Role
of sonic hedgehog in cyclopamine-induced holoprosencephaly
Raj
P. Kapur, PhD, Department of Pathology
Co-Investigator: Henk Roelink, Department of Biological Structure;
Investigators
hypothesized that the teratogen cyclopamine and structurally related
alkaloids might interfere with the intercellular signals mediated
by the protein sonic hedgehog (SHH). Genetic perturbations of the
SHH gene produce holoprosencephaly and the active form of SHH is
covalently-linked to cholesterol through a biosynthetic process
that might be vulnerable to steroidal agents, like cyclopamine.
Investigators focused on chick embryos as a model system for in
vivo manipulations. Cyclopamine treatment induces holoprosencephaly
reproducibly, whereas the other agents tested do not. In addition,
changes (e.g., absent floor plate, diminished and displaced motor
neurons) consistent with loss of SHH-mediated signals are evident
in the neural tubes of embryos treated with cyclopamine, but not
those treated with the other alkaloids. These observations are consistent
with the hypothesis that cyclopamine reduces SHH biosynthesis from
its endogenous source in the notochord.
A collaboration
with David Raible (UW Biostructure) was established to examine effects
of cyclopamine on embryogenesis in zebrafish. Preliminary studies
indicate that cyclopamine interferes with ventral patterning in
the caudal neural tube and somites, but not in the forebrain. The
alterations observed in the affected sites are consistent with disruption
of established functions of SHH in vivo.
Investigators
are presently working to confirm some of these preliminary findings
and to examine other potential targets for cyclopamine action, such
as cells that respond to SHH inductive signals. The plan is to study
other teratogenic and non-teratogenic steroids with a particular
interest in how these agents interfere with SHH processing.
Resulting
Grant Support
Based
on preliminary findings, investigators submitted an RO-1 proposal
with Henk Roelink as the principle investigator and Raj Kapur as
a co-investigator. This proposal has been reviewed and will be funded
by the NIEHS (RO1ES09201).
Polymorphisms
in biotransformation enzymes and risk of lung cancer: a population-based
study
Thomas
L. Vaughan, MD, MPH, Epidemiology, UW
This
project investigates whether inherited polymorphisms in two biotransformation
enzymes, CYP1A1 and microsomal epoxide hydrolase (mEH), are associated
with varying risks of lung cancer in humans. These enzymes are involved
in the metabolism of polycyclic aromatic hydrocarbons in tobacco
smoke. Several previous studies indicate that polymorphisms in these
enzymes may affect their activity or production, and the sparse
epidemiologic evidence to date suggests that persons with one or
both of the variant alleles may be at increased risk of lung cancer.
This
proposal builds upon two ongoing NIH grants at the Fred Hutchinson
Cancer Research Center. These grants are funding the collection
of detailed exposure information and blood samples on population-based
cases and controls; blood processing; DNA extraction; GSTM1,
GSTT1 and GSTP1 genotyping; and GSTP1 protein
expression. The present proposal seeks to assay DNA on a subset
of the participants (250 cases and 250 controls matched on smoking
status) for their mEH and CYP1A1 genotypes. Odds ratios associated
with the variant genotypes will be calculated, after adjusting for
the potential confounding effects of tobacco use and other risk
factors for lung cancer. Vaughan's laboratory also will investigate
modifications of any gene associations by various risk factors,
and by the genotype of the other biotransformation enzymes.
DNA
extraction of 500 subjects has been completed.
This
study will provide preliminary data that will be critical to funding
larger studies investigating the roles of multiple biotransformation
enzymes in lung cancer.
Inhibition
of cell cycling in the developing CNS by methylmercury: Role of
p21
Elaine
M. Faustman, PhD, Environmental Health, UW
Methyl
mercury (MeHg) is a global environmental pollutant and well recognized
developmental neurotoxicant in humans, primates, and rodents, causing
cell loss and microcephaly. Histologic evidence suggests that cell
cycle inhibition, not cell death, underlies the reduction in numbers
of central nervous system (CNS) cells. This suggests a central role
for cell cycle inhibition in MeHg neurodevelopmental toxicity.
