99 research outputs found

    Mechanism of action studies on the FR-9000482 class of antitumor antibiotics

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    1997 Summer.Includes bibliographical references.The interactions of members of the FR-900482 class of antitumor antibiotic agents with DNA has been examined. Importantly, the first in vitro demonstration of nucleic acid interstrand cross-linking has been reported and the DNA base pair sequence specificity of the cross-linking event has been elucidated. These agents demonstrate a high degree of selectivity for 5'-CG-3' sequences of DNA. As such, bio-mechanistic analogy between these compounds and the clinically employed compound Mitomycin Chas been shown. Efforts have also examined extensively the ability of these agents to give rise to orientation isomers of each respective cross-link and their different properties. DNA-protein cross-linking by these agents has also been examined. A sequence-specific DNA-peptide binding motif has been identified which undergoes drug-mediated DNA-protein cross-linking. This is the first reported instance of a mitosene based-minor groove DNA-protein cross-link event. Significantly, the motif examined is characteristic of tissues which bear striking similarity to those of cancerous cell lines

    A Sensitive High Performance Liquid Chromatography (HPLC) Assay for the Quantification of Doxorubicin Bound to DNA

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    Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of intracellular active form of doxorubicin. Thus, we have developed a high performance liquid chromatography (HPLC) methodology in order to quantify the concentrations of doxorubicin that are bound to DNA in tumors and tissues as an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate bound drug; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2 um, 2.0 x 100 mm) analytical column; and a gradient mobile phase of 0.1% formic acid in water and acetonitrile. The assay has a lower limit of quantification (LLOQ) of 10 ng/mL and is shown to be linear up to 3,000 ng/mL. We demonstrated the suitability of this assay for doxorubicin bound to DNA in vivo by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72 hours after administering PEGylated liposomal doxorubicin (Doxil®; PLD) IV at 6 mg/kg. This HPLC assay allows for a sensitive and simple intracellular quantification of doxorubicin as compared to other methods and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle carriers.Doctor of Philosoph

    DNA Damage Recognition Proteins and Their Involvement in Cisplatin Resistance

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    cis-Diamminedichloroplatinum(II) (CDDP) is a chemotherapeutic agent widely used in the treatment of various types of cancer. Its mechanism of cytotoxicity is unclear although it is believed that DNA is the critical target. CDDP binds to DNA forming a variety of adducts including intrastrand adducts, interstrand adducts, monofunctional adducts and DNA-protein crosslinks. This thesis presents evidence that there are protein(s) present in mammalian cells which recognise CDDP-damaged DNA, To assay for these DNA damage recognition proteins (DDRPs) conditions for two very separate assays were developed. The gel mobility shift assay, which detects protein complexes under non-denaturing conditions, identified two retardation complexes which bound to CDDP damaged DNA in human, murine and feline tumour cell extracts. Binding of these complexes is shown to CDDP treated oligonucleotide of 54 base pairs but not to a CDDP treated oligonucleotide of 27 base pairs, therefore suggesting binding is dependent on having normal DNA duplex. The other system used in the detection of the DDRPs is the South-Western assay. This allowed the detection of proteins of sizes 25, 50, 100KD binding to CDDP treated DNA. The proteins in the South-Western system are run under denaturing conditions. It is not entirely clear as to whether the proteins detected in both systems are the same or whether they represent entirely different species. CDDP has been reported to bind to DNA and cause areas of singlestrandedness around the adducts. The results presented in this thesis demonstrate that the 50KD and 100KD DDRP which bind to CDDP treated double-stranded DNA may also have an affinity for single-stranded DNA. The 25KD DDRP, however, only recognises double-stranded DNA treated with CDDP suggesting that it is recognising the CDDP adducts and not the areas of single-strandedness generated around the adducts. Resistance to CDDP proves a major problem area in treatment regimes. Many cell lines resistant to CDDP have been derived in vitro by multiple exposures to the drug. Many mechanisms of resistance to CDDP have been suggested from these lines. If a role of the DDRPs was to process damage in the DNA then cell lines resistant to CDDP may show an increase in expression of the DDRPs. This thesis presents evidence that an ovarian tumour cell line resistant to CDDP in comparison to its parental line shows an increase in the binding to the 50KD and 100KD DDRPs. Work in chapter 5 presents the isolation of CDDP resistant cell lines, by acute exposure to the drug, with an increase of up to seven fold resistance levels. Evidence is presented for the resistant clones being of a mutational origin. Resistant variants occur at a frequency of 3.2x10e-6 per viable cell. This frequency can be increased to 3.4x10e-5 by treatment of the cells with the chemical mutagen ethyl methane sulphonate, EMS. The CDDP resistant phenotype is maintained after six months growth in drug free medium. This single step selection may provide clones which are more clinically relevant than the lines isolated by multiple exposures to CDDP. They may therefore provide a superior model for the study of drug resistance mechanisms to CDDP. However examination of the DDRPs showed no detectable difference in the resistant clones derived from the A2780 human ovarian tumour cell line. The thesis therefore presents evidence of the existence of DDRPs in mammalian cells. The role of these damage recognition proteins will be discussed

    Molecular and cellular pharmacology of novel pyrrolo [2,1-c] [1,4] diazepine-based anticancer agents.

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    The pyrrolo 2,1-c 1,benzodiazepines (PBDs) are a family of naturally occurring antitumour antibiotics which includes anthramycin, DC-81, tomaymycin and sibiromycin. They exert their biological activity through covalent binding to the exocyclic N2 group of guanine in the minor groove of DNA and block transcription in a sequence-specific manner. These PBD monomers span three DNA base pairs and have a preference for binding to purine-G-purine triplets. The PBDs have been used as a scaffold to attach other moieties, leading to novel sequence-selective DNA minor groove alkylating agents. In addition, as part of a rational approach to producing more efficient and selective DNA interstrand crosslinking agents, two PBD monomers have been linked together to form PBD dimers. The research in this thesis is a study of the molecular and cellular pharmacology of several series of novel PBD-containing agents including novel PBD dimers with different linker lengths, PBD-nitrogen mustard conjugates, PBD-polyamide conjugates and C2-aryl PBD monomers. Cytotoxicity in human tumour cell lines, efficiency of DNA interstrand crosslinking in naked linear plasmid DNA, and DNA sequence specificity were assessed. DNA interstrand crosslink formation and repair in cells were also measured. Resulting from this work Q2lQT-exo-unsaturated PBD dimers have been characterised to be highly cytotoxic and efficient in producing interstrand crosslinks both in naked DNA and in cells that are not repaired up to 48 hours. Only two of the PBD-nitrogen mustard conjugates showed some interaction with DNA although several members of this group showed significant cytotoxicity. A PBD-tri-pyrrole conjugate was found to bind preferentially to the sequence 5'-AGATTATC. Novel C2-aryl PBD monomers were shown to bind selectively to 5'-purine-G-purine sequences and demonstrated significant cytotoxicity. In addition, a method utilizing fluorescently end-labelled oligonucleotides was developed and validated to screen libraries of PBD-containing molecules synthesised on beads by combinatorial chemistry. This method allowed the isolation and discrimination of beads containing compounds, which have a high affinity for specific DNA sequences

    The role of base excision repair proteins in the cellular responses to the anticancer drug cisplatin

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    Thesis (Ph.D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2000.Includes bibliographical references.by Maria Kartalou.Ph.D

    Ruthenium (II) complexes as potential chemotherapeutic agents

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