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The cytochromes P450 (CYPs), epoxide hydrolases (EHs) and glutathione S-transferases (GSTs) are three of the major families of enzymes involved in the metabolism of xenobiotics in the human body. Immunohistochemical analysis revealed a high frequency of expression of xenobiotic metabolising enzymes in all tumour types studied, in contrast to corresponding normal tissue which displayed only low levels of expression. Further examination of the CYP1 family was carried out by immunoblot analysis. All breast tumours studied were found to express CYP1B1, and not CYP1A1 or CYP1A2. Moreover, CYP1B1 was identified in a number of kidney tumours but not in corresponding normal kidney, indicating that CYP1B1 may be a tumour-specific form of CYP, RT-PCR, in combination with restriction digestion and DNA sequencing, was used to identify CYP mRNA species present in several tumour types. Although CYP1A1 mRNA was identified in breast carcinomas, CYP1B1 was found to be the most frequently expressed form of the CYP1 family in this tissue. CYP3A mRNA was also displayed by several breast tumours, and demonstrated by sequencing to be CYP3A5. A similar situation to breast tumours was observed in tumours of the gastro-intestinal and urinary tracts, with CYP1B1 being the most frequently expressed form of the CYP1 family, and only a small number of samples displaying evidence of CYP1A mRNA. The effects of the expression of xenobiotic metabolising enzymes in tumours may be complex, and depend upon the relative amounts of active protein present, but it is likely that they will exert an influence on both the development of carcinogenesis and the anti-cancer drug resistance of tumours.

2,5-Diphenyloxazole (PPO) was tested as a potential alternate inducer for the aryl hydrocarbon hydroxylase (AHH) system. Its apparent lack. of carcinogenicity and toxicity provide a possible system for investigation of enzyme systems related to chemical carcinogenesis without exposure of the researcher to potent carcinogenic compounds. These studies found PPO to be an inducer of AHH in cultured human lymphocytes. When PPO was utilized as a substrate for the AHH assay system, the major metabolites produced were strongly fluorescent. A simple fluorometric assay was developed which employed PPO as the substrate and which measured constitutive activity more efficiently than similar assays using benzo(a)pyrene as the substrate. Quantitation of both basal and induced lymphocyte AHH metabolism of PPO may be applicable to human population studies and may provide a tool to determine possible genetic variables with respect to carcinogen metabolism related to cancer risk.

