The Toxicologist - Society of Toxicology

membrane localization and subsequent signal transduction via modulation of Ras
farnesylation. This work was reviewed by EPA and approved for publication but
does not necessarily reflect official Agency policy.
952 ANALYSIS OF MOLECULAR, CELLULAR, AND
BIOCHEMICAL CHANGES IN THE LIVER OF MALE
B6C3F1 MICE TREATED WITH NITRAPYRIN—A
NITROGEN STABILIZER.
M. J. LeBaron 1 , M. R. Schisler 1 , H. L. Kan 1 , J. Thomas 1 , D. L. Eisenbrandt 2
and B. B. Gollapudi 1 . 1 The Dow Chemical Company, Midland, MI and 2 Dow
AgroSciences, Indianapolis, IN.
The purpose of this study was to investigate potential modes-of-action (MoA) contributing
to nitrapyrin (2-chloro-6-(trichloromethyl)pyridine)-induced hepatocellular
tumors in B6C3F1 mice. In this MoA study male B6C3F1 mice were fed 0,
75, 250, or 400 mg nitrapyrin/kg body weight/day (mkd) for 7 or 14 days. Another
group was given nitrapyrin for 14 days then switched to a control diet for 21 days
to investigate recovery. Body weight decreased slightly in animals given 400 mkd
with treatment-related decreases in serum cholesterol at 250 and 400 mkd after 7 or
14 days of treatment, which were within normal limits after the recovery period.
Mice given 250 or 400 mkd nitrapyrin for 7 or 14 days had increases in liver
weights, consistent with the histopathologic findings of centrilobular/midzonal hypertrophy
with cytoplasmic eosinophilia. Analysis of hepatocellular proliferation
via BrdU incorporation indicated a clear dose- and hepatolobular zone-related induction
of S-phase DNA synthesis. Recovery group animals had no treatment-related
histopathologic liver alterations. Gene expression analysis of the liver indicated
a direct, robust dose-related increase in the Cyp2b10/CAR-associated
transcript with an associated increase in Cyp2b10 protein. The lack of associative
enzyme activity suggested that nitrapyrin administration resulted in mechanismbased
(suicide) inhibition of the enzyme. Gene expression analysis indicated no activation
of AhR, PXR, or PPAR-α signaling pathways. In summary, nitrapyrin-induced
effects were consistent with the causal key events related to CAR-mediated
rodent liver tumorigenesis and were completely reversible upon treatment cessation.
Moreover, these data provided convincing evidence that key events and hepatocellular
tumors do not occur at or below 75 mkd. A Human Relevance
Framework evaluation supports that this phenobarbital-like MoA for mouse liver
tumors associated with high doses of nitrapyrin is not relevant to humans.
953 THE FRY TUMOR SUPPRESSOR ENCODES AN
INHIBITOR OF EPITHELIAL MESENCHYMAL
TRANSITION.
J. Graham 1 , X. Ren 2 and H. Zarbl 1 . 1 Toxicology, UMDNJ-RWJMS, Piscataway,
NJ and 2 UC, Berkeley, CA.
We previously identified Fry, the rat ortholog of the Drosophila furry gene, as a
Mammary Carcinoma Susceptibility locus. FRY is reduced > 40% in human breast
cancer cell lines relative to the nontransformed MCF10A mammary epithelial cell
line, suggesting that decreased FRY may contribute to carcinogenesis. To evaluate
the function of the Fry gene, we stably transfected the triple-negative, human
MDA-MB-231 mammary tumor cell line with the wild type (wt) allele from Cop
(231wCFry). In Matrigel, the 231wCFry cells formed polarized mammospheres resembling
those formed by the nontumorigenic MCF10A cells, while parental
MDA-MB-231 cells formed disorganized clusters. When injected into nude mice,
tumors formed by the 231wCFry cells were 8-fold smaller, encapsulated and noninvasive
in sharp contrast to MDA-MB-231. Gene-expression profiling revealed
that Fry regulates gene networks that maintain cellular and tissue organization, differentiation,
motility and proliferation. The Wnt/β-catenin signaling pathway,
which maintains cell polarization, was most significantly changed by Fry.
Immunocytochemistry revealed that the levels and subcellular distribution of βcatenin
and integrins in 231wCFry, both markers of epithelial cell differentiation,
more approximated those in MCF10A than in MDA-MB-231, suggesting Fry restored
a differentiated phenotype to the cancer cells in vitro. Consistent with a role
in suppression of tumor progression, analysis of microarray data for 2,200 human
breast tumor samples (Oncomine 3.0 Cancer Profiling Database) revealed that FRY
was decreased in aggressive breast cancers. In summary, ectopic expression of wt
Cop Fry in tumor cells enhanced cell polarization, adhesion, and differentiation,
while suppressing tumor growth and invasiveness. These observations suggest that
Fry expression can reverse or suppress epithelial mesenchymal transition (EMT), a
process involved in the acquisition of invasive and metastatic phenotypes, making
FRY a significant new target for therapeutic interventions.
