in the nuclei of a human glioblastoma by immunohistochemistry. Finally, Ansari and coauthors [7] confirmed by Western blotting analysis of nuclear extracts from glioma cells throughout the cell cycle that nuclear accumulation (translocation) of p50 and p65 perfectly correlates with their respective DNA-binding activities.

Genomic analyses of GBM tissues have revealed variable amplification of several genes that either through their products (proteins) and/or the activation of signaling cascades contribute to the upregulation of the NF-{kappa}B pathway. This includes the following specific genes with varying frequencies of amplication in GBM: (a) GPR56 gene, which in functional genomic assays is associated with transcriptional upregulation of NF-{kappa}B reporter constructs [8]; (b) epidermal growth factor receptor (EGFR) gene [9,10], which can activate NF-{kappa}B through the PI3'-K/Akt pathway that leads to phosphorylation of I-kappaBalpha on serines 32 and 36, thereby promoting the nuclear translocation of the p65 subunit [11]; (c) murine double minute 2(MDM2) gene [12–14], which has been shown to upregulate NF-{kappa}Bp65 expression both transcriptionally and at the protein level [15,16]; (d) fibroblast growth factor-inducible 14 (FN14) gene [17], which regulates glioma cell survival via NF-{kappa}B pathway activation and bcl-xL and bcl-w expression and is associated with the translocation of NF-{kappa}Bp65 from the cytoplasm to the nucleus [18]; and (e) osteopontin gene [14], whose gene product induces NF-{kappa}Bp65 nuclear accumulation, NF-{kappa}B·DNA binding, and transactivation leading to promatrix metalloproteinase-2 activation [19]. Additionally, the deletion or mutation of phosphatase and tensin homolog deleted from the chromosome 10 (PTEN) gene in some GBM tumors [20–24] could facilitate the constitutive activation of NF-{kappa}B-dependent transcription [25]. These genomic correlates of a constitutively activated NF-{kappa}B pathway in GBM are depicted in Fig. 2Go.

The therapeutic implications of these aforementioned observations for GBM include the incorporation of known inhibitors of the NF-{kappa}B pathway into the treatment protocol, especially in a combinatorial fashion with radiation and/or chemotherapy. Such agents might include: small interfering ribonucleic acid to NF-{kappa}B (siRNA/NF-{kappa}Bp65) [26] to inhibit the translational synthesis of NF-{kappa}Bp65 protein; curcumin, non-steroidal anti-inflammatory drugs (NSAIDs–aspirin, sulindac, sulfasalazine, and celecoxib), resveratrol, and farnesyl transferase inhibitors, all of which appear to act as antagonists of inhibitor kappa kinase (IKK) [6,27–32] in the NF-{kappa}B pathway; proteasome inhibitors such as bortezomib (Velcade) [26]; decoy oligonucleotides to bind NF-{kappa}B and to prevent its nuclear translocation [33]; and rofecoxib to inhibit the DNA binding capacity of NF-{kappa}B [34,35]. These therapeutic interventions are depicted in Fig 2Go.

As proof of concept for the role of an activated NF-{kappa}B pathway and for these potential therapeutic agents in GBM, the following observations are submitted: Lee and co-workers [36] showed that sulindac and its metabolites inhibited the invasiveness of human glioblastoma cells via down-regulation of Akt and matrix metalloproteinase (MMP)-2. Feldkamp et al [37] demonstrated substantial growth inhibition in 2 of 3 human GBM xenografts in response to a farnesyl transferase inhibitor. Yin and co-authors [38] reported that the proteasome inhibitor PS-341 (Velcade, bortezomib) caused cell growth arrest and apoptosis in human GBM cell lines and primary GBM explants. Robe and co-workers [6] demonstrated in vitro and in vivo (anti-xenograft) activity of theNF-{kappa}B inhibitor sulfasalazine against human glioblastomas, including a reduced expression of cyclin D1. Tuettenberg et al [39] combined continuous low-dose chemotherapy with temozolomide and rofecoxib in patients with newly diagnosed GBM and achieved an antiangiogenic efficacy in those tumors characterized by high angiogenic activity. Finally, Weaver and co-authors [40] reported on the ability of chemotherapy to induce a marked increase in active intranuclear NF-{kappa}B in human glioma cell lines and potentiation of these chemo-therapeutic agents following antagonism of NF-{kappa}B. Currently, phase I-II trials incorporating sulfasalazine [41] and a phase I trial incorporating Velcade [42] into the therapies of recurrent or progressive gliomas are underway.

