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2008 36: 972 originally published online 31 October 2008Toxicol Pathol
Bih-Rong Wei, Jennifer B. Edwards, Shelley B. Hoover, Heather S. Tillman, L. Tiffany Reed, Robert C. Sills and R. Mark
-Catenin Accumulation in Hepatocellular Carcinomas of Diethylnitrosamine-Exposed Rhesus
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Altered β β-Catenin Accumulation in Hepatocellular
Carcinomas of Diethylnitrosamine-Exposed Rhesus Macaques
BIH-RONG WEI,1JENNIFER B. EDWARDS,1SHELLEY B. HOOVER,1HEATHER S. TILLMAN,1
L. TIFFANY REED,1ROBERT C. SILLS,2AND R. MARK SIMPSON1
1Molecular Pathology Unit, Laboratory of Cancer Biology and Genetics, Center for Cancer Research,
National Cancer Institute, Bethesda, Maryland, USA
2Cellular and Molecular Pathology Branch, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina, USA
Chemical exposures are important risks for development of hepatocellular carcinoma (HCC). One such chemical, diethylnitrosamine (DENA), is
present in food products as well as in industrial and research settings. Further examination of tumors induced by DENA may yield clues to human
risk. HCC from seven rhesus macaques exposed to DENA was selected from a tissue archive to examine for evidence of Wnt/β-catenin signaling
events, which are frequently associated with HCC. DENA exposure durations ranged from 8 to 207 months, and total accumulated dose ranged from
0.7 to 4.08 mg. Unexposed colony breeder macaques served as controls. Previously unrecognized HCC metastases were discovered in lungs of three
macaques. Overexpression of β-catenin and glutamine synthetase was detected by immunohistochemistry in six confirmed primary HCC and all
metastatic HCC, which implicated Wnt/β-catenin activation. Concomitant β-catenin gene mutation was detected in one primary HCC; similar find-
ings have been reported in human and rodent HCC. Neither β-catenin mutation nor β-catenin overexpression appeared to influence metastatic poten-
tial. Accumulation of intracellular proteins involved in Wnt/β-catenin signaling during HCC oncogenesis in rhesus macaques exposed to DENA
appears to include other mechanisms, in addition to mutation of β-catenin gene.
biological specimen banks; sequence analysis; DNA; carcinogens; mutagens; signal transduction pathway
that are linked with the development of HCC include heavy
alcohol intake, use of oral contraceptives, genetic disorders,
and exposures to chemical carcinogens (Calvisi et al. 2001).
Pathogenesis of HCC can involve molecular alterations such as
overexpression of growth factors or oncogenes, inactivation of
tumor suppressor genes (e.g., p53 and Rb), and mutation acti-
vation of oncogenes (e.g., K-Ras) (Laurent-Puig and Zucman-
Rossi 2006). Genetic modifications that activate Wnt/β-catenin
signaling pathway have also been implicated in both human
and rodent HCC (Laurent-Puig and Zucman-Rossi 2006;
Thompson and Monga 2007; Kim, Sills, and Houle 2005).
β-catenin is a dual-function protein involved in cell adhe-
sion and gene transcription (Katoh 2007; Daugherty and
Gottardi 2007; Thompson and Monga 2007). During home-
ostasis, β-catenin complexes to E-cadherin at the plasma mem-
brane and plays an important role in maintaining cell-cell
adhesion. Free cytoplasmic β-catenin is phosphorylated at the
N-terminal by axin/GSK-3β/APC complex and subsequently
degraded by the ubiquitin-proteasome system. Therefore, the
level of free cytoplasmic β-catenin remains low. When Wnt
signaling is activated, GSK-3β activity is inhibited. This
impairs degradation of β-catenin and causes the accumulation
of β-catenin in the cytoplasm. Free cytoplasmic β-catenin then
translocates into the nucleus and interacts with T cell
factor/lymphoid enhancing factor transcription factors, result-
ing in up-regulation of its target genes (Giles, van Es, and
Hepatocellular carcinoma (HCC) is the most common
hepatic malignancy and the third most common cause of can-
cer death in human beings (Parkin, Bray, and Devesa 2001;
Roberts and Gores 2005). Chronic infection with hepatitis B
and C viruses as well as the exposure to aflatoxin B1 are main
risk factors believed to be responsible for approximately 80%
of human HCC (Bosch, Ribes, and Borras 1999). Other factors
This research was supported by the Intramural Research Program, Center
for Cancer Research, National Cancer Institute (NCI), Bethesda, MD. Heather
S. Tillman was supported by an NCI Cancer Research Training Award
Fellowship while a fourth-year veterinary medical student, University of
Georgia, Athens. Dr. Tillman’s current address is Diagnostic Center for
Population and Animal Health, Michigan State University, East Lansing, MI.
L. Tiffany Reed was supported by an NCI Cancer Research Interns in
Residence Training Award while a fourth-year veterinary medical student,
University of Georgia, Athens. Dr. Reed’s current address is the School of
Veterinary Medicine, Purdue University, West Lafayette, Indiana.
Address correspondence to: R. Mark Simpson, Center for Cancer
Research, National Cancer Institute, 37 Convent Drive, 2000, Bethesda, MD
20892, USA; phone: 301-435-7176; e-mail: email@example.com.
Abbreviations:HCC, hepatocellular carcinoma; DENA,
trosamine; NHP, nonhuman primates; GS, glutamine synthetase; IHC,
immunohistochemistry; H&E, hematoxylin and eosin; bp, nucleotide base
pairs; PCR, polymerase chain reaction; Ser33, serine amino acid 33; Pro, pro-
line; DAB, 3,3’-diaminobenzidine tetrahydrochloride.
Toxicologic Pathology, 36: 972-980, 2008
Copyright © 2008 by Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
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