The Toxicologist - Society of Toxicology

plays an important role in survival, drug resistance and angiogenesis. The human
STAT3 promoter region has several cis-elements including CRE/ATF, SBE and GC
boxes. Studies in this laboratory showed that Sp proteins (Sp1, Sp3 & Sp4) that
bind GC-rich sequences are overexpressed in many tumor types and are associated
with poor prognosis in pancreatic cancer patients. Since Sp proteins also regulate
genes associated with survival, angiogenesis and growth, we investigated the possible
interactions between Sp proteins and STAT3 and genes regulated by these important
trans-acting factors. Results from individual knockdown (RNA interference)
of Sp1 (siSp1), Sp3 (siSp3), and Sp4 (siSp4) and combined knockdown Sp1,
Sp3, and Sp4 (siSp) on expression of STAT3 were examined in Panc28 and L3.6pL
cells. Transfection of Panc28 and L3.6pL cells with siSp1 or siSp4 decreased expression
of pSTAT3 and STAT3 but siSp3 had no effect on STAT3 expression.
However, the effect of combined knockdown (siSp) of all 3 Sp proteins was much
more effective than individual Sp knockdown in both cell lines. In contrast, knockdown
of STAT3 (siSTAT3) did not effect expression of Sp1, Sp3 or Sp4 however
siSp decreased expression of several putative STAT3 regulated genes such as Cyclin
D1, survivin and VEGF (vascular endothelial growth factor). Thus results of Sp1,
Sp3, and Sp4 knockdown by RNA interference demonstrate that both pSTAT3
and STAT3 are Sp-regulated genes in pancreatic cancer cells suggesting that agents
(betulinic acid, curcumin and celastrol) targeting Sp transcription factors will also
decrease STAT3.
957 PEROXISOME PROLIFERATOR-ACTIVATED
RECEPTOR (PPAR)γ IN MAMMARY SECRETORY
EPITHELIAL CELLS STOPS DMBA-MEDIATED BREAST
TUMOURIGENESIS.
A. J. Apostoli 1 , N. Peterson 1 , R. Rubino 1 , S. SenGupta 1 , M. Schneider 1 and C.
Nicol 1, 2 . 1 Pathology & Molecular Medicine, Queen’s University, CRI, Kingston, ON,
Canada and 2 Division of Cancer Biology & Genetics, Queen’s University, CRI,
Kingston, ON, Canada.
One in every 28 women will die from the growth and spread of breast cancer. Our
previous studies with peroxisome proliferator-activated receptor (PPAR)γ heterozygous
mice suggest PPARγ normally suppresses 7,12-dimethylbenz[a]anthracene
(DMBA)-induced breast tumour progression, but the mechanisms and cell types
involved remain unknown. Given the proposed transdifferentiation between mammary
secretory epithelial (MSE) cells and adipocytes, we hypothesize that MSEspecific
PPARγ signaling normally acts to prevent DMBA-mediated breast tumour
progression. Here we generated Cre/loxP-based, MSE cell-specific PPARγ knockout
(PPARγ-MSE KO) mice. Eight week old female PPARγ-MSE KO mice and
their congenic wildtype (PPARγ-WT) controls were mated to induce pregnancy
and allowed to lactate for 3 days to ensure complete PPARγ deletion in MSE cells.
All lactating dams were then treated by gavage once/week for 6 weeks with 1 mg
DMBA. At week 7, females were randomized to receive either a normal chow diet
(DMBA Only: PPARγ-WT, n=10 and PPARγ-MSE KO, n=17) or one supplemented
with a PPARγ activating drug (DMBA+ROSI: PPARγ-WT, n=12 and
PPARγ-MSE KO, n=10) for the study duration (25 weeks). Our data suggests that
DMBA Only-treated PPARγ-MSE KO mice had a decreased median survival
(week 17 vs 23 respectively), a 2-fold decrease in mammary tumour latency
(p25 weeks) and mammary tumour volumes decreased
(PPARγ-MSE KO: ~3-fold and PPARγ-WT: ~3-fold) compared to DMBA Onlytreated
mice. These results suggest that normal PPARγ signaling in MSE cells contributes
to the suppression of DMBA-mediated breast tumourigenesis. They also
add evidence supporting a protective role for PPARγ activation in stopping environmental
chemical-mediated breast tumour progression.
