Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 121-123). / Microfiche. / xvii, 123 leaves, bound ill. 29 cm

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2009. / Title from title page screen (viewed on August 6, 2009). Research advisor: Alex Sparreboom, Ph.D. Document formatted into pages (ix, 79 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 74-78).

Thesis (Ph. D.)--University of Florida, 1984. / Description based on print version record. Typescript. Vita. Includes bibliographical references (leaves 236-269).

Thesis (Ph.D.) -- McMaster University, 2001. / Includes bibliographical references (leaves 166-205). Also available via World Wide Web.

Lung cancer is the leading cause of cancer and cancer deaths worldwide, with non-small-cell lung carcinomas (NSCLC) representing 85% of all diagnosed lung cancers. Platinum-combination chemotherapy is the current standard treatment for NSCLC, however, associated toxicities and resistance limit its efficacy. Our laboratory previously identified activating transcription factor 3 (ATF3), a stress-inducible gene whose elevated and sustained expression can trigger apoptosis to a wide variety of stressors, as a key regulator of cisplatin cytotoxicity as well. Thus, enhanced and sustained induction of ATF3 by combining platins with other ATF3 inducers potentially represents an effective therapeutic strategy. A chemical library screen identified vorinostat and topotecan as ATF3 inducers that also enhance cisplatin cytotoxicity. ATF3 plays a significant role in cisplatin, vorinostat and topotecan and their combinations cytotoxicity. Importantly, vorinostat and topotecan induced synergistic cytotoxicity with cisplatin in NSCLC cell lines and their cisplatin resistance sub-lines with enhanced ATF3 expression observed. Our study suggests a potential novel therapeutic approach where ATF3 inducing agents in combination with platins represents a rational combination based therapeutic strategy.

Despite their ubiquitous use, Pt(II) anti-cancer drugs still suffer from many issues such as off-drug target effects, renal and nephrotoxicity as well as acquired and intrinsic drug resistance. To obtain a better understanding of how to mitigate these deleterious effects can be mitigated we first must know all the targets of these drugs. Highlighted in this dissertation is previous work performed by groups exploring the localization of Pt in cells using fluorescence microscopy. While Pt drugs such as cisplatin contain no native fluorescence, a great deal of work has been done to covalently modify complexes with fluorescent tags. From studies using this technique, it been reported that Pt can target a number of compartments within the cell ranging from the nucleus to the cytoplasm. With each different derivative being observed in varied cell lines it becomes difficult to deconvolute a universal pattern to where Pt localizes, furthermore, the connected fluorophore could also bias Pt localization. To add general functionality and eliminate the bias of a pre-tethered fluorophore our lab has developed a number of different azide and alkyne-modified complexes that append a “reactive handle” to Pt compounds. This modification allows for use of the bioorthogonal azide-alkyne click reaction we are able to observe Pt localization after treatment. The focus of this work includes method development to conjugate a fluorophore to our Pt complexes in vitro and in cell cultures. We examined a number of different cell lines and observed frequent localization in the nucleolus of the cell. Also in this work is the development of methods to append multiple fluorophores to each Pt site to increase our ability to visualize these complexes in cells. Finally, we have also constructed a new Pt-azide that exhibits slower exchange kinetics due to a chelating exchangeable group. The use of this new complex will enable studies to determine whether changing the leaving group results in differential localization of Pt drugs in cells.

Methods to map small-molecule interactions with cellular RNAs are important for understanding endogenous activation, such as in riboswitches, as well as the potential for exogenous compounds to target RNA. Cisplatin is one of the most widely used of the platinum anticancer drugs that are prescribed in approximately 40-50% of all chemotherapy treatments (Dyson and Sava, 2006; Harper et al., 2010). Despite nearly 40 years of experience with this class of drugs, we still lack a comprehensive understanding of the targets of Pt compounds and their effects on cells. Pt(II) compounds are well-known DNA and RNA crosslinking agents, but the latter area is under-studied. In order to better understand the impacts of cisplatin and other platinum(II)-derived small molecules on cellular RNA, we have developed a technique we call “Platinum-seq,” which couples reverse transcription mapping of platinated RNAs to high-throughput sequencing. Chapter 1 is a study of cisplatin and a novel click-functionalized platinum compound (2-ADAP Pt) binding to the HDV ribozyme, a small catalytic RNA. Chapter 2 moves our platinum mapping approaches from low-throughput, sequencing gel based methods into next-generation sequencing for high-throughput analysis of all platinum sites in cellular RNA, a method we have named “Platinum-seq.” Chapter 3 is a study of differential gene expression of Saccharomyces cerevisiae treated with cisplatin and a second novel platinum(II) compound (azaplatin), using data acquired from the work in Chapter 2. Chapter 4 describes recent efforts to implement pre-enrichment of sequencing targets using click chemistry followed by DNA hybridization, in order to enrich for platinated fragments before sequencing library construction. Together, this work represents a significant step forward in advancing analysis of Pt(II) binding to cellular RNA, a potentially important target for this widely used class of anticancer compounds. Methods developed here are broadly applicable to genome-wide identification of platinum accumulation on DNA as well, which has not been pursued despite the extensive use of these compounds.