In summary, our morphoproteomic studies provide additional evidence for the presence of a constitutively activated NF-{kappa}B pathway in GBM and thereby support the incorporation of agents that interrupt this pathway into the therapy of patients with GBM.


   Acknowledgments
 
The authors thank Laurie Kneller-Walter, HT (ASCP) for technical assistance, and Sharon Coup-Stroh for secretarial support and help with the graphics.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Burger PC, Scheithauer BW. Tumors of the central nervous system. In: Atlas of Tumor Pathology, 3rd series, fascicle 10 (Rosai J, Sobin LH, Eds), Armed Forces Institute of Pathology, Washington, DC, 1994; pp 49–70.
  2. Grossman SA, Batara JF. Current management of glioblastoma multiforme Semin Oncol 2004;31:635–644.
  3. Zhang PL, Pellitteri PK, Law A, Gilroy PA, Wood GC, Kennedy TL, Blasick TM, Lun M, Schuerch C III, Brown RE. Overexpression of phosphorylated nuclear factor-kappaB in tonsillar squamous cell carcinoma and high-grade dysplasia is associated with poor prognosis. Mod Path 2005;18:924–932.[Medline]
  4. Brown, RE. Morphoproteomics: exposing protein circuitries in tumors to identify potential therapeutic targets in cancer patients. Expert Rev Proteomics 2005; 2:337–348.[Medline]
  5. Guo CB, Chen F, Gao ZW. DNA binding activity of nuclear factor-kappa B in human astrocytoma. Aizheng 2004;23:1207–1209.[Medline]
  6. Robe PA, Bentires-Alj M, Bonif M, Rogister B, Deprez M, Haddada H, Khac M-T, Jolois O, Erkmen K, Merville M-P, Black PM, Bours V. In vitro and in vivo activity of the nuclear factor-{kappa}B inhibitor sulfasalazine in human glioblastomas. Clin Cancer Res 2004;10:5595 5603.[Abstract/Free Full Text]
  7. Ansari S, Safak M, Del Valle L, Enam S, Amini S, Khalili K. Cell cycle regulation of NF-{kappa}B-binding activity in cells from human glioblastomas. Exp Cell Res 2001;265:221–233.[Medline]
  8. Shashidhar S, Lorente G, Nagavarapu U, Nelson A, Kuo J, Cummins J, Nikolich K, Urfer R, Foehr E. GPR56 is a GPCR that is overexpressed in gliomas and functions in tumor cell adhesion. Oncogene 2005;24:1673–1682.[Medline]
  9. Hui AB, Lo KW, Yin XL, Poon WS, Ng HK. Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization. Lab Invest 2001;81:717–723.[Medline]
  10. Ohgaki H. Genetic pathways to glioblastomas. Neuropathology 2005;25:1–7.[Medline]
  11. Le Page C, Koumakpayi IH, Lessard L, Saad F, Mes-Masson AM. Independent role of phosphoinositol-3-kinase (PI3K) and casein kinase II (CK-2) in EGFR and Her-2-mediated constitutive NF-kappaB activation in prostate cancer cells. Prostate 2005;65:306–315.[Medline]
  12. Biernat W, Kleihues P, Yonekawa Y, Ohgaki H. Amplification and overexpression of MDM2 in primary (de novo) glioblastomas. J Neuropath Exp Neurol 1997; 56:180–185.[Medline]
  13. Schiebe M, Ohneseit P, Hoffman W, Meyermann R, Rodemann HP, Bamberg M.Analysis of mdm2 and p53 gene alterations in glioblastomas and its correlation with clinical factors. J Neuro-Oncology 2000;49:197–203.[Medline]
  14. Markert JM, Fuller CM, Gillespie GY, Bubien JK, McLean LA, Hong RL, Lee K, Gullans SR, Mapstone TB, Benos DJ. Differential gene expression profiling in human brain tumors. Physiol Genomics 2001;5:21–33.[Abstract/Free Full Text]
  15. Gu L, Findley HW, Zhou M. MDM2 induces NF-kappaB/p65 expression transcriptionally through Sp1-binding sites: a novel, p53-independent role of MDM2 in doxorubicin resistance in acute lymphoblastic leukemia. Blood 2002;99:3367–3375.[Abstract/Free Full Text]
  16. Hu Q, Zhou MX, Liu SY, Zhang LQ, Liu AQ, Guo YJ, Song Y. Regulation of NF-kappaB/P65 by MDM2 in acute lymphoblastic leukemia in childhood. Zhonghua Erke Zazhi [Chinese] 2003;41:921–924.
