Thesis (M.S.)--University of Texas at Dallas, 2008. / Includes vita. Non-Latin script record Includes bibliographical references (leaves 39-40)

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Chitin, a homopolymer of β (1-4)-linked N-acetylglucosamine, is one of the most abundant biopolymers in nature. It is widely distributed in the exoskeleton of crustaceans and insects, in the cell walls of most fungi and some algae. Chitin is an extremely insoluble material with limited industrial applicability. The deacetylated derivative of chitin, chitosan, is a water soluble cationic biopolymer having a broad range of applications (Hirano, 1999). Chitosan is naturally found in the cell wall of Zygomycetes, in the ascospore of Saccharomyces cerevisiae (Briza et al., 1988) and in the cyst wall of Entamoeba invadens (Das et al., 2006). Chitosan biosynthesis requires the coordinated action of chitin synthase (E.C.2.4.1.16) and chitin deacetylase (E.C.3.5.1.41) (Davis & Bartnicki, 1984). Chitin synthase polymerizes N-acetyl glucosamine precursor molecules into chitin and chitin deacetylase catalyzes the deacetylation of the nascent chitin chains. The chitin deacetylase enzymes are members of the family 4 of carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/~cazy/CAZY] (Couthino et al., 1999), which includes several members sharing a conserved region in the primary structure assigned as the NodB homology domain(Caufrier et al., 2003) or polysaccharide deacetylase domain. Chitin deacetylase was first identified and partially purified from extracts of the fungus Mucor rouxii. Since then, chitin deacetylase has been purified from several fungi and chitin deacetylase open reading frames have been cloned from a few microorganisms including M. rouxii (Kafetzopoulos et al., 1993), Colletotrichum lindemuthianum (Tokuyasu et al., 1999; Shresta et al., 2004), Phycomyces blakesleeanus (GenBank AB046690), Schizophillum commune (GenBank AF271216), Blumeria graminis (GenBank AAK84438), Saccharomyces cerevisiae (Christodoulidou et al., 1999) and Schizosaccharomyces pombe (Matsuo et al., 2005). The structure and the catalytic mechanism of chitin deacetylase from C. lindemuthianum were recently studied (Blair et al., 2006). Chitin deacetylase plays a role in the cell wall biosynthesis in M. rouxii and Absidia coerulea (Gao et al., 1995). In C. lindemuthianum and Aspergillus nidulans, it was suggested that chitin deacetylase participates in plant-pathogen interactions to promote plant invasion (Tsigos et al., 2000). In S. cerevisiae, chitin deacetylase is essential for the ascospore cell wall rigidity and the resistance against lytic enzymes (Christodoulidou et al., 1996). The use of chitin deacetylase enzyme for the industrial deacetylation of chitin awaked a great interest. Different fungal strains were screened and compared for their ability to produce a chitin deacetylase secreted, active on insoluble substrates and showing low inhibition with acetate, a product of reaction. Rhizopus circinans proved to be a good chitin deacetylase producer with the targeted characteristics. The second part of the work was to isolate the cDNA encoding for the chitin deacetylase of R. circinans. The native enzyme was purified to homogeneity for sequencing the N-Terminal extremity. The enzyme was purified in only two steps from the culture supernatant of R. circinans. Then, the purified enzyme was sequenced and the first nine amino acids were identified. In the same way, a R. circinans cDNA library was also constructed. The cDNA library was screened using two approaches: on the one hand with radiolabeled homologous probe and on the other hand by PCR with primers designed for the 5 extremity, on the basis of the deduced sequence of the N-Terminal extremity of the native enzyme and for the 3 extremity, from the deduced R. oryzae chitin deacetylase. Two cDNA sequences (D2 and I3/2) with homology to fungal chitin deacetylase genes were isolated with the radiolabeled probe and one sequence (RC+) by PCR approach. The sequences were analyzed and characterized. The three sequences possessed several characteristics of chitin deacetylase sequence: homology with known chitin deacetylase cDNA, the presence of the deacetylase polysaccharide domain, and the same potential glycosylation sites than M. rouxii chitin deacetylase. The cDNA D2, I3/2 and RC (RC sequence is the mature protein sequence of RC+ sequence) were expressed in the yeast Pichia pastoris to confirm their potential chitin deacetylase activity. Numerous constructions were tested. A poly-histidine tag was cloned to facilitate the further purification of the recombinant enzyme. Only the RC sequence showed a high chitin deacetylase activity. Several hypotheses were emitted to explain the low chitin deacetylase activity level measured with the inserts D2 and I3/2. The recombinant RC protein was purified to homogeneity in one step, and partially characterized.

