Cisplatin-induced nucleosome and RNA polymerase II modification mediate cellular response to the drug

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2004.Vita.Includes bibliographical references.(cont.) with recombinant material. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level. Chapter 3: Nucleotide Excision Repair of Site-Specifically Platinum-Modified Tetrasomes. The nucleosome, the basic structural unit of chromatin, is composed of a histone (H3/H4)₂ tetramer flanked by two H2A/H2B dimers, around which is wrapped 146 base pairs (bp) of DNA. The (H3/H4)₂ tetramer plays a central role in the structural integrity and positioning of the nucleosome core particle. Site- specifically platinated tetrasomes were prepared to investigate the modulating effects of histone tetramers on excision repair of cisplatin-intrastrand cross-links. In addition, (Pt(DACH))²⁺-modified tetrasome was prepared to investigate the effect of spectator ligands on excision repair of platinated tetrasomes. The NER results reveal that the (H3/H4)₂ tetramer is sufficient to block excision of both cisplatin-DNA and Pt(DACH))²⁺-DNA adduct in vitro. The efficiency of excision of cisplatin-modified tetrasomal DNA is about half (53%) that of the [Pt(DACH)]²⁺-modified tetrasome. Chapter 4: Cisplatin Adducts Change the Rotational Positioning of DNA on the Nucleosome ...Chapter 1: Cellular Processing of Platinum Anti-Cancer Drugs --Identifying Pathways for Chemogenotherapeutic Drug Design. Cisplatin, carboplatin, and oxaliplatin are widely used in cancer chemotherapy. Platinum-DNA adducts, formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. This review focuses on recently discovered cellular pathways that are activated in response to cisplatin, including those involved in regulating drug uptake, the signaling of DNA damage, cell cycle checkpoints and arrest, DNA repair, and cell death. Such knowledge of the cellular processing of cisplatin adducts with DNA provides valuable clues for the rational design of more efficient platinum-based drugs as well as the development of new therapeutic strategies. Chapter 2: Nucleotide Excision Repair from a Site-Specifically Platinum-Modified Nucleosome. Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a d(GpG) or a d(GpTpG) intrastrand cross-link. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is [approximately] 2-fold higher than the level observedby Dong Wang.Ph.D