DESIGN, SYNTHESIS, AND EVALUATION OF DITERPENONES AS POTENT CHEMOPREVENTIVE AGENTS FOR AFB1 INDUCED CARCINOGENISIS IN HUMAN LIVER CELLS By Miguel A. Zuniga, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Major Director: Qibing Zhou, Ph.D Assistant Professor Department of Chemistry Terpene quinone methides (TPQMs) have been isolated from a variety of plants and show broad activities against bacteria, fungi, and cancerous cell lines. The biological activity has been attributed to the reactive electrophilic QM moiety, this structural feature has long been recognized as an intermediate in organic synthesis and in certain biosynthetic processes. It has been shown that quinone methide structures play a key role in the chemistry of several classes of antibiotic drugs and antitumor compounds such as mitomycin C and anthracyclines. The goal of this study was to understand the basis of QM bioactivity so that terpene catechols as analogs of natural TPQMs precursors can be designed as effective chemopreventive agents.In order to investigate the oxidation pathway of terpene QM precursors, a homoconjugated diterpene catechol was synthesized. A review of the literature revealed that Cu2+ -induced oxidation of simple catechols proceeds through a two-step one electron transfer process, and o-quinone is the sole oxidation product. In contrast, our studies showed direct p-QM formation from a diterpene catechol and no o-quinone oxidation products were observed. Furthermore, the Cu2+-induced oxidation pathway of our homoconjugated diterpene catechol revealed multiple p-QM formations under aqueous conditions. The implications of these findings suggest that terpene QM precursors can cause extensive DNA damage through in situ generated hydroxyl radicals or by DNA alkylations with p-QMs. To elucidate the Cu2+-induced DNA damage mechanism, a series of catechol analogues of natural terpene QM precursors were designed to investigate potential effects of stereochemistry, substitutiional, and functional groups on nucleobase alkylation and production of reactive oxygen species. The results of these tests suggested that production of ROS was the dominant mechanism for the observed DNA damage in the Cu2+-induced oxidation regardless of stereo and structural differences of catechols or subsequent oxidation products as QM or quinone. From the DNA damage study we found that the presence of NADH significantly enhanced the extent of DNA damage by oxidation of these catechols. More specifically, in the case of alkene catechols, our results showed that DNA damage was independent of the concentration of catechols, thus providing ample evidence for production of ROS through the redox cycle of catechols/quinones. Additional support for the formation of hydroxyl radical and futile redox cycling was clearly demonstrated by comparison of the fragmentation pattern with that of a Fenton reaction. The identify of the ROS was also shown to be in the form of a Cu(I)OOH complex by radical scavenging and metal chelation experiments. Cis-terpenones were first shown to have chemoprotective activity by Dr. Zhou and colleagues. In collaboration with their efforts to identify the mode of action of cis-terpenones, another project to achieve an isotope labeled cis-terpenone was undertaken. The isotope study was employed to obtain and experimentally demonstrate the feasibility of incorporating a radioactive label in cis-terpenone for the future studies of cis-terpenone metabolism. An analysis of the deuterium labeled cis-terpenone from the isotope exchange reaction showed that the isotope was being incorporated into multiple positions through scattering processes. This non-radioactive isotope study made it possible to optimize the conditions prior to using a radioactive tritium label, which will be a requisite for future metabolic studies.As an extension of this work, a structure activity relationship (SARs) study was undertaken with a focus on improving the physiochemical properties and chemical stability of cis-terpenones. The primary purpose of this study was to attempt to explain the reason for the observed protective effects of cis-terpenones against AFB1. Considerable efforts were made to introduce an unsaturated double bond in the structure of cis-terpenone by an intramolecular Pd (II) catalyzed Heck reaction. Unfortunately, this method was unsuccessful and which was attributed to the disconnection of our starting material during the formation of an enolate intermediate. A second model study to generate desired coumarin and ditepene related structures was investigated with a Diels Alder [4+2] cycloaddition reaction. After numerous attempts, we found that the successfull synthesis of these compounds was highly dependent on the temperature, solvent, and the use of stabilizers in the reaction. Finally, the targeted diterpene analogs were screened for protective effects against AFB1 by the MTT cell viability assay. However, these preliminary data showed that additional structural features and key modifications are still required to better correlate the structure with the mechanism of chemoprotection.

There is growing evidence that RNAs compete for binding and regulation by a finite pool of microRNAs (miRs), thus regulating each other through a competing endogenous RNA (ceRNA) mechanism. My dissertation work focused on systematically studying ceRNA interactions in cancer by reverse-engineering context-specific miR-RNA interactions and ceRNA regulatory interactions across multiple tumor types and study the effects of these interactions in cancer. I attempted to use ceRNA interactions to explain how genetic and epigenetic alterations are propagated to target established drivers of tumorigenesis. Using bioinformatics analysis of primary tumor samples and experimental validation in cell lines, I have investigated the roles that mRNAs and noncoding RNAs can play in tumorigenesis via ceRNA interactions. Specifically, I studied how RNAs target tumor-suppressors and oncogenes as ceRNAs, and attempted to accounting for some of the missing genomic variability in tumors.

Neuroblastoma is a heterogeneous pediatric malignancy originating from the developing sympathetic nervous system, with poor long-term survival for high-risk patients (~40%). About half of advanced neuroblastomas harbor high-level amplification of the MYCN gene, and these tumors show few, if any, additional driver lesions. Despite significant increase in the body of knowledge of genetics in neuroblastoma, all the high-risk patients follow similar therapeutic procedures and little advancement has been made on molecular target based therapies. The major challenge is to dissect the complexity and heterogeneity of these tumors to find driver genes and activated pathways that are essential for the survival of these cancer cells. We used an integrated systems biology approach to define the core regulatory machinery responsible for maintenance of an aggressive neuroblastoma phenotypic state. In the first part of the thesis, I will discuss our computational approach to decipher the tumor heterogeneity by subtype classification, followed by identification of master regulator protein modules for three distinct molecular subtypes of high-risk neuroblastomas, which were validated in a large independent cohort of cases. We propose that such modules are responsible for integrating the effect of mutations in upstream pathways and for regulating the genetic programs and pathways necessary for tumor state implementation and maintenance. The second part of the thesis is focused on experimental validation of putative master regulators in the subtype of neuroblastomas associated with MYCN amplification. By using RNAi screening followed by experimental and computational analyses to elucidate the interdependencies between the top master regulators, we identified TEAD4-MYCN positive feedback loop as a major tumor maintenance mechanism in this subtype. While MYCN regulates TEAD4 transcriptionally, TEAD4 regulates MYCN through transcriptional and post-translational mechanisms. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally orchestrate 70% of the subtype-specific gene expression signature. TEAD4 gene expression was associated with neuroblastoma patient survival independently of age, tumor stage and MYCN status (P=2.1e-02). In cellular assays, MYCN promoted growth and repressed differentiation, while TEAD4 activated proliferation and DNA damage repair programs, the signature hallmarks of MYCN-amplified neuroblastoma cells. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk neuroblastoma pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. The critical role of the core master regulator module in controlling tumor cell viability, both in vitro and in vivo, and its clinical relevance as a prognostic factor highlights TEAD4 as a novel and highly effective candidate target for therapeutic intervention. In this thesis, we demonstrate that interrogation of tumor specific regulatory networks with patient-derived gene expression signatures can effectively elucidate molecular subtypes as well as the core transcriptional machinery driving subtype specific hallmarks. This approach enables identification of oncogenic and non-oncogenic dependencies of high-risk neuroblastoma and is applicable to other tumor subtypes.

Orientador: João Lauro Viana de Camargo / Banca: Luis Fernando Barbisan / Banca: Heloisa Maria de Siqueira Bueno / Resumo: Um ensaio de média-duração em múltiplos órgãos de roedores, baseado no paradigma iniciação-promoção da carcinogênese, foi proposto por pesquisadores japoneses como alternativa ao ensaio convencional de longaduração para detecção de cancerígenos químicos. Esse ensaio alternativo, denominado DMBDD (acrônimo para os 5 agentes iniciadores da carcingênese neste protocolo), foi originalmente padronizado com a linhagem de ratos Fischer 344. Em 1996, o IBAMA adotou oficialmente uma variação (DMBDDb), proposta por nosso laboratório, como fonte de evidência do potencial cancerígeno de praguicidas agrícolas. O protocolo adotado pelo IBAMA tem algumas particularidades, como o uso de ambos os gêneros de ratos da linhagem Wistar e dois grupos controle positivo (tratados com fenobarbital - FB ou com 2'-acetoaminofluoreno -2'-AAF). Este protocolo foi utilizado ao longo de seis anos em nosso laboratório (TOXICAN) para a realização de cinco bioensaios sob contratos com empresas do setor agroquímico. O presente estudo consiste da revisão dos diagnósticos de três órgãos desses ensaios - o fígado, rins e intestinos - escolhidos porque foram os que apresentaram mais lesões na análise de cada um daqueles ensaios. A capacidade indutora enzimática dos agentes do protocolo foi avaliada pela expressão imunohistoquímica das enzimas hepáticas CYP 2B1/2B2 e 1A2. Os resultados indicam atividade promotora do FB, embora menos evidente que a do 2'-AAF, particularmente nos ratos machos. Apesar da alta variabilidade da linhagem de rato Wistar , este estudo permitiu estabelecer um banco de informações sobre as lesões que caracteristicamente são encontradas naqueles órgãos dos animais Wistar submetidos ao protocolo DMBDDb. / Abstract: A medium-term multi-organ rat bioassay based on the initiation-promotion carcinogenesis paradigm has been proposed by Japanese researchers as an alternative to the conventional long-term assay for chemical carcinogenesis detection. This alternative bioassay, designated DMBDD (after the five carcinogenic initiators of this protocol), was originally standardized with the male Fischer 344 strain of rats. In 1996, the Brazilian Agency for the Environment (IBAMA) officially recognized a variation (DMBDDb), proposed by our laboratory, as a valid source of evidence of the carcinogenic potential of agrochemicals. The protocol adopted by IBAMA has some modifications, such as the use of both sexes of the Wistar strain of rats and two positive controls (Phenobarbital - PB, 2'-acetoaminofluorene - 2'-AAF). During six years, five different bioassays managed under contract with agrochemical companies were developed by our laboratory (TOXICAN). This study presents the revised results obtained from three organs of this protocol - liver, kidney and intestines -, chosen because they most frequently presented lesions through those assays analyses. Besides, the induction of the CYP 2B1/2B2, 1A2 isoforms was also immunohistochemically evaluated in the liver. Our results document the promoting activity of PB, otherwise less evident than 2'-AAF, especially in male rats. Although a high variability of the Wistar rat strain tested was evident, this study allowed building up a data bank of characteristic lesions in those selected organs of Wistar rats under the DMBDDb protocol treatment. / Mestre

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.