Cellular responses against deoxyribonucleic acid damaged by platinum anticancer drugs

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Vita.Includes bibliographical references.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.(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.(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.(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.by Yongwon Jung.Ph.D