Understanding the functions of HMGB proteins in the mechanism of action of cisplatin

• Main Author: Park, Semi, Ph. D. Massachusetts Institute of Technology
• Other Authors: Stephen J. Lippard.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2013
• Subjects: Chemistry.
• Online Access: http://hdl.handle.net/1721.1/78437

Thesis (Ph. D. in Inorganic chemistry)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012. === Cataloged from PDF version of thesis. Vita. === Includes bibliographical references. === High mobility group box (HMGB) proteins are DNA-binding proteins that regulate many important DNA-related processes. They are known to recognize the major lesion present in cisplatin-modified DNA, and have been assumed to increase cisplatin cytotoxicity by "shielding" the damaged site from the cellular repair apparatus. This thesis will describe the work in the area of molecular biology and biochemistry to improve our understanding of the functions of HMGB proteins in the mechanism of action of cisplatin. In Chapter 1, the molecular biology of cisplatin and HMGB proteins is described based on previously reported in vivo and in vitro experiments. The interaction of HMGB proteins and platinated DNA is reviewed based on several structural studies of platinum-DNA adducts of cisplatin, HMG box motif, and the binding complex of the two. Several cell-based studies supporting a repair-shielding hypothesis, suggesting an inhibitory function of HMGB proteins in the repair of cisplatin damage will be introduced with a focus on HMGB1, the most vigorously investigated HMGB protein. Additionally, other HMGB 1-mediated processes that may be related to cisplatin-triggered cell death will be discussed. Lastly, the correlation between testis-specific HMGB proteins and the cisplatin hypersensitivity of testicular germ cell tumors will be discussed. Although some HMGB proteins including HMGB 1 inhibit the repair of platinated damage on DNA in vitro, experiments conducted in live cells reveal conflicting correlation between the expression level of HMGB1 and cisplatin cytotoxicity. Chapter 2 describes studies in cultured human cancer cells aimed at examining the intracellular repair of platinum-modified DNA and the influence of HMGB1 on the repair processes. The expression of HMGB1 is artificially down-regulated by RNAi. HeLa and A549 cell lines present different trends in cisplatin cytotoxicity upon HMGB1 knockdown. Intracellular repair of cisplatin is lower in knockdown cells regardless of the parental cell line. This result stands in opposition to what is expected from the repair-shielding hypothesis. In addition, the repair of different cisplatin adducts was investigated in fibroblast cells deficient in one of the nucleotide excision repair proteins in order to understand the repair mechanisms of each adduct. Chapter 3 presents an in vitro study investigating the redox-dependence of the binding interaction of HMGB1 to the cisplatin-1,2-d(GpG) intrastrand cross-link. Two cysteine residues in HMGB1 domain A form a reversible disulfide bond under mildly oxidizing conditions. Both HMGB 1 domain A and full-length HMGB 1 presented significantly weaker binding to a DNA probe containing a 1,2-d(GpG) intrastrand cross-link when in their oxidized state compared to their fully-reduced form. Mutagenesis studies on the cysteine residues revealed that this redoxdependence originates from disulfide bond formation. Footprinting analysis of a platinated DNA probe bound to oxidized or reduced domain A showed that the asymmetric binding mode of domain A to platinated DNA is not significantly altered by oxidation. These results suggest that the cellular redox environment can influence the interaction of HMGB 1 with the platinated DNA. In Chapter 4, the binding properties and repair inhibitory function of HMGB4 to cisplatin-modified DNA are described. Based on its testis-restricted expression profile and sequence similarity with HMGB 1, we propose that HMGB4 functions as a cisplatin cytotoxicity enhancer in TGCT. To verify this hypothesis, HMGB4 and its binding domains were generated recombinantly and interactions with cisplatin-modified DNA were investigated in vitro. The binding properties of HMGB4 are quite similar to those of HMGB 1 except for a few differences: i) full-length HMGB4 has stronger binding affinity than full-length HMGB1, because of a lack of a C-terminal acidic tail. ii) binding of HMGB4 domain A is much more symmetric with respect to the platinated lesion than that of HMGB 1 domain A. Furthermore, HMGB4 presented stronger repair inhibition capacity than HMGB1 at an equimolar concentration. These results support the hypothesis that HMGB4 enhances cisplatin cytotoxicity in TGCT. Chapter 5 will be the conclusion chapter of this thesis. Works in this thesis will be summarized with what we can learn from those results. In additions, future directions in the study of HMGB proteins will be suggested. Appendices A and B describe the incomplete work on HMGB4 in live cells. Appendix A delineates the expression profile of human HMGB4 established by western blot analysis. Human HMGB4 presented a testis-preferred expression. In Appendix B, attempts to establish a HMGB4 knockdown testicular cancer cell line are described. === by Semi Park. === Ph.D.in Inorganic chemistry