The long-term goal of this research is to improve cancer therapy by combining knowledge about the mechanism of action of cisplatin with synthetic chemistry to produce new compounds and strategies for the selective destruction of tumors. Cisplatin and carboplatin are leading anticancer drugs and, for certain cancers, a paradigm for successful chemotherapy. Detailed information about how they function will facilitate clinical protocols, by optimizing pathways whereby they destroy cancer cells and by attacking resistance mechanisms, as well as the design of more effective drugs. A guiding hypothesisis that the selective toxicity of cisplatin for tumor versus normal cells, and its greater efficacy for tissues such as solid testicular tumors, are a consequence of the formation and survival of specific adducts with DNA, its acknowledged biological target. The major cisplatin/carboplatin adducts, intrastrand d(GpG), d(ApG) and d(GpNpG) cross-links, where N is an intervening deoxynucleotide, account for >950/0 of drug binding. They block transcription by RNA polymerase I1 and trigger cell death. Working against this process is nucleotide excision repair (NER) of the adducts. The binding of cellular proteins, such as HMGB1, to DNA across from cisplatin cross-links shields them from NER and sensitizes cells to the drug. HMGBl levels are elevated following administration of steroid hormones to breast or ovarian cancer cells containing the receptor, potentiating cisplatin- and carboplatin-induced cell death. A clinical trial is testing whether this process occurs in human ovarian cancer patients. The strategy is being extended by synthesizing new platinum compounds having a chemically linked hormone and by investigating prostate cancer cells. Tumor tissue is targeted with platinum conjugates that bind cellular receptors. Additional cisplatin-DNA recognition proteins are being discovered with the use of platinated-DNA analogs having a tethered, photo-activatable cross-linking unit. Upon irradiation, the cross-linker captures damage-recognition proteins, which are isolated and identified by mass spectrometry. Their role in potentiating cisplatin activity will be investigated by RNA interference and immunoprecipitation strategies. Cisplatin treatment of cells induces chemical modification of nucleosome core histone proteins, which facilitates NER. Disruption of this process in cancer cells affords an additional sensitization tactic. Animal xenograft experiments and a phase I pilot study will continue to be a significant part of our program as we seek to develop our compounds and strategies to the point where they can ultimately be used to deliver improved cancer treatment.
The specific aims of the research are as follows: (a) To prepare and characterize site-specific 1,2-intrastrand d(GpG) DNA cross-links of active platinum anticancer drugs, investigating the potential role of flanking nucleotides in forming such adducts. Structures of platinated duplexes and their complexes with HMG-domain proteins will be determined. (b) To investigate the kinetics and thermodynamics of HMG-domain protein binding to cisplatin-modified DNA. Since the interaction has not been optimized, we proposed initially to modify the HMG domain to stabilize the complex significantly. Subsequently recognizing the difficulty of introducing such constructs selectively into cancer cells, we modified the objective to synthesize platinum complexes that would, when bound to DNA, carry an intercalating arm that would cross from the major into the minor groove and fill the hydrophobic notch created opposite the 1,2-intrastand cross-link. Parallel studies will examine other structure specific recognition proteins (SSRPs) of functional significance. (c) To evaluate key cellular responses to the platinum drugs we focus on NER, the main pathway for removal of cisplatin-DNA cross-links. The interference of this process by HMG-domain md other cellular proteins, including nucleosome histone core proteins, w7ill be investigated. (d) To prepare site-specifically platinated DNA and investigate the ability of thc drug to inhibit transcription, key steps of which require SSRPs. Of particular interest is to evaluate transcription-coupled repair (TCR) as a major contributor to the death of the cancer cell following cisplatin treatment. Inhibition of transcription by unrepaired cisplatin-DNA adducts may trigger a cascade of events leading to cell death. Few details are available about this process. (e) To determine whether special DNA sequences, such as that in telomeres, could be targeted by the active platinum drugs. Since preliminary work indicated such not to be the case, we instead modified this aim and initiated a study of cisplatin-DNA adducts in nucleosomes. Our first goals are to prepare site-specifically platinated nucleosomes, investigate their processing by cellular proteins, and to determine the effect of core histone modification on the properties of the resulting complexes. (f) To understand cellular responses to platinum compounds in such a manner as to improve human cancer therapy. The success of cisplatin in treating testicular cancer is proposed to involve higher endogenous levels of HMG-domain proteins in these tumors, and this hypothesis will be evaluated. A Phase I Pilot Study at the DFCI combining steroid hormone with carboplatin or cisplatin treatment will allow our conjecture to be tested in a clinical setting. We seek to prepare and study new platinum complexes with appended agents to evoke better cellular response to the drug and promote selective uptake into tumor tissue. These compounds will be investigated against cancer cells in xenografts and human mammary tumor transplanitns mice.
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