NJCCR Predoctoral Fellowship
NIEHS Center Grant P30ES005022, NCI Grant RO1CA77222
954 REDUCTION OF PTEN DOSE IMPAIRS DNA REPAIR
AND PREDISPOSES TO UVB-INDUCED SKIN
TUMORIGENESIS.
Y. He 1 , M. Ming 1 , L. Feng 2 , C. Shea 1 , K. Soltani 1 , B. Zhao 2 , W. Han 1 , R.
Smart 3 , C. Trempus 2 and J. Pritchard 2 . 1 Medicine/Dermatology, University of
Chicago, Chicago, IL, 2 Laboratory of Pharmacology, National Institute of
Environmental Health Sciences, National Institute of Health, Research Triangle Park,
NC and 3 Department of Environmental and Molecular ToxicologyGroup, North
Carolina State University, Raleigh, NC.
Non-melanoma skin cancer is the most common cancer in the US. The major environmental
risk factor causing skin cancer is the UVB radiation in sunlight
through damaging DNA. The tumor suppressor PTEN, a critical regulator for
multiple cellular processes, is frequently mutated or deleted in many human cancers.
Here we show that target reduction of PTEN levels in epidermal keratinocytes
is a predisposing factor for UVB-induced skin tumorigenesis in mice. In contrast to
the response to ionizing radiation, PTEN down-regulation prolongs UVB-induced
arrest of growth and activation of the DNA damage checkpoint Chk1 pathway.
PTEN down-regulation impairs the capacity of global genomic nucleotide excision
repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic
DNA lesions. At the molecular level, PTEN loss suppresses the expression of the
key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38
axis. Reconstitution of the XPC levels in PTEN-inhibited cells restores the GG-
NER capacity. In human skin neoplasia, PTEN is significantly down-regulated in
both premalignant actinic keratosis and malignant squamous cell carcinoma, suggesting
a critical role of PTEN in both tumor development and progression.
Therefore, these findings demonstrated that PTEN is an essential genomic gatekeeper
in the skin through positively regulating XPC-dependent GG-NER following
UVB damage.
955 TOLFENAMIC ACID INHIBITS GROWTH AND
SURVIVAL OF MULTIPLE TUMORS AND
DOWNREGULATES SPECIFICITY PROTEIN (SP)
TRANSCRIPTION FACTORS.
G. Chadalapaka 1 , I. Jutooru 1 , S. Sreevalsan 1 , S. Pathi 1 and H. Wilson 2 . 1 VTPP,
Texas A&M University, College Station, TX and 2 Department of Small Animal
Clinical Sciences, Texas A&M University, College Station, TX.
Cancer is the second leading cause of death in United States. In spite of improvements
in conventional anticancer therapies, the number of cancer deaths hasn’t
been significantly reduced. Current research in our laboratory focuses on the anticancer
activity and toxicology of tolfenamic acid, a mechanism-based non-steroidal
anti-inflammatory drug (NSAID) that targets downregulation of specificity protein
(Sp) transcription factors. RNA interference studies support the vital role of specificity
protein (Sp) transcription factors as an important target for anticancer agents
since Sp1, Sp3 and Sp4 proteins are overexpressed in most tumors and regulate expression
of genes required for cancer cell survival, metastasis (survivin, bcl-2,
NFkB), growth (cyclin D1, epidermal growth factor receptor-EGFR, c-MET) and
angiogenesis (vascular endothelial growth factor [VEGF]) and their receptors.
Tolfenamic acid targets downregulation of Sp proteins in pancreatic, colon and
esophageal cancer cells and the effects were investigated in SJCRH30 and RD
human rhabdomyosarcoma cancer cells and a series of canine melanoma, mammary
cancer and osteosarcoma cells. In this study, tolfenamic acid (50-200 μM)
significantly inhibited growth of rhabdomyosarcoma and different canine cancer
cell lines; western blot analysis showed that Sp1, Sp3 and Sp4 proteins were overexpressed
in these cells and treatment with 50-200 μM Tolfenamic acid decreased expression
of Sp1, Sp3, Sp4 and several Sp-regulated gene products including VEGF,
EGFR, p65 (NFkB), c-Met and survivin. These results demonstrate that tolfenamic
acid, a drug currently in use for treating acute and chronic pain, may also be an important
new anticancer compound for treatment of human and canine cancers.
956 ROLE OF SPECIFICITY PROTEIN (SP)
TRANSCRIPTION FACTORS IN REGULATION OF
STAT3 IN PANCREATIC CANCER CELLS.
I. D. Jutooru 1 , G. Chadalapaka 1 and S. H. Safe 1, 2 . 1 Veterinary Physiology and
Pharmacology, Texas A&M University, College Station, TX and 2 Institute of
Biosciences & Technology, Houston, TX.
Signal Transducer and Activator of Trancription (STAT) is activated by various cytokines
and growth factors resulting in dimerization and phosphorylation of
STAT’s. Activated STAT binds to specific DNA-response elements and promotes
cell growth, development, inflammation and immune responses. Constitutive activation
of STAT3 is detected in most cancer types including pancreatic cancer and
SOT 2011 ANNUAL MEETING 203