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Cellular responses against DNA damaged by platinum anticancer drugs

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dc.contributor.advisor Stephen J. Lippard. en_US Jung, Yongwon, 1977- en_US
dc.contributor.other Massachusetts Institute of Technology. Dept. of Chemistry. en_US 2006-07-31T15:30:01Z 2006-07-31T15:30:01Z 2005 en_US 2005 en_US
dc.description Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005. en_US
dc.description Vita. en_US
dc.description Includes bibliographical references. en_US
dc.description.abstract The anticancer activity of platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin is mediated by their ability to attack DNA such that generated adducts trigger numerous cellular responses. A better understanding of these processes is critical for developing more effective therapeutic approaches, which can increase the anti-cancer activity of the drugs while minimizing side effects and extending successful treatment to a wider range of human cancers. Chapter 1 provides the current comprehension of early cellular responses to platinum-DNA adducts. The event primarily occurs through platinum-DNA adduct recognition by a number of cellular proteins. Among proteins that recognize platinum-DNA lesions, one class constitutes proteins that selectively recognize severely distorted DNA generated by the platinum adduct formation. The TATA-binding protein (TBP) and high mobility group protein HMGB1, both highly abundant and vital proteins, are reported to bind cisplatin- damaged DNA and to be involved in mediating the cytotoxic activity of platinum-based agents. Chapters 2 and 3 discuss the structural and kinetic properties of both proteins binding to platinated DNA. en_US
dc.description.abstract (cont.) The TBP protein recognizes the TATA box element of transcriptional promoters and recruits other initiation factors. TBP binds with high affinity (Kd = 0.3 nM) to DNA containing site-specific cisplatin 1,2-intrastrand d(GpG) cross-links. The ko and koff values for the formation of these TBP complexes are 1-3 x 10⁵ M⁻¹s⁻¹ and [approx.] 1-5 x 10⁻⁴ sec⁻¹, respectively, similar to the corresponding values for the formation of a TBP-TATA box complex. When TBP was added to an in vitro nucleotide excision repair (NER) assay, it specifically shielded cisplatin-modified 1,2-(GpG) intrastrand cross-links from repair. HMGB1, a highly conserved non-histone DNA- binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A with a dissociation constant Kd of 120 nM. Interaction of the C- terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. en_US
dc.description.abstract (cont.) Another group of proteins that encounter platinum-damaged DNA are involved with DNA function and therefore are in frequent contact with DNA. DNA, RNA polymerases and histone proteins inevitably run into platinum-DNA adducts. Transcription inhibition by DNA adducts of cisplatin is considered to be one of the major routes by which this anticancer drug kills cancer cells. Stalled RNA polymerases at platinum-DNA lesions evoke various cellular responses such as nucleotide excision repair, polymerase degradation, and apoptosis. The consequences of RNA polymerase blockage by platinum lesions are discussed in the next two chapters. T7 RNA polymerase and site-specifically platinated DNA templates immobilized on a solid support were used. Polymerase action is inhibited at multiple sites in the vicinity of the platinum lesion. The stalled polymerase can be dissociated from the DNA by subsequent polymerases initiated from the same template. The immediate consequences of human RNA polymerase (Pol) II arrest at the site of DNA damaged by cisplatin were studied in whole cells and cell extracts, with a particular focus on the stability of stalled Pol II and its subsequent ubiquitylation. en_US
dc.description.abstract (cont.) Pol II was completely blocked by a cisplatin intrastrand cross-link and the stalled polymerase was quite stable in nuclear extracts as well as in cisplatin-treated HeLa cells. The stalled Pol II proteins were transcriptionally active and capable of resuming transcription beyond the DNA adduct following its chemical removal from the template. A series of experiments revealed that lysines other than Lys-48 of ubiquitin are involved in Pol II ubiquitylation following DNA damage. Only a fraction of ubiquitylated Pol II dissociates from damage sites and that it is rapidly destroyed by proteosomes. In the final chapter, hapten-conjugated platinum compounds were studied in an effort to follow DNA damaged by platinum agents in living cells. Platinum complexes containing a desthiobiotin moiety (DTB-Pt) with different linkers were synthesized and characterized in vitro and in cells. DNA damaged by DTB-Pt was strongly interacted with streptavidin-coated beads. Moreover, less than 1 fmol of DTB-Pt DNA adduct can be detected by using a simple dot blot analysis. en_US
dc.description.statementofresponsibility by Yongwon Jung. en_US
dc.format.extent 240 p. en_US
dc.format.extent 12652366 bytes
dc.format.extent 12662521 bytes
dc.format.mimetype application/pdf
dc.format.mimetype application/pdf
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. en_US
dc.subject Chemistry. en_US
dc.title Cellular responses against DNA damaged by platinum anticancer drugs en_US
dc.title.alternative Cellular responses against deoxyribonucleic acid damaged by platinum anticancer drugs en_US
dc.type Thesis en_US Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Dept. of Chemistry. en_US
dc.identifier.oclc 65192495 en_US

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