In der vorliegenden experimentellen Arbeit wurde in in-vitro-Modellen der Zusammenhang zwischen Expression der Tumorantigene MAGE-A (melanoma-associated antigenes) und der Wirksamkeit von Chemotherapeutika untersucht. Die MAGE-Antigene MAGE-A1 bis MAGE-A12 (ohne A7) kommen in diversen malignen Tumoren vor; neben Melanomen auch in Tumoren der Lunge, Brust, Prostata, Ovarien, Harnblase, des Gastrointestinal-Trakts und des Kopf-Hals-Bereichs. Bereits vielfach wurden Zusammenhänge zwischen MAGE-A-Tumorantigen-Expression und einer erhöhten Tumorinvasivität, Zellproliferation, Metastasierungsrate und kürzerem Überleben hergestellt. In dieser Arbeit gelang nun der erstmalige Nachweis, dass MAGE-A-Tumorantigene die chemotherapeutische Wirksamkeit beeinflussen. Zunächst gelang der Nachweis, dass die Expression von MAGE-A11 mit geringer Cisplatin-Wirksamkeit korreliert. Eine im Anschluss generierte MAGE-A11 überexprimierende Zelllinie zeigte ein durchschnittlich um 9 % schlechteres Ansprechen auf Cisplatin als die Kontrollzelllinie. / The purpose of the present study was to examine whether there is a correlation between the expression of MAGE-A tumorantigenes (melanoma-associated antigenes) and the efficacy of chemotherapeutic agents. The tumorantigenes MAGE-A1 to MAGE-A12 (without MAGE-A7) occur in different tumors, such as melanoma, tumors of the lung, chest, prostata, ovary, gastro-intestinal tract and area of head-and-neck. Previous studies showed correlations between MAGE-A expression and tumor invasivity, cell proliferation, malignancy and survival. The present study proofs for the first time that there is a significant correlation between MAGE-A11 expression and a lower efficacy of Cisplatin. For this study we generated a MAGE-A11 overexpressing cell line which showed a 9% lower efficacy of Cisplatin.

Four cases of extremity cancers received effective management with cisplatin and etoposide via isolated limb perfusion. They demonstrated minimal, if any, soft tissue damage. This result counters the theory that a caustic response is a prerequisite for successful therapy. This characteristic allows for simultaneous surgical resection with regional, isolated limb perfusion of potent cytotoxic agents without increased morbidity from tissue necrosis, a common consequence of previously used drugs. There is no apparent affect on wound healing, even in cases involving extensive, radical operative procedures.

Platinum(II) anticancer medications essentially react with DNA forming kinks inthe double helix of DNA and causing apoptosis. It has also been noted that theseanticancer medications react with methionine and cysteine in the body. With the new discoveries of selenium containing amino acids including selenomethionine and selenocysteine, new research is ongoing to see what types of products can be formed from these amino acids. Our research reacts [Pt(Met-S,N)Cl2] 2+ with selenomethionine to determine what types of products are produced. Monochelates including [Pt(SeMet-Se,N)Cl2] 2+ have formed two isomers, as well as other products that insinuate both selenomethionine and methionine binding with the platinum to form various [Pt(SeMet- Se,N)(Met-S,N)]2+ products. When initially reacting 6 mM [Pt(Met-S,N)Cl2] 2+ with 3 mM SeMet, the monochelates of both are produced without forming any free methionine which would suggest that there is free platinum in our solution creating the SeMet monochelate. When adding additional SeMet to the solution the same products are formed that are created when reacting 6 mM [Pt(Met-S,N)Cl2] 2+ and 6 mM SeMet. The 1H NMR spectrum for these products imply a product of [Pt(SeMet-Se,N)(Met-S,N)] 2+. Also, reactions with [Pt(en)(ox)] 2+ and SeMet were conducted and produced various products at two different pH’s. A [Pt(SeMet-Se,N2] 2+ product was formed at lower pH and produced free ethylenediamine, however at a higher pH only [Pt(en)(SeMet-Se,N)]2+ was produced.