1,721,029 research outputs found
Functional consequence of plasmid DNA modified site-specifically with 7-deaza-deoxyadenosine at a single, programmable site
No full textReplacement of a single dA nucleotide positioned at a programmed site in a DNA plasmid with its 7-deaza-analog is described together with its complete resistance to restriction enzymatic cleavage.National Cancer Institute (U.S.) (Grant CA34992
Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions
No full textFDA-approved platinum-based anticancer drugs, cisplatin, carboplatin, and oxaliplatin, are some of the most effective chemotherapies in clinical use. The cytotoxic action of these compounds against cancer requires a combination of processes including cell entry, drug activation, DNA binding, and transcription inhibition resulting in apoptotic cell death. The drugs form Pt lesions with nuclear DNA, leading to the arrest of key cellular functions and triggering a variety of cellular responses. DNA probes containing Pt−DNA conjugates are important tools for studying the molecular mechanisms of these processes. In order to facilitate investigation of specific Pt−DNA lesion processing within live cells, we devised a strategy for constructing plasmids containing a single site-specific Pt−DNA adduct. The method involves the use of nicking restriction enzymes to create closely spaced tandem gaps on the plasmid followed by removal of the intervening doubly nicked DNA strand to form a short single-stranded gap. Synthetic platinated oligonucleotides were incorporated into the gapped plasmid construct to generate a covalently closed circular platinated plasmid in good yield. We discuss the application of this methodology to prepare plasmids containing a platinum 1,2-d(G*pG*) or 1,3-d(G*pTpG*) intrastrand cross-link, two notable adducts formed by the three clinically approved drugs.National Cancer Institute (U.S.) (CA34992
Transcription Inhibition by Platinum DNA Cross-links in Live Mammalian Cells
No full textWe have investigated the processing of site-specific Pt−DNA cross-links in live mammalian cells to enhance our understanding of the mechanism of action of platinum-based anticancer drugs. The activity of platinum drugs against cancer is mediated by a combination of processes including cell entry, drug activation, DNA-binding, and transcription inhibition. These drugs bind nuclear DNA to form Pt−DNA cross-links, which arrest key cellular functions, including transcription, and trigger a variety of responses, such as repair. Mechanistic investigations into the processing of specific Pt−DNA cross-links are critical for understanding the effects of platinum−DNA damage, but conventional in vitro techniques do not adequately account for the complex and intricate environment within a live cell. With this limitation in mind, we developed a strategy to study platinum cross-links on plasmid DNAs transfected into live mammalian cells based on luciferase reporter vectors containing defined platinum−DNA lesions that are either globally or site-specifically incorporated. Using cells with either competent or deficient nucleotide excision repair systems, we demonstrate that Pt−DNA cross-links impede transcription by blocking passage of the RNA polymerase complex and that nucleotide excision repair can remove the block and restore transcription. Results are presented for 3800-base pair plasmids that are either globally platinated or carry a single 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand cross-link formed by either cis-{Pt(NH3)2}2+ or cis-{Pt(R,R-dach)}2+, where {Pt(NH3)2}2+ is the platinum unit conveyed by cisplatin and carboplatin and R,R-dach is the oxaliplatin ligand, R,R-1,2-diaminocyclohexane.National Cancer Institute (U.S.) (grant CA032134)National University of Singapore (Overseas Postdoctoral Fellowship
Monofunctional Platinum-DNA Adducts Are Strong Inhibitors of Transcription and Substrates for Nucleotide Excision Repair in Live Mammalian Cells
Full text linkTo overcome drug resistance and reduce the side effects of cisplatin, a widely used antineoplastic agent, major efforts have been made to develop next generation platinum-based anticancer drugs. Because cisplatin–DNA adducts block RNA polymerase II unless removed by transcription-coupled excision repair, compounds that react similarly but elude repair are desirable. The monofunctional platinum agent pyriplatin displays antitumor activity in mice, a cytotoxicity profile in cell cultures distinct from that of cisplatin, and a unique in vitro transcription inhibition mechanism. In this study, we incorporated pyriplatin globally or site specifically into luciferase reporter vectors to examine its transcription inhibition profiles in live mammalian cells. Monofunctional pyriplatin reacted with plasmid DNA as efficiently as bifunctional cisplatin and inhibited transcription as strongly as cisplatin in various mammalian cells. Using repair-defective nucleotide excision repair (NER)-, mismatch repair-, and single-strand break repair–deficient cells, we show that NER is mainly responsible for removal of pyriplatin–DNA adducts. These findings reveal that the mechanism by which pyriplatin generates its antitumor activity is very similar to that of cisplatin, despite the chemically different nature of their DNA adducts, further supporting a role for monofunctional platinum anticancer agents in human cancer therapy. This information also provides support for the validity of the proposed mechanism of action of cisplatin and provides a rational basis for the design of more potent platinum anticancer drug candidates using a monofunctional DNA-damaging strategy.National Cancer Institute (U.S.) (Grant Number CA034992
TOOLS FOR INVESTIGATING CELLULAR RESPONSES TO OXIDATIVE STRESS ARISING FROM THE INDUCTION OF REACTIVE OXYGEN SPECIES IN THE INTRACELLULAR ENVIRONMENT
No full textPh.DDOCTOR OF PHILOSOPHY (FOS
INVESTIGATION OF ASYMMETRIC PLATINUM (IV) COMPLEXES AS ANTICANCER PRODRUGS
Full text linkPh.DDOCTOR OF PHILOSOPH
RETHINKING PLATINUM ANTICANCER DRUG DESIGN: TOWARDS TARGETED AND IMMUNO-CHEMOTHERAPEUTIC APPROACHES
Full text linkPh.DDOCTOR OF PHILOSOPH
NOVEL FLUORESCENT PROBES FOR THE INVESTIGATION OF PLATINUM-BASED ANTICANCER COMPLEXES AND REACTIVE OXYGEN/NITROGEN SPECIES IN CELLS
No full textPh.DDOCTOR OF PHILOSOPH
MECHANISTIC INSIGHTS INTO PLATINUM (IV) PRODRUG COMPLEXES OF CISPLATIN FOR CANCER THERAPY
Full text linkPh.DDOCTOR OF PHILOSOPH
Role of endonucleases XPF and XPG in nucleotide excision repair of platinated DNA and cisplatin/oxaliplatin cytotoxicity
Full text linkResistance of tumor cells to platinum anticancer agents poses a major problem in cancer chemotherapy. One of the mechanisms associated with platinum-based drug resistance is the enhanced capacity of the cell to carry out nucleotide excision repair (NER) on platinum-damaged DNA. Endonucleases XPF and XPG are critical components of NER, responsible for excising the damaged DNA strand to remove the DNA lesion. Here, we investigated possible consequences of down-regulation of XPF and XPG gene expression in osteosarcoma cancer cells (U2OS) and the impact on cellular transcription and DNA repair. We further evaluated the sensitivity of such cells toward the platinum anticancer drugs cisplatin and oxaliplatin.National Cancer Institute (U.S.) (Grant Number CA034992.)National University of Singapore.German Academic Exchange Service (DAAD
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