Preimplantation genetic diagnosis: Analysis of gene expression, nuclear DNA and cytoplasmic DNA

Preimplantation genetic diagnosis (PGD) following in vitro fertilization (IVF) offers couples at risk for transmitting genetic disorders the opportunity to identify affected embryos prior to replacement. In particular, embryo gender determination permits screening for X-linked diseases of unknown etiology. Analysis of embryos can be performed by polymerase chain reaction (PCR) amplification of material obtained by micromanipulation. This approach provides an alternative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. ^ Lately, the focus of preimplantation diagnosis and intervention has been shifting toward an attempt to correct cytoplasmic deficiencies. Accordingly, it is the aim of this investigation to develop methods to permit the examination of single cells or components thereof for clinical evaluation. In an attempt to lay the groundwork for precise therapeutic intervention for age related aneuploidy, transcripts encoding proteins believed to be involved in the proper segregation of chromosomes during human oocyte maturation were examined and quantified. Following fluorescent rapid cycle RT-PCR analysis it was determined that the concentration of cell cycle checkpoint gene transcripts decreases significantly as maternal age increases. Given the well established link between increasing maternal age and the incidence of aneuploidy, these results suggest that the degradation of these messages in aging oocytes may be involved with inappropriate chromosome separation during meiosis. ^ In order to investigate the cause of embryonic rescue observed following clinical cytoplasmic transfer procedures and with the objective of developing a diagnostic tool, mtDNA concentrations in polar bodies and subcellular components were evaluated. First, the typical concentration of mtDNA in human and mouse oocytes was determined by fluorescent rapid cycle PCR. Some disparity was noted between the copy numbers of individual cytoplasmic samples which may limit the use of the current methodology for the clinical assessment of the corresponding oocyte. ^

Immunomodulation by shark cartilage extracts

The immune system is composed of innate and adaptive mechanisms. Innate immune responses are significantly modulated by immunomodulatory factors that act through the induction of specific patterns of cytokine production in responding cells. Human leukocytes have been shown to respond to substance(s) present in acid extracts of commercial shark cartilage (SC). Shark cartilage is a food supplement taken by consumers as a prophylaxis and for the treatment of conditions ranging from arthritis to cancer. No reliable scientific evidence in the literature supports the alleged usefulness of shark cartilage supplements, but their use remains popular. Cartilage extracts exhibit immunomodulatory properties by inducing various inflammatory, Th1-type cytokines and potent chemokines in human peripheral blood leukocytes (HPBL) in vitro. The objectives of the study were to (1) to determine the nature of the active component(s), (2) to define the scope of cellular response to SC extract, and (3) to elucidate the molecular mechanisms underlying bioactivity. Results showed that there are at least two cytokine-inducing components which are acid stable. One anionic component has been identified as a small (14-21 kDa) glycoprotein with at least 40% carbohydrate content. Shark cartilage stimulated HPBL to produce cytokines resembling an inflammatory, Th1 polarized response. Leukocyte-specific responses consist of both initial cytokine responses to SC directly (i.e., TNF-α) and secondary responses such as the IFN-γ response by lymphocytes following initial SC stimulation. Response of RAW cells, a murine macrophage cell line, indicated that TNF-á could be induced in macrophages of another mammalian species in the absence of other cell types. The results suggest that the human monocyte/macrophage is most likely to be the initial responding cell to SC stimulation. Stimulation of cells appears to engage at least one ligand-receptor interaction with TLR 4, although the role of TLR 2 cannot be ruled out. Initial activation is likely followed by the activation of the JNK and p38 MAPK signal transduction pathways resulting in activation, release, and translocation of transcription factor nuclear factor κB (Nf-κB). This dissertation research study represents the first in-depth study into characterizing the bioactive component(s) of commercial shark cartilage responsible for its immunomodulating properties and defining cellular responses at the molecular level.

The role of the transcription factor Ets1 in melanocyte development

Melanocytes, pigment-producing cells, derive from the neural crest (NC), a population of pluripotent cells that arise from the dorsal aspect of the neural tube during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The deletion of the transcription factor Ets1 in mice results in hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The goal of the present study was to establish the temporal requirement and role of Ets1 in murine melanocyte development. In the mouse, Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick cranial NC, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of melanocytes, enteric ganglia, and other NC derivatives. ^ Using a combination of immunofluorescence and cell survival assays Ets1 was found to be required between embryonic days 10 and 11, when it regulates NC cell and melanocyte precursor (melanoblast) survival. Given the requirement of Ets1 for Sox10 expression in the chick cranial NC, a potential interaction between these genes was investigated. Using genetic crosses, a synergistic genetic interaction between Ets1 and Sox10 in melanocyte development was found. Since Sox10 is essential for enteric ganglia formation, the importance of Ets1 on gut innervation was also examined. In mice, Ets1 deletion led to decreased gut innervation, which was exacerbated by Sox10 heterozygosity. ^ At the molecular level, Ets1 was found to activate a Sox10 enhancer critical for Sox10 expression in melanoblasts. Furthermore, mutating Ets1 at a site I characterized in the spontaneous variable spotting mouse pigmentation mutant, led to a 2-fold decrease in enhancer activation. Overexpression and knockdown of Ets1 did not affect Sox10 expression; nonetheless, Ets1 knockdown led to a 6-fold upregulation of the transcription factor Sox9, a gene required for melanocyte and chondrocyte development, but which impairs melanocyte development when its expression is prolonged. Together, these results suggest that Ets1 is required early during melanocyte development for NC cell and melanoblast survival, possibly acting upstream of Sox10. The transcription factor Ets1 may also act indirectly in melanocyte fate specification by repressing Sox9 expression, and consequently cartilage fate.^

Evaluation of the cytotoxic and cytostatic activities of medicinal plants used by Peruvian healers against cancer-related symptoms

Twelve plants used medicinally in Callejon de Huaylas, Department of Ancash, northeastern Peru were selected and screened in vitro for cytotoxic and cytostatic activities. Traditional preparations, aqueous extracts and organic extracts (methanol:dimethyl chloride) were tested against murine leukemia P388 cells using flow cytometry. Seventy-five percent or more of the traditional and aqueous extracts were cytostatic at concentrations of 1mg/ml. For organic extracts, cytostatic activity ranged from 8.3% (at 6.25 μg/ml) to 58.3% (at 100 μg/ml). Quinchamalium procumbens, Ophryosporus chilca and Baccharis genistelloides showed strong activity. Extracts of Brachyotum rostratum, Monnina salicifolia, and Orthrosanthus chimboracensis were particularly interesting, since they were cytostatic but not cytotoxic at concentrations of 0.5 mg/ml. These Andean plants merit further analysis. The high percentage of activity found among the traditional preparations suggests that the traditional medical knowledge of Callejon de Huaylas healers deserves respect and merits further research. ^

Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Novel insights into the mechanisms of regulation of tyrosine kinase receptors by Ras Interference 1

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBP&agr;. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.

Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.

Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.

Establishment of a PCR and restriction enzyme assay for the detection of the hemochromatosis gene in normal subjects and diabetic patients

The purpose of this study was to establish a polymerase chain reaction (PCR)-restriction enzyme assay for detecting the hereditary hemochromatosis (HHC) mutation, C282Y, in gestational and gestational diabetic subjects in South Florida. DNA samples from 43 gestational subjects were amplified by PCR, digested with RsaI, and analyzed by electrophoresis. An allelic frequency of 2.33%, or 4.65% heterozygosity, was observed. The assay is successful and applicable to future studies on HHC and gestational diabetes. ^

Fas-FasL expression and interactions in mouse tumor cell lines: Implications for tumor immune escape

The Fas system, comprising the Fas receptor (Fas/Apo-1/CD95) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. FasL exists as transmembrane and soluble forms and induces apoptosis on crosslinking with Fas receptor. Recent evidence indicated that tumor cells exploit this system for their immunologic escape that includes the loss of Fas and the gain of FasL expression. In the present study, nine mouse tumor cell lines of diverse origin were examined immunocytochemically for the expression of Fas and FasL. Nine of nine cell lines expressed FasL, and five of nine cell lines expressed Fas. FasL expression in these tumor cell lines was demonstrated to be functional by its induction of apoptosis in Fas-sensitive target cells in coculture experiments. These results suggest that FasL may be a prevalent mediator of immune privilege in mouse malignancies, and support the recently proposed "counterattack model" for local elimination of tumor-reactive immune cells by tumor cell-derived FasL.^ Culture supernatant of four cell lines expressing FasL showed cytotoxic effect on Fas-sensitive target cells, indicating the possibility of secreted FasL in the medium. The Fas-expressing cell lines were sensitized to anti-Fas antibody cytotoxicity following treatment with IL-2 and IFN-$\gamma$, suggesting cytokine stimulation as an effective target for future immunotherapeutic strategies. ^

Anti-diarrheal plants of central Anatolia: Do they inhibit diarrhea-causing bacteria?

Infectious diarrhea results in 2 to 5 million deaths worldwide per year, and treatments that are safe, effective, and readily available are under investigation. The field of medicinal ethnobotany focuses on plants that are used by different cultural groups for treating various diseases and evaluates these plants for efficacy and cytotoxicity. In the present study, ethnobotanical research was conducted with Central Anatolian villagers in Turkey. Folk concepts and etiologies surrounding diarrhea were analyzed, as were salient plant-based remedies for diarrhea. Reviewing the literature, 91 plant species were described as anti-diarrheal in all of Turkey. In Central Anatolia, villagers described 35 species. For continued research via bactericidal and bacteriostatic bioassays, 15 plants were selected. Methanolic and aqueous extracts of medicinally used plant parts were evaluated for inhibitory properties against 10 diarrhea-causing bacteria in the first bioassay, and later 21 bacteria in a second assay utilizing spectrophotometry. The cytotoxic properties were also evaluated in an Alamar Blue Assay using HepG-2, PC-3, and SkMEL-5 human cell lines. While several extracts showed bactericidal and bacteriostatic properties, the methanolic extract of R. canina galls inhibited the most bacteria at the lowest concentrations. They were not cytotoxic. Thus, R. canina methanolic gall extracts were selected for bio-assay guided fractionation. Antibacterial activity was maintained in the third fraction which was composed of almost pure ellagic acid. The bioassay was repeated with standard ellagic acid, and the polyphenol retained potency in inhibiting multiple bacterial strains. Several other extracts showed promise for safe, effective anti-bacterial remedies for diarrhea.

The potential role of environmental exposures and genomic signaling in development of central nervous system tumors

The etiology of central nervous system tumors (CNSTs) is mainly unknown. Aside from extremely rare genetic conditions, such as neurofibromatosis and tuberous sclerosis, the only unequivocally identified risk factor is exposure to ionizing radiation, and this explains only a very small fraction of cases. Using meta-analysis, gene networking and bioinformatics methods, this dissertation explored the hypothesis that environmental exposures produce genetic and epigenetic alterations that may be involved in the etiology of CNSTs. A meta-analysis of epidemiological studies of pesticides and pediatric brain tumors revealed a significantly increased risk of brain tumors among children whose mothers had farm-related exposures during pregnancy. A dose response was recognized when this risk estimate was compared to those for risk of brain tumors from maternal exposure to non-agricultural pesticides during pregnancy, and risk of brain tumors among children exposed to agricultural activities. Through meta-analysis of several microarray studies which compared normal tissue to astrocytomas, we were able to identify a list of 554 genes which were differentially expressed in the majority of astrocytomas. Many of these genes have in fact been implicated in development of astrocytoma, including EGFR, HIF-1α, c-Myc, WNT5A, and IDH3A. Reverse engineering of these 554 genes using Bayesian network analysis produced a gene network for each grade of astrocytoma (Grade I-IV), and ‘key genes’ within each grade were identified. Genes found to be most influential to development of the highest grade of astrocytoma, Glioblastoma multiforme (GBM) were: COL4A1, EGFR, BTF3, MPP2, RAB31, CDK4, CD99, ANXA2, TOP2A, and SERBP1. Lastly, bioinformatics analysis of environmental databases and curated published results on GBM was able to identify numerous potential pathways and geneenvironment interactions that may play key roles in astrocytoma development. Findings from this research have strong potential to advance our understanding of the etiology and susceptibility to CNSTs. Validation of our ‘key genes’ and pathways could potentially lead to useful tools for early detection and novel therapeutic options for these tumors.