Now that scientists have solved
the code for the human genome and are moving forward to sequence the genomes
of other organisms, a significant challenge lies in how to harness this vast
amount of new information to benefit society. Recognizing that cross-communication
and cooperation among different research labs is essential to realizing the
potential of genomic science for toxicology, the NIEHS earmarked $37 million
to establish the Toxicogenomics Research Consortium (TRC) in November 2001.
image credit: UNC-CH |
A major objective of the TRC is to link investigator-initiated research projects
at five academic centers--the Massachusetts Institute of Technology, Duke
University, the University of North Carolina at Chapel Hill (UNC-CH), Oregon
Health & Science
University, and the Fred Hutchinson Cancer Research Center/University of
Washington--in a collaborative effort to understand how organisms respond to
chemical exposures
and other kinds of environmental stress. At UNC-CH, investigator-initiated
research projects focus on different model systems--from mice and cell lines
to humans--all with a common, unifying focus on environmental challenges
that cause DNA damage or oxidative stress, leading to cancer.
With the goal of finding molecular signatures that can be used for early
risk assessment, these projects employ microarray tools to assay for gene expression
responses to cancer-related chemicals. Some use the same compounds that cancer
patients are already receiving, to gather information on how different populations
may react to certain drugs.
Dampening the Background Noise
In one TRC projects, David W. Threadgill, an assistant professor of environmental
sciences and engineering, uses mouse models to investigate differences in response
to various tumor-inducing and chemotherapeutic chemicals from one mouse strain
to another. In his model, all the mice of one strain are essentially identical,
providing an unlimited number of individuals that have the same genotype. This
experimental approach enriches the opportunity to separate out what is "noise" in
the data from what is truly meaningful. Threadgill's lab is currently profiling
about a dozen mouse strains, some of which are susceptible to certain types
of cancer while others are resistant.
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A perfect likeness. UNC-CH researcher David W. Threadgill uses
strains of mice in which all the animals have the identical genotype to
study cancer susceptibility from exposure to a variety of environmental
toxicants. Some of the mouse strains are resistant to cancer, while others
are more susceptible to the disease.
image credit: Arnold Greenwell/EHP |
Among organisms, there is a wide range of susceptibility to the effects of
different toxicants. The classes of chemical compounds that cause cancer--including
alkylating agents, DNA-damaging agents, and toxicants that affect the cell
cycle--vary in the mechanisms by which they cause the disease. There is also
extensive variation in the way different cancer patients respond to treatment
and how they handle the toxic effects of different chemical therapies.
To better understand why this is so, Threadgill exposes different mouse strains
to various chemicals over a variety of dose regimens and time spans, then compares
the results to a control group. After exposure, tissue is harvested from the
liver, colon, and breast for extraction of RNA that is converted into cDNA.
The cDNA is hybridized to microarray chips containing 17,000 mouse genes. If
a gene is turned on or up as a result of exposure, it will fluoresce more brightly
than the same gene on the corresponding chip for the control group. Analysis
of the signals reveals which genes increase their expression level as a result
of exposure to a particular toxicant and which genes are turned down.
Threadgill explains, "Lots of genes are turned on, up, or down. Comparison
of the signals from different strains reveals which genes are unique to certain
classes of compounds, at what dose levels and time treatments." His lab also
examines direct causes of the associated physical changes and their correlation
with prior knowledge of how certain classes of compounds affect tissue. He
has detected significant strain-dependent differences in gene expression in
both baseline and treated colon tissue. He says, "The characterization of these
differences in the context of disease and treatment end points should reveal
new insights into the mechanistic causes of the cancer end points."
Screening thousands of genes and expression changes presents a huge computational
challenge to discern what the critical signatures are. Biostatisticians have
begun to mine the data collected by Threadgill over the first two and a half
years of his project to evaluate thousands of signals and identify similarities
and differences between treatment time courses, exposure levels, and individual
strains. Knowing which genes are significant is vital to finding prognostic
markers for whether an individual is susceptible to effects from certain compounds
and for evaluating what makes certain individuals more susceptible than others.
Layers of Response
In a coordinated experimental design, Charles M. Perou, an assistant professor
of genetics, is screening some of the same compounds as Threadgill in his investigations
of the transcriptional response in breast cancer using human cell lines from
breast tumors and immortalized breast epithelial cell lines (that is, publicly
available cell lines that have been maintained over time for experimental use).