Histone Deacetylase Inhibitors (HDACi) are a new class of chemotherapeutics which have shown promise in pre-clinical and clinical settings. HDACi have been shown to act by re-programming gene expression, with the transcription of some genes such as p21WAF1 being activated, while others like SRC and BCL2L1 are repressed. The mechanism behind HDACi gene expression changes remains unknown; although it has been shown to involve a direct interaction with gene promoters. Using a quantitative qRT-PCR approach, the effect of various HDACi on the transcription of p21WAF1, SRC and BCL2L1 was examined. TSA and apicidin led to an up regulation of p21WAF1 mRNA levels while c-Src and Bcl-xL mRNA levels were downregulated. Short c-Src mRNA transcripts were unaffected following TSA and apicidin treatments, despite the full length transcripts being repressed. Repression of full length c-Src and Bcl-xL mRNA transcripts was not seen following treatment with MS-275 and MGCD0103, although p21WAF1 mRNA expression was induced. ChIP experiments revealed that following HDACi treatment, histone acetylation levels and RNA Polymerase II occupancy increased in the promoter regions of both the SRC and BCL2L1 genes. RNA Polymerase II occupancy lasted less than 15 minutes in the 3’ regions of the gene following treatment with apicidin and TSA, but was more long-term following MS-275 and MGCD0103 treatment. The protein phosphatase inhibitor Calyculin A completely blocked HDACi mediated repression of c-Src and Bcl-xL mRNA, suggesting a role for protein phosphatases in the mechanism behind HDACi. It is therefore hypothesized that HDACi work through at least two different mechanisms. Whether or not an HDACi leads to gene repression depends on its ability to disrupt an HDAC/protein phosphatase complex and not on their HDAC specificities. The disruption of the complex leads to the release of an active protein phosphatase. The released phosphatase can then presumably act on various factors changing a gene from an active to paused state, possibly through promoter proximal pausing. HDACi unable to disrupt this complex are unable to induce gene repression. Collectively, these studies highlight not only the complexity of HDACi mediated effects within the cell, but also present a new explanation behind HDACi mediated gene repression.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 135-159). Also available in electronic version. Access restricted to campus users.

Barbituric acids have been historically classified as compounds that act on the central nervous system, and as such provide therapeutic uses as anxiolytics, sedatives, hypnotics, and anticonvulsants. Recent investigations of barbituric acid derivatives have provided scientists with information that barbituric acids may have applications in antibacterial, anti-chlamydial, anti-viral, as well as anti-cancer treatments. Additionally, recent literature accounts have indicated that barbituric acid derivatives may also act as immune modulators. The recent explorations of barbiturates and their potential anti-cancer and immune modulating properties are the subject of this work. Novel synthetic approaches to the development of new barbituric acid derivatives were explored thoroughly, and the mechanisms of these novel syntheses were detailed by experiment and spectroscopic characterizations. In many cases the reaction procedures were designed for large scale, efficient syntheses, that are directly applicable to pharmaceutical production of these potentially valuable therapeutic compounds. Several new products unique to barbituric acid reactions were characterized spectroscopically. Barbituric acid derivatives were the subject of biological evaluation, and the results are reported in this work. Overall, unique synthetic approaches to the production of novel barbituric acid derivatives were accomplished to create several new classes of barbiturates with potential applications in cancer treatment.

