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October 01, 2012 (Vol. 32, No. 17)

Gene Expression Analysis Reshapes Biomedical Research

  • Fetal Alcohol Disorder

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    Research has shown that alcohol induces aberrant changes in gene expression and in the epigenome of embryos. One result is the misregulation of proteins such as serotransferrin, which was found in greater amounts in mouse embryos exposed to alcohol. The increase (A) was confirmed by immunolocalization of serotransferrin to the branchial arches (BA1) and somites, particularly in the heart (boxed area), as compared to control (B). Misregulated proteins are known to be involved in nervous system development and function. [International Journal of Proteomics, Hindawi Publishing, 2012, doi:10.1155/2012/867141]

    An interesting example is provided by the fetal alcohol spectrum disorder, a multisystem condition that is causally linked to maternal alcohol use during pregnancy, and is characterized by physical, behavioral, and cognitive deficits, including developmental delay, growth deficiency, craniofacial dysmorphology, and modifications that affect other organs and systems at various degrees.

    Previously, Dr. Zhou and colleagues revealed that ethanol exposure alters the cellular DNA methylation program during early neural tube development, and identified over 2,100 epigenetically changed genes in which the cytosines are differentially methylated in alcohol-treated embryos.

    To better understand the protein expression changes that occur as a result of epigenetic and genetic changes in the fetal alcohol spectrum disorder, Dr. Zhou, in collaboration with Stephen Mason, Ph.D., an adjunct researcher who is also in the department of anatomy and cell biology at the Indiana University School of Medicine, used whole-embryo cultures to examine the alcohol-signature protein profile across all cell and tissue types in mice at the early neural developmental stage during neurulation.

    “This study is a continuation of our previous studies,” explains Dr. Zhou.

    The team identified 40 protein spots that were differentially expressed between alcohol-treated and control cultures. Several of these proteins, confirmed by mass spectrometry, fulfill key roles in the cell cycle and the ubiquitin-proteasome pathway.

    The results indicated that epigenetic and genetic changes occurring as a result of alcohol exposure impact protein expression during neurulation. The biological functions that were perturbed were linked to tissues and organs that originate, during development, from all three embryonic layers.

    “Analyses of epigenetic modifications and those that survey gene expression, protein expression, and metabolic perturbations are required to investigate the molecular and cellular changes as a whole, to eventually understand the causal mechanisms that differ between two distinct states, such as between the child with developmental delay caused by drinking during pregnancy and the healthy child,” explains Dr. Zhou.

    “We are trying to understand how gene expression is regulated, how its deregulation causes disease, and ultimately how we can correct those deregulated states to treat disease,” says Bing Zhang, Ph.D. assistant professor of biomedical informatics at the Vanderbilt University School of Medicine.

    Dr. Zhang’s group has explored the possibility of defining a gene expression signature that can be used to make prognostic and therapeutic decisions in patients with colorectal cancer. This malignancy, currently the third leading cause of cancer mortality worldwide, is stratified into four stages (I to IV), with a higher stage being assigned to more serious disease.

    Cure, which occurs in approximately 95% of patients with stage I disease treated surgically, is difficult to accomplish in patients with stage IV disease, who also require chemotherapy.

    “For stage II and III patients, which represent a large population, the question is whether we need to provide chemotherapy, because previous clinical trials suggest that it is not required for certain patients, but it is beneficial for others,” explains Dr. Zhang.

    Histological features, such as tumor size, lymph node positivity, and metastatic dissemination have been used to perform such predictions in the past, but often they were not reliable.

    “Our idea was to use gene expression analysis to better predict the prognosis of stage II and III colon cancer patients,” explains Dr. Zhang.

    A challenging aspect is that different gene expression signatures from different studies often do not overlap with each other. “We tried to find common biological themes across previously published signatures, and by integrating them into biological networks, we generated a gene expression signature that is more biologically meaningful and, additionally, has a good prognostic value,” says Dr. Zhang.

    This approach revealed that genes with mechanistically important roles in colorectal cancer may be used to develop reliable prognostic models that accurately predict recurrences and the response to adjuvant chemotherapy. Gene expression-based stratification of these two cancer stages is crucial for increasing survival and the quality of life, while minimizing the chemotherapy-associated toxic effects and the financial costs involved.

  • 3D Systems

    While historically the biology of specific cell types was examined by using homogenous monolayer cultures grown in vitro, this experimental approach tends to be an oversimplification. As opposed to the culture systems that often use a single cell type, organs consist of a complex network of multiple cell types that also includes extracellular matrix, growth and signaling factors, and other components.

    Dynamic bidirectional signaling events within this microenvironment assume key functions in tissue maintenance and in suppressing or promoting tumor development. The microenvironment is one of the most important factors that emerged in recent years to shape cellular behavior, and three-dimensional (3D) cell culture systems, which more faithfully reflect the in vivo conditions, have increasingly found applications in many biomedical areas.

    Nils Cordes, M.D., Ph.D., professor of molecular and cellular radiobiology at the Dresden University of Technology, and colleagues have comparatively examined the gene expression profiles in two exponentially growing human cancer cell lines—lung carcinoma and squamous cell carcinoma—grown in a 3D extracellular matrix scaffold or as a conventional two-dimensional monolayer.

    “The idea behind this experiment was that in vitro cell cultures that we are working with do not always reflect the in vivo situations, and we wanted to know whether the differential gene expression shapes tumor cell sensitivity to radiation and chemotherapeutics,” explains Dr. Cordes.

    The technique revealed significant gene expression changes between the two conditions.

    “We showed that there was a differential gene expression pattern, between the 3D and the 2D conditions, in genes encoding ECM proteins, integrins, cell shape proteins, and proteins linked to morphology, but not to DNA repair,” notes Dr. Cordes.

    Moreover, under the 3D growth conditions, cells showed an increased resistance to radiotherapy and chemotherapy as compared to the 2D conditions, pointing toward the importance of the growth conditions in shaping gene expression. Important in this context is another study from Dr. Cordes’ group, in which differences in cell cycling, oxygen levels, and radiation dosimetry were ruled out as critical determinants of higher tumor cell survival levels under 3D growth conditions.

    Few advances have reshaped biomedical and clinical areas within such a short time, and to such a great extent, as the ones that marked gene expression analysis. Technological developments, their intimate integration with many basic science disciplines, and a growing number of clinical applications have been defining this field for the past decade.

    While collecting increasingly sophisticated and complex datasets has undoubtedly played an instrumental role, perhaps an even greater impact was exerted by the development and implementation of novel conceptual frameworks, a theme that provides a central learning point and was so relevantly expressed in Sir William Bragg’s words: “The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them.”

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