This may allow for comparative analyses of results across different model systems
that could lead to new insights into the resulting biological insults from
exposure to specific toxicants. Cell lines are treated with different regimens,
and then examined for gene expression changes using microarrays.
Perou is also studying the effects of these drugs on real breast tumors of
patients before, during, and after chemotherapy for comparison of similarities
or differences in experimental cell lines with tumors in patients. In a paper
in the June 2004 issue of Cancer Research, Perou's lab reported that
a small but significant set of genes were identified as being changed in both in
vitro cells and living tumors, and that these are likely to be very important
in cellular response to toxicants. In this study, 20-30 changes observed
in the expression profiles identified potential candidate genes for a general
response to these compounds.
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Molecular mysteries. UNC-CH researchers are elucidating how susceptibility
to breast cancer is impacted by a “generic” stress response
that is similar across genes. Such a generic response underlies characteristic
drug-related responses, which molecular signatures indicate vary from person
to person.
image credit: Image Source |
Perou and colleagues have discovered that although the compounds tested have
different mechanisms and targets in cells, the overall cellular response to
the different drugs is the same--a sort of dominant generic stress response
that is similar across genes. The secondary drug-specific response pattern
is layered on top of this generic stress response pattern. Perou says, "Arrays
do a great job of implicating genes in certain processes in response to different
drugs, but the genes involved in the response are the same. . . . This leads
to the hypothesis that some people with reduced ability to respond might be
more susceptible because of variants in these generic stress response genes."
Perou's work has also uncovered unique molecular signatures that distinguish
two subtypes of breast cancer. Patients with one type will do well under the
standard drug therapies and survive. However, for those with the other type,
prognosis for survival if given standard treatment is not good; more aggressive
treatment such as radical mastectomy and radiation would be needed, and even
then the chances of survival are not nearly as good. This complex overlapping
pattern of drug-specific response genes embedded within the common response
will be reported in a second paper submitted for publication.
Perou is particularly interested in the generic stress phenomenon because
individuals who have susceptible variants of these genes could be unable to
respond to many different toxicants in their environment. He says, "Microarrays
provide a big list of candidate genes. The challenge is to figure out [which
genes] are the passengers and [which] are the drivers, then apply that information
to population studies to identify genetic variants that are hyperactive and
correspond to susceptibility." Through the TRC, he says, it may ultimately
be possible to link findings and design experiments such that scientists can
identify a common set of genes in mice and humans for independent validation
for susceptibility. "As we learn more about evolutionarily conserved genes
across species that are involved in reactions to environmental chemicals," Perou
says, "we will have more confidence in the scientific findings."
Confidence Building
William K. Kaufmann, director of the UNC-CH Program in Toxicogenomics and
director of the Genetic Susceptibility Research Core in the UNC Center for
Environmental Health and Susceptibility, studies expression of tumor suppressor
genes in the cell cycle. Kaufmann is interested in how changes in the DNA at
the ends of chromosomes provide life span checkpoint monitors that signal the
time for aging cells to die. Experimental evidence indicates that responses
similar to those involved in cell senescence occur in breast, liver, and colon
cells exposed to carcinogenic compounds. Such changes are accompanied by reproducible
gene expression patterns.
Kaufmann says, "When the same unexpected patterns of response are seen in
lines from several different individuals, our confidence that the response
to stress is biologically meaningful increases." Knowledge gained from the
characterization of modulations in expression of genes as a result of exposure
to different carcinogens could one day provide a fingerprint to enable design
of precise treatment strategies for specific individuals exposed to different
toxicants.
As the robustness of microarray technology for gene expression profiling
improves and becomes more standardized, scientists will be able to employ molecular
signatures to tailor therapies as well as conduct exposure risk assessment.
Kaufmann concludes that in spite of sometimes feeling overwhelmed by too many
genes and the need for a bigger computer, the UNC-CH microarray research projects
are producing a data-rich picture, and are spawning whole new areas of discipline
to look for patterns of change. Such individual scientific discoveries and
applications of the principles of toxicogenomics are resulting in a serendipitous
research crossover that will continue to spur insights, leading to faster,
broader, and more effective applications for public health.
Mary Eubanks