Preliminary
experiments indicate that MeHg can cause a biphasic inhibition of
primary CNS cell proliferation with an S-phase inhibition after
12 hours of chronic exposure in vitro, followed by inhibition
at the G2/M phase. Under similar exposure conditions, MeHg also
appears to induce expression of cell cycle regulatory genes, including
p21, associated with cell cycle arrest. These data are consistent
with a hypothesis that altered expression of cell cycle regulatory
genes underlies the observed inhibition of cell proliferation upon
exposure.
The
project will evaluate the role of p21 and altered cell cycling
in response to MeHg exposure during development using p21-deficient
and normal mice. Because p21 is involved in halting cell
cycle progression in response to cell injury, p21-deficiency may
enhance sensitivity to MeHg during development. Pregnant female
mice will be exposed to MeHg, their uteri will be removed, and CNS
tissue analyzed by flow cytometry for cell cycling. Cells will also
be sorted by flow cytometry according to cell cycle phase, collected
and analyzed for alterations in p21 gene expression and protein
level. These analyses will provide key information regarding the
events at the molecular level which may underlie MeHg-induced cell
cycle inhibition during CNS development.
Mitochondrial
toxicants and the mammalian stress response
Sam
A. Bruschi, PhD, Department of Medicinal Chemistry, UW
This
project is isolating mammalian homologs of yeast proteins necessary
for a retrograde communication phenomenon between organelles and
the nucleus. The RTG genes (RTG1, RTG2, and RTG3)
switch on and upregulate nuclear genes in response to functional
perturbations in the mitochondria and/or peroxisomes. Given the
large number of environmental chemicals and therapeutic agents that
alter mitochondrial function, the possibility was raised the mammalian
homologs of RTG genes may be useful biomarkers to assess alternations
to organelle function.
PCR
was used to clone RTG genes directly from Saccharomyces
cerevisiae genomic DNA. Restriction digests were used to determine
that both RTG1 and RTG2 were inserted in the correct
orientation in all but one of the colonies selected from each group.
However, of the forty colonies of RTG3 screened, all sequences
were inserted backwards. The biological significance, if any, of
this observation is unknown. Each selected colony was amplified
and purified as a working stock.
The
investigators confirmed the identity of these inserts by using the
SC2 DNA sequencing facility. Slot blot experiments indicated that
each RTG probe, in addition to the GAPDH probe (a positive control),
hybridized specifically to both yeast DNA and mouse DNA. As expected,
the relative signal strength for each probe was diminished with
mouse DNA in comparison to yeast DNA. Southern blot experiments
for each genomic DNA (E. coli., C. elegans, S. cerevisiae,
mouse and human) are continuing. Preliminary results confirm that
non-isotopic labeling procedures used are sensitive enough to detect
single copy sequences. Moreover, specific RTG1 cross-hybridization
with a higher molecular fragment in C. elegans DNA indicates
that a high homology sequence is evident in this species.
Mechanisms
Of Ethanol-Induced Neuronal Apoptosis
Zhengui
Xia, PhD, Department of Environmental Health, UW
Alcohol
abuse causes several physiological effects including fetal alcohol
syndrome resulting from maternal ethanol exposure in the developing
fetus. However, the exact mechanisms of ethanol-induced neurotoxicity
have not been defined. To determine if ethanol induces cell death
in primary neurons, Xia's laboratory cultured cortical neurons from
newborn rats in BEM-based medium containing 10% fetal calf serum.
Six days later, neuron cultures were treated with 1% or 2% ethanol
for 48 hours, and cell viability examined by MTT metabolism. Preliminary
data showed that ethanol can induce cell death in primary cortical
neurons under normal culture conditions. Ethanol previously has
been shown to promote apoptosis in cerebellar granule neurons by
inhibiting the trophic effect of NMDA.. To the principal investigator's
knowledge this is the first report that ethanol, added to normal
healthy neuron cultures in the presence of serum, can induce cell
death.
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