Browsing by Subject "Adenosine Triphosphate"
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Item Aberrant DNA Methylation and Cancer: A Global Analysis of Promoter Hypermethylation in Human Lung Cancers(2006-12-20) Shames, David S.; Minna, John D.Tumor-acquired alterations in DNA methylation include both genome-wide hypomethylation and locus specific hypermethylation. Global loss of DNA methylation destabilizes chromatin architecture, augments genomic instability, and may reactivate repetitive element expression. Promoter hypermethylation often coincides with loss of heterozygosity at the same loci, and together these events can result in loss of function of the gene in tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers are unknown; however, it is known that certain genes are methylated with high frequency in selected tumors, whereas others are methylated across most types of tumors. The objective of the work described below was to use global profiling platforms (RNA and DNA) to identify epigenetically modulated genes that may be involved in cancer pathogenesis and bring these to the point where they could be developed as targets for diagnostic and treatment strategies. Using a global expression profiling approach and pharmacological inhibition of the DNA methyltransferases, 132 genes were identified that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine (5-aza) in multiple non-small cell lung cancer cell lines, and are expressed in untreated immortalized human bronchial epithelial cells. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (N=20) and adjacent non-malignant tissue showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. Promoter methylation of eight of these genes were studied in breast cancers (N=37), colon cancers (N=24), and prostate cancers (N=24) along with counterpart non-malignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. The data presented below suggest that while tumors differ in their molecular genetic phenotypes and pathogenesis, there may be underlying similarities in the pathways they follow toward malignancy. Some of these similarities may be reflected in the methylation programs tumor cells engage, which in turn, provides an opportunity to exploit for therapeutic applications and diagnosis. The approaches described herein entail a systematic and reproducible method to identify novel methylation markers in a variety of cancers, and the results of these studies provide a basis for developing a generic set of methylation markers for early detection screening across common epithelial cancers.Item Characterization of the Reaction Cycle of Mj0796: A Model Archaeal Adenosine Triphosphate- Binding Cassette Transporter Nucleotide Binding Domain(2006-12-20) Moody, Jonathan Edward; Thomas, Philip J.Adenosine-triphosphate binding cassette (ABC) transporters couple nucleotide hydrolysis to vectorial transport of solutes across lipid bilayers. These proteins, found in all kingdoms of life, have been implicated in a variety of human genetic disorders and engender drug resistance in cancer cells and infectious prokaryotes. Despite the widely varying solutes transported by these protein machines, a conserved functional mechanism is suggested by the high degree of amino acid conservation found in the nucleotide binding domains of all ABC transporters. Using two model archaeal ABC transporter nucleotide binding domains, MJ0796 and MJ1267, from Methanocaldococcus jannaschii the highly conserved Walker A, Walker B, and LSGGQ motifs were probed using site-directed mutagenesis. Catalytic carboxylate mutants, MJ0796-E171Q and MJ1267-E179Q, exhibited nucleotide-dependent dimerization upon analytical gel filtration and equilibrium centrifugation experiments. This self-association was negatively affected by changes in the electrostatic environment, as shown using alanine substitutions at these loci as well as altering the ionic conditions of the experiments. The MJ0796-E171Q protein was crystallized, and its structure solved to 1.9 angstrom resolution. The structure reveals an ATP sandwich dimer with two nucleotides bound at the dimeric interface, with each binding site composed of Walker A and B residues from one monomer and LSGGQ residues from the opposing monomer. A proposed reaction cycle based upon the MJ0796-E171Q dimer structure was probed using Walker A, Walker B, and LSGGQ point mutants. Mixtures of the Walker A mutant MJ0796-K44A with LSGGQ mutant MJ0796-S147F, both hydrolytically deficient in isolation, did not exhibit activity. In stark contrast, mixtures of MJ0796-S147F and MJ0796-E171Q did exhibit 25% wild type activity, suggesting a mechanism whereby two nucleotide binding events and a single hydrolysis event complete the minimal reaction cycle. This also suggests that during wild type hydrolysis, two nucleotides are hydrolyzed per cycle. These heterodimeric mutant mixtures were further analyzed using tryptophan fluorescence emission and anisotropy. Mixing experiments were performed using a full transporter system, the lipoprotein release machinery from Escherichia coli. A modified ABC transporter reaction cycle is presented.