958 MITOCHONDIAL UNCOUPLING PROTEIN 3
ANTAGONIZES TUMOR PROMOTION BY ALTERING
CANONICAL KERATINOCYTE GROWTH AND
DIFFERENTIATION SIGNALING PATHWAYS.
S. M. Nowinski 1 , J. E. Rundhaug 2 , O. Rho 1 , J. Digiovanni 1 and E. M. Mills 1 .
1 Division of Pharmacology and Toxicology, College of Pharmacy, The University of
Texas at Austin, Austin, TX and 2 Science Park - Research Division, The University of
Texas MD Anderson Cancer Center, Smithville, TX.
Compared to normal tissues, nearly all malignancies exhibit increased glycolysis
and decreased oxygen consumption. In contrast, mitochondrial uncoupling proteins
drive futile mitochondrial respiration that is uncoupled from ATP synthesis.
We previously showed that mice expressing a keratin 5 - uncoupling protein 3 (K5-
UCP3) transgene in the epidermis are completely protected from the formation of
skin carcinomas in response to a DMBA/TPA chemical carcinogenesis regimen. To
204 SOT 2011 ANNUAL MEETING
explore the mechanism of UCP3 induced cancer resistance, our lab inter-bred K5-
UCP3 animals with Tg.AC mice that express a cutaneous oncogenic v-Ha-Ras
transgene. Whereas wild type Tg.AC mice form tumors in response to TPA treatment
alone, K5-UCP3/Tg.AC animals are protected from TPA-induced tumorigenesis,
indicating that UCP3 likely inhibits tumor promotion. The data support the
hypothesis that UCP3 blocks promotion by increasing keratinocyte differentiation,
resulting in the loss of tumorigenic cells through normal skin turnover. Indeed, K5-
UCP3 mice exhibit increased skin turnover and increased markers of differentiation
in vivo and in vitro. Rhod-2 staining revealed elevated cytoplasmic calcium in K5-
UCP3 cells, which is a known important intermediate event in normal keratinocyte
differentiation. Several signaling pathways implicated in keratinocyte differentiation
are also augmented in K5-UCP3 cells, including Protein Kinase Cδ phosphorylation
(Western Blotting) and Notch 3 signaling (q-PCR). Increased keratinocyte
differentiation should correspond to growth arrest. Interestingly, Akt phosphorylation
is modified in K5-UCP3 cells, while other growth pathways are largely unaffected.
These data reveal novel roles for UCP3 in keratinocyte biology, and suggest
that mitochondrial uncoupling is a promising new approach to cancer prevention.
959 EFFECTS OF LOW LEVELS OF BISPHENOL A IN
HUMAN ENDOTHELIAL CELLS.
H. Andersson and E. Brittebo. Pharmaceutical Biosciences, Faculty of Pharmacy,
Uppsala, Sweden. Sponsor: M. Stigson.
Epidemiological evidence report an association between exposure to bisphenol A
and cardiovascular disease and diabetes 1 . Alterations in endothelial cell functions
play an important role in the development of cardiovascular disorders and diabetes
and it is therefore important to study the cell specific effects of bisphenol A in this
cell type.
The aim of the present study was to investigate the effects of low levels of bisphenol
A in human umbilical vein endothelial cells (HUVEC). The role of nuclear and cytosolic
receptors previously associated with the effects of bisphenol A was also investigated.
HUVEC were treated with 0.1-1000 nM bisphenol A for 6 or 24 hours
and markers of endothelial dysfunction, inflammation and angiogenesis where assayed
using qRT-PCR, western blot and fluorometric assays measuring nitric oxide
and reactive oxygen species. The results revealed that 6 hours incubation of
HUVEC with low dose bisphenol A increased the production of nitric oxide (10
nM; P