  17. Tran NL, McDonough WS, Donohue PJ, Winkles JA, Berens TJ, Ross KR, Hoelzinger DB, Beaudry C, Coons SW, Berens ME. The human Fn14 receptor gene is up-regulated in migrating glioma cells in vitro and over-expressed in advanced glial tumors. Amer J Pathol 2003; 162:1313–1321.[Abstract/Free Full Text]
  18. Tran NL, McDonough WS, Savitch BA, Sawyer TF, Winkles JA, Berens ME. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling system regulates glioma cell survival via NF{kappa}B pathway activation and bcl-xL/bcl-w expression. J Biol Chem 2005;280:3483–3492[Abstract/Free Full Text]
  19. Philip S, Kundu GC. Osteopontin induces nuclear factor kappB-mediated promatrix metalloproteinase-2 activation through I kappa B alpha/IKK signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways. J Biol Chem 2003;278:14487–14497.[Abstract/Free Full Text]
  20. Knobbe CB, Reifenberger J, Reifenberger G. Mutation analysis of the Ras Pathway genes NRAS, HRAS, KRAS and BRAF in glioblastomas. Acta Neuropathol 2004; 108:467–470.[Medline]
  21. Arslantas A, Artan S, Orner U, Muslumanoglu H, Durmaz R, Cosan E, Atasoy MA, Basaran N, Tel E. The importance of genomic copy number changes in the prognosis of glioblastoma multiforme. Neurosurg Rev 2004;27:58–64.[Medline]
  22. Ohgaki H. Genetic pathways to glioblastomas. Neuropathology 2005;25:1–7.[Medline]
  23. Rich JN, Hans C, Jones B, Iversen ES, McLendon RE, Rasheed BK, Dobra A, Dressman HK, Bigner DD, Nevins JR, West M. Gene expression profiling and genetic markers in glioblastoma survival. Cancer Res 2005;65:4051–4058.[Abstract/Free Full Text]
  24. Fukushima T, Favereaux A, Huang H, Shimizu T, Yonekawa Y, Nakazato Y, Ohagki H. Genetic alterations in primary glioblastomas in Japan. J Neuropathol Exp Neurol 2006;65:12–18.[Medline]
  25. Agarwal A, Das K, Lerner N, Sathe S, Cicek M, Casey G, Sizemore N. The AKT/I-kappaB kinase pathway promotes angiogenic/metastatic gene expression in colorectal cancer by activating nuclear factor-kappa B and beta-catenin. Oncogene 2005;24:1021–1031.[Medline]
  26. Lun M, Zhang PL, Pellitteri PK, Law A, Kennedy TL, Brown RE. Nuclear-factor-kappaB pathway as a therapeutic target in head and neck squamous cell carcinoma: pharmaceutical and molecular validation in human cell lines using velcade and siRNA/NF-{kappa}B. Ann Clin Lab Sci 2005;35:251–258.[Abstract/Free Full Text]
  27. Yamamoto Y, Gaynor RB. Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer. J Clin Invest 2001;107: 135–142.[Medline]
  28. Takada Y, Bhardwaj A, Potdar P, Aggarwal BB. Non-steroidal anti-inflammatory agents differ in their ability to suppress NF-kappaB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene 2004; 23:9247–9258.[Medline]
  29. Aggarwal S, Takada Y, Singh S, Myers JN, Aggarwal BB. Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-kappaB signaling. Int J Cancer 2004;111:679–692.[Medline]
  30. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin:preclinical and clinical studies. Anticancer Res 2003;23:363–398.[Medline]
  31. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y.Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 2004;24:2783–2840.[Abstract/Free Full Text]
  32. Takada Y, Khuri FR, Aggarwal BB. Protein farnesyl-transferase inhibitor (SCH66336) abolishes Nf-kappaB activation induced by various carcinogens and inflammatory stimuli leading to suppression of NF-kappaB-regulated gene expression and up-regulation of apoptosis. J Biol Chem. 2004;279:26287–26299.[Abstract/Free Full Text]
  33. Gill JS, Zhu X, Moore MJ, Lu L, Yaszemski MJ, Windebank AJ. Effects of NF-kappaB decoy oligonucleotides released from biodegradable polymer microparticles on a glioblastoma cell line. Biomaterials 2002;23:2773–2781.[Medline]
  34. Brown RE. The NF-{kappa}B pathway and the successful application of anti-inflammatory and angiostatic therapy in Langerhans’ cell histiocytosis. Brit J Haematol 2005; 130:147–148.[Medline]
  35. Niederberger E, Tegeder I, Schafer C, Seegel M, Grosch S, Geisslinger G. Opposite effects of rofecoxib on nuclear factor-kappaB and activating protein-1 activation. J Pharmacol Exper Therapeut 2003;304:1153–1160.[Abstract/Free Full Text]
  36. Lee HC, Park IC, Park MJ, An S, Woo SH, Jin HO, Chung HY, Lee SJ, Gwak HS, Hong YJ, Yoo DH, Rhee CH, Hong SI. Sulindac and its metabolites inhibit invasion of glioblastoma cells via down-regulation of Akt/PKB and MMP-2. J Cell Biochem 2005;64:597–610.
  37. Feldkamp M, Lau N, Roncari L, Guha A. Isotype-specific Ras·GTP-levels predict the efficacy of farnesyl transferase inhibitors against human astrocytomas regardless of ras mutational status. Cancer Res 2001;61: 4425–4431.[Abstract/Free Full Text]
  38. Yin D, Zhou H, Kumagai T, Liu G, Ong JM, Black KL, Koeffler HP. Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM). Oncogene 2005;24:344–354.[Medline]
  39. Tuettenberg J, Grobholz R, Korn T, Wenz F, Erber R, Vajkoczy P. Continuous low-dose chemotherapy plus inhibition of cyclooxygenase-2 as an antiangiogenic therapy of glioblastoma multiforme. J Cancer Res Clin Oncol 2005;131:31–40.[Medline]
  40. Weaver KD, Yeyeodu S, Cusack JC Jr, Baldwin AS Jr, Ewend MG. Potentiation of chemotherapeutic agents following antagonism of nuclear factor kappaB in human gliomas. J Neuro-Oncol 2003;61:187–196.[Medline]
  41. Robe PA, Martin D, Albert A, Deprez M, Chariot A, Bours V. A phase 1–2, prospective, double blind, randomized study of the safety and efficacy of Sulfasalazine for the treatment of progressing malignant gliomas: study protocol of [ISRCTN45828668]. BMC Cancer 2006;6:29.[Medline]
  42. Reardon DA, Wen P. Therapeutic advances in the treatment of glioblastoma: rationale and potential role of targeted agents. Oncologist 2006;11:152–164.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brown, R. E.
Right arrow Articles by Law, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brown, R. E.
Right arrow Articles by Law, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS