The Toxicologist - Society of Toxicology

400 ALTERATIONS OF SIGNALING PROTEIN
INTERACTIONS IN RESPONSE TO INCREASING DOSE.
C. Kinzer, H. Williams, X. Gao, N. Rubenstein, A. Cook, J. Vrana and J.
Boyd. Bennett Department of Chemistry, West Virginia University, Morgantown, WV.
Although current understanding of signal transduction within biological networks
is based upon changes in protein phosphorylation over time, cells are also responsive
to increasing doses of inhibitors independent of time. By assessing the functional
response of a cellular network to increasing doses of inhibitors, additional information
can be gained about the relationships and roles of proteins in response to
cellular stress. To determine the direction of information flow in a cellular network
in response to stress, the phosphorylation activity of several protein kinases within
the mitogen-activated protein kinase (MAPK) pathways were monitored. HepG2
cells were exposed to increasing doses of deguelin, and KCN. Correlation and protein
activity were quantified using multiplex phosphoprotein assays on lysates obtained
400 minutes after dosing. The resulting correlation analysis across all dosing
intervals was in general agreement with accepted temporal models of signal transduction.
However, correlation values compared on a dose-to-dose level differed significantly
from the temporal model and revealed surprising changes in specificity
among protein kinases. This preliminary analysis into interactions of signaling proteins
in response to increasing dose offers a unique way of framing cellular network
relationships in biochemical networks, and holds the potential to aid in the eventual
prediction of toxicity by identifying cellular strategies for adaptation and survival.
401 IMPACT OF ENERGY METABOLISM ON THE DESIGN
OF KINASE-TARGETED THERAPEUTICS.
A. Cook, H. Williams, X. Gao and J. Boyd. Bennett Department of Chemistry,
West Virginia University, Morgantown, WV.
Protein kinases play key roles in signaling pathways that regulate cellular functions
such as proliferation and apoptosis. Due to the potential regulatory control over
such important biological processes, interest in kinase-targeted chemotherapeutics
has grown exponentially in the drug discovery arena. Current drug design is aimed
at developing substrate-competitive kinase inhibitors, which are thought to offer
higher selectivity and sensitivity over ATP-competitive kinase inhibitors that must
compete with intracellular ATP concentrations. However, what has not been explored
as comprehensively is the impact that these inhibitors can have on energy
metabolism. This study seeks to investigate the activity of two different AKT and
GSK3 inhibitors, substrate-competitive versus an ATP-competitive, in relation to
glycolysis and oxygen consumption. Our results suggest that beyond the effects of
inhibiting metabolically-linked proteins, ATP-competitive inhibitors may alter intracellular
interpretation of available ATP which leads to decreases in compensatory
energy metabolism and could result in greater toxicity.
402 cAMP-DEPENDENT PATHWAY(S) INCREASE P27 KIP -
CYCLIN D1 THROUGH THE RAP-GTP/B-RAF MAPK
SIGNALING PATHWAY IN RENAL CANCER.
N. Mastrandrea, K. Y. Tham, J. D. Cohen, T. J. Monks and S. S. Lau.
Pharmacology/Toxicology, University of Arizona, Tucson, AZ.
The Eker rat (Tsc-2EK/+) bears a germline mutation in the tuberous sclerosis-2
(Tsc-2) tumor-suppressor gene, and can undergo spontaneous and chemically induced
loss of heterozygosity. Renal tumors null for tuberin, the Tsc-2 gene product,
derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ) treated Tsc-
2EK/+ rats, display elevated p27 and cyclin D1 levels. Similar elevations were observed
in TGHQ transformed QTRRE renal epithelial cells derived from Tsc-
2EK/+ rats, which also exhibit high ERK, B-Raf and Raf-1 kinase activities. p27
signaling pathways, especially those associated with its binding partner, cyclin D1,
are relatively unknown. In QTRRE cells cAMP signaling regulates both p27 expression
and Rap-GTP/B-Raf activation of the MAP kinase cascade. We now report
that dibutyryl cAMP (db-cAMP; 1mM) or the phosphodiesterase inhibitor,
theophylline (0.6 mg/ml), produced a time-dependent increase in p27 protein levels
(1.4, 2.0, 3.0 fold, and 1.6, 2.8, 2.4 fold at 2, 10 and 24 hrs, respectively). A
concomitant increase in cyclin D1 levels was also observed in db-cAMP and theophylline
treated QTRRE cells. Inhibition of Raf kinases, with either sorafenib (50
μM) or B-Raf siRNA resulted in MAPK down-regulation of p27. Moreover, following
a 48 hr treatment of QTRRE cells with p27 siRNA there was an equivalent
88% decrease in both p27 and cyclin D1 protein levels; similarly, a 48 hr treatment
with cyclin D1 siRNA resulted in a concomitant 50% decrease in p27 protein levels,
suggesting that the p27-cyclin D1 complex promotes preservation of both proteins.
Collectively these data reveal that the cAMP/Rap1b/B-Raf pathway modulates
the expression of p27 and cyclin D1 in Tsc-2 gene regulated-renal cancer.
86 SOT 2011 ANNUAL MEETING
Therefore, the loss of tuberin and engagement of the cAMP pathway may independently
direct p27-cyclin D1 stabilization during renal tumor formation.
(P30ES006694, T32ES007091, AstraZeneca Studentship)
403 EFFECT OF P, P’-DDE ON JAK2, STAT1α, AND NFκB
ACTIVATION IN MACROPHAGES J774A.1.
N. A. Torres-Aviles 1 , L. C. Acosta-Saavedra 1 , A. L. Luna 1 , E. K. Silbergeld 2 and
E. S. Calderon-Aranda 1 . 1 Department .Toxicologia, Cinvestav, Mexico, Mexico and
2 BSPH, Johns Hopkins University, Baltimore, MD.
DDE(dichloro-diphenyl-dichloroethylene) is the most persistent metabolite of
DDT since it has been found in high concentration in the serum of people of region
where DDT was used. In macrophages J774A.1 activated with IFN-γ or LPS,
p,p’-DDE reduced nitric oxide (NO -) production and iNOS gene expression, as
well as bactericide ability against Mycobacterium microti, suggesting that both
pathways share molecules targeted by p,p’-DDE. The iNOS gene transcription is
regulated by NFκB and STAT1α,activated by LPS and IFN-γ pathway, respectively,
however, it has shown that in dendritic cells, among others, JAK2, a kinase
involved in canonical activation of STAT1α, participates in the activation pathway
of NF-κB. The objective of this study was to assess whether JAK2 is a molecular
target of p,p’-DDE in macrophages activated with LPS, to explain, at least partially,
the mechanism of decreasing of NO - production. Methods In J774A.1
macrophages activated with LPS was evaluated the effect of one hour pre-exposure
to 2.5 μg/ml p,p’-DDE on the activation of JAK2, STAT-1α and NF-κB by
Western blot, and NO -, determined by the Griess method. Results shown that the
p,p’-DDE inhibits NO - production in LPS-activated cells and that JAK2 activation
is involved in LPS-induced production of NO -; in non exposed cells LPS induces
phosphorylation of JAK2/STAT-1α, while p,p’-DDE in activated cells, overinduced
JAK2/STAT-1α, but reduced NF-kB activation induced by
LPS,suggesting that this effect is non-dependent of over-activation of JAK2; p,p’-
DDE in non activated cells induces JAK2/STAT-1α pathway. Taken together our
results show that the inhibition of NO - production is at least partially dependent
of the inhibition of the iNOS gene activation via of NF-κB, but this effect is not related
with the effect of p,p’-DDE on JAK2, but more studies about the mechanisms
of inhibition of p,p’-DDE in the functional activation of macrophages are needed.
This work was supported by Conacyt-46297.
404 A ROLE FOR STRESS-RESPONSIVE SIGNALING IN THE
REGULATION OF ERK OSCILLATIONS.
T. J. Weber 1 , H. Shankaran 2 , K. M. Waters 2 , W. B. Chrisler 1 and R. L. Sontag 1 .
1 Cell Biology & Biochemistry, PNNL, Richland, WA and 2 Computational Biology,
PNNL, Richland, WA.
Deregulation of feedback control processes by stress-responsive signaling can play
an important role in pathophysiology. We have recently demonstrated that the extracellular
signal regulated kinase (ERK) displays persistent nuclear-cytosolic oscillations
in response to tyrosine kinase receptor ligands (EGF, bFGF) in mouse and
human cell systems. ERK oscillations are inhibited by ≥ 0.32 hydrogen peroxide
and 10 cGy X-irradiation, doses that do not produce observable decrease in cell viability
using a neutral red assay. Antibiotics used to select for transduced cells expressing
selectable markers inhibit ERK oscillations in both reversible (normal immortalized
human keratinocytes) and irreversible (primary human keratinocytes)
fashion. We confirmed that X-irradiation induces an increase in phospho-p38 levels
by Western blot and p38 is frequently activated in response to a broad range of
cell stressors. Growth factor-dependent ERK oscillations can be restored in a subset
of non-oscillating cells by an inhibitor of p38 kinase (SB203580). Collectively,
these observations demonstrate that 1) ERK oscillations are inhibited by hydrogen
peroxide and X-irradiation at low dose exposures, 2) ERK oscillations can be restored
in non-oscillating cells by a p38 kinase inhibitor and 3) standard practices
for maintaining and engineering cells can disrupt ERK oscillations potentially contributing
to inter-laboratory biological variability. The disruption of ERK oscillations
represents a putative new mode-of action by which toxicological agents can
perturb biological systems.
405 HARMINE MODULATES DIOXIN-INDUCED CYP1A1
ENZYME THROUGH TRANSCRIPTIONAL AND POST-
TRANSLATIONAL MECHANISMS.
M. El Gendy and A. El-Kadi. Faculty of Pharmacy and Pharmaceutical Sciences,
University of Alberta, Edmonton, AB, Canada.
Dioxins are widespread environmental contaminants that induce the carcinogenactivating
enzyme cytochrome P450 1A1 (CYP1A1) through an aryl hydrocarbon
receptor (AhR)-dependent mechanism. Harmine is a common β-carboline present
in several medicinal plants such as Peganum harmala. Harmine possesses antitumor