Zahlreiche Studien schreiben Histondeacetylase-Inhibitoren einen Anti-Tumor-Effekt auf verschiedene hämatologische und solide Tumoren durch Apoptoseinduktion, vermehrte Zelldifferenzierung und verminderte Zellproliferation zu. Als Mechanismus wird eine Einflussnahme auf die Genexpression durch Modulation von Histondeacetylasen und deren Auswirkung auf den Acetylierungsstatus von Histonen und Nicht-Histon-Proteinen angenommen. Ziel dieser Arbeit war es, die Auswirkungen des Histondeacetylase-Inhibitors LBH589 auf Proliferation und Differenzierung von Kolonkarzinomzellen und dessen Metastasenzellen in Zellkulturexperimenten zu untersuchen. Die Untersuchungen wurden an Zellen der Zellkulturlinien SW480 (kolorektales Karzinom) und SW620 (Metastase des kolorektalen Karzinoms) durchgeführt. Für die Zellproliferation wurden die Zellen nach entsprechender Vorbehandlung in einer Neubauer-Zellzählkammer ausgezählt. Zur Feststellung des Verlaufs der Zelldifferenzierung diente die Bestimmung der Intestinalen Alkalischen Phosphatase als Marker. Unter LBH589-Inkubation kam es zu einer Hemmung der Zellproliferation sowohl bei SW480-Zellen als auch bei SW620-Zellen. Allerdings ergab sich kein signifikanter Unterschied bei der Auswertung der Kontrolllösungen mit jeweils äquimolaren Mengen DMSO. Daher konnte dem HDAC-Inhibitor LBH589 im Rahmen dieser Arbeit kein sicherer Effekt auf die Inhibition der Zellproliferation zugeschrieben werden. LBH589 hatte keinen nachweisbaren relevanten Einfluss auf die Differenzierung von Zellen der beiden Zelllinien. Allenfalls konnte ein leicht hemmender Einfluss auf die Zelldifferenzierung gezeigt werden, der jedoch nicht signifikant ausfiel. Weitere Untersuchungen sind anzustreben, um den Verlauf der Zellproliferation und weiterer Differenzierungsmarker unter dem Einfluss von LBH589 sowie äquimolaren Mengen DMSO detaillierter zu charakterisieren. Zukünftige Arbeiten zu Histondeacetylase-Inhibitoren und deren Effekt auf Zellen des kolorektalen Karzinoms, sowie Histondeacetylase-Inhibitoren in der Kombinationstherapie von kolorektalen Tumoren sind sicher sinnvoll. / A lot of studies have shown an anti-tumor-effect of histone deacetylase inhibitors on various hematological and solid tumors by induction of apoptosis, induction of differentiation an inhibition of proliferation. These effects are supposed to be caused by influencing gene expression through modulation of histone deacetylases and the acetylating status of histones and non-histone-proteins. In this study the influence of the histone deacetylase inhibitor LBH589 on proliferation and differentiation in colon cancer cells and its metastasis was analysed. The studies were done with cells from cell culture lines SW480 (colorectal carcinoma) and SW620 (metatasis of the colorectal carcinoma). For proliferation the cells were counted in a Neubauer-chamber and for differentiation the intestinal alkaline phosphatase was determined. Under the influence of LBH589 an inhibition of proliferation was shown in both cell lines. But there was no significant difference to the results shown by the control substance of equimolar amounts of DMSO. So there could no certain effect be related to LBH589 on cell inhibition. LBH589 has shown no influence on differentiation in both cell lines, at the most there was a light non-significant inhibition on differentiation. Further studies are needed to characterize the influence of LBH589 and equimolar amounts of DMSO on proliferation and other markers of differentiation